SCIENTIFIC / TECHNICAL REPORT submitted to EFSA
Defining Environmental Risk Assessment Criteria for Genetically Modified
Insects to be placed on the EU Market1
Prepared by
Mark Benedicta, Michael Eckerstorfera, Gerald Franzb, Helmut Gaugitscha,
Anita Greitera, Andreas Heissenbergera, Bart Knolsa, Sabrina Kumschickc,
Wolfgang Nentwigc and Wolfgang Rabitscha
a
Environment Agency Austria
b
International Atomic Energy Agency
c
University of Bern
1
CT/EFSA/GMO/2009/03. Accepted for Publication on 10 September 2010
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Abstract
Global efforts towards the development of genetically modified (GM) arthropods have
progressed to a stage where some might possibly be placed on the EU market within the next
decade. Risk assessment issues, therefore, need to be addressed, and adequate and
comprehensive risk assessment guidelines need to be developed.
This report first describes the ongoing developments in the field of GM-arthropods
(transformed species, development purposes, and construction of GM-arthropods), and
subsequently identifies potential adverse effects as well as methods to investigate these.
Crucial arthropod characteristics and necessary baseline information are discussed, and the
surrogate and modelling approach evaluated for their usefulness regarding the environmental
risk assessment (ERA) of GM-arthropods. Expertise needed is presented in terms of scientific
disciplines, expertise fields, research institutes and individual experts.
It is concluded that the ERA of GM-arthropods should consider various issues regarding the
genetic modification, the respective species and the receiving environment. Potential risks
could be identified concerning gene flow and its consequences, effects on target and nontarget organisms, management practices and measures, biogeochemical processes and human
health. Since potential risks depend on the method used for modification, the purpose of the
GM-arthropod and the species itself, it is recommended to follow a case-by-case approach for
the ERA of GM-arthropods.
2
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Summary
This study was carried out based on the technical specifications set out in the tender
CFT/EFSA/GMO/2009/01 of the European Food Safety Authority (EFSA). The aim of this
report is to provide background information in the area of risk assessment of genetically
modified (GM) arthropods which are possibly to be placed on the market in the European
Union (EU) within the next decade. In order to achieve the objectives of the study, a thorough
collection of data on already existing GM-arthropods, as well as onarthropods that might be
modified and possibly released in the near future was conducted. This exercise also included
already available comparable conventional applications, like the sterile insect technique (SIT).
There are several different purposes for which the genetic modification of arthropods can be
used. For some of these purposes, developments and possible applications are already
available, such as the production of certain organic compounds using silk worms, or genetic
modification for scientific purposes (Drosophila sp.). However, these applications do only
take place in contained facilities (e.g. laboratory). Other applications include the control of
infectious diseases through population replacements with GM-arthropods which no longer
have the ability of transmitting diseases. This technology is only at an early stage of
development and no studies are available concerning possible hazards, which might be caused
by a release. The use of beneficial arthropods as biocontrol agents is also discussed.
The main application of GM-arthropods to be released is pest control via population
suppression or elimination. The use of SIT, by sterilising insects using radiation, has a long
history with a vast body of experience, especially with fruit flies. This technology and the
"Release of Insects carrying a Dominant Lethal" (RIDL) are the most advanced applications
that use GM-insects. The genetic modifications used cover marker genes for sexing in the
production of sterile insects or efficiency control after the release, as well as modifications
causing sterility or conditional lethality.
GM-arthropods of possible relevance for the EU in the next 10 years were identified and
described in detail. Ten species were considered for the identification of possible hazards of
GM-arthropods in connection with their likely transformation and application for the
environmental risk assessment (ERA). Possible effects were described and methods for their
investigation summarised. In principle, these hazards were gene transfer (vertical and
horizontal including their consequences), effects on target organisms (triggering adaptive
processes, population elimination or host range changes), effects on non-target organisms
(predators and symbionts), and effects on biodiversity, pollination and biogeochemical
processes (such as decomposition). Impacts on management practices and measures, and
effects on human health were also discussed. All these possible effects were evaluated
regarding their severity and likelihood to pose a risk.
For the identification of assessment endpoints and methodologies, key parameters of GMarthropods were defined and described. Methods for their assessment were proposed and
evaluated using SWOT analysis. Necessary baseline information regarding habitat type or the
ecology of the GM-arthropod is proposed. The usefulness of the surrogate and modelling
approach in the ERA of GM-arthropods are discussed.
3
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
With four case study species, i.e. the mosquitoes Aedes aegypti and Aedes albopictus, and the
fruit flies Ceratitis capitata and Bactrocera oleae, the analysis of ERA aspects is summarised
and presented, exploring the most important aspects and issues to be considered in the ERA of
GM-arthropods.
In addition, scientific disciplines and fields of expertise that might feed an ERA of GMarthropds are presented and described. Also existing expertise on this issue was collected
(literature as well as individual professionals and research institutes) and presented in two
databases supplementing this report: one containing relevant literature and the other
containing names and contact details of experts and institutions, that may provide input in the
development of ERA guidance of GM-arthropods or who can actually be involved in the ERA
themselves. Both databases are provided by the contractor to EFSA in electronic format.
Key words: insect, arthropod, transgenic, genetically modified, environmental risk
assessment, European Union, GM, EU, ERA
4
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table of contents
Abstract .................................................................................................................................................... 2 Summary .................................................................................................................................................. 3 Table of contents ...................................................................................................................................... 5 Background .............................................................................................................................................. 9 Terms of reference.................................................................................................................................. 11 Acknowledgements ................................................................................................................................ 13 Introduction and objectives .................................................................................................................... 14 Methods .................................................................................................................................................. 15 1. Information search ......................................................................................................................... 15 1.1. Expert interviews .................................................................................................................. 15 1.2. Internet and citation databases .............................................................................................. 16 2. Selection of GM-arthropods .......................................................................................................... 17 3. SWOT Analysis ............................................................................................................................. 18 GM-arthropods ....................................................................................................................................... 19 4. Definition of GM-arthropod .......................................................................................................... 19 5. Purpose of GM-arthropods ............................................................................................................ 20 5.1. Production of products of interest ......................................................................................... 20 5.2. Population replacement and incapacitating vectors .............................................................. 20 5.3. Population suppression, containment or eradication ............................................................. 22 5.3.1. Sterile Insect Technique (SIT) .......................................................................................... 22 5.3.2. Release of Insects carrying a Dominant Lethal (RIDL) ................................................... 25 5.4. GM-arthropod applications employing RNAi based immunity
to vectored disease agents .................................................................................................... 26 5.5. Beneficial arthropods as biological control agents ............................................................... 27 5.6. Other applications ................................................................................................................. 28 6. Construction of GM-arthropods .................................................................................................... 29 6.1. The transformation system .................................................................................................... 29 6.2. Principal molecular components ........................................................................................... 31 6.3. Transgenic strains ................................................................................................................. 33 6.3.1. Genetic sexing strains ....................................................................................................... 33 6.3.2. Marker strains ................................................................................................................... 36 6.3.3. Conditional lethality strains .............................................................................................. 36 6.4. Common features of the described applications ................................................................... 36 7. GM-arthropods worldwide ............................................................................................................ 37 7.1. Coleoptera ............................................................................................................................. 43 7.1.1. Tribolium castaneum (Red flour beetle) ........................................................................... 43 7.2. Diptera (Culicidae)................................................................................................................ 43 7.2.1. Aedes aegypti (Yellow fever mosquito) ........................................................................... 43 7.2.2. Aedes albopictus (Asian tiger mosquito) .......................................................................... 44 7.2.3. Aedes fluviatilis................................................................................................................. 45 7.2.4. Anopheles albimanus (New world malaria mosquito) ...................................................... 45 7.2.5. Anopheles arabiensis ........................................................................................................ 46 7.2.6. Anopheles gambiae s.s. (African malaria mosquito) ........................................................ 46 7.2.7. Anopheles stephensi (Indo-Pakistan malaria mosquito) ................................................... 47 7.2.8. Culex quinquefasciatus (Southern house mosquito)......................................................... 47 7.3. Diptera (Tephritidae) ............................................................................................................ 48 7.3.1. Anastrepha ludens (Mexican fruit fly) ............................................................................. 48 7.3.2. Anastrepha suspensa (Caribbean fruit fly) ....................................................................... 48 7.3.3. Bactrocera dorsalis (Oriental fruit fly) ............................................................................ 48 7.3.4. Bactrocera oleae (Olive fruit fly) ..................................................................................... 49 5
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
7.3.5. Bactrocera tryoni (Queensland fruit fly) .......................................................................... 50 7.3.6. Ceratitis capitata (Mediterranean fruit fly) ...................................................................... 50 7.4. Diptera (other) ....................................................................................................................... 51 7.4.1. Cochliomyia hominivorax (New world screwworm; Calliphoridae) ................................ 51 7.4.2. Drosophila spp. (Fruit flies; Drosophilidae) .................................................................... 51 7.4.3. Lucilia cuprina (Green bottle fly; Calliphoridae) ............................................................. 52 7.4.4. Musca domestica (House fly; Muscidae) ......................................................................... 52 7.4.5. Stomoxys calcitrans (Stable fly; Muscidae) ..................................................................... 53 7.5. Hymenoptera ......................................................................................................................... 53 7.5.1. Apis mellifera (Honey bee) ............................................................................................... 53 7.5.2. Athalia rosae (Turnip sawfly) .......................................................................................... 53 7.6. Lepidoptera ........................................................................................................................... 54 7.6.1. Bicyclus anynana (Squinting bush brown) ....................................................................... 54 7.6.2. Bombyx mori (Silk moth) ................................................................................................. 54 7.6.3. Cydia pomonella (Codling moth) ..................................................................................... 54 7.6.4. Pectinophora gossypiella (Cotton pink bollworm) .......................................................... 55 7.7. Acari...................................................................................................................................... 55 7.7.1. Metaseiulus occidentalis (Western predatory mite) ......................................................... 55 7.8. Crustacea ............................................................................................................................... 56 7.8.1. Parhyale hawaiensis ......................................................................................................... 56 7.8.2. Procambarus clarkii (North American crayfish).............................................................. 56 8. GM-arthropods of possible relevance for the EU in the next 10 years .......................................... 56 8.1. Aedes aegypti (Yellow fever mosquito) ................................................................................ 62 8.2. Aedes albopictus (Asian tiger mosquito) .............................................................................. 64 8.3. Anopheles gambiae species complex: A. arabiensis and A. gambiae s.s. ............................. 67 8.4. Bactrocera oleae (Olive fruit fly) ......................................................................................... 69 8.5. Ceratitis capitata (Mediterranean fruit fly) .......................................................................... 71 8.6. Lucilia cuprina (Green bottle fly) ......................................................................................... 73 8.7. Stomoxys calcitrans (Stable fly) ........................................................................................... 74 8.8. Cydia pomonella (Codling moth) ......................................................................................... 76 8.9. Pectinophora gossypiella (Cotton pink bollworm) ............................................................... 77 Risk assessment of GM-arthropods ........................................................................................................ 79 9. Specific areas of potential risk to be addressed in the ERA of GM-arthropods ............................ 79 9.1. Adverse effects associated with gene flow ........................................................................... 81 9.1.1. Vertical gene flow to populations of the same or sexually compatible species................ 81 9.1.2. Horizontal gene transfer ................................................................................................... 85 9.2. Interactions of the GM-arthropod with the target organisms ................................................ 88 9.2.1. Triggering adaptive processes in the target population .................................................... 88 9.2.2. Host range ......................................................................................................................... 89 9.3. Interactions of the GM-arthropod with non-target organisms .............................................. 90 9.3.1. Effects on predators and parasitoids ................................................................................. 91 9.3.2. Biodiversity ...................................................................................................................... 92 9.3.3. Pollination......................................................................................................................... 93 9.4. Impact on specific agricultural management practices and management
measures to control arthropods vectoring diseases .............................................................. 94 9.5. Effects on biogeochemical processes .................................................................................... 96 9.6. Effects on human health........................................................................................................ 97 10. Methods to investigate adverse effects of GM-arthropods........................................................ 99 10.1. Vertical gene flow ................................................................................................................. 99 10.2. Host range ........................................................................................................................... 100 6
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
10.3. Symbionts ........................................................................................................................... 101 10.4. Predation ............................................................................................................................. 101 10.5. Biodiversity ......................................................................................................................... 102 10.6. Pollination ........................................................................................................................... 102 10.7. Water bodies ....................................................................................................................... 102 10.8. Soil / decomposition ........................................................................................................... 102 11. Keyparameters for the ERA of GM-arthropods ...................................................................... 103 12. Methods for the assessment of key parameters ....................................................................... 105 12.1.1. Fertility rate .................................................................................................................... 107 12.1.2. Mating competitiveness .................................................................................................. 107 12.1.3. Longevity ........................................................................................................................ 108 12.1.4. Development time........................................................................................................... 109 12.1.5. Egg hatching rate ............................................................................................................ 110 12.1.6. Larval survival ................................................................................................................ 110 12.1.7. Pupal survival ................................................................................................................. 110 12.1.8. Adult emergence ............................................................................................................. 111 12.1.9. Size/weight ..................................................................................................................... 111 12.1.10. Flight ability ................................................................................................................... 111 12.1.11. Altered biochemistry ...................................................................................................... 112 12.1.12. Abiotic stress resistance.................................................................................................. 113 12.1.13. Vector competence ......................................................................................................... 114 12.1.14. Arthropod density ........................................................................................................... 114 12.1.15. Migration behaviour ....................................................................................................... 116 12.1.16. Habitat interactions ......................................................................................................... 117 12.1.17. Climate interactions ........................................................................................................ 117 12.1.18. Food interactions ............................................................................................................ 118 12.1.19. Altered host range ........................................................................................................... 118 12.1.20. Sensitivity to insect pathogens........................................................................................ 119 12.1.21. Predator interactions ....................................................................................................... 120 12.1.22. Insecticide resistance ...................................................................................................... 120 13. Baseline Information ............................................................................................................... 121 13.1. Habitat type ......................................................................................................................... 121 13.2. Ecology of GM-arthropods within their habitat .................................................................. 122 13.3. Baseline data ....................................................................................................................... 123 14. Surrogate Approach................................................................................................................. 124 15. Modelling Approach ............................................................................................................... 126 16. Implications for the implementation of an ERA of GM-arthropods ....................................... 129 16.1. Summarised analysis of ERA aspects ................................................................................. 130 16.2. Issues to be considered in an ERA of GM-arthropods ........................................................ 136 Expertise for ERA of GM-arthropods .................................................................................................. 138 17. Scientific Disciplines and fields of expertise .......................................................................... 138 17.1. Biology................................................................................................................................ 140 17.2. Molecular biology ............................................................................................................... 142 17.3. Genetics............................................................................................................................... 142 17.4. Entomology ......................................................................................................................... 143 17.5. Ecology ............................................................................................................................... 143 17.6. Chemistry ............................................................................................................................ 145 17.7. Medicine ............................................................................................................................. 145 17.8. Agricultural science ............................................................................................................ 146 17.9. Toxicology .......................................................................................................................... 146 7
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
17.10. Statistics/Informatics........................................................................................................... 146 17.11. Biosafety ............................................................................................................................. 147 17.12. Others .................................................................................................................................. 147 18. Research institutes and academics .......................................................................................... 148 18.1. Publication Database ........................................................................................................... 148 18.2. Experts and Institutions Database ....................................................................................... 148 Cross-cutting considerations ................................................................................................................ 153 19. Paratransgenesis ...................................................................................................................... 153 Conclusions .......................................................................................................................................... 155 References ............................................................................................................................................ 158 Appendices ........................................................................................................................................... 183 Abbreviations ....................................................................................................................................... 200 8
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Background
In the field of genetically modified organisms (GMOs), the European Food Safety Authority
(EFSA) provides information to applicants in the form of scientific opinions on the safety of
GMO market registration applications, and produces guidance documents for the risk
assessment of GMOs. These guidance documents guide and assist applicants in the
preparation and presentation of GMO market registration applications submitted under
Regulation (EC) No 1829/2003 on GM food and feed, and Directive 2001/18/EC on the
deliberate release into the environment of GMOs.
These guidance documents are available on-line and contain scientific and technical support
for the risk assessment (RA) of:
−
GM-plants and derived food and feed (EFSA 2006b),
−
GM-plants containing stacked transformation events (EFSA 2007),
−
GM-microorganisms and their derived products intended for food and feed use (EFSA
2006a).
Also scientific opinions of the EFSA panel on genetically modified organisms (EFSA GMO
panel) are provided on the world-wide-web e.g.:
−
Statistical considerations for the safety evaluations of GMOs (EFSA 2010a)
−
Scientific opinion on guidance for the risk assessment of GM-plants used for non-food
or non-feed purposes (EFSA 2009)
In February 2007, the European Commission asked EFSA to produce a guideline on the safety
evaluation of GM-animals, addressing both aspects of environmental and food/feed safety.
To follow-up on the European Commission’s request and taking into account ongoing
international scientific progress on the subject, EFSA decided to address in parallel both
aspects of the environmental safety and the safety assessment of food and feed products
derived from GM-animals. The present report deals with the first aspect.
EFSA intended to focus on environmental safety aspects related to the placing on the EU
market of GM-animals and contracted studies on GM-fish, GM-insects and GM-mammals
and -birds on the definition of environmental risk assessment criteria. For GM-insects the
results are delivered in the form of a report by a consortium led by Umweltbundesamt
(Environment Agency Austria). Environment Agency Austria formed a consortium with the
University of Bern (Switzerland) and included the International Atomic Energy Agency
(IAEA) in the project team.
Subsequently, this report will be used as a background document by the EFSA working group
and the EFSA GMO panel.
In line with the technical specifications of the tender call, the present report provides
information on scientific disciplines and fields of expertise that might feed an ERA of GMinsects to be commercially released into the EU environment and on research institutes and
professionals having expertise on the subject. Also potential hazards are identified and
information on relevant criteria to be considered when performing an ERA of GM-insects
9
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
provided. Assessment endpoints and methodologies are identified and evaluated and crosscutting issues as well as knowledge gaps considered.
The project team was composed of Mark Benedict, Michael Eckerstorfer, Helmut Gaugitsch,
Anita Greiter, Andreas Heissenberger, Bart Knols, Wolfgang Rabitsch (all Environment
Agency Austria), Sabrina Kumschick, Wolfgang Nentwig (both University of Bern) and
Gerald Franz (IAEA). The project team was completed by a review panel that provided
additional scientific input and consisted of Eliana Fontes (Embrapa – Brazilian Enterprise for
Agricultural Research), Fred Gould (North Carolina State University) and Gabor Lövei
(Aarhus University).
10
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Terms of reference
In accordance with the scope of the project “Defining environmental risk assessment criteria
for genetically modified insects to be placed on the EU market” (project ID
CFT/EFSA/GMO/2009/01) environmental risk assessment criteria were defined in the form
of a report and cover the following points:
Collecting information on relevant scientific disciplines and fields of expertise and on
research institutes and academics:
−
Task 1.1: Definition of scientific disciplines and fields of expertise that might feed an
environmental risk assessment of GM-insects to be commercially released into the
environment (see chapter 17).
−
Task 1.2: Collection of information on research institutes and academics having
expertise in the scientific disciplines and fields identified (see chapter 18).
Identification of potential hazards and their associated exposures:
−
Task 2.1: List of GM-insects that could be commercially released into the environment
in the EU in the nearby future (see chapter 8).
−
Task 2.2: List and characterisation of the types of relevant receiving environments
across the EU where those GM-insects might be released (see chapter 8).
−
Task 2.3: Identification of all potential adverse effects to the biotic and abiotic
environment ensuing from the commercial release into the environment of those GMinsects (see chapter 9).
−
Task 2.4: Discussion of the consequences of each potential adverse effect identified
for all insect-trait-environment combinations listed (see chapter 9).
−
Task 2.5: Discussion of the likelihood of occurrence of each potential adverse effect
for the insect-trait-environment combinations (see chapter 9).
−
Task 2.6: Review of experimental procedures, approaches and methodologies used to
study the environmental consequences and the likelihood of occurrence of the adverse
effects discussed (see chapter 10).
−
Task 2.7: Extraction from the deliverables obtained relevant criteria to be considered
when performing an environmental risk assessment of GM-insects to be commercially
released into the environments (see chapter 16.2).
Identification of assessment endpoints and methodology:
−
Task 3.1: Identification of crucial insect characteristics at different life-history stages,
environmental variables and genotype-environment interactions to be considered when
evaluating the net survival and reproductive fitness, spread, migrations, persistence
and invasiveness of GM-insects, as well as ecological interactions with other
organisms (see chapter 11).
−
Task 3.2: Assessment of the experimental designs, approaches and methodologies
used for generating empirical information on the survival and reproductive fitness,
11
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
spread, migration, persistence and invasiveness of GM-insects as well as ecological
interactions with other organisms (see chapter 12).
−
Task 3.3: Assessment of using surrogate non-GM-insects with similar phenotypic
characteristics than those of GM-insects for generating empirical information on the
survival and reproductive fitness, spread, migration, persistence and invasiveness of
GM-insects, as well as on ecological interactions with other organisms (see chapter
14).
−
Task 3.4: Assessment of prospective models for predicting the survival and
reproductive fitness, spread, migration, persistence and invasiveness of GM-insects, as
well as their interactions with other organisms and for extrapolating risk estimates for
phenotypes tested in laboratory and/or semi-natural environments both to other
phenotypes and more complex and multiple environments (see chapter 15).
−
Task 3.5: Identification of the kinds of baseline information about receiving
environments that are to be considered when conducting an environmental risk
assessment of commercially released GM-insects (see chapter 13).
−
Task 3.6: Extraction from the deliverables obtained relevant criteria to be considered
when performing an environmental risk assessment of GM-insects commercially
released into the environment (see chapter 16.2).
The report will serve as a basis for guidance on the environmental risk assessment of GManimals, to be prepared by an EFSA working group and the GMO Panel of EFSA.
12
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Acknowledgements
This contract was awarded by EFSA to: Environment Agency Austria as contractor.
Environment Agency Austria acts as lead partner of a consortium together with the University
of Bern.
Contract title: Defining Environmental Risk Assessment Criteria for Genetically Modified
Insects to be placed on the EU Market.
Contract number: CT/EFSA/GMO/2009/03.
The project team wants to thank the members of the review panel - Eliana Fontes, Fred Gould
and Gabor Lövei - as well as the members of the EFSA steering committee - Sharon Cheek,
Yann Devos, Jozsef Kiss, Nancy Podevin and Elisabeth Waigmann - for their input and
helpful suggestions. It also thanks all the experts who replied to the questionnaire which was
distributed in the course of the project to renowned scientists to assess the current knowledge
in relevant fields of expertise. Input was provided by Nidchaya Aketarawong, Luke Alphey,
David Andow, Kate Aultman, Jeff Bale, Detlef Bartsch, Camilla Beech, Romeo Bellini,
Christophe Boëte, Kostas Bourtzis, Ray Cannon, Lim Li Ching, Jenny Cotter, Andrea
Crisanti, Paul De Barro, Paul Eggleston, Guido Fava, Heather Ferguson, Eliana Fontes, Fred
Gould, Pierre-Henry Gouyon, Alfred Handler, David Heron, Hans Herren, Angelika Hilbeck,
Marjorie Hoy, Andrea Hubery, Anthony James, Stephanie James, Katia Komitopoulou, PaulHenning Krogh, Frantisek Marec, Thomas Miller, John Mumford, Javaregowda Nagaraju,
Stefan Rauschen, Guy Reeves, Paul Reiter, Robert Rose, Thomas Scott, Frantisek Sehnal,
Beverly Simmons, Steve Sinkins, Willem Takken, Sujinda Thanaphum, Yeya Toure, John
Turner and Ernst Wimmer.
13
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Introduction and objectives
INTRODUCTION
The study was carried out following EFSA tender CFT/EFSA/GMO/2009/01. The aim of this
report is to provide a scientific basis for the RA of GM-arthropods which may be placed on
the market in the EU within the next decade. The tender was based on a request by the
European Commission to EFSA to produce a guideline for the safety assessment of GManimals. This report deals with one group of GM-animals, namely GM-arthropods, and covers
the topics as defined in the terms of reference (see above).
This report consists of three parts:
1.
The written report including the collection of scientific information on GMarthropods, possible hazards, and assessment methodology.
2.
A literature database
3.
A database containing information on relevant experts and institutions.
The latter two are submitted to EFSA in an electronic format.
The report contains information on current development of GM-arthropods, their possible
receiving environment and applications, which may be expected for the EU. In addition, some
cross-cutting considerations are included.
Besides the description of the GM-technology, of the possibilities regarding the application of
GM-arthropods and of the biology of the relevant species, the main possible adverse effects
are identified. The report is completed by discussing assessment endpoints and methodology
and issues to be considered for the ERA of GM-arthropods.
OBJECTIVES
Overall objective
To define criteria for ERA of GM-arthropods, which may be placed on the market in the EU
within the next decade, and to provide a basis for the development of a guidance document for
the RA of GM-arthropods.
Specific objectives
To collect information on current knowledge on GM-arthropods, their development and
practical application, and to set-up a collection of information on relevant fields of expertise
and experts, which could be involved in the RA of these GM-arthropods.
To identify potential hazards, and to discuss the likelihood of their occurrence and possible
consequences as well as to collect information on methods to study possible effects of GMarthropods on the environment.
To identify assessment endpoints, and to collect information on assessment methodologies.
14
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Methods
In the following chapters methods used to collate the information presented in this report are
described.
1.
Information search
In order to provide comprehensive information on different issues serving as the basis for
various chapters of this report (collection of information on scientific disciplines and fields of
expertise, research institutes and academics as well as on GM-arthropods and relevant
literature), the knowledge of the project team was augmented with first hand information
provided by the members of the review panel and a number of renowned experts. In addition a
thorough search of internet resources and scientific literature was undertaken. Both
approaches are complementary, thereby ensuring the presentation of exhaustive information
and the reflection of state-of-the-art scientific and technical information from multiple
research disciplines.
1.1.
Expert interviews
Excessive information can be found in scientific databases and on the internet, and many
experts are currently involved in research on GM-arthropods. To condense the data available,
it was necessary to use an efficient and effective approach and to focus on the most important
and most relevant institutions and professionals as well as literature.
Members of the project team and the review panel recommended renowned experts that where
contacted and asked to respond to a questionnaire developed by the project team (see
Appendix A). Those experts were also asked to recommend additional experts that could
contribute to this project, leading to a second and third round of contacted experts. The
following questions were asked:
−
Which GM-insects/GM-arthropods are in your opinion of relevance for the EU and/or
to be potentially released in the next 10 years?
−
Which scientific disciplines and fields of expertise that you know of are currently
involved in tasks relevant for the RA of GM-insects/GM-arthropods?
−
Which scientific disciplines and fields of expertise could in your opinion contribute to
an ERA of GM-insects/GM-arthropods?
−
Which institutes and academics that you know of are currently involved in tasks
relevant for the RA of GM-insects/GM-arthropods?
−
Which institutes and academics could in your opinion contribute to an ERA of GMinsects/GM-arthropods?
−
Which experts should be contacted and asked to answer this questionnaire?
−
Please list scientific literature that you think is of relevance for the ERA of GMinsects/GM-arthropods
−
Please list all relevant guidance documents that you know of.
15
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
With this approach experts, mostly from the EU (e.g. United Kingdom, Italy, Greece,
Germany, Denmark, France), but also from the USA, South America, Africa and Asia were
identified and contacted. Their fields of expertise cover a wide range and include e.g.
evolutionary biology, genetics and molecular biology, medicine, entomology and ecology. 49
experts returned the questionnaire and provided important information and input. The
questionnaires were screened for correctness by the project team (e.g. some fields of expertise
and recommended literature did not match the scope of this report, some recommended
experts stated in their reply, that they are not longer involved in research on GM-arthropods).
The contacted experts were also asked to fill in a form providing input for the expert-database
supporting the conclusion that they could contribute with their expertise to an ERA of GMarthropods.
1.2.
Internet and citation databases
In addition to the information provided by the contacted experts, a structured internet search
and a database search of the scientific literature (ISI Web of Knowledge, that searches
amongs others in journals, books, conference proceedings and patent databases) with relevant
keywords (see table 1) were conducted.
The search was based on the following research questions:
−
Which arthropods have been genetically modified?
−
Which institutes and professionals are involved in research/development of GMarthropods?
−
Which guidance documents are available concerning the risk assessment of (GM)arthropods?
−
Which scientific literature is of relevance for an ERA of GM-arthropods?
As key elements arthropod, genetically modified and risk assessment were definded leading to
the following search terms: arthropod, insect, genetically modified, GM, genetically
engineered, transgenic, SIT, sterile insect technique, RIDL, release of insects carrying a
dominant lethal, ERA and environmental risk assessment. Table 1 shows the result of the
literature search.
Retrieved references were screended for there relevance since e.g. a lot of results covered
GM-crops and their influence on target or non-target organisms. Full references were
imported into a Reference Manager Database® (see 18.1). Only English references were
retrieved.
The world-wide-web was also searched using Google. Keywords were the same as identified
above. The reason was to check if an application or research topic or an important research
institution was overlooked by the experts or the search on ISI web of knowledge. Because of
time constraints handsearch of electronic and paper full text journals was not conducted.
16
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 1: Literature search
Database: ISI web of knowledge
Searched fields: topic
Period searched: all
Search date: 18.-22. January 2010
Search term
Genetically modified AND insect
Genetically modified AND arthropod
GM AND insect
GM AND arthropod
Genetically engineered AND insect
Genetically engineered AND arthropod
Transgenic AND insect
Transgenic AND arthropod
Transformation AND insect
Transformation AND arthropod
(SIT OR sterile insect technique) AND insect
(SIT OR sterile insect technique) AND arthropod
(RIDL OR release of insects carrying a dominant
lethal) AND insect
(RIDL OR release of insects carrying a dominant
lethal) AND arthropod
(ERA OR environmental risk assessment) AND
arthropod
ERA OR environmental risk assessment) AND insect
Number of records retrieved
2,801
107
368
20
580
26
5.081
221
2,940
133
1,121
40
25
0
124
791
Search strings were kept simple in order to keep the amount of references retrieved on a confinded limit.
Only the first 250 references were screened for relevance.
In addition to this basic search the members of the project team conducted additional searches
for the preparation of the various chapters of this report, e.g. to retrieve relevant publication
on modelling approaches, as well as information on specific species discussed in chapters 7
and 8.
2.
Selection of GM-arthropods
As stated above, various resources were used to provide a comprehensive list of GMarthropods worldwide (see table 4).
This list was shortened to contain only those GM-arthropods that are far advanced in the
research and development pipeline and for which a level might be reached whereby potential
releases in open field settings within the EU in the next decade may be anticipated. For the
reduction of the list certain, the following inclusion and exclusion criteria were defined:
−
Recorded within the confines of the EU;
−
Established within the EU;
−
Common, expanding, and/or spreading in the EU;
−
Release programme of non-GM-arthropod candidate species available worldwide.
17
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
The selected species are deemed relevant concerning potential releases of GM-applications
and to facilitate the characterisation of potential hazards (see table 5).
In a next step, specific case study examples (see table 9) were selected from the short-list to
include both an agriculture pest and a species of relevance within the field of public health
(i.e. for disease vector control). The criteria for this selection were the following:
−
Advanced status of development (GM-traits available);
−
High likelihood of notification for application;
−
Availability of high quality data;
−
Species not only relevant for overseas territories.
On the basis of these case study species, implications for the implementation of an ERA of
GM-arthropods are discussed.
3.
SWOT Analysis
A key activitiy was an assessment of methods used to generate empirical information on the
survival and reproductive fitness of GM-arthropods as well as on their spread, migration,
persistence, and invasiveness, whilst taking into account interactions with other organisms
(see chapter 12).
Based on the list of key parameters for the description of crucial arthropod characteristics,
environment variables and genotype-environment interactions compiled by the project team
(see chapter 11), methods were proposed and evaluated using a SWOT analysis. SWOT
analysis is a management tool for assessing Strengths, Weaknesses, Opportunities, and
Threats of a project or enterprise. However, it is increasingly used in environmental studies
and can provide important and meaningful information, e.g. for evaluating and comparing
different methods.
SWOT analysis distinguishes between internal and external factors or variables. Internal
factors (strengths and weaknesses) in the context of this report describe the information an
authority receives when evaluating a notification. It indicates what kind of information the
authority can expect and what is lacking. Strengths in that context are advantages (e.g. sound
data, ease of collection). Weaknesses are disadvantages (e.g. poor data, uncertainty). External
factors (opportunities and threats) present a more general view on a certain method,
evaluating also its context which is capable of influencing the authority handling the
application. Opportunities in that respect are e.g. latest or possible developments or factors
facilitating the respective method. Threats are general factors complicating the respective
methods e.g. high costs or poor availability of experts.
The SWOT analysis was conducted by the experts of the project team taking into account
GM-arthropods of possible relevance for the EU within the next 10 years (see chapter 8).
18
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
GM-arthropods
This part of the report contains background information needed as a basis for the risk
assessment of GM-arthropods (see chapters 9 - 16). After a definition of the term GMarthropod, their purpose is discussed (including SIT, RIDL and RNAi applications) and
information on their construction given. Existing GM-arthropods are presented, and those
GM-species of possible relevance for the EU within the next 10 years discussed in detail.
4.
Definition of GM-arthropod
This report addresses important issues for ERA of GM-arthropods to be placed on the EU
market, concentrating on GM-arthropods that may possibly be notified for this purpose within
the next decade, until 2020.
According to current EU legislation, a GMO is defined as: ‘An organism, with the exception
of human beings, in which the genetic material has been altered in a way that does not occur
naturally by mating and/or natural recombination’ (DIR 2001/18/EC; Article 2, (2)).
This report considers unconfined environmental releases of GM-arthropods according to the
meaning of Directive 2001/18/EC; Article 2, (3), covering ‘any intentional introduction into
the environment of a GMO or a combination of GMOs for which no specific containment
measures are used to limit their contact with and to provide a high level of safety for the
general population and the environment’.
Applications for placing GMOs on the market for commercial reasons will be considered as
well as applications for unconfined large scale environmental releases of GM-arthropods,
which are conducted by public institutions and international bodies irrespective of commercial
interests. Placing on the market in this context means ‘making available to third parties,
whether in return for payment or free of charge (DIR 2001/18/EC; Article 2, (4))’.
Certain types of applications discussed in this report, e.g. applications to reduce vector
competence in arthropod species or population suppression, another approach called
paratransgenesis is followed, which is based on the use of GM-symbiotic bacteria or GMarthropod-viruses (Coutinho-Abreu et al., 2010). Since this approach does not lead to any
genetic modification of the host arthropod itself, these applications need to be assessed
according to the EFSA GMO panel guidance document for RA of environmental releases of
GM-microorganisms (GMMs). Thus paratransgenic applications are not within the remit of
this report. However, such applications will pose specific issues with regard to the ERA of
GMMs and since it will be of importance to further address these issues in the framework of
development of ERA-guidelines for GMMs (EFSA 2006a), some input is provided for crosscutting considerations (see chapter 19).
19
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
5.
Purpose of GM-arthropods
There are four main application strategies for GM-arthropods that are currently being
developed or envisaged. Firstly, arthropod modifications developed for the production of
useful products or higher production thereof, besides enrichment or medicinal applications.
Second, arthropods can be modified in such manner that they become incapacitated to
transmit diseases and such traits may be driven into target populations, essentially rendering
the vector population benign. The third application integrates GM-approaches into classical
sterile insect technique programmes and the fourth application transforms beneficial
arthropods for biocontrol purposes.
5.1.
Production of products of interest
For the production of useful products the following applications can be imagined:
−
Scale insects, cochineal bug (Dactylopius confusus): Strains producing special
pigments or increased production of the pigment.
−
Silk worm (Bombyx mori): Strains producing silk with special characteristics, or
strains producing more silk based on a sexing system that eliminates male moths, since
females produce bigger cocoons (Tomita et al., 2003; Pew, 2004).
−
Honey bee (Apis mellifera): Strains producing honey with special characteristics.
−
Mediterranean fruit fly (Ceratitis capitata): Strains producing protein as cheap food
for aquaculture, etc. Also mass production of specific peptides for medicinal or
industrial applications (e.g. human growth hormone).
Since those applications are either speculative, in the early stages of development or not
intended for release, those examples are not further discussed within this report.
5.2.
Population replacement and incapacitating vectors
This strategy is primarily meant for disease transmitting insect species (for a review see
Marshall and Taylor, 2009). The ultimate goal is to replace all insects of a population with
GM-arthropods that have been modified in a way that they can no longer serve as disease
vectors (Ito et al., 2002). For fruit flies and similar pest insect species no trait is
known/identified that could be utilised in a population replacement strategy nor is an effector
available that would reduce the pestiferous nature of the insect. The replacement is facilitated
by the spread of the transgene through a drive mechanism, e.g. a hyper-active transposable
element (TE). The most recent example is the Medea element. Medea is an artificial TE that
spreads rapidly in a Drosophila laboratory population (Chen et al., 2007a). Another source for
gene drive are homing endonuclease genes (HEGs) (Deredec et al., 2008). However, the
population replacement strategy is still strictly theoretical, i.e. the feasibility was only
demonstrated on a very small scale in the laboratory and not on any scale in a realistic (semi-)
field situation. Only recently guidelines for initial testing were proposed, if HEG-based gene
drive systems should become available (Benedict and Robinson, 2008). They require
considerable amounts of basic research that needs to be done before a larger scale – but still
20
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
confined – test can be considered. The guidelines do not include considerations with regard to
RA of open field releases of GM-arthropods of any scale.
One of the key problems of this strategy is the drive mechanism. On the one hand, a very
effective, highly mobile system would be required to obtain sufficient spreading of the
transgene in the population. On the other hand, the introduction of transgenic inserts with a
high mobility increases the potential risks of such applications, including the undesired
transmission of transgenes to other (closely) related species.
There are further problems associated with gene-drive strategies. Firstly, it is known that
some transgene insertions exert a negative impact on the viability/fitness of the host. The
degree of this influence is dependent on the position of the transgene in the host genome.
Using conventional TE technology, transgenes will be inserted at random positions, so it is
difficult to predict the consequences of the insertion, e.g. concerning the spread in the target
population. It is particularly difficult to assess the effects of the individual transgenic
insertions at different genomic loci present in the individual GM-arthropods in a population.
HEGs on the other hand reproducibly insert at specific sites and could potentially insert with
presumably minimal effects on fitness. Secondly, it is not known whether the target
population, or at least some individuals, have the ability to control the mobility of the
transformation vector that could prevent its spread in the target population. Thirdly, it is very
difficult, if not impossible, to recall the released transgene. Considering the current state-ofthe-art in this technology and the many unresolved problems related to its successful use and
the associated risks it is very unlikely that an application for the release of GM-arthropods for
this strategy will be notified in the near future, especially whithin the next 10 years.
Incapacitated vectors are incapable of transmitting disease. The rationale is that transmission
of diseases does not increase the fitness of the vector. Therefore, eliminating its capacity to
transmit harmful agents would have minimal effect on its biology and less ecological
perturbation and would be more sustainable. To accomplish that, several ways have been
evaluated. Ito et al. (2002) generated GM-mosquitoes (Anopheles) that express antiparasitic
genes (SM1 peptide) in their midgut epithelium, Moreira et al. (2002) worked on the
expression of bee venom phospholipase (PLA2) gene as an effector gene to block the
development of the malaria parasite in Anopheles stephensi and Kokoza et al. (2010) worked
on the expression of two effector molecules in Aedes aegypti with additive antipathogen
action Additional effector molecules capable of blocking pathogen transmission are e.g.
discussed in Speranca and Capurro (2007) and Coutinho-Abreu (2010).
Three accomplishments must be reached in order to implement such a technology: a) the
ability to transform the insect, b) to develop an effector that prevents amplification of the
pathogen, and c) a means to drive the transgene through wild populations to near-saturation.
The first target has been accomplished, the second is in under development with varying
levels of successes, but means to accomplish the third by any kind of drive mechanism remain
absent (Ito et al., 2002). Another obstacle is the genetic diversity and mutability of the
pathogen. Plasmodium e.g. has a very plastic genome and therefore the use of only one
effector gene may lead to parasite resistance (Ito et al, 2002; Moreira et al., 2002) whereas the
use of multiple effector molecules may reduce the risk of development of resistant parasites.
Also reduced fitness of GM-mosquitoes remains problematic (Marrelli et al., 2006).
21
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
It is unlikely that this particular application of transgenesis will be implemented in the EU
within a decade. The reasons are several-fold: a) useful effectors to prevent disease
transmission in Europe are not available (Speranca and Capurro, 2007). The best tested
effector is against dengue transmission in Aedes aegypti, b) for agricultural pests for which
transgenesis has been achieved, direct damage and not disease transmission is the primary
harm, c) extensive testing will be required to measure the spread of a transgene in wild
populations, considering also horizontal gene transfer and resistance development and to
determine its safety and integrity (Moreira et al., 2002). Because even first generation GMinsects have not been developed, it is extremely improbable that this technology will be
adopted anywhere anytime soon.
5.3.
Population suppression, containment or eradication
This purpose is important for arthropod plant pests or arthropods capable of transmitting
human or animal diseases.
5.3.1.
Sterile Insect Technique (SIT)
SIT is based on massive releases of sexually compatible male arthropods which can introduce
sterility into a target pest population. Usually this is facilitated by treating mass reared
individuals with irradiation (e.g. gamma rays) to introduce chromosome damages in their
germline cells, which in turn prevent the generation of viable offspring upon mating with wild
females of the pest population. The dosage of the irradiation treatment should be such that the
competetiveness of the released animals remains high enough whilst ensuring that a
sufficiently high percentage of offspring dies or is rendered infertile.
Four methods have been used to create sterile insects for SIT: hybrid sterility, chemosterilization, gamma irradiation and X-rays. Hybrid sterility can be achieved by crossing
closely related species but has fallen out of fashion due to the fact that virgin females and
males of two strains must be crossed to produce insects for release. This is extremely
inefficient, and the effect of hybridization on mating competitiveness of the offspring remains
questionable, and will not be discussed further. Chemo-sterilants (DNA alkylating agents)
have been used successfully in insects and vertebrates for sterilisation. While they are
effective, care must be taken handling them in the production facility, and one report of
sterility in a predator (spider) that consumed chemo-sterilised mosquitoes chilled the
prospects for further use. This phenomenon, however, has not been studied in sufficient detail
to assert with certainty that chemosterilants have no future potential. Gamma irradiation is
accomplished by exposing insects to a source of either 135Cs or 60Co. Such sources are held in
shielded machines and are the current method of choice. The radiation damage is not specific
to the germ cells however: somatic damage occurs to all of the soma. This may reduce the
vigour of released insects. High-energy X-ray irradiators are gradually replacing gamma
sources as they are more easily shipped and do not necessitate permanent installation of an
irradiation source. They also offer high capacity at competitive cost.
Knipling developed a theoretical model of the SIT in the early 1940s (Klassen, 2003), but it
was not until 1954 that the technique was successfully implemented with the elimination of
the New World screwworm Cochliomyia hominivorax from the island of Curaçao
(Baumhover et al., 1955). Since then, in line with Knipling’s basic model, the SIT has on
22
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
numerous occasions been used in field programmes as an effective and very powerful method
of insect pest management. Examples are the eradication of the:
−
New World screwworm, Cochliomyia hominivorax, from the USA down to Panama
and the elimination of an invasive population from Libya (Wyss, 2000; Bowman,
2006);
−
Mediterranean fruit fly, Ceratitis capitata, from the southern part of the USA down to
Guatemala and from Chile (Ortiz et al., 1987; Robinson, 2002; Gonzalez and
Troncoso, 2007);
−
Melon fruit fly, Bactrocera cucurbitae, from Okinawa island (Japan) (Kakinohana et
al., 1997);
−
Australian fruit fly, Bactrocera tryoni, from western Australia (Meats, 2007);
−
Tsetse fly, Glossina austeni, from Zanzibar (Unguja) island (Tansania) (Vreysen et al.,
2000).
In addition, globally, many SIT programmes are on-going that are not aimed at eradication,
but suppression of the target population and as prophylactic measures to prevent the
introduction or reintroduction of the respective pest species (since arthropods are highly
mobile they have the ability to immigrate from surrounding areas. Pest organisms like fruit
flies are also distributed along international trade routes). Since 1996 the SIT is also applied
in the Mediterranean basin. So far, three mass rearing facilities for the production of sterile
Mediterranean fruit flies exist in that area with a combined maximum capacity of ca. 570
million males per week (Madeira, Portugal; Valencia, Spain; Israel).
In the SIT large numbers of insects are reared in specialised facilities. In case of the use of a
sexing strain they are sorted by their sex, the males exposed to ionising radiation and released
into the target area (in case of the Mediterranean fruit fly at a rate of ca 100,000 males per
km2 per week). Ionising radiation causes redundant dominant lethal mutations (mostly
chromosome breaks). The random nature (i.e. every sperm carries a different set of lethal
mutations) and the type of damage caused (chromosome rearrangements) ensure that the
target population cannot become resistant against radiation-induced sterility. Rare instances of
behavioural resistance (assortative mating) caused by the mass rearing have occurred, but
have not generally prohibited effective programmes. After mating between the released,
irradiated males and the females in the target population, eggs are laid (although in many
cases at a reduced rate), but offspring die during embryonic development. In some insects
there is a significant amount of F1 sterility among the rare individuals that do survive.
Repeated releases of irradiated insects are required to either suppress the target population to
reduce damage below economic thresholds or to achieve elimination from the target area. The
choice between these two strategies depends, among other aspects, on the possibility to
establish quarantine barriers to protect an elimination zone against reinfestations. For
established pests with a wide distribution (as opposed to local infestations as a result of rare
invasion events) the elimination strategy can only be used successfully if an effective
quarantine can be established and maintained. Part of the quarantine is the
blocking/controlling of trade in and out of the elimination zone. However, trade barriers are
not allowed within the EU and therefore all currently ongoing SIT programmes in the EU (for
23
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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Mediterranean fruit flies) are aimed at regional control and not at eliminating the pest from a
specific area. This implies that sterilised insects are released continuously, i.e. the sterile
insects are used like a bio-pesticide. Reinfestation is a constant threat to treatment areas. A
good example is California where the Mediterranean fruit fly was eradicated several times,
but despite intense quarantine efforts reinfestations occured, especially in the Los Angeles
area. For several years, an alternative strategy therefore has been applied (Preventative
Release Programme). After creating a pest-free zone, irradiated Mediterranean fruit flies are
released constantly but at a reduced rate so that any newly introduced wild Mediterranean
fruit flies immediately encounters sterile insects and cannot establish sizeable populations.
Some other important aspects of the SIT are:
−
The SIT is strictly species-specific as it depends on the mating of individuals. Even in
areas where several fruit fly pests are present, no cross-mating occurs because all of
these species are distinct and maintain pre-copulatory isolation barriers. The
exceptions to this rule are species that belong to species complexes, e.g. the
Bactrocera dorsalis complex.
−
SIT is used as an area-wide approach and releases are also performed proactively and
routinely. If an area contains the defined population (even if only localised) the entire
area is treated regularly and not only in reaction to pest outbreaks. This approach is
clearly different from the application of insecticides where pest populations
reproducing in areas around the field are not treated, or cannot be treated for the
following reasons: in case of fruit flies, areas with wild host plants, abandoned
orchards or private backyards cannot be reached with insecticides and provide a
constant source for re-infestations of the cultivated areas.
−
Potentially hazardous chemical and biological residues are reduced to a minimum or
eliminated. The released insects are sterile (but not radioactive) and the goal is to
prevent the establishment of a breeding population in the target area. Roughly 3 days
after the end of a release well over 90 % of the released Mediterranean fruit flies can
no longer be detected. It is assumed that these insects fall prey to spiders, ants, wasps
etc.), and are consumed by these predators before or after death. The impact of the
release of GM-arthropods on these predatory or parasitic species, therefore, needs to
be considered.
−
The SIT is used most efficiently as one component in Area-Wide Integrated Pest
Management (AW-IPM) programmes (Hendrichs et al., 2007), e.g. due to its nonchemical nature the SIT can be integrated very efficiently with other biological control
strategies against the target pest or against other pest species present in the target area.
Additional measures include, for example, field sanitation and others mechanical
procedures.
−
The level of sterility induced in a pest population corresponds to the irradiation dose.
Some programmes insist on very high levels of sterility, e.g. Probit 9 (99.9978 %
sterility), which means that for example the Mediterranean fruit fly has to be irradiated
with 140 Gy. However, at such high levels of irradiation the mating competitiveness
of released flies is reduced. Therefore, contemporary SIT programmes use a lower
24
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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dose and accept to release partially sterile males (95-99 % sterility, reviewed by Bakri
et al. (2005)). The net efficiency (i.e. the combination of competitiveness of the males
and the sterility level) in the field is higher than in case of full sterility. Especially for
lepidopteran species the radiation dose required to obtain full sterility is very high (e.g.
for Codling moth 400 Gy for males and 100 Gy for females to obtain 98 % or more
sterility (Vreysen et al., 2009)). Consequently, the somatic damage is also very high
and the viability of the moths is reduced significantly. In such cases, a modification of
the SIT could be used (F1 or inherited sterility). In this approach, partially sterilised
moths are released but mating with the target population introduces sufficient
chromosomal lethality to render the F1 generation practically sterile (Vreysen et al,
2009).
Transgenic sexual sterility has been accomplished in mosquitoes and in the Mediterranean
fruit fly. Other efforts are underway to interfere with proper sperm function. None of these
approaches can be implemented in their current form, but it is likely that they will within 3-5
years. Gamete integrity is also being affected by HEGs by cutting rDNA, but this system
cannot be controlled in a way that would make it useful for releases. However, within some
years, it is likely that better control will be possible.
Genetic modification of arthropods for SIT is considered to reduce the costs of SIT
implementation, e.g. through biological marking so that released arthropods can be
distinguished from feral ones (e.g. by using certain transgenic marker genes) and to facilitate
the production of novel strains with enhanced SIT performance. Using genetic modification
several components could be optimized including a genetic sex-separation system, efficient
mass production and competitive sexually sterile males.
5.3.2.
Release of Insects carrying a Dominant Lethal (RIDL)
Another possibility for population suppression, containment or elimination is the release of
GM-arthropods causing sterility in the F1 generation, because they carry a dominant lethal
gene (RIDL), which is passed on and activated in the offspring (Thomas et al., 2000; Horn
and Wimmer, 2003).
This strategy is meant to replace the application of ionising radiation for the induction of
sterility in offspring of released GM-insect. The GM-insect carry, at least in the currently
available strains, only one dominant lethal gene. Like in the SIT, RIDL strains are massreared (but not irradiated) followed by the release into the target area. During production, a
dominant lethal gene that the strains carry is suppressed by rearing the insects e.g. in the
presence of tetracycline which specifically inhibits the respective promoter used to express
the gene. The released insects mate with specimens of the target population and thereby
transmit the lethal gene. In the absence of the inhibitor, the transgene is activated and causes
lethality at some stage during the development of the offspring. Depending on the constructs
used, i.e. on the type of promoter and the lethal gene, this lethality will be expressed only in
females or in both sexes. The type of the transgenic construct will also determine at what
developmental stage the offspring will die. Like the population replacement strategy, RIDL
has been proven to work on a small scale in the laboratory and in studies of Aedes albopictus
under confined conditions in Malaysia (pers. comm. Luke Alphey, Oxitec). The fact that only
one lethal gene is used may become problematic when RIDL strains are applied under more
25
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
realistic conditions, i.e. against entire pest populations with a high level of genetic diversity. It
cannot be excluded that the target population becomes resistant against the lethal gene; a
scenario equivalent to the development of insecticide resistance in insect pests. In the RIDL
system the lethal gene and its control element(s) have to interact with the host genome to be
functional and numerous mechanisms are known that potentially abolish the lethal effect
(Handler et al., 2004). For some arthropod species, e.g. mosquitoes, it is considered that late
lethality is more effective provided that the adults die before they can transmit viable
pathogens (Yakob et al., 2008). However, this implies that the offspring of the mating
between the released arthropods and the wild population carry the transgene and survive
beyond the embryo stage (in some cases until the pupal or even adult stage (Fu et al., 2010)).
For fruit flies such an approach would be detrimental as it would result in significant damage
of larvae to the agricultural produce.
5.4.
GM-arthropod applications employing RNAi based immunity to vectored
disease agents
RNA interference (RNAi) as a means to post-transcriptionally silence the expression of
specific genes has been studied in arthropods and has been shown to function in some
arthropod species which are of relevance with regard to GM-arthropod applications for
release into the environment (Huvenne and Smagghe, 2010). I.e in mosquitoes and
leafhoppers it is possible to activate the RNAi pathway by expression of inverted repeats of a
target gene to mediate the assembly of double stranded RNAs, which induce the silencing
mechanism (Dong and Friedrich, 2005; Brown and Catteruccia, 2006). The technique was
used to study the biochemistry of the arthropods and to identify molecular targets for blocking
the establishment of disease agents (e.g. viruses and malaria parasites) in these arthropods.
According to the findings that alpha and flaviviruses trigger the RNAi pathway as an innate
immunity response in mosquitoes, it has been explored if the RNAi mechanism could be used
to interfere with vectorial competence in arthropods, specifically in Aedes and Anopheles
mosquitoes (Sanchez-Vargas et al., 2009). In different approaches, the efficacy of the system
is studied to either block the expression of proteins which play an important role for vector
competence or to directly block viral replication in the mosquito host (reviewed in CoutinhoAbreu et al. (2010)). With this latter strategy, Franz et al. (2006) could impair vector
competence of Aedes aegypti mosquitoes for the dengue virus by expression of an inverted
repeat RNA derived from a protein coding sequence of dengue type 2 virus. They also
hypothesised that such an approach could provide a powerful tool for further development of
population replacement strategies to combat transmission of the Dengue virus by mosquito
vectors. GM-mosquitoes that use RNAi as a means to suppress the innate immunity of Aedes
aegypti mosquitoes against Sindbis virus to reduce their survival rate after infection have
recently been developed (Khoo et al., 2010).
In the laboratory, GM-mosquito strains have also been rendered refractory to malaria parasites
by RNAi approaches (Brown and Catteruccia, 2006). However, concerns remain that the
strategy may not be equally effective following field releases of such GM-mosquitoes. Firstly
RNAi cannot completely suppress protein expression. Second, using a single silencing target
will probably not be effective with a highly diverse population of mosquitoes as encountered
under open field conditions. Furthermore, the long-term stability of the trait, which would be
26
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
instrumental to achieve effectiveness, has to be investigated further. Another issue is the
fitness cost associated with the induced changes (Brown and Catteruccia, 2006). However, the
development of tissue and infection-specific regulation of activation of an RNAi response
could address some of these concerns.
RNAi approaches have also been discussed with regard to the construction of genetic sexing
strains for SIT applications in mosquitoes and Ceratitis fruitflies (Brown and Catteruccia,
2006).
However, the release of GM-arthropods based on RNAi technology in the near future seems
unlikely, considering various issues that will have to be resolved.
5.5.
Beneficial arthropods as biological control agents
Some beneficial arthropods are natural enemies of pest species and can be used as biological
control agents. The goal of developing GM-strains of these species is to enhance their
effectiveness in several ways (e.g. resistance to pesticides, tolerance to temperature extremes,
extended lifespan) to improve pest management programmes (Pew, 2004). Some of those
genetic improvements were already accomplished by traditional genetic methods but
recombinant DNA techniques could make improvements more efficient and less expensive:
e.g. a certain useful gene can be inserted into a number of beneficial species and there are no
limitations to the genetic variability within a species since more genes are available for use
(Hoy, 2000).
So far, the only GM-beneficial arthropod is Metaseiulus occidentalis, the Western predatory
mite. It served as a model organism for transformation using a novel maternal microinjection
method injecting the plasmids directly into the abdominal region of the mite. Since
Metaseiulus occidentalis is also an important biological control agent, one transformation
goal was to improve the effectiveness of the species, but the GM-strain was unstable under
field conditions. Besides the lack of RA guidelines for permanent release into the
environment, there is also only a limited number of suitable genes for the improvement and
no further attempts have been made (Hoy, 2009). However, microinjection could be adapted
to other beneficial arthropods and genetic transformation may improve the efficiency of
Metaseiulus occidentalis as a biological control agent.
Biological control agents are often not native to the environment in which they are released.
In that case, the application of a GM-arthropod must be accompanied by the respective
process with regard to alien species for biological control purposes, which is strictly regulated
in the EU as well as on member state level (FAO, 1996; EPPO, 2000; van Lenteren et al.,
2006).
27
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
5.6.
Other applications
Some other applications for GM-arthropods are theoretically possible, like the release of
sterilised GM-arthropods as vectors of pathogens for agricultural pests (auto-dissemination)
or as vectors of pheromones (e.g. for mating disruption of moth pest species), but no
respective GM-applications have been developed to date.
−
Auto-dissemination: in this approach (patented by the company Exosect,
Southampton, UK) the released sterile arthropods in a SIT programme are coated with
biocides (EntostatTM) or pheromones against other pest species present in the target
area (Chandler, 2005).
−
Mating disruption for moth pest: in this approach female pheromone formulations are
distributed by the released GM-arthropods in the pest-infested area. Male moths are
attracted to these chemicals and mating in the pest population is reduced, resulting in a
decline of the population size.
Another application could be the release of sterilised GM-arthropods as vectors of
vaccination. This strategy has been proposed and patented by Crampton et al. (1994). The
vaccine is produced and transmitted to humans by the released mosquitoes. However, lack
over the control of vaccination dose (i.e. the number of bites received), as well as the inability
for humans to reject or decline vaccination, make this method highly questionable in terms of
future application. Similar efforts like for the improvement of biological control agents are
expected for beneficial arthropods including resistance to insecticides and diseases (e.g. for
Apis mellifera). Those approaches have not advanced to a level whereby an application within
the next 10 years can be expected.
28
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
6.
Construction of GM-arthropods
6.1.
The transformation system
The goal of GM-technology is to generate modified organisms, in the specific area of
concern, either by manipulating existing traits/characteristics or by introducing new traits.
One of the basic components necessary in this technology is the transformation vector, i.e. the
tool to achieve stable integration of DNA into the target genome. At present, TEs (mobile
elements, transposons) are most commonely used for the transformation of arthropods are and
only rarely other means are used (e.g. virus vectors).
TEs occur naturally in all organisms, from bacteria to humans. When their sequence is intact,
they have the ability to excise themselves from one position and to insert into a new position
of the host genome. Their structure is usually relatively simple. In most TEs relevant for
genetic modification, two primary components are necessary and sufficient for mobility: two
inverted repeat sequences at the borders of the element and an enzymatic component, the
transposase, encoded by the main part of the element.
The use of such elements for transformations of arthropods was pioneered in Drosophila
(Spradling and Rubin, 1982). There the P element was engineered for stable integration into
the target genome. At the technical level this was achieved by separating the two principal
components onto two separate elements: one element contains only the inverted repeats, a
marker gene and a multi-cloning site for additional transgenes (together called the
“transformation vector”) and the other element carries a transposase gene, which is not
inserted in the genome of the GM-arthropod during the transformation process (the element
called “helper”). For production of sufficient amounts of vector DNA for germline
transformation, both are maintained in bacterial plasmids (standard cloning vectors). These
two plasmids are micro-injected together into early embryos where the transposase gene on
the helper plasmid is expressed. The transposase then recognises the inverted repeats on the
second plasmid and catalyses the integration of the transformation vector into the target
genome. During the following cell divisions the helper plasmid is diluted and lost. Thus only
the chromosomally integrated transformation vector is maintained and stably inherited. In this
way any transgene inserted between the two inverted repeats can be introduced into an
arthropod. Such “binary systems” are also used for most target non-drosophilid species.
However, the P element does not function outside of Drosophila and therefore other TEs had
to be identified for use in other arthropods.
The first successful transformation of a non-drosophild species was reported for Ceratitis
capitata using the Minos element (Loukeris et al., 1995a). The first mosquito species
transformed was Aedes aegypti using the Hermes element (Coates et al., 1998) and the first
anopheline mosquito transformed was Anopheles stephensi, also using the Minos element
(Catteruccia et al., 2000a). For practical reasons the transformation vector also carries a
marker to identify individuals which have been transformed successfully after microinjection. Initially, genes encoding the wild type protein of a visible mutation (e.g. the white
eye colour mutation) were used as markers, but today virtually all vectors contain a gene
encoding one of the many different fluorescent proteins.
29
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Most applications of GM-technology that are likely to be implemented initially require stable
insertions. Table 2 lists all TEs that have been engineered to date to create suitable
transformation vectors for arthropods. GM-insect technology was initially developed based on
the model of P element transposition. However, since the P element can be easily
remobilised, one point of concern for GM-arthropods was the stability of the transformation
vector in the target genome. Most of the TEs used as transformation vectors in nondrosophilid species are members of larger families of related elements. To avoid crossmobilisation of the transformation vector by related endogenous elements present in the target
organism new techniques have been developed that “cripple” the vector after it has been
integrated. Either one or both inverted repeats are removed thereby removing the target site(s)
required for transposase binding. This was developed for transformation vectors based on the
piggyBac element which is currently the most widely used vector (Handler et al., 2004;
Dafa'alla et al., 2006). These stabilised vectors ensure that also at very high insect mass
rearing levels, as required for the SIT, the transgene remains at its original insertion site and
does not multiply in the target organism, and it has been unexpectedly difficult to remobilise
(especially piggyBac) insertions in species examined thus far (O'Brochta et al., 2003;
Sethuraman et al., 2007). PiggyBac has become one of the most popular vectors because it
has been useful for transformation of every arthropod species in which it has been tested.
An additional improvement related to transformation vectors is the development of targeted
integration systems. At present, integration of the transgene occur at random locations in the
target genome. A stable integration of the transgene into the host genome is required for SIT
and RIDL. However, for “population replacement”-applications instability is required, i.e. a
fully functional TE with an extremely high transposition activity/efficiency.
Conventional TEs (piggyBac, Hermes, Minos) are expected to integrate at random locations in
the target genome. This can have, depending on the particular integration site, negative effects
for the function of the transgene (no or reduced function) and/or for the host organism (from
reduced viability/fitness to lethality). Systems are under development where in a first step a
specific DNA sequence is inserted in the host genome and several of these anchor sites are
evaluated for optimal performance with respect to instability or any other unintentional
adverse effects as mentioned above. If that has been demonstrated, such sites can serve as a
defined “docking site” for additional transformation events (Schetelig et al., 2009).
Additionally systems (e.g. HEGs) are under development where a specific DNA sequence is
first inserted in the host genome and after release it inserts into specific locations in wild
individuals (Burt, 2003).
The insertion of genes into the same target sites in the genome has been demonstrated for two
types of applications: efficient and reproducible production of GM-arthropod strains for
laboratory research and, site-specific integration of transgenic elements in GM-arthropods
designed for environmental release, which are mobile and can “jump” to other genomic loci
with the same recognition sequence.
The first case is a laboratory convenience that facilitates to compare the expression of
different genes in the same chromosomal milieu, e.g. in laboratory strains of Drosophila
(Nimmo et al., 2006; Amenya et al., 2010). However, this technology has also been
successfully applied for the Mediterranean fruit fly (Schetelig et al., 2009).
30
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 2.
Transposable elements available for use as transformation vector in GMarthropods
Name
Original host
Reference
piggyBac
Trichoplusia ni
Cary et al. (1989)
Minos
Drosophila hydei
Franz and Savakis (1991)
Hermes
Homer
Musca domestica
Bactrocera tryoni
Warren et al. (1994)
Pinkerton et al. (1999)
Hopper
Bactrocera dorsalis
Handler (2003)
Herves
Anopheles gambiae s.s.
Arensburger et al. (2005)
Hobo
Drosophila melanogaster
Handler and Gomez (1996)
hAT family of elements
Mariner elements
Mos1
Drosophila mauritiana
Medhora et al. (1991)
Himar1
Haematobia irritans
Robertson and Lampe (1995)
Tn5
prokaryotic composite transposon
Goryshin and Reznikoff (1998)
The second case is site-specific insertion of a transgene into particular native genomic
sequences. In the first case, HEGs have been applied in the African malaria mosquito
Anopheles gambiae s.s. (Windbichler et al., 2007; Deredec et al., 2008). These DNA elements
are mobile, but their insertion sites are specific and can exist in as few copies as only one per
genome. Efforts are underway to modify the site specificity to create new target sites, e.g. in
genes related to fecundity. Also transposase enzymes that create insertions into the same
genomic site at high frequencies have been engineered (Maragathavally et al., 2006).
However, it is not yet known how extensively target site recognition and insertion function
can be modified with this approach.
6.2.
Principal molecular components
Various molecular components have been isolated over the last couple of years from an
increasing number of arthropod species. These can be used in a “mix and match” approach
depending on the required purpose (see chapter 5). However, the following list (table 3) is not
exhaustive and can only serve to provide an overview of some of the main components that
have been used to date. It needs to be stressed that developments in this area are so rapid that
it is virtually impossible to exactly predict which particular vector system will be used in
future applications. Thus any ERA will have to be done on a case-by-case basis taking into
account the specific components that are actually present in the GM-arthropod in question.
Table 3 gives an overview of the molecular components used for genetic modification of
arthropods.
31
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 3.
Molecular components for genetic modifications
Component
Purpose
Fluorescent proteins
Green Fluorescent
Protein (GFP)
EGFP
Origin
Reference
Aequorea victoria (jellyfish)
Prasher et al. (1992)
Tsien (1998)
ECFP, EYFP
Horn et al. (2002)
DsRed
Discosoma striata
Matz et al. (1999)
Handler and Harrell
(2001); Lorenzen et al.
(2002)
Berghammer et al.
(1999)
Promoters
Polyubiquitin
Constitutive
Host
3xP3
Eye-specific
Actin5C
Constitutive
Drosophila melanogaster
(artificial promoter based on
Pax-6/eyeless promoter)
Drosophila melanogaster
ß2 Tubulin
Testes-specific
Host
Serendipity (sry α)
Early embryogenesis
Host
Pinkerton et al. (2000);
Catteruccia et al. (2000a)
Catteruccia et al. (2005);
Scolari et al. (2008)
Schetelig et al. (2009)
Heatshock Hsp70
(hsp23, hsp70 and
hsp83)
MSSP
Heat induction
Drosophila melanogaster;
host
Loukeris et al. (1995a);
Kalosaka et al. (2006)
Male serum specific
Host
Host
Komitopoulou et al.
(2004)
Komitopoulou et al.
(2004); Chen et al.
(2007b)
Lombardo et al. (2005)
Host
Lombardo et al. (2005)
Drosophila melanogaster
Allen and Christensen
(2004)
VgT2
AgApy
D7r4
Act88F
Host
Female salivary gland
specific
Female salivary gland
specific
Flight muscle specific
Effector genes
hidAla5
Cell death gene
Host
Bergmann et al. (1998)
AgCP[SM1]4
Malaria-refractoriness
mPLA2
Malaria-refractoriness
Synthetic gene expressing 4
units of SM1 peptide
Expression of bee venom
phospholipidase
Ito et al. (2002); Moreira
et al. (2004)
Rodrigues et al. (2008)
Other components
Tetracycline-dependent
activator/responder
system (tTA/tRE)
Tetracyclinedependent on/off
switch
The tetracycline transactivator
(tTA) protein created by
fusing one protein,
TetR(tetracycline repressor),
found in Escherichia coli
bacteria with another protein,
VP16, produced by the
Herpes simplex virus
Gossen and Bujard
(1992)
32
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Component
Polyadenylation site
Purpose
To add poly-A tail
Origin
SV40
Reference
Lanford et al. (1986)
Genes with sex-specific
splicing (e.g.
transformer,
doublesex)
transformer
Sex-specific
expression of effector
gene via sex-specific
splicing
Female to male
conversion via RNA
interference
To reduce position
effect
Host
Fu et al. (2007)
Host
Saccone et al. (2007)
Drosophila melanogaster
Sarkar et al. (2006)
Gypsy insulators
Fluorescent proteins are used as markers for transgenesis and for released arthropods as a selectable marker for sexing.
Promoters serve as elements to control gene activity and effector genes cause the desired modification of the GMarthropod.
In many cases it was observed that the use of a recombinant promoter or effector similar to
those present in the target species is more efficient than the one isolated from other species.
Therefore, it is most likely that a strain constructed for the use in an applied, large-scale
release programme will contain only the endogenous homologues (see references in table 3).
6.3.
Transgenic strains
There are currently three main applications for GM-arthropod, a) genetic sexing strains
(GSS), b) strains containing marker genes and c) conditional lethality strains (or combinations
of these).
6.3.1.
Genetic sexing strains
Genetic sexing strains (GSS) can be used for sex separation for plant pests (e.g.
Mediterranean fruit fly) as well as for arthropod species capable of transmitting diseases (e.g.
malaria). Bisexual release in SIT is inadvisable because released females contribute nothing to
the effectiveness of the respective programme. Although e.g. bisexual release of the
screwworm did not prevent successful implementation of SIT, it is certain that if a sexseparation strain had been available that it would have increased the efficiency of the
programme. Release of females is either inadvisable because of their ability to transmit
pathogens (even when sterile), pestiferousness or ability to damage crops. Removal of
females from the released arthropods has been accomplished by physical methods including
size differences between male and female pupae (mosquitoes) or pupa color using automated
sorting systems (Mediterranen fruit fly). These methods share one disadvantage in that the
female insects must be cultured nearly to maturity before separation after which most are
destroyed, thus reducing production efficiency: rairing of both sexes requires roughly twice as
much space, nutrient media and disposal costs, than rearing males only.
By generating strains which facilitate exclusive production/release of males the efficiency of
the SIT, i.e. the sterility achieved in the field, was increased by a factor of 3 to 4 because
mating among the released medflies is avoided (McInnis et al., 1994). Other benefits of the
male-only strains are reduced costs for production (e.g. for rearing diet), handling (e.g.
manpower/equipment for sterilisation), release (e.g. transportation for release) and monitoring
(e.g. number of trapped insects to be screened). Furthermore, the problem of “sterile stings” is
33
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solved, i.e. released females sting the fruit despite the fact that they are sterile and, thereby,
reduce the market price of the fruit. Today, all 12 Ceratitis capitata mass rearing facilities in
operation world-wide (2 of them in Europe providing relevant experience on this issue) use
GSS and their combined maximum production capacity is 3.500 million males per week.
All genetic sexing strains for Ceratitis capitata in use today are based on modifications
obtained through classical genetics, i.e. they do not involve any genetic modification.
However, it is envisaged that GM-technology could be used to develop strains that have
certain advanced features. This includes the possibility to generate more efficient sexing
strains and the possibility to add markers to the released flies. Current GSS are, due to their
genetic structure, 30 to 50 % sterile, which reduces their productivity and increases mass
rearing costs. In principle it should be possible to develop transgenic GSS that circumvent this
problem. A stable marker with good visibility would replace the current, to some extent
problematic, practice that the flies are dyed with a fluorescent powder before they are released
into the field to be able to distinguish them from the wild flies. Transgenic strains carrying an
internal fluorescent marker have been already developed. Depending on the nature of the
transgenic construct such markers could replace the fluorescent dyes or serve as sperm marker
to determine the mating status of females trapped in the field. It is also possible to combine
such markers with the currently available non-transgenic GSS. However, so far none of the
transgenic strains was characterised sufficiently or provides sufficient advantage over current
GSS to be used for the implementation in an active Mediterranen fruit fly SIT programme. At
present, GM-Ceratitis capitata strains are primarily developed by the public sector
(universities, government organizations). There are only two small private companies
involved in this area; Oxitec Ltd. (Oxford, UK) and Minos Biosystems (Crete, Greece).
In order to separate the sexes earlier (thus preventing the disadvantages that come with the
need to rear the females nearly to maturity as described above), genetic sexing strains have
been devised in which a dominant selectable marker is linked to the male-determining locus
by screening random chromosome rearrangements. This process has resulted in the most
successful GSS, the Mediterranean fruit fly temperature sensitive lethal. Creating GSSs is
sometimes simple, but in most cases, creating such strains is intricate and the necessary
chromosome rearrangements may result in semi-sterility of the strains, which makes
production less efficient. Furthermore, these systems break down due to recombination and
require careful maintenance. Transgenic strains are expected to have lower breakdown rates,
but the rates at which this will occur are unknown and can only be determined during massproduction trials. GSS also require some selectable marker that is identified from within the
species. The advantage of the GM-approach is that it allows sex-specificity and a selectable
marker to be incorporated into a single transgenic construct, so the location of the insertion is
not critical and potentially useful strains can be generated and identified quickly. The same
construct, or species-specific variants, can also be constructed fairly quickly for use in various
species. A GM-approach also allows the developer to select from various effectors that are
active early in development.
To create more stringent and versatile sex-separation systems, GM-approaches are being
developed and tested. These generally use a sex-specific promoter or splicing to produce a
product to eliminate females, i.e. the same molecular components as described above for plant
pest. Male arthropods produced by this method will then be radiation-sterilised. As noted
34
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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above these are seldom completely sterile, so some introduction of transgene material into the
environment could occur (Papathanos et al., 2009). Such technology would be relevant to any
GM-arthropods released in SIT programmes.
To develop a sexing system, three principal components are required:
1.
On/Off switch: Females are required to maintain the mass rearing colony and only a
certain fraction of the production will be sexed for release. Currently, the only
component that achieves sufficient accuracy as an on/off switch is the tTA/tRE
system. In an existing transgenic GSS for the Mediterranean fruit fly (Morrison et al.,
2009) the accuracy was 100 % even at slightly elevated rearing levels.
2.
Female specificity: Here either a female-specific promoter or the sex-specific splicing
of a gene from the sex determination cascade can be used (Viktorinova and Wimmer,
2007).
3.
Lethal gene: In principle, any lethal gene could be used. However, preference is given
to endogenous genes, e.g. genes involved in apoptosis. In the Mediterranen fruit fly
GSS mentioned above no additional lethal effector gene is required because the full
expression of the tTA gene in the absence of tetracycline is sufficient to cause
lethality.
An alternative approach is the convertion of females into males. Rather than eliminating
females, a strategy, based on RNAi against the transformer gene, to change XX females into
males has been demonstrated in Ceratitis capitata (Salvemini et al., 2009). This allows
greater efficiency of production because all embryos produced can be changed into males
before conventional sexual sterilisation by irradiation. However, it must first be demonstrated
that the XX males are indeed fully functional and competitive with respect to mating.
The technical challenge is to identify, develop and combine these three components in such a
way that:
−
The lethality occurs as early as possible to minimize the costs for rearing the females.
−
The accuracy is 100 % or at least very close to 100 %. This could be of lower
importance for fruit flies but is very important for disease-transmitting arthropods.
−
Males fitness should not be affected negatively to maintain full productivity (and this
applies also to the females in the colony rearing) as well as optimal performance and
mating competitiveness in the field.
−
In combination with the transformation vector (or the remnants of it), the transgenic
construct must be stable even at very high levels of mass rearing.
35
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
6.3.2.
Marker strains
The development of marker strains is much easier due to the fact that in most cases the marker
used for the detection of successful transformation can also be used for the field application.
Most fluorescent markers are sufficiently strong and stable to allow easy detection of GMarthropods even one or more weeks after death. This is a prerequisite because traps in the field
are usually inspected only once a week. In cases where the marker is required to determine the
mating status of trapped females, an additional fluorescent protein marker under the control of
a testes-specific promoter is required.
A variation on this application combines sex-specific marking (see below) with devices for
selecting males from among a mixed-sex population. The method (Catteruccia et al., 2005)
introduces a transgene expressing a fluorescent protein under the control of a male-specific
promoter into the mosquito. Only males express the transgene marker, and they can be
identified and sorted using Complex Object Parametric Analyzer and Sorter (COPAS)
technology which is similar to flow cytometry for larger objects. This method is simple but
requires culturing equal numbers of males and females, most of the latter of which must be
discarded as production waste.
6.3.3.
Conditional lethality strains
Irradiation of males prior to release not only requires handling but creates some level of
somatic damage, which can reduce the quality (viability and competitiveness) of the released
arthropods. It also requires the use of a gamma-cell irradiator or X-ray machine. The former
are increasingly difficult to obtain and transport to (remote) field sites. The latter technology
is offering promise, but has only recently become operational. The unintended damage of both
methods to the irradiated animals is similar. This is why transgenic methods are being
developed for sexual sterilization (Catteruccia et al., 2009). It is anticipated that such systems
will eliminate the general loss of vigour that results from irradiation and will often provide
more complete sexual sterility.
6.4.
Common features of the described applications
All the examples for the use of GM-techniques mentioned above have the following
considerations in common: The procedure to generate GM-strains for a practical application
involves many evaluation steps, i.e. the strategy will be very similar to the one that was
applied, when the current GSS for the Mediterranean fruit fly were developed. Without the
availability of defined docking/integration sites (still under development) many independent
lines will have to be created because of the random integration of the transgene. The
characteristics of these will differ, depening on the influence of the chromosomal environment
(position effect). The first round of evaluations will determine which line(s) are optimal with
respect to the desired features. The selected lines from this step will be subjected to small
scale evaluations where some biological traits like viability, mating competitiveness and to
some extent stability are measured. It is likely that also at this step some lines will have to be
rejected. The remaining lines will be introduced into a low level mass rearing (ca. 100.000
individuals per generation). At this stage, standard quality control parameters
(FAO/IAEA/USDA, 2003) will have to be determined. This way also the cost/benefit
36
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
considerations for potential end-users will become apparent, which will influence whether a
new strain is worth implementing. Only after a new strain has passed all these test stages it
can be considered for use in a release programme. However, even if a strain is accepted, the
respective end-user will have to perform additional evaluations before it is introduced into
mass rearing for release.
7.
GM-arthropods worldwide
Worldwide various species have been genetically modified for various purposes or are under
development, e.g. for basic research or for pest control (see chapter 5). Table 4 lists those
species and provides an overview of the genetic modifications, the transformation systems,
the intended use and the current status of development. In addition, a short description for
each of the species is given in order to provide some background information about its
importance and control measures (conventional as well as transgenic). Thus this chapter
serves also as a basis for the selection of those GM-arthropods that could possibly be
commercially released into the environment in the EU in the forthcoming decade.
Germline transformation of all species in table 4 has been accomplished, in some cases only
as a proof of principle demonstration. In a few species, the intended use is a laboratory model
(e.g. Drosophila spp., Bicyclus anynana) and no releases are intended: pestiferousness is not
the primary cause of interest, and will not be discussed in detail.
For many species in table 4, conventional control methods are available including
conventional pesticides, biopesticides, trapping, source reduction and conventional SIT. The
technical capacity of the existing interventions to exert some level of control is not the reason
for developing GM-approaches. Alternatives are favoured because of cost, degree of control
desired, acceptability for social or aesthetic reasons, health effects and environmental safety.
This is true for GM-alternatives as well. They must provide benefits in the above categories in
order to make them attractive.
The list of GM-arthropods provided is based on expert knowledge and an intensive search of
both scientific databases and the world-wide-web (see chapter 1). Although the aim was to
provide a list as complete as possible, it may be that some species (e.g. for basic research
purposes) are missing. On the one hand, scientific databases are not completely
comprehensive themselves and on the other hand research might be ongoing of which the
findings have not been published yet. Nevertheless, those GM-arthropods that might be of
relevance for a commercial release in the EU in the next 10 years are covered. Due to the
limited timeframe, these selected species and applications would be the most important in
therms of economical relevance or public health importance and therefore experience
extensive research and developments.
37
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 4.
GM-arthropods worldwide
Species name
Common name
Modification
Transformation
system2
Red flour beetle
eG/Y/CFPs,
vermilion eye colour,
developmental genes
Hermes, Minos,
piggyBac,
Intended use
Current status
References
Basic studies especially
of development and
osmoregulation,
laboratory model
Easily transformed and
cultured with numerous
mutations available.
Lorenzen et al.
(2003); Pavlopoulos
et al. (2004);
Siebert et al. (2008)
Insects
Coleoptera (beetles)
Tribolium
castaneum
Culicidae, Diptera (mosquitoes)
Aedes aegypti
Yellow fever
mosquito
Aedes albopictus
Asian tiger mosquito
Aedes fluviatilis
No release plans known.
Markers (eGFP,
hydroxykyneurenine),
anti-dengue effectors,
anti-Plasmodium
effector, (docking
sites), neo, flightless
female
Mos1, piggyBac,
Tn5,
Hermes,mariner
Population suppression
via SIT/RIDL,
introducing refractoriness
into populations
Plans for release of
RIDL strain males are
underway.
Adelman et al.
(2002); Yakob et al.
(2008); Fu et al.
(2010)
Fluorescent protein
piggyBac
Population suppression
via SIT/RIDL,
introducing refractoriness
into populations
First transformation
now being submitted for
publication.
No release plans known
but likely candidate for
RIDL.
Lobo et al. (2001);
Bellini et al. (2007)
eGFP, antiPlasmodium effector
piggyBac,
Model system
No release plans known.
Rodrigues et al.
(2006)
2
In most species germline transformations was performed using a binary system in which a vector plasmid is injected into preblastoderm embryos along with a transposase-producing helper plasmid
or purified transposase RNA.
38
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Species name
Common name
Modification
Anopheles
albimanus
New world malaria
mosquito
eGFP
Transformation
system2
piggyBac
eGFP
piggyBac
Anopheles
arabiensis
Intended use
Current status
References
No release plans known.
Perera et al. (2002)
Model system
Conventional SIT
planned
Helinski et al.
(2008); Robinson et
al. (2009)
Anopheles gambia
s.s.
African malaria
mosquito
Fluorescent protein,
homing endonuclease
induced sexual
sterility, malespecific marker, antiPlasmodium effectors
piggyBac
Population suppression
via SIT and variants,
introducing refractoriness
into populations
Contained cage trials for
male sterility planned in
US (2010) and Italy
(2011).
Miller et al. (1987);
Arensburger et al.
(2005)
Anopheles stephensi
Indo-Pakistan malaria
mosquito
eGFP, DsRed, antiPlasmodium
effectors, testes
expression
Minos, piggyBac
Sex-separation, docking
sites, anti-Plasmodium
effector
No release plans known.
Catteruccia et al.
(2000b)
Culex
quinquefasciatus
Southern house
mosquito
eGFP
Hermes
Transgenesis
demonstration, flight
muscle expression
No release plans known.
Allen et al. (2001)
CopGreen, PhiYFP
and J-Red
piggyBac
SIT
No release plans known.
Condon et al.
(2007)
Tephritidae, Diptera (true fruit flies)
Anastrepha ludens
Mexican fruit fly
Anastrepha
suspensa
Caribbean fruit fly
DsRed
piggyBac
Temperature sensitive
lethal for sexing, SIT
No release plans known.
Handler and Harrell
(2001)
Bactrocera dorsalis
Oriental fruit fly
DsRed, white eye
piggyBac
Sperm marking
No release plans known.
Handler and
McCombs (2000)
Bactrocera oleae
Olive fruit fly
eGFP
Minos
Sex conversion for SIT
No release plans known.
Koukidou et al.
39
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Species name
Common name
Modification
Transformation
system2
Intended use
Current status
References
(2006)
Bactrocera tryoni
Queensland fruit fly
hobo
bacterial neomycin
phosphotransferase
II (NPT)
Test of transformation
vector
No release plans known.
Raphael et al.
(2004)
Ceratitis capitata
Mediterranean fruit
fly
eGFP, white eye
RIDL
Minos, piggyBac,
Hermes
Sex separation, RIDL,
sperm marking, sex
conversion, SIT
Non-transgenics
produced commercially
or released worldwide
Loukeris et al.
(1995b); Salvemini
et al. (2006);
Saccone et al.
(2007)
eGFP
piggyBac
Sex separation and
sterility for SIT
Non-transgenics
produced commercially
and released in Panama
Benedict and
Robinson (2003);
Allen et al. (2004);
Handler et al.
(2009)
Numerous and
diverse
Numerous
Basic biological studies
esp. developmental,
evolutionary,
neurobiology. Laboratory
model.
No release plans known.
Medhora et al.
(1991); Loukeris et
al. (1995a)
SIT, sterility and sexing
No release plans known.
Scott et al. (2004)
Other Diptera (flies)
Cochliomyia
New world screw
hominivorax
worm
Drosophila spp.3
Lucilia cuprina
Green bottle fly
eGFP
piggyBac
Musca domestica
House fly
Neomycin, eGFP
Hermes, piggyBac
Stomoxys calcitrans
Stable fly
eGFP
Hermes
No release plans known.
SIT
No release plans known.
3
Species include Drosophila melanogaster, erecta, mauritiana, mohaviensis, pseudoobscura, sechellia, simulans, sukukii, virilis, willistoni, yakuba
40
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. O'Brochta et al.
(2000)
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Species name
Common name
Hymenoptera (wasps, bees, ants)
Apis mellifera
Honey bees
Athalia rosae
Coleseed sawfly
Lepidoptera (moths and butterflies)
Bicyclus anynana
Squinting bush brown
Modification
Transformation
system2
eGFP
Intended use
Current status
References
Tc1-element
Marker for detection of
viable Apis mellifera.
transformants
Test for sperm mediated
transformation system,
no commercial
applications
Pew (2004)
eGFP
piggyBac
Tet-OFF
No release plans known.
Sumitani et al.
(2003)
eGFP
Hermes, piggyBac
Model organism
No release plans known.
Marcus (2004)
eGFP
piggyBac
GAL4/UAS, Production
of recombinant
proteins and improved
silk, pharmaceuticals
Numerous contained
applications for
commercial
production of silks
and proteins.
SIT programme in
Canada using nontransgenic
Radiation sterilised
marker strain being
released in USA,
Non-transgenics
produced and
released for SIT in
USA and Mexico.
Prudhomme and
Couble (2002);
Imamura et al.
(2003)
Initial field test
completed. No release
Hoy (2000); Pew
Bombyx mori
Silk moth
Cydia pomonella
Codling moth
Pectinophora
gossypiella
Cotton pink bollworm
RIDL, eGFP, DsRed
piggyBac,
Marker for SIT, RIDL
Western predatory
LacZ
none
Feasibility study for
transformation through
Sex separation, improved
SIT
Bloem et al. (2007);
Marec et al. (2007)
Benedict and
Robinson (2003);
Wang et al. (2010)
Other arthropods
Acari (mites)
Metaseiulus
41
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Species name
Common name
occidentalis
mite
Crustacea (crustaceans)
Parhyale
None
hawaiensis
Procambarus
clarkii
North American
crayfish
Modification
Transformation
system2
Intended use
Current status
References
microinjection,
Model organism for risk
issues
plans known.
(2004)
DsRed
Minos
Model organism e.g. for
developmental studies
No release plans known.
Pavlopoulos and
Averof (2005)
neoR
Replicationdefective pantropic
retroviral vector
LSRNL-(VSV-G)
Feasibility study for
crayfish transformation
No release plans known.
Sarmasik et al.
(2001)
42
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
7.1.
Coleoptera
7.1.1.
Tribolium castaneum (Red flour beetle)
Tribolium castaneum is a global pest of stored grains. Flour beetles are long-lived, resistant to
insecticides and often abundant in silos. Because this species infests a food product (larvae
feed on damaged grain), costs related to the infestation include the direct damage to the
commodity, its presence in food products and the need to minimise harmful chemical residues
used for pest control in the products.
Protectants are applied when the grain is being stored. Insecticides used for this purpose
include pyrethroids, carbamates and organophosphates. Resistance to malathion has been
detected in this species. Increased regulation and extensive testing limit the rate of
development of new insecticides; therefore efforts are underway to develop alternatives
including biopesticides, insect growth regulators (IGRs), entomopathogenic fungi, pheromone
traps and plant products. Inert desiccating dusts have also been demonstrated to be effective,
but their use is not widespread. Because these pests occur in structures, fumigation can also
be used to control them. Phosphine is the most common fumigant and controlled CO2 and N2
atmospheres can also be implemented. For the same reason that fumigants and controlled
atmospheres can be effective, it is possible to cool the grain to the extent that development
halts. The costs and suitability of such methods must be considered in light of the fact that
grains are often stored for several months.
Tribolium castaneum is used as a laboratory model (see table 4). Because genetic control
would involve releasing GM-arthropods into the commodity, undesirable contamination
would result. Standards for food contamination with insect parts would restrict this approach
to inoculative methods. This is in contrast to e.g. mosquitoes or fruit flies in which the GMarthropods isolated from direct contact with the target of protection – people or fruits.
Additional information is available in Lorenzen et al. (2003), Siebert et al. (2008) and on the
world-wide-web (The University of Kentucky, 2003; Baldwin and Fasulo, 2010).
7.2.
Diptera (Culicidae)
7.2.1.
Aedes aegypti (Yellow fever mosquito)
Aedes aegypti has a global distribution and is most abundant in urban areas where larvae
develop in artificial containers holding water. The species is the primary vector of dengue,
yellow fever and Chikungunya viruses. Adult females feed on blood and both sexes feed on
natural sugar sources. Generation time is usually less than 3 weeks.
Several methods of control are available, but despite of their variety none is adequate, even
when applied in well-organised, expensive programmes. Available methods for control are:
adulticiding with insecticides, ovitrapping, source reduction in the form of draining artificial
and natural larval sites, space spraying (fogging) the interior or surroundings of homes and
other resting structures, larviciding using insect growth regulators, monomolecular surface
films, insecticides and microbial pesticides with Bt toxin from Bacillus thuringiensis
israelensis (Bti). Biological control using fish or copepods is also possible. Contemporary
control is also largely based on personal protection methods, notably the use of topical
repellents and personal protection via bednets, but have little effect on mosquito abundance.
In Europe fogging (mostly with pyrethroids) has been applied against heavy infestations in
parts of Italy and France. In France, surroundings of houses of residents infected with
Chikungunya virus were space-sprayed and breeding sites treated with the biopesticide Bti. In
43
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Italy, at times of heavy infestations, Bti tablets are sometimes handed out to residents, for use
in their gardens or on balconies. Other methods, like the use of insect growth regulators (e.g.
diflubenzuron) have been used in southern Europe (e.g. Greece). An overview of mosquito
control activities by country was published recently (Becker, 2007).
A large program in India in the 1960-1970s funded by WHO and the Indian Council for
Medical Research was moving aggressively to control this and other species by variations of
SIT when political opposition ended the program.
SIT and release of translocation strains that cause semi-sterility has been attempted against
this species on various occasions without success, mainly due to fitness costs, the operational
difficulty of irradiation and the density-dependent nature of the mosquito population (the first
genetic control trials using classical (i.e. non-GM) genetic control were conducted fifty years
ago, with Aedes aegypti, in Florida, USA (Dame et al., 2009)). In addition adult mosquitoes
seem to be less robust than other SIT species like Ceratitis capitata. The concept has not been
abandoned, with the hope that improved rearing and handling procedures of mosquitoes will
make genetic control viable. This is represented most prominently by the RIDL technology
(see table 4). A variety of other genetic control tactics have been deployed against this
species, as reviewed by Curtis (2006).
Additional information is available in Almeida et al. (2007), Lee et al. (2008), Yakob et al.
(2008), Fu et al. (2010) and on the world-wide-web (Oxitec, 2010a).
7.2.2.
Aedes albopictus (Asian tiger mosquito)
Aedes albopictus is a highly invasive mosquito originating from South-East Asia. It will likely
saturate its potential global distribution within the next few years and few regions that are
favourable for it to become established will be able to prevent an invasion. There are two
forms: a tropical form and a more temperate form capable of surviving the winter through egg
diapause. The likelihood of establishment is determined in part by the origin of the
mosquitoes that are introduced into a particular destination. Its habitats and habits are similar
to Aedes aegypti (see chapter 7.2.1), but with a notable daytime feeding propensity. This puts
also individuals who have well-sealed housing at risk for exposure to this species and the
diseases it transmits.
Available conventional methods for control are the same as for Aedes aegypti (see chapter
7.2.1). Over the recent years, attempts to control populations of Aedes albopictus have been
undertaken in Italy, by using classical SIT (Bellini et al., 2007). During a pilot project in
2004, in a 10 ha area in and around Rimini, Northern Italy, some fifty thousand irradiationsterilised males were released, with a small but noticeable impact (in terms of induced
sterility). Since then, efforts have intensified with increased mass-production and releases.
These efforts, though small in scale, demonstrate the feasibility of genetic control against this
species inside the EU. Successful eradication of Ae. albopictus infestations has occurred in
France, Switzerland and Italy. In Sardinia and Pisa, the species was eradicated between 1996
and 1997. Eradication was also accomplished in a few municipalities in the regions of Veneto
and Piemonte. In all of these cases intensive searches for breeding sites, followed by
treatment with Bti, was sufficient to eliminate the invasion. The scale of infestations,
however, was always limited (Scholte and Schaffner, 2007).
44
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
A small trial to introduce inability to diapause was performed in Illinois in the early 1990s
and evidence of an increase in this trait resulted. As with Aedes aegypti, hope for genetic
control using technology similar to RIDL is the current focus of development (see table 4).
More information is available in Hawley (1988), Bellini et al. (2007) Benedict et al. (2007)
and on the world-wide-web (GISD, 2009a; Oxitec, 2010a).
7.2.3.
Aedes fluviatilis
The distribution of Aedes fluviatilis (see table 4) extends from southern Mexico to northern
Argentina on the eastern side of the Andes mountains. Aedes fluviatilis blood feeds on both
humans and animals, but is not considered to be a vector of either yellow fever or dengue.
Therefore, it is a laboratory model only for studying e.g. avian malarias.
No efforts to control this species by genetic means have been performed, have been described
or are expected in the next decade.
Information on Aedes fluviatilis is for example available on the world-wide-web (WRBU,
2010a).
7.2.4.
Anopheles albimanus (New world malaria mosquito)
Anopheles albimanus is a tropical to sub-tropical species and considered an important vector
of malaria in Central America. It extends from the extreme southern USA through Central
America into northern South America and is found on Caribbean islands. It feeds on both
feral and domesticated animals as well as humans. Like many anophelines, larvae develop in
shallow fresh water bodies. These may be natural (stream pools, river margins, estuaries) or
artificial (hoof prints, ponds). They are most common in sunlit pools. Adults rest in shaded,
cool and dark areas during the day and feed during the night. The generation time is usually
less than three weeks. Unlike Aedes mosquitoes, eggs are not capable of diapausing or
surviving desiccation.
Extensive organised control of this species is not widespread though several methods are
available: adulticiding with insecticides, source reduction in the form of draining artificial and
natural larval sites, indoor residual spraying of homes, larviciding using insect growth
regulators, surface films, insecticides and Bti toxin. Biological control using fish and
bioinsecticides are also possible. Personal protection via bednets, house screening and
repellents also reduce personal risk but do not significantly reduce vector abundance.
Two large SIT programs against this species were conducted in El Salvador in the 1970s. The
first was highly successful and resulted in local elimination of this vector. The second was an
expanded feasibility study with limited benefit. Efforts were abandoned with the advent of
civil war. No intent to revive SIT in any form against this species has been reported since (see
table 4). It remains a natural target however since germline transformation is simple and many
islands exist on which SIT would be attractive because reintroduction could be controlled.
More information is available in Charles and Senevet (1953), PAHO (1993) and Perera et al.
(2002).
45
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
7.2.5.
Anopheles arabiensis
Anopheles arabiensis is widely distributed in Sub-Saharan Africa and surrounding islands
including La Rèunion and Mauritius. The biological traits are similar to its sibling species,
Anopheles gambiae s.s., which is discussed in chapter 7.2.6. Important differences include the
following: It extends into more arid areas than Anopheles gambiae s.s. and its blood-feeding
preference is less anthropophagic and more opportunistic (zoophagic). A final notable
contrast with Anopheles gambiae s.s. is that it often rests outside of dwellings so that homefocused control such as insecticide-treated bednets and indoor residual spraying are less
effective against this species than they are against Anopheles gambiae s.s. which rests
indoors.
The most common “control“ measures occur incidentally to what is essential personal
protection: indoor residual spraying of insecticides (DDT or synthetic pyrethroids) and
insecticide treated curtains and bednets. The latter are usually treated with a long lasting
formulation of a pyrethroid insecticide. Mosquito coils and space sprays provide some
protection but have little impact in terms of population suppression. Larviciding is not widely
practiced. Experimental methods include fungus-impregnated resting sites and wall coverings
to prevent mosquito entry or to expose them to these biopesticides when passing through.
The International Atomic Energy Agency is developing a conventional SIT programme
against this species. A research component included production of GM-Anopheles arabiensis
(see table 4), but there are no plans to release these. This programme has field sites on La
Rèunion Island and in the Northern State of Sudan.
Additional information on Anopheles arabiensis is available in Morlais et al. (2005), Helinski
et al. (2008) and Robinson et al. (2009).
7.2.6.
Anopheles gambiae s.s. (African malaria mosquito)
Anopheles gambiae s.s. is the most important vector of malaria in Africa where it transmits all
species of human malaria resulting in a majority of the estimated 1 to 3 million deaths per
year. It is limited in distribution to sub-Saharan Africa as far south as northern South Africa
and has invasive potential as demonstrated by a calamitous invasion of northern Brazil in the
1930s. It was eliminated by an intensive larviciding campaign using the toxic chemical Paris
Green; otherwise, it would likely have spread throughout northern South America, Central
America and the Caribbean. It is highly anthropophagic/philic. Like all other anophelines, it
has no capacity for egg diapause, and the generation time is less than 3 weeks. It rests indoors
and in other peri-domestic shaded cool areas.
The most common “control” measures are the same as for Anopheles arabiensis. Spraying of
insecticides and insecticide treated curtains and bednets have been demonstrated to reduce
wild populations – a fact which reflects the mosquito`s strong anthropophagic character.
Anopheles gambiae s.s. is clearly a high value target for modification because it is literally the
most dangerous animal in the world. SIT has been considered for this species in Burkina Faso
where a small but unsuccessful attempt with sterile hybrids was undertaken during the 1970s.
The species is also the subject of attempts to accomplish transgenic sterilisation, sex
separation and Plasmodium resistance. Modern GM-methods are currently the only methods
being developed for genetic control. Paratransgenic methods using Wolbachia and GM-Asaia
bacteria are also under development (see table 4).
46
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Additional information is available in Coluzzi et al. (1979).
7.2.7.
Anopheles stephensi (Indo-Pakistan malaria mosquito)
Anopheles stephensi is one of the most important malaria vectors in Asia where it is
distributed from eastern China westward to Egypt. It can be found both in urban and rural
areas. Like Aedes, it can be found in artificial containers and water catching structures used
for household water: cisterns, wells, tubs and fountains. In rural areas, they are found in
stream pools, margins, seepages, irrigation channels etc. It is an anthropophagic species and a
primary malaria vector.
Like most Anopheles control, it utilizes broad spectrum measures including space spraying,
bednets, residual insecticides and source reduction. Larval sites and potential blood sources
are widespread, so it is difficult to identify and eliminate individual sources of this species.
In India, this species is confined to urban environments, surrounded by rural areas where
Anopheles culificacies serves as the main vector of malaria. Occurrence in these urban islands
has been considered ideal in terms of genetic control because treatment of a relatively small
area with large numbers of inhabitants will be very cost-effective. Curtis (2003) has proposed
such ‘islands’ for genetic control (using SIT or RIDL).
There are no current efforts to revitalize these efforts. It is, however, a popular anopheline to
study in the laboratory. Because it is easily transformed and cultured, it has been one of the
most advanced species for developing GM-applications (see table 4), but not particularly
because of its competence as a vector (and mostly in conjunction with non-human malarias).
Information on Anopheles stephensi is available for example on the world-wide-web (WRBU,
2010b; Allmosquitoes.com 2010).
7.2.8.
Culex quinquefasciatus (Southern house mosquito)
Culex quinquefasciatus originated in North America, but is now widespread throughout the
tropics and subtropics. It is a common urban mosquito capable of developing in numerous
sites including highly polluted water bodies contaminated with animal and human faeces.
Therefore, it can develop in e.g. storm water runoff, cesspools and latrines. Culex species
often blood feed on both humans and animals (especially birds), serving as a conduit for
viruses that are maintained primarily in animal reservoirs such as West Nile virus. Culex
species often harbour Wolbachia that cause cytoplasmic incompatibility manifested as sexual
sterility between various mosquito isolates. Eggs are laid in discrete rafts, rather than
dispersed as anopheline and aedine eggs.
Draining larval sites is effective and often possible. However, many of the sites are small and
have domestic functions or result from activities of wild animals and are either necessary or
isolated. Insecticides including pyrethroids, carbamates and organophosphates can be used,
but resistance is widespread and alternatives are needed. Efforts to develop and apply
biological controls including Bacillus thuriengiensis and Bacillus spheaericus offer options as
do covering small protected larval sites (latrines) with polystyrene beads.
A few small efforts to implement genetic control have been made (see table 4), but there are
no current plans to do so. While germline transformation has been accomplished, it is not easy
to do. Cytoplasmic incompatibility may offer a simpler entree to produce strains suitable for
47
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
SIT and advances are being made in this area in Aedes species, some technology of which
will be transferable.
Additional information is available in Allen et al. (2004) and on the world-wide-web (GISD,
2006a).
7.3.
Diptera (Tephritidae)
7.3.1.
Anastrepha ludens (Mexican fruit fly)
Anastrepha ludens is limited to tropical and subtropical parts of the Americas. It occasionally
infests areas in the southern USA after which intensive elimination campaigns are conducted.
It is primarily a pest of citrus and mango although it also infests pear, peach and apple among
other fruits. Adults are long-lived, surviving even up to a year.
Like many agricultural pests, larvar being hidden inside a grain or fruit provides good
protection against insecticides. Furthermore, unlike some fruit flies, this species is not lured
by sex pheromones. It can be trapped using protein and carbohydrates as baits. Spraying
insecticide treated protein baits is also effective and the use of tents with cloth of a certain
mesh size (to keep the fruit fly in but let the natural predators escape). SIT is also used on an
area-wide basis. Burying unharvested fruit also reduces infestations.
Anastrepha ludens is the subject of classical SIT control (see table 4), so efforts to transform
it sexually, sterilise it and sex it are likely, but have not been implemented.
More information is available in Condon et al. (2007) and on the world-wide-web (University
of Florida, 2001a).
7.3.2.
Anastrepha suspensa (Caribbean fruit fly)
Anastrepha suspensa (see table 4) is distributed in Florida, Cuba, Hispañola and Puerto Rico.
It is closely related to Anastrepha ludens (Mexican fruit fly). The economic damage to fruits
is not as widespread as that of e.g. Ceratitis capitata (Mediterranean fruit fly), but some fruits
are severely affected e.g. guavas, Surinam cherries and roseapples. Its size ranges from ½ to
twice that of a House fly. The life stages and habitats are otherwise similar to other fruit flies
with females ovipositing in fruit, larvae developing in the flesh and dropping to the ground to
pupate.
Pesticide bait sprays are the method of choice to control this species.
Information is for example available on the world-wide-web (University of Florida, 2001b;
Weems and Heppner, 2001; EPPO, 2006).
7.3.3.
Bactrocera dorsalis (Oriental fruit fly)
Bactrocera dorsalis is a member of a species complex composed of very closely related
species and the exact taxonomy of this complex is not fully resolved yet. Bactrocera dorsalis
is widespread throughout Asia and now established in Hawaii. There are occasional outbreaks
in the mainland USA prompting rapid strong elimination programs. The life stages and habits
are generally similar to the other fruit flies discussed in this report. Adults are sexually mature
in about nine days. A female can lay more than 3,000 eggs during her lifetime, but 1,200 to
48
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
1,500 eggs per female are typical. A wide range of ripe fruits are preferred for oviposition
including citrus, mango, peach, papaya and guava.
Biological control using parasitoids and SIT has been helpful (see table 4). In addition,
insecticide-impregnated fiberblocks or cotton containing the male attractant methyl-eugenol
are effective. Steiner traps baited with a lure and toxicants are also used to monitor the
presence and control of the flies. As is usual for fruit flies, burying unharvested mature fruit
reduces populations.
Additional information on Bactrocera dorsalis is for example available on the world-wideweb (University of Florida, 1999a).
7.3.4.
Bactrocera oleae (Olive fruit fly)
Bactrocera oleae is a pest of olives throughout most of the Mediterranean and an actual (or
potential) pest wherever the climate is suitable for olives. Unlike many fruit flies, which will
develop on a wide variety of fruits, the larvae feed exclusively on olives. Therefore,
abundance and damage is highest in commercial plantings of the host plant. This species has
spread to olive growing areas of California, Africa, Asia and Mexico. The number of
generations per year is limited by climatic conditions. In cooler climates, flies overwinter as
pupae beneath the soil.
Currently, the standard control method is to treat the olive trees repeatedly with bait spray
containing a food lure and organophosphates. It is applied when trap captures begin to
increase in early summer. When an advanced fruit infestation is detected cover sprays with
insecticides can be applied. The last area-wide spray is allowed 30 days before the olive fruit
collection starts. Unless the new crop in olive plantations is treated repeatedly by insecticidal
cover or bait sprays from June/July to October/November, the entire olive fruit production can
be wiped out by mid fall. Alternative methods have been developed and tested but none of
these were sufficiently effective. This included the release of parasitoids (e.g. Opius
concolor), pheromones, food lure, visual lures and mass trapping.
The SIT may represent an alternative to the massive application of insecticides. Since 1961
extensive research has been devoted to the development of the SIT against the Olive fruit fly.
The effort lasted for about two decades, especially in Greece, until it was abandoned because
of many difficulties with mass rearing, the quality of the mass reared flies and, finally, the
rather poor results from pilot field applications. The two most important problems are the
very high cost of the artificial diet and the difference in mating time between the wild and the
released flies, i.e. most mass reared olive flies mate earlier in the day than their wild
counterparts and therefore the sterility cannot be induced efficiently into field populations. In
recent years alternative, less expensive ingredients for the diet have been tested successfully.
The viability of SIT has been promoted by recent advances in fly culturing (see table 4). The
typical transgenic improvements for SIT (sex-separation and sterility) would improve its
efficiency.
More information is for example available on the world-wide-web (Weems and Nation, 1999;
University of Florida, 1999b; Oxitec, 2010b).
49
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
7.3.5.
Bactrocera tryoni (Queensland fruit fly)
Bactrocera tryoni is native to Australia, the focus of its pestiferousness. It occurs in climates
ranging from temperate to tropical. Fruits affected are peach, apple, citrus, plum and
numerous other fruits. There is significant risk of invasion in similar climates including
Florida. It became established in Perth, Western Australia but was eliminated from that area
by baits, male lures and SIT. This fruit fly does not breed continuously but overwinters in the
adult stage. The total life cycle requires two to three weeks in summer and up to two months
in the fall. Adult females live many months, and four or five overlapping generations may
occur annually. Adults may live a year or even longer.
Males can be attracted to insecticide-laced lures, bait spays, insecticide treatment (e.g.
Malathion) and also fruit destruction/burying is often a method of control.
SIT has been practiced, so the attendant needs for sex separation and sexual sterilisation
would be natural targets for GM-application (see table 4).
Information on Bactrocera tryoni is available amongst others in Raphael et al. (2004) and on
the world-wide-web (University of Florida, 2002).
7.3.6.
Ceratitis capitata (Mediterranean fruit fly)
Ceratitis capitata, the Mediterranean fruit fly, originated in originated in East Africa. From
there it has spread relatively recently to other parts of the world including the Mediterranean
basin, the American continent (up to California and Florida), and Australia. Due to its
invasiveness the Mediterranean fruit fly is considered one of the most important quarantine
pests and its presence in a particular region/country has a significant impact on the export of
fruits. Larvae feed on the fruits of many plants including citrus, apricot, and strawberry.
Damage results from fruit tissue piercing during oviposition, development of the larvae inside
the fruit and introduction of secondary pathogens by the ovipositing female. Females may lay
several hundred eggs. This species can overwinter as a pupa if the winters are relatively mild.
Mere piercing of the fruit by the ovipositing females reduces the market value for fresh fruits.
The standard control measure against the Mediterranen fruit fly is the spraying of insecticides.
Commonly, 4-12 spray applications per year are required. Increasingly stringent restrictions
on currently employed insecticides by importing regulatory agencies demands that alternative,
non-chemical control measures are being developed and applied to fruits and vegetables
exported from the region. However, some of the non-chemical strategies that are used
successfully against some other fruit fly species, e.g. Male Annihilation Technique (MAT) or
the use of parasitoids, do either not function satisfactorily for the control of this species.
Currently, the only viable and environmentally safe method that is available commercially as
an alternative to insecticides is the SIT.
Because of the high economic importance of this species, conventional SIT has been used in
area-wide control programs and as a prophylactice measure in California. It is a likely
candidate for sexual transformation of females to males, sexual sterilisation and sex
separation by transgenesis (see table 4). All currently available GSS do not involve
transgenics but the development of GM-strains is ongoing.
More information is available in Saccone et al. (2007) and on the world-wide-web (University
of Florida, 2001c; Oxitec, 2010c; Invasive.org 2010).
50
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
7.4.
Diptera (other)
7.4.1.
Cochliomyia hominivorax (New world screwworm; Calliphoridae)
The native range of Cochliomyia hominivorax included all of tropical and subtropical North
America before its elimination from that region. Now it is confined to the Caribbean and
northern South America. Where it is present, it is a serious pest of livestock (and feral
mammals) resulting in loss of weight, hide damage and death. It occasionally infests humans.
Females lay up to 500 eggs in vertebrate wounds such as navels of new born calves and
injuries. The young larvae burrow into and feed on flesh for three to seven days causing a
condition called myiasis after which they are mature and drop to the ground where they
pupate. Females can lay up to 3,000 eggs during their life and have considerable dispersal
capacity – up to 200 km.
Control by SIT has been extremely effective. A barrier against reinvasion of Central and
North America is now maintained by aerial release of radio-sterilised flies over the Darien
Gap in Panama thus providing an example of how quarantine must be included as an effective
control measure. As a demonstration of the power of this technology, it was the intervention
of choice when Cochliomyia homnivorax was accidentally introduced into Libya in the late
1980s. However, this was made possible only by an existing SIT programme in the Americas
that could provide sterile flies. An effective surveillance system for its presence in elimination
zones is conducted by herdsmen who report its presence to veterinarians. Measures such as
treating fresh wounds with prophylactic chemicals in the form of smears (coumaphos,
lindane, or ronnel) is possible but obviously requires attention to each animal and wound.
Animals can also be treated in toto by spraying with ronnel or coumaphos or dipped in the
latter.
Elimination by area-wide SIT is the control of choice. Germline transformation has been
accomplished, but the success of SIT using bisexual release and declining rates of release
necessary to maintain the barrier has led to an entrenchment of existing technology (see table
4). In the absence of this technology, it would be difficult to control this pest as it is extremely
dispersed and highly mobile.
Additional information can be found for example in Dame (1984), Benedict and Robinson
(2003) and on the world-wide-web (Merck, 2008a; Oxitec, 2010d).
7.4.2.
Drosophila spp. (Fruit flies; Drosophilidae)
The Drosophila species that are considered in this report are all model animals for laboratory
studies including development, evolution and gene regulation (see table 4). The following
refers generally to drosophilids, but those that have been transformed are of greatest interest:
Drosophila melanogaster, erecta, mauritiana, mohaviensis, pseudoobscura, sechellia,
simulans, suzukii, virilis, willistoni and yakuba. They are distributed worldwide in a wide
variety of habitats though the number of species is greater in the tropics. Many are pesta and
specifically associated with human activities around homes and farms. The most prominent
species of study, Drosophila melanogaster is often referred to simply as “the fruit fly”
although tephritids are the true fruit flies (see chapter 7.3). Their pestiferousness is usually
minimal although specific cases of problematic infestations exist.
51
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Drosophilids can be controlled if necessary by spraying insecticides on larval sites, space
spraying and screening. When they infest decaying plants, they can be reduced by burying or
burning the material.
Numerous methods to develop genetic control are possible for Drosophila melanogaster;
however, the pestiferousness is inadequate to motivate further development and implemention
of genetic control.
More information on Drosophila are provided for example in Medhora et al. (1991) and
Loukeris et al. (1995a).
7.4.3.
Lucilia cuprina (Green bottle fly; Calliphoridae)
Lucilia cuprina is a largish fly with an iridescent green colour which immediately
distinguishes it from House flies and Stable flies. While named also Australian sheep blow
fly, it is also found in North America and Africa. It flourishes in warm and humid, but not hot
environments. The veterinary problem results from the larval stage that feeds on decaying
flesh of mammals causing losses, particularly in sheep production. Irritation (rubbing and
biting) results and the volatiles produced by damage from larvae in turn attract more flies to
lay eggs in the wound. Adults are strong fliers, dispersing as far as 15 km.
Affected areas are often where faeces and urine stain the wool, so measures to reduce this are
effective. Some preventative measures are surgical e.g. tail docking or shearing practices.
Insecticides including pyrethroids, organophosphates, and insect growth regulators are also
helpful. These must be used cautiously since regulations control the amount of insecticide
residues that are permissible in the wool. Fly traps are also effective when combined with
sheep management practices.
SIT has been investigated against this species. Considerable aberration and classical genetic
efforts have developed potential strains for release (see table 4).
Information is available for example in Mahon (2001) andScott et al. (2004) and on the
world-wide-web (Queensland government, 2005, Government of Western Australia, 2010).
7.4.4.
Musca domestica (House fly; Muscidae)
Musca domestica is distributed worldwide. It is a filth fly signifying that it is often associated
with areas with poor sanitation, but it is omnivorous and common anywhere where suitable
food is available. Adults do not feed on blood or piece skin but rather salivate on food items
and consume the dissolved matter. Females can lay 500 eggs during their lifetime. Because of
the foul diets that houseflies prefer, they mechanically transmit parasitic and bacterial diseases
including Giardia, Entamoeba and Ascaris.
Numerous methods are available including mechanical and electrical grid traps, sticky traps,
insecticide space sprays and baits. Because it thrives where waste is abundant, sanitation can
reduce or eliminate problem levels of flies. Musca domestica is omnivorous; so removing all
sources where flies can proliferate is difficult.
SIT has been proposed and methods for separating sexes, pupae from larvae etc. have been
developed (see table 4). Adult females are believed to mate only once, and although this is not
a prerequisite for successful SIT, this makes them attractive for SIT.
52
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Information on Musca domestica is available for example on the world-wide-web (Illinois
Department of Public Health, 2010).
7.4.5.
Stomoxys calcitrans (Stable fly; Muscidae)
The Stable fly, Stomoxys calcitrans is distributed worldwide in temperate and tropical areas.
Both males and females feed on blood of humans and other mammals. Bites are painful, but
they transmit diseases only by mechanically e.g. anthrax, infectious equine anaemia and surra.
They can be quite annoying and bites are painful to people and animals. Their livestock harm
is primarily due to biting and blood loss which results in irritation, reduced weight gain and
milk production. Larvae develop in wet decaying vegetation such as hay, seaweed, silage and
semi-dry dung. Stable flies are capable of long distance movement so breeding sites are often
remote from the location at which they cause problems.
Sanitation is the primary means of control. Removal and disposal of stray straw and hay from
the vicinity of livestock is effective.
Conventional sex-separation strains have been developed using dominant markers and
chromosome rearrangements (see table 4) and the species is easily colonized and cultured.
SIT against this species has been proposed numerous times but currently there are no efforts
to develop an operational programme. Unlike most SIT programmes, males cause damage, so
releasing them would be problematic.
More information is provided in Patterson et al. (1981) and on the world-wide-web (Merck,
2008b).
7.5.
Hymenoptera
7.5.1.
Apis mellifera (Honey bee)
Apis mellifera is a beneficial insect with an important economic value as a pollinator and e.g.
responsible for 15 to 30 percent of the food U.S. consumers eat. Beside this function Apis
mellifera produces various products of interest like honey, beewax or propolis. It has a near
global distribution that includes Europe, North- and South America, Australia and South-East
Asia.
Honeybees are currently undergoing a worldwide decline due to infestations of parasitic mites
(e.g. Acarapis woodi, Varroa destructor), ravages of various viruses and susceptibility to
pesticides and insecticides. This decline is causing big problems in agriculture and substantial
economic losses (ZipcodeZoo.com, 2009a).
One goal of genetically modifying Apis mellifera is to create an insecticide-resistant strain
(see table 4), but so far only tests for sperm-mediated transformation are ongoing (Pew,
2004).
7.5.2.
Athalia rosae (Turnip sawfly)
Athalia rosae originated in Europe, but has currently a global distribution. It is a pest of
cruciferous and umbelliferous plants including winter rape. Caterpillar-like larvae consume
the underside of leaves of the plants on which eggs are laid. There are up to three generations
per year depending on the weather. Adults are capable of long-distance wind-assisted
dispersal. Both sexes feed on plants.
53
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
The pest was thought to have been eradicated in the 1940s, but has re-emerged as a serious
pest problem. Control measures include cultural practices (eradication of weeds, under-winter
plowing, destroying plants remains and planting trap crops) as well as insecticide applications
to plants. It is difficult to control, in part due to the rapid growth of the plants on which it
feeds and the diverse acceptable diets.
There have been no attempts to use e.g. SIT against this species. However, the haploid
determination of male sex appears to offer opportunities, particularly if inoculative releases
could be accomplished (see table 4).
Additional information on Athalia rosae is for example available on the world-wide-web
(AgroAtlas, 2010; INRA, 2010).
7.6.
Lepidoptera
7.6.1.
Bicyclus anynana (Squinting bush brown)
Bicyclus anynana is an African species, which is a subject of transgenic modification as a
model system (see table 4). It displays wet and dry seasonal colour change (polyphenism) and
other environmentally influenced plasticity, the study of which provides insight into gene
regulation/environment interactions.
Additional information can be found in Marcus et al. (2004), Brakefield et al. (2009) and in
the world-wide-web (AnAge, 2010).
7.6.2.
Bombyx mori (Silk moth)
Bombyx mori, the primary caterpillar used for silk production, is distributed worldwide
wherever there are interested individuals who wish to culture it. It is a completely
domesticated descendant of Bomyx mandarina (Asian distribution) with which it can
hybridise, but no feral form exists. The primary diet of the caterpillar consists of white
mulberry leaves. Larvae feed continuously, and the fifth stage larvae wrap themselves in a
cocoon of silk from which adults emerge 2-3 weeks later. Adults cannot fly.
Bombyx mori is not a pest species either in artificial or natural settings.
Genetic control is not warranted, however numerous GM-applications for producing various
types of silks and pharmaceutical proteins are being developed (see table 4).
Additional information is for example available in Prudhomme and Couble (2002).
7.6.3.
Cydia pomonella (Codling moth)
Cydia pomonella is an agricultural pest, the larva being known as the common apple worm or
maggot. It is native to Europe and was introduced to North America, where it has become one
of the regular pests in apple orchards. It is found almost worldwide, generally wherever
apples and other tree fruits are cultivated. Adult female lay their eggs close to the fruit so the
larvae can eat on the fruit immediately after hatching for about three weeks. Two to three
generations per year are possible when the climatic conditions are conducive.
Cydia pomonella is conventionally controlled by insect growth regulators, broad spectrum
insecticides or mating disruption.
54
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
The small number of generations and predictable emergence of virgin adults makes this
species a natural target for SIT (see Table 4). It is currently being controlled in Canada using
this method.
Information on Cycia pomonella is for example contained in Bloem et al. (2007), Marec et al.
(2007) and on the world-wide-web (USDA, 1999).
7.6.4.
Pectinophora gossypiella (Cotton pink bollworm)
Pectinophora gossypiella is distributed worldwide wherever cotton is grown but it originates
from India. Eggs are laid in cotton bolls where the larvae feed and destroy the cotton by
chewing through the fibers and feeding on the seeds. This directly damages the fibers and also
provides a means of entry for secondary infestations of insects and fungal infections.
The inaccessibility of the boll interior makes control difficult; however insecticide use is of
some value. Bt cotton has been particularly effective against this species (though not in all
countries). Cultural control can be accomplished by plowing down standing plants post
harvest. Soaking fields with water in irrigated plantings also drowns remaining larvae;
however those that do survive can overwinter to emerge the following spring. Pheromone
mating disruption is also practiced.
A large integrated control programme is underway including conventional SIT in the USA
and Mexico. This programme uses a combination of pheromone mating disruption, Bt cotton
and SIT. Trial releases using a fluorescent protein marked GM-strain are underway in
Arizona. Such marking provides a long-lasting alternative to fluorescent dust (see table 4).
Additonal information is for example available in Benedict and Robinson (2003), USDA
(2008) and on the world-wide-web (NCC, 2001; University of California, 2010; Oxitec
2010e).
7.7.
Acari
7.7.1.
Metaseiulus occidentalis (Western predatory mite)
Metaseiulus occidentalis is a beneficial arthropod due to its effective predation on pest mites
in agricultural crops and is therefore also of economic importance (e.g. in suppressing spider
mites in apple orchards). In Europe this mite is distributed in middle as well as south and
south-eastern Europe but absent from Scandinavia.
After Metaseiulus occidentalis developed resistance against organophosphorus this species
became a model organism and a strain resistant against carbaryl, organophosporus and
sulphur was developed in the laboratory by conventional methods and released in pest control
programmes (Hoy, 2009).
This species became also a model organism in the field of genetic modification (see Table 4).
A new technique, maternal microinjection, was tested leading to initial field tests with the
GM-mite but no further research is ongoing. In future, GM-strains could be produced leading
to an improved performance as a biological control agent (Presnail and Hoy, 1992) e.g.
allowing the application of this mite and the use of insecticides of the same time.
55
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
7.8.
Crustacea
7.8.1.
Parhyale hawaiensis
The amphipod crustacean Parhyale hawaiensis is a model organism in evolutionary biology.
A GM-strain was produced using the Minos transposable element (see table 4) since using
genetic modification could benefit further studies of gene functions in crustaceans
(Pavlopoulos and Averof, 2005).
7.8.2.
Procambarus clarkii (North American crayfish)
Procambarus clarkia is an alien species in Europe. Native to North America it was introduced
for aquaculture purposes and soon became a serious pest. It not only threatens indigenous
crayfish species (e.g. Astacus astacus, the European crayfish) by direct and indirect
competition but also transmits the crayfish plague, a fungal disease that spreads rapidly in
Europe and against which Procambarus clarkii is immune. It reduces native biodiversity and
alters food webs and ecosystem processes.
Attempts to contain the spread of Procambarus clarkii have been made but sofar without
success. A new approach could be the sterilization of males leading to population decline
when released. Additional studies are needed to perfect the technique and to calculate the
number of males needed to be released (Aquiloni et al., 2009). Procambarus clarkii also
served as a model species in crustacean transformation (see table 4). The transfer of superior
genetic traits like disease resistance into economically important crustacean species for
commercial aquaculture could be possible (Sarmasik et al., 2001).
8.
GM-arthropods of possible relevance for the EU in the next 10 years
In order to select those GM-arthropods that are far advanced in the research and development
pipeline and could possibly be of relevance for the EU within the next 10 years selection
criteria were defined as described in chapter 2. Table 5 shows the result of this selection
process.
56
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 5.
Selection of GM-arthropods relevant for the EU within the next 10 years
GM-arthropods worldwide
GM-arthropods of possible relevance for the EU during the next 10 years
Release programmes with
non-GM-arthropods
available world wide
Conclusion
Red flour beetle
Y
Y
Y
N
N
ZipcodeZoo.com (2009b)
Y
Y
Y
Y4
Y
Y
Y
Y
Y4
Y
Y
Y
N
N
N
Almeida et al. (2007); Yakob et al.
(2008); WRBU (2010c)
Benedict and Robinson (2003);
Benedict et al. (2007); GISD
(2009a), WRBU (2010e)
WRBU (2010a)
Y
Y
Y
N
N
Y
Y
4
Y
Charles and Senevet (1953)
Morlais et al. (2005)
4
Y
Julvez (1989)
Expanding
Common name
Established in EU
Species name
References
Recorded in EU
Selection criteria
Coleoptera (beetles)
Tribolium castaneum
Culicidae, Diptera (mosquitoes)
Aedes aegypti
Yellow fever mosquito
Asian tiger mosquito
Aedes albopictus
Aedes fluviatilis
New world malaria mosquito
Anopheles albimanus
1
Y
Anopheles arabiensis
Anopheles gambiae s.s
1
Y
African malaria mosquito
Y
Y
Y
Y
Anopheles stephensi
Indo-Pakistan malaria mosquito
N
N
N
N
N
ZipcodeZoo.com (2009c)
Culex quinquefasciatus
Southern house mosquito
Y
N
N
Y4
N
Benedict and Robinson (2003);
GISD (2006a)
Tephritidae, Diptera (true fruit flies)
Anastrepha ludens
Mexican fruit fly
N
N
N
Y
N
EPPO (2006b)
Anastrepha suspensa
Caribbean fruit fly
Y
Y
N
Y4
N
Holler and Harris (1993); EPPO
(2006a); The Carribean pest
57
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
GM-arthropods worldwide
GM-arthropods of possible relevance for the EU during the next 10 years
Conclusion
References
Release programmes with
non-GM-arthropods
available world wide
Expanding
Common name
Established in EU
Species name
Recorded in EU
Selection criteria
information network (2010a)
Habu et al. (1984); EPPO (2006c);
Orankanok et al. (2007); EPPO
(2010)
ZipcodeZoo.com (2009d); Oxitec
(2010b)
GISD (2006b); Jessup et al. (2007)
Bactrocera dorsalis
Oriental fruit fly
Y
Y
N
Y
N
Bactrocera oleae
Olive fruit fly
Y
Y
Y
Y4
Y
Bactrocera tryoni
Queensland fruit fly
Y
Y
N
Y
N
Ceratitis capitata
Mediterranean fruit fly
Y
Y
Y
Y
Y
Cochliomyia hominivorax
New world screwworm
N
N
N
Y
N
Drosophila spp.2
Y
Y
Y
N
N
Lucilia cuprina1
melanogaster is the common fruit
fly or vinegar fly, but these are
not true fruit flies
Green bottle fly
Y
Y
Y
Y4
Y
Mahon (2001)
Musca domestica
House fly
Y
Y
Y
N
N
ZipcodeZoo.com (2009f)
Barnes et al. (2004); Dantas et al.
(2004); Guillen and Sanchez (2007);
GISD (2009b)
Other Diptera (flies)
58
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Benedict and Robinson (2003);
ZipcodeZoo.com (2009e)
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
GM-arthropods worldwide
GM-arthropods of possible relevance for the EU during the next 10 years
Conclusion
Stomoxys calcitrans
Stable fly, biting House fly
Y
Y
Y
Y4
Y
Expanding
Common name
Established in EU
Species name
Release programmes with
non-GM-arthropods
available world wide
References
Recorded in EU
Selection criteria
Patterson et al. (1981);
ZipcodeZoo.com (2009g)
Hymenoptera (wasps, ants, bees)
Apis mellifera
Honey bee
Y
Y
Y
N
N
Athalia rosae
Turnip sawfly, coleseed sawfly
Y
Y
Y
N
N
N
N
N
N
N
3
N
ZipcodeZoo.com (2009a)
ZipcodeZoo.com (2009h)
Lepidoptera (moths and butterflies)
Bicyclus anynana
Squinting bush brown
Bombyx mori
Silk moth
N
N
N
N
Cydia pomonella
Codling moth
Y
Y
Y
Y
Y
Pectinophora gossypiella1
Cotton pink bollworm
Y
Y
Y
Y
Y
Western predatory mite
Y
N
N
Y
N
ZipcodeZoo.com (2009j); Hoy
(2009)
N
N
N
N
N
ITIS (2010)
Benedict et al. (2008);
ZipcodeZoo.com (2009i)
The Caribbean pest information
network (2010b)
Benedict and Robinson (2003)
Acari (mites)
Metaseiulus occidentalis
Crustaceae (crustaceans)
Parhyale hawaiensis
59
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
GM-arthropods worldwide
GM-arthropods of possible relevance for the EU during the next 10 years
Red swamp crayfish
Y
Y
Y
Conclusion
Established in EU
Procambarus clarkii
References
Release programmes with
non-GM-arthropods
available world wide
Common name
Expanding
Species name
Recorded in EU
Selection criteria
N
N
1
Sarmasik et a. (2001); Aquiloni et al.
(2009)
only relevant in overseas territories
Species include e.g. Drosophila melanogaster, erecta, mauritiana, mohaviensis, pseudoobscura, sechellia, simulans, virilis, willistoni, yakuba
3
no open field release
4
no ongoing releases
N=no, Y=yes
2
60
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
The 30 GM-arthropods listed in table 4 (counting Drosophila only once) were reduced to a
list of 10 GM-arthropods of possible relevance for the EU in the next 10 years (four of them
only relevant for overseas territories).
Tribolium castaneum, Drosophila spp. and Parhyale hawaiensis were not considered relevant
since they are model organisms and no release programme is available or anticipated. The
same holds true for Musca domestica, Apis mellifera, Athalia rosae and Procambarus clarkia
were no release programme is available at the moment. Aedes fluviatilis and Anopheles
albimanus do not occur in the EU and are geographically restricted to parts of Central and
South America. Their invasive capacity is considered low. Anopheles stephensi is an Asian
malaria vector. Its invasive capacity is also considered low. Culex quinquefasciatus is a
common and widely distributed mosquito in the tropics and sub-tropics. It is not present in the
EU (except in overseas territories).
Anastrepha ludens was not considered relevant, since this species is not recorded in the EU.
Anastrepha suspensa, Bactrocera dorsalis, and Bactrocera tryoni were not considered as of
possible relevance since those species are not expanding in the EU. Cochliomyia
hominivorax, Bicyclus anynana, Bombyx mori and Metaseiulus occidentalis are also not
considered relevant since they do not occur or are at least not established within the confines
of the EU.
Serving as the basis for the identification of potential hazards and their associated exposures
these ten species (highlighted in table 5) will be presented in the following parts of this report
in more detail. The descriptions of the species focus on the respective importance, give a
general description including life cycle, habitat and climate requirements. Also the global
distribution and occurrence in Europe are described, as well as the genetic modification and
the purpose of development to control the agricultural or medical/veterinary pest species.
More details are presented for Aedes aegypti, Aedes albopictus, Bactrocera oleae and
Ceratitis capitata since those were selected as case study species for demonstrating the results
of the identification of potential hazards and their associated exposures (see chapter 2 and 16).
The anticipated maximum area of release of the species described below is normally
congruent with the area where the respective species occurs naturally. Therefore, no separate
descriptions of area of release and area of distribution are given, since the information would
be the same for both. The habitats of the ten selected GM-arthropods cannot be described
clearly by applying the EUNIS habitat type classification. This tool is primarily designed to
fit natural vegetation units and ecosystem types. The described mosquito species, however,
survive in small bodies of water which can be found in all type of ecosystems including manmade and highly disturbed (urban) habitats. Fruit flies, Codling moth, the Green bottle fly,
Stable fly and the Cotton pink bollworm are pests in agricultural habitats, some of them with
a very broad host range; therefore they also can occur in many or most parts of the
agricultural landscape. Finally, the Green bottle fly, following sheep keeping in its
distribution, is present on pastures or around buildings, without any strong connection to
specific habitat types.
61
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
8.1.
Aedes aegypti (Yellow fever mosquito)
Importance
The Yellow fever mosquito – also called the Egyptian tiger mosquito – is historically the
primary vector for viruses that cause dengue and yellow fever in humans (ECDC, 2007). In
Asia, this species is also considered the principal vector of the Chikungunya virus. These
diseases are transmitted by females during blood-feeding. Males do not play a major role in
viral transmission although they may be transovarially infected and pass on infections
horizontally during mating. Females feed on blood which is used to support the development
of eggs. Aedes aegypti is described as anthropophilic because it prefers to take its blood from
humans. Its diurnal biting activity, with peaks of activity at mid-morning and late afternoon,
practically excludes effective interventions like insecticide-treated bednets, limiting personal
protection to the use of topical repellents besides larval/adult mosquito control using
insecticides and source reduction. Dengue fever is primarily a human disease for this reason,
but yellow fever in its native environment is spread between humans and non-human primates
because mosquitoes that feed on carrier monkeys living in jungle canopies can transmit the
virus to humans through intermediate hosts. Transmission between humans is caused by
repeated biting of several individuals when the mosquito injects saliva that acts as an
anticoagulant. Additional information is provided by Angelini et al. (2007) and Almeida
(2007).
Between 1881 and 1910, several large outbreaks of dengue virus occurred in Greece, but the
most severe epidemic struck the country in 1927-1928. It affected more than a million people,
with some 1500 fatal cases (Chastel, 2009). It appears that large-scale application of residual
insecticides for malaria control (notably DDT) after World War II led to elimination of Aedes
aegypti from most parts of Europe. The species has recently been incriminated as a potential
vector of Rift Valley Fever virus in the Mediterranean Basin (Moutailler et al., 2008). The
island of Madeira has seen an increase in Aedes aegypti populations, with increased concern
of dengue transmission.
General description
Aedes aegypti is a medium-sized blackish mosquito easily recognised by a silvery-white
pattern of scales formed like a lyre on its front back. Segments 1 to 4 of the hind tarsi possess
broad basal white rings, segment 5 is white. The colouration of both sexes is similar. Like all
mosquito larvae Aedes aegypti has a well-developed head with mouth brushes used for filterfeeding, a large thorax with no legs and a segmented abdomen. The pupa is comma-shaped
and is commonly called "tumbler".
Life cycle
The mosquito has four distinct life stages: egg, larva, pupa and adult. The first three stages are
aquatic. The females lay desiccation-resistant eggs on the surface of the water in tree holes,
cans or other water-holding containers. The larvae live in the water and shed their skin four
times growing larger after each moulting. Larvae feed on aquatic microbiota. On the fourth
moult the larva changes into a pupa. The pupal stage is a resting, non-feeding stage. It takes
about two days before the adult is fully developed. The total time for development through all
four instars is dependent upon water temperature and food supply, and typically ranges from 4
to 10 days. The species is summer-active in the north and active all year in the south. Unlike
62
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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some other Aedes species, Aedes aegypti does not overwinter in the egg stage in colder
climates, but southern populations remain reproductively active during winter and are
periodically inactive during cold periods. Longevity is affected by larval nutrition,
temperature and humidity. On average, females live up to one month, males shorter.
Habitat and climate
Artificial or natural water containers (water storage containers, water bodies on flat roofs or
gutters of buildings, flower pots, etc.) that are within or close to places where humans live are
used as larval habitats. Old automobile tires provide excellent larval habitats and an adult
resting site as they effectively collect and retain rain water for a long time (Reiter, 1998).
Larvae die at temperatures below 10 °C and above 44 °C. Adults are killed by temperatures
below freezing and fitness is reduced at temperatures below 5 °C.
Global distribution
Originating from Africa, Aedes aegypti is now typically found throughout the tropical and
subtropical regions of the world, but it has also been introduced and spread far into temperate
climatic areas and even into the Arctic (Eritja et al., 2005).
EU distribution
Roughly said, Aedes aegypti can survive wherever temperature conditions are suitable for the
species (over 5-10 °C). Aedes aegypti is a peridomestic species found not far from human
dwellings. This species is particularly abundant in urban and peri-urban areas. The anticipated
maximum area of release is in continental Europe restricted to the Mediterranean part but also
to larger urban areas. In Europe it has been found in the following countries: Albania,
Armenia, Azerbaijan, Azores (Portugal), Bosnia and Herzegovina, Bulgaria, Canary Islands,
France, Georgia, Greece, Italy, Portugal, Romania, Spain, and Ukraine (WRBU, 2010b).
Genetic modification
Numerous modifications have been accomplished or are under development. All would likely
be made via TEs as either random insertions or using docking site technology. Aedes aegypti
is easily manipulated in the laboratory, making it a favourite for studies. It can be equally
easily transformed with several TEs and both fluorescent and an eye colour marker. The
desiccation capability of eggs makes this a simple species for maintaining numerous strains.
For Aedes aegypti, the bisex OX513A strain, and a female-flightless strain, OX3604C
(fsRIDL) have been developed (Oxitec, 2010). In addition, strains can be produced that
express marker genes, which would facilitate detection of released males (see also chapter
8.1). RIDL, however, has only been functional in small-scale laboratory trials and it remains
unknown what the impact of mass-rearing (e.g. 1 million males per day) will be on the
stability of the strain.
Purpose of development
Beyond basic biological and model studies, both population suppression (SIT/RIDL) and
replacement of populations with disease-refractory types are possible (see table 4). The
likelihood of the application of the latter is not high until the basic characteristics of mosquito
releases are better understood. Two particular effects are being developed: conditional
lethality (or incapacitation) of females and male sexual sterility. Although it is possible that
63
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
incapacitated females might be released (e.g. flightless), it should be anticipated that the first
releases will consist of males only. These might be radiation sterilised.
8.2.
Aedes albopictus (Asian tiger mosquito)
Importance
Aedes albopictus is one of the 100 world's most invasive species according to the Global
Invasive Species Database (GISD, 2005b). It is an aggressive outdoor diurnal biter that has a
very broad host range and feeds on humans, livestock, amphibians, reptiles and birds
(DAISIE, 2009). It can transmit pathogens and viruses, such as the West Nile Virus, Yellow
fever virus, St. Louis Encephalitis (SLE), Dengue fever (ECDC, 2007), and Chikungunya
fever to name a few important diseases, but has been incriminated as a vector of at least 22
arboviral diseases. A recent review by Weaver and Reisen (2010) details the expansion of
several arboviruses, claiming both the rural and urban expansion of both Aedes aegypti and
Aedes albopictus as a major cause for the increased impact of such diseases on public health.
The problems associated with disease transmission by Aedes albopictus have largely been
confined to tropical and sub-tropical regions. However, its expansion in the United States has
led to its role as a vector of West Nile Virus. In Europe, the high mosquito densities
experienced in Northern Italy (Ravenna region) in 2007 led to its role as a vector of
Chikungunya virus that was introduced by a single traveller from India (Rezza et al., 2007).
This outbreak affected more than 200 people with one fatality, but has not sustained itself.
Nevertheless the Asian tiger mosquito has become a significant pest in many communities
because it closely associates with humans (rather than living in wetlands) and increased travel
to disease-endemic areas by tourists residing within the EU has resulted in an increase in
returning travellers carrying (asymptomatically) viruses that can be transmited by Aedes
albopictus in Europe. The combined expansion and size of mosquito populations and increase
in viremic non-symptomatic travellers returning from endemic countries thus can be
considered an increased public health threat for the EU. Moreover, because of its diurnal
blood-feeding habit, this mosquito poses a severe nuisance, necessitating the use of personal
protection measures like skin repellents during daytime.
General description
Aedes albopictus is about 4 to 10 mm in length with a striking white and black pattern. The
males are roughly 20 % smaller than the females, but otherwise morphologically very similar.
A single silvery-white line of tight scales begins between the eyes and continues down the
dorsal side of the thorax. This characteristic marking is the easiest and best way to identify the
Asian tiger mosquito. Mosquito larvae have a well-developed head with mouth brushes used
for feeding, a large thorax with no legs and a segmented abdomen. Like Aedes aegypti, the
pupa is comma-shaped.
Life cycle
The mosquito has four distinct life stages, which consist of egg, larva, pupa and adult. The
first three stages occur in water. The females lay desiccation-resistant eggs on the surface of
the water in tree holes, tires or other water-holding containers. The larvae live in the water
and shed their skin four times growing larger after each moulting. The larva feed on microorganisms and organic matter in the water. On the fourth moult the larva changes into a pupa.
64
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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The pupal stage is motile but prefers resting and is non-feeding. It takes about two days
before the adult is fully developed. They rely on rainfall and e.g. plant watering to raise the
water level and inundate the eggs for hatching. Like other mosquito species, only the females
require blood to develop their eggs. Apart from that, they feed on nectar and other plant juices
like the males.
Habitat and climate
Aedes albopictus is a tree hole mosquito, and so its breeding places in nature are small,
restricted, shaded bodies of water surrounded by vegetation (Hawley, 1988). It inhabits
densely vegetated rural areas. However, its ecological flexibility allows it to colonise many
types of man-made sites and urban regions. It may reproduce in flower pots, bird baths, cans,
abandoned containers and water recipients. Tires are particularly beneficial for mosquito
reproduction as they are often stored outdoors and effectively collect and retain rain water for
a long time. Decaying leaves from neighbouring trees in the water produce chemical
conditions similar to tree holes, which provide an excellent substrate for breeding. Aedes
albopictus can also establish and survive throughout non-urbanised areas lacking any artificial
containers, raising additional public health concerns for rural areas. In the warm and humid
tropical regions, they are active during the entire year, however, in temperate regions they
hibernat. Eggs from temperate strains can even tolerate snow and temperatures below
freezing. In addition, adult tiger mosquitoes can survive throughout the winter in suitable
microhabitats. Furthermore, in these strains, the combination of short photoperiods and low
temperatures can induce the females to lay diapausing eggs which can hibernate. This feature
of diapause, which most other tropical mosquitoes lack, may be one of the keys to the success
of Aedes albopictus. Hibernation is necessary north of the +10 °C January isotherm. However,
under artificial climatic conditions, as maintained in greenhouses, survival is possible on a
year-round basis, even when the outdoor environment is inhospitable. This situation is
experienced in the Netherlands, where introduction of Aedes albopcitus has been observed
since 2005, as a result of shipments of Lucky Bamboo plants from southern China. Since
then, specimens have been caught in monitoring traps also during the winter months.
Global distribution
Aedes albopictus, originally indigenous to south-east Asia, and some islands of the western
Pacific and Indian Ocean, has spread during recent decades to Africa, the middle-east, Europe
and the Americas (north and south) after extending its range eastwards across Pacific islands
during the early 20th century (Knudsen 1995; Eritja, 2005). The majority of introductions are
apparently due to transportation of dormant eggs in tires (Reiter, 1998). Although Aedes
albopictus is native to tropical and subtropical regions, it is successfully adapting itself to
cooler regions (Urbanelli et al., 2000).
EU distribution
Historically, the Asian tiger mosquito was confined to South-East Asia and islands of the
Western Pacific and Indian Ocean (Gratz, 2004). Globalisation and increases in global
transport since the 1970s have facilitated its spread across the planet, currently making it the
world’s most invasive insect species. Mostly through transportation of dormant egg stages, it
had invaded 28 countries by 2007 (Benedict et al., 2007). Invasions in the EU and other
(European) countries close to the EU are listed in table 6. It first emerged in Albania in 1979,
where it was evidently introduced through a shipment of goods from China. To date, Aedes
65
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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albopictus have been found in nearly all Mediterranean and Central European countries
(Scholte and Schaffner, 2007).
Table 6. Invasions in Europe of Aedes albopictus
Year of invasion
Before 1979
1990
1999
2000
2003
2003
2003
2004
2004
2005
Country
Albania
Italy
France
Belgium
Greece
Switzerland
Israel
Spain
Croatia
Netherlands
Reference
Adhami and Reiter (1998)
Sabatani et al. (1990)
Schaffner and Karch (2000)
Schaffner et al. (2004)
Benedict, et al. (2007)
Flacio et al. (2004)
Benedict et al. (2007)
Aranda et al. (2006)
Klobucar et al. (2006)
Scholte and Schaffner (2007)
A recent report from the European Centre for Disease Prevention and Control (ECDC, 2009)
listed established homogenous populations of Aedes albopictus from Albania, Croatia,
France, Greece, Monaco, Montenegro, Italy, San Marino, Slovenia, Spain and Vatican City. It
was observed once in 2007 in Germany, but its establishment there is not yet proven. It has
also been introduced into Belgium (2000), but did not become established there. A special
situation exists in the Netherlands, where it has been observed only inside greenhouses. For
southern Switzerland, recent data suggest an onward spread, while the mosquito is present in
isolated areas in Bosnia and Herzegovina, but no further details are available.
The anticipated maximum area includes all Mediterranean and Central European countries,
the UK but also adjacent northern and eastern areas, depending on the model used to predict
future expansion and establishment (see also chapter 15). Taking the IPCC climate change
scenarios (minimal impact scenarios) as a basis, most changes for 2010 are anticipated in two
areas: in Central Europe (including the southernmost parts of Sweden), and in the Balkans. In
the longer term (2030), this ‘Central European zone’ will reach as far as the Baltic States and
cover large parts of southern Sweden. However, the ‘Balkan zone’ will not expand but even
shrink, with parts of Romania and Bulgaria becoming unsuitable for Aedes albopictus. When
taking into account maximum climate change impact scenarios, both the short- (2010) and the
long-term (2030) changes are similar and show a significant further eastward extension,
suggesting that most of Europe would become favourable for Aedes albopictus establishment.
It is concluded that the temperate strains of this species have become firmly established
within the EU, and that expansion is likely, with a future possible distribution encompassing
most of Europe (favourable scenarios) or at least the Mediterranean Basin and Eastern parts of
Europe (conservative scenarios).
Genetic modification
Successful germline transformation of this species has been accomplished and it is very likely
that numerous modifications will quickly follow. Its invasive nature and daytime biting
characteristic will make it a likely target for genetic control. All would possibly be made via
TEs as either random insertions or using docking site technology. Only piggyBac
transformation has been accomplished at present. This species is not as easily manipulated in
the laboratory as Aedes aegypti, but this will not likely prevent rapid development. At present
66
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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the only GM-application under development against this species is the RIDL technique for
which the female-flightless RIDL strain OX3688 has been developed (Oxitec, 2010). An
additional GM-application would be to mark males for release through expression of malespecific fluorescent proteins like GFP or DsRDed.
Purpose of development
Both population suppression (SIT/RIDL) and replacement of populations with diseaserefractory types will be possible (see table 4). Two particular effects developed in Aedes
aegypti could be applied: conditional lethality (or incapacitation) of females and male sexual
sterility. Although it is possible that incapacitated females might be released (e.g. flightless),
it could be anticipated that the first releases will consist only of males. These might be
radiation sterilised. Funding was provided recently for further research on Aedes albopictus,
the development of mass-rearing technology, as well as the advancement of RIDL towards
field implementation. Studies in contained environments are currently undertaken in
Malaysia, with open field releases pending. There are no similar activities at present in the
EU.
8.3.
Anopheles gambiae species complex: A. arabiensis and A. gambiae s.s.
Species within the Anopheles gambiae complex are morphologically similar but differ
substantially in their behavioural and ecological characteristics, as do the chromosomal and
molecular forms within Anopheles gambiae. From the viewpoint of genetic modification,
Anopheles gambiae s.s. and Anopheles arabiensis are similar, with interference with malaria
transmission being the main goal for modification. As such, it was decided not to separate the
two Anopheles species from table 5 regarding the species descriptions.
Importance
Human malaria in sub-Saharan Africa is mainly transmitted by mosquito vectors of the
Anopheles gambiae complex. The complex consists of about seven species that vary in their
ability to transmit malaria. Anopheles arabiensis, together with Anopheles gambiae s.s., are
the most efficient and most broadly distributed vectors of human malaria in sub-Saharan
Africa (Onyabe and Conn, 2001; Morlais et al., 2005). Although malaria control efforts are
increasing globally, its impact on development is still substantial, with an estimated 280
million cases per annum and >800 thousand deaths, particularly in sub-Saharan Africa, where
90 % of the mortality occurs in children below the age of five and pregnant women (WHO,
2009).
General description
The different species within the Anopheles gambiae complex are morphologically
indistinguishable. Anopheles mosquitoes in general can be distinguished from other mosquito
genera by the palps, which are as long as the proboscis, and by the presence of discrete blocks
of black and white scales on the wings. Adult Anopheles can also be identified by their typical
resting position, males and females with their abdomens sticking up in the air rather than
parallel to the surface.
Life cycle
In contrast to aedine mosquitoes, female Anopheles predominantly oviposit in transient sunlit
pools of clear and fresh water. These eggs are not desiccation resistant and can only survive
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for days to weeks in dried-up habitat. Larvae live in the water and shed their skin four times
growing larger after each moulting. Larvae feed on aquatic microbiota. On the fourth moult
the larva changes into a pupa. The pupal stage is a fairly inactive, non-feeding stage, in which
the mosquito develops into a winged, reproductive adult. It takes about two days before it is
fully developed. The key determinant for aquatic growth and development is the water
temperature, with average time from egg to adult being 8-10 days in most tropical settings.
Habitat and climate
Whereas Anopheles gambiae s.s. is often the predominant vector when malaria transmission is
at its highest level during the rainy season, Anopheles arabiensis shows a greater tolerance to
dry environments, and sustains transmission during the drier months of the year as well as it
being the main vector in Sahelian savannas.
Global distribution
Anopheles arabiensis is found throughout the Afrotropical region except for the Equatorial
forest belt (Coetzee et al., 2000). It is the sole vector of malaria on the Cape Verdian islands
(Cambournac et al., 1982), and present but not a vector on La Réunion island. It also occurs in
Yemen and Saudi Arabia. Anopheles gambiae s.s. is widely distributed throughout subSaharan Africa. This taxon is further subdivided into five chromosomal and two molecular
forms with subtle differences in their biology and ecology. Importantly, these taxa display
substantial levels of reproductive isolation that may affect the success of genetic control
programmes.
EU distribution
Neither species occurs in continental Europe. Among the European overseas territories
Anopheles arabiensis is found in La Réunion, whereas Anopheles gambiae s.s. occurs in the
Comoros (Mayotte) (Leong et al., 2003).
Genetic modification
Stable germline transformation of Anopheles gambiae s.s. with the piggyBac TE was first
reported in 2001 (Grossman et al. 2001). There is no published report of germline
transformation of Anopheles arabiensis, although this was accomplished in the IAEA
laboratories in 2006.
Purpose of development
GM-mosquitoes are being developed for either population replacement strategies or for
population suppression (Marshall and Taylor, 2009). Although to date neither Anopheles
gambiae s.s. nor Anopheles arabiensis have been rendered refractory to human malaria
parasites through genetic modification, it is anticipated that the knowledge generated with
other anophelines and rodent malaria (Ito et al., 2002) may ultimately pave the way for such
systems. Although the driving of refractoriness genes has focused on TEs for a considerable
period (James, 2005), they pose various difficulties. More recently, the focus has been on
Medea elements, HEGs, engineered underdominance, as well as the endosymbiont Wolbachia
and meiotic drive. None of these systems are currently operational for these species of malaria
vectors.
As for population reduction, GM-approaches for use within the SIT have been developed,
notably an efficient sexing mechanism based on the use of testes-specific expression of
68
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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fluorescent proteins that can be visualised to aid automatic sorting of males. This system has
been developed for the Asian vector Anopheles stephensi, Anopheles gambiae s.s. and
Anopheles arabiensis.
8.4.
Bactrocera oleae (Olive fruit fly)
Importance
The Olive fruit fly, Bactrocera oleae, is a serious pest of olives in most of the countries
around the Mediterranean Sea and generally in areas where olive trees are grown. The larvae
feed exclusively on the olives mesocarp and thereby cause significant economic losses (FAO,
2010). The damage caused by tunnelling of larvae in the fruit results in about 30 % loss of the
olive crop in Mediterranean countries, especially in Greece and Italy where large commercial
production occurs. Economic thresholds for the Olive fruit fly in table olives are extremely
low. Generally infestation levels of 1 % or less are required for high quality production. For
oil production the problem is secondary contamination with bacteria and fungi, acidification
as a result of Olive fruit fly infestation (causes taint and devalues the oil). Also fruit drop in
the field occurs due to Olive fruit fly infestations causing up to 100 % crop failure.
Furthermore, Bactrocera oleae can transmit a bacterium (Pseudomonas savastanoi pv. oleae)
to olive trees, which causes olive knot disease.
General description
The Olive fruit fly is one of the smaller species in the genus. The adult female is
approximately 5 mm long and has a wing length of approximately 10 mm. The wings are
mostly transparent and marked with brown spots, including a spot at the wing tips. The thorax
is black, with a silvery pubescence dorsal surface stripped with three narrow parallel black
lines. The humeri, or shoulders, and an area above and below the base of the wings are
yellow. The inner portion of the scutellum is black and the posterior portion is yellow. The
abdomen is black, covered with a scattered grey pubescence. The basal segments are marked
with pale transverse bands and an irregular parallel bar or blotch of reddish-brown occupying
the centre of the apical segments. The terminal segment is reddish-yellow. The sheath of the
ovipositor is black, with the ovipositor reddish in colour.
Life cycle
The female usually mates once and copulates at the very end of the photophase just before the
onset of darkness. Beginning in June, females actively seek and oviposit in early maturing
olive fruits until late summer. From 10 to 12 eggs may be laid daily, usually one per olive
fruit (unless the high population density or small fruit production forces the females to deposit
eggs in fruits already infested), and about 200 to 250 eggs are laid in a lifetime. The female
punctures the fruit with the ovipositor and lays an egg beneath the skin. The legless larva
feeds upon the fruit tissue, causing the fruit to drop off the tree. Larvae feed during 10-20
days from June/July to October, depending on the time of egg laying and temperature. Then
the larvae emerge from the olive fruit and pupate in the soil. Duration of the life cycle varies
from one to six or seven months (depending on the climate), enabling a maximum of 5
generations per year. In the Mediterranean climate of southern European countries, the Olive
fruit fly usually develops 3-4 generations per year, with more generations in southern regions
(e.g. Crete) and fewer in northern regions (e.g. central Italy). The winter is spent in the pupal
stage several centimetres below the soil and leaf litter (in southern regions with mild winters
69
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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as an adult as well). The adult flies emerge from March to May, depending upon the latitude
and temperature. Male flies produce an auditory stridulatory sound or signal during courtship.
Females can mate several times during their lifetime and can be found until October.
Habitat and climate
The habitat of Bactrocera oleae is restricted to olive plantations. In Europe these occur in the
Mediterranean area where the olive tree is considered to be the indicator plant for
Mediterranean habitats and climate. Usually the limitations of olive plantations are given by
the latitude of 30° north.
Global distribution
As a monophagous feeder on olives, Bactrocera oleae originates in native olive tree areas,
namely the coastal areas of the eastern Mediterranean Basin (the adjoining coastal areas of
southeastern Europe, western Asia and northern Africa) as well as northern Iran at the
southern end of the Caspian Sea (Segura et al., 2008). As the olive tree was introduced for
cultivation into new areas, also its pest was transported there. Therefore, Bactrocera oleae is
now also found in eastern and southern Africa, the Canary Islands, India, and apparently
wherever olives occur in the Eastern Hemisphere (Nardi et al., 2005). In the Western
Hemisphere, it has recently invaded California, first detected in West Los Angeles in fall
1998 and by 2001 has spread throughout California, Arizona and Western Mexico.
EU distribution
The species is native to Europe and present wherever olive trees are cultivated, largely in the
Mediterranean Basin and therefore this region is the anticipated maximum area of release. It is
known to be present in Albania, Balearic Islands, Canary Islands, Corsica, Crete, Croatia,
Cyprus, France, Greece, Italy, Malta, Portugal, Sardinia, Serbia, Sicily, Spain and Turkey. For
certain Mediterranean countries, e.g. Greece, it is considered as the most serious insect pest to
agriculture as a whole.
Genetic modification
While only a demonstration of germline transformation has been accomplished, RNAi has
been used to masculinise females. It is likely that accomplishing this by transgenesis will be
attempted. In addition it is believed that the problem with the difference in mating time could
be solved if GSS would be available for the Olive fruit fly. However, the genetic knowledge
for this species is extremely poor (e.g. not a single mutation is known) and this excludes the
possibility to develop the same type of GSS as it is available today for the Mediterranean fruit
fly. In this case the only alternative is to use molecular techniques to generate GSS. Some of
the molecular components that could be used for this are available or are under development.
Some GM-Olive fruit fly strains exist but the overall status is much less advanced than in the
Mediterranean fruit fly.
Purpose of development
Olive fruit fly populations can be controlled via the release of sterile insects but a principal
requirement is the availability of a sexing technology for the elimination of females. Maleonly strains are required because it has been shown that mass-reared olive flies mate at a
different time of the day than wild flies. Consequently, in bi-sexual releases the released
males and females mate with each other, i.e. the sterility is not transmitted effectively into the
70
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
target population. In male-only releases the males are forced to mate with the wild females. A
second limitation of bi-sexual releases is the fruit damage caused by the released females. The
most likely method to generate sexing strains for the Olive fruit fly is transgenesis (see Table
4). Methods are either available or under development where the females are either killed or
converted into males. The required sterility can either be induced by classical irradiation (SIT)
or by transmitting dominant lethal mutations into the target population (RIDL).
8.5.
Ceratitis capitata (Mediterranean fruit fly)
Importance
The Mediterranean fruit fly, Ceratitis capitata, is one of the world’s most destructive fruit
pests and a highly invasive species and is listed among the 100 world's most invasive species
(GISD, 2005b). It is a generalistic feeder and can affect more than 260 plant species (Liquido
et al., 1991), many of which are of commercial importance. Although it is considered a major
pest of citrus, often it is a more serious pest of deciduous fruits, such as peach, pear and apple.
The larvae feed upon the pulp of host fruits and eventually reduce the whole fruit to a juicy,
inedible mass. In some of the Mediterranean countries only the earlier varieties of citrus are
grown, because the flies develop so rapidly that late season fruits are too heavily infested to
be marketable. Some areas have had almost 100 % infestation in stone fruits. Harvesting
before complete maturity of the fruit is practiced in Mediterranean areas, which are generally
infested with this fruit fly. In addition to the direct damage to fruit production, the presence of
Ceratitis capitata in certain areas has significant impact on global trade. Countries without the
Mediterranean fruit fly demand considerable and expensive post harvest control measures
before they accept import of fruit from infested areas. In some cases the presence of Ceratitis
capitata can result in a complete ban of fruit exports.
General description
The adult Ceratitis capitata is 4 to 5 mm long (Thomas et al., 2001). The general colour of
the body is yellowish with a tinge of brown, especially the abdomen, legs, and some of the
markings on the wings. The oval shaped abdomen is clothed on the upper surface with fine,
scattered black bristles, and has two narrow, transverse, light coloured bands on the basal half.
The larvae are typically elongate, cream coloured, cylindrical maggot-shaped and grow up to
about 10 mm in lenght. The oblong, dark brown pupae reach about 5 mm length. Pupae are
enclosed in a hard brown cuticle and are immobile.
Life cycle
Eggs of Ceratitis capitata are deposited under the skin of a fruit that is just beginning to ripen,
often in an area where some break in the skin has already occurred. Several females may use
the same deposition hole with 75 or more eggs clustered in one spot. Each female will deposit
2 to 10 eggs. Eggs hatch in 1.5 to 3 days in warm weather. Larvae begin feeding almost
immediately after hatching. They pass through three instars before they emerge from the fruit.
They may emerge in large numbers just after daybreak and pupate in the soil. The pupa is
immobile and contained within a hard cuticle. Adults emerge from the pupal cases in large
numbers early in the day during warm weather and more sporadically in cooler weather. The
timespan required for Ceratitis capitata to develop from egg to adult is temperaturedependend; with 32 °C it takes about 16 days, with 24 °C 30 days and with 18 °C 100 days;
under tropical conditions it normally takes 21-30 days.
71
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Habitat and climate
As a generalist feeding on fruits, Ceratitis capitata can be found in a variety of orchards and
orchard-like plantations. Best climates are subtropical and Mediterranean climates but they
also tolerate cooler climates of the temperate zone. Ceratitis capitata regularly cause damage
in northern France, Germany, Switzerland and Austria but cannot survive the winter there.
Global distribution
The world-wide distribution of this pest is described in White et al. (1992). The
Mediterranean fruit fly, is believed to have originated from East Africa (Gasperi et al., 2002)
and references therein). Based on molecular data these authors suggest that it has spread from
there only recently to the Mediterranean Basin (it was first recorded in Spain in 1842), the
American continent (first record from 1905, in Argentina) and to Hawaii (first record from
1910). After spreading throughout the Mediterranean area Ceratitis capitata has infested
Australia (first record from 1897).
EU distribution
In Europe the largest established populations are found in the Mediterranean Basin, i.e.
Portugal, Spain, France, Italy and Greece (White et al., 1992; EPPO, 2010b). This region is
the centre of a vast fruit and vegetable industry which not only feeds the expanding
population in this region, but which also exports great quantities of fresh fruits and vegetables
to Northern Europe and elsewhere. For example, one-third of the world’s citrus production
and exports originate in the Mediterranean Basin. In central Europe Ceratitis capitata can be
detected occasionally but due to the climatic conditions no permanent populations have been
established.
Genetic modification
Because of its global economic importance and the fact that there are effective non-GM SIT
programmes against this species, it has been one of the most common targets of genetic
modification, only second to the silkworm. Currently male-only strains based on classical
genetics are used successfully. There is the hope that GM-approaches will improve the
quality/effectiveness of such systems either by adding a good marker to the current systems or
by developing new sexing strategies. Ceratitis capitata is easily transformed with several
transposable elements and many effector genes are available including fluorescent markers,
lethal genes and sex determination genes. The required sterility can either be induced by
classical irradiation (SIT) or by transmitting dominant lethal mutations into the target
population (RIDL).
Purpose of development
The overall goal is to reduce costs either directly (reduced production/release/monitoring
costs) or indirectly by increasing the efficiency of the released flies in the field. It is possible
to improve currently available sexing strains by adding a reliable marker (e.g. fluorescent
protein) to be able to distinguish the released flies and the wild flies. Alternatively, it could be
envisaged that other types of sexing mechanisms could be developed via transgenesis. In
principle it should be possible to create strains with molecular techniques that can be mass
reared more effectively or that perform better in the field. All ongoing SIT programmes use
irradiation to sterilise the released insects. The RIDL was proposed as alternative (see table
4), i.e. the radiation-induced sterility would be replaced by the release of fertile insects
72
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
carrying either a dominant lethal gene or a gene affecting the viability of the female offspring
in the field (e.g. flightless gene). However, the suitability of these approaches has not been
validated in field tests yet.
8.6.
Lucilia cuprina (Green bottle fly)
Importance
Lucilia cuprina causes a condition known as 'sheep strike'. The female lays her eggs in open
wounds on sheep. The emerging larvae cause large lesions, which can be fatal. Blowfly strike,
or flystrike is a serious welfare problem in the animal industry. This cutaneous myiasis or
infestation not only causes severe discomfort or stress to the animal, but will also cause death
when left untreated. Ewe lambs and female sheep are primarily affected and are struck
predominately in the rear quadrant of the animal due to faecal staining. Due to the difficulty
in controlling these flies, there are considerable losses in the sheep industry every year. There
is also increasing concern about insecticide use and the surgical procedures done to control
Lucilia cuprina, making this not only an animal welfare issue but also an economical one.
The maggots of Lucilia cuprina rapidly grow while consuming the living tissue of the sheep,
thereby poisoning the sheep with ammonia secretions. Sheep show signs of skin irritation by
rubbing and biting the affected areas during the first few days after the eggs have been laid.
This causes an inflammatory response resulting in severe irritation and pyrexia. Once a
flystrike has started other flies are attracted to the site. Although treatment is available, the
delayed response time due to symptoms allows wool breakage in the affected area and skin to
become tender overall. There are many predispositions to the flystrike that make a host more
favourable, including an infection with dermatophilosis and footrot, both of which can be
treated and prevented. In some animals a weak resistance can develop, but this immune
response is often associated with a decrease in productivity. Lucilia cuprina also infests
freshly dead carrion and is well known due to its importance in forensic entomology.
General description
Lucilia cuprina is a species of blow fly characterised by a metallic outer appearance and
reddish eyes. They usually have a shiny green or greenish/blue abdomen with bronze/coppery
reflections. Therefore Lucilia species are also known as the bronze bottle flies. Their body
shape is round to oval and their length varies from 4.5–10 mm. As typical flies, they have
only one pair of membranous wings, the second pair became completely reduced to tiny
halteres which are used for flight stabilisation. Adults are easy to distinguish due to bristles on
the meron (a triangular plate on the side of the thorax), in addition to the arista, the prominent
hair on the terminal antennal segment being plumose, or feathery. Lucilia cuprina are most
easily identified by their strong dorsal bristles (setae) and their black thoracic spiracle.
Life cycle
Flies have four stages of growth: egg, larvae, pupa, and adult. When infesting carrion, adult
Lucilia cuprina arrive early, appearing hours or even minutes after death to lay their eggs.
The eggs then hatch into larvae which immediately begin to feed and grow. After about five
days, larvae enter the pupal stage. It is described as an inactive stage, although many changes
occur during this part of the flies’ life cycle. The whole process can take from eleven to
twenty-one days depending on environmental conditions including temperature and
nutritional availability. In most cases warmer temperatures and better nutrition lead to a faster
73
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
life cycle. Lucilia cuprina can produce between four and eight generations per year depending
mostly on ambient temperatures.
Habitat and climate
This fly likes warmer weather with their optimal temperature being around 29 °C. It can fly
up to ten miles and can be found on anything ranging from carrion to decaying fruit. Larvae
are often found in shaded regions of carrion, while the adults prefer bright, open areas.
Global distribution
Although also known as the Australian sheep blowfly origins are linked to Afrotropical and
oriental regions of the world. Today it can be found throughout the world in various warm
locations. Australia is one of the many places Lucilia cuprina is found, and the place where it
has been known to cause the highest economic impact. Its wide distribution is due to
movement patterns and the movement of humans and livestock within the last century.
EU distribution
Lucilia cuprina is often confused with a species with which hybridisation is possible in the
lab, Lucilia sericata (Stevens and Wall, 1996). However, it seems clear that Lucilia cuprina is
not found in continental Europe. In the overseas territories it may inhabit New Caledonia and
French Polynesia but available reports are not consistent (Kurahashi and Fauran 1980; Bishop
Museum, 1993; Duponte and Burnham Larish, 2003; French Polynesian NPPO, 2008).
Genetic modification
A series of field trials with partially sterile insects was conducted in Australia from 1976 to
1990 (Mahon, 2001). During these trials sexing strains, modified with classical genetics, were
used. Therefore, improvements in sex separation and sexual sterilisation can be expected if
the appropriate molecular techniques would be available. To date, germline transformation
has been demonstrated (Heinrich et al., 2002) with th intention to develop this system into a
sex-separation method (Scott et al., 2004).
Purpose of development
Population suppresion via variations on SIT could be expected.
8.7.
Stomoxys calcitrans (Stable fly)
Importance
The Stable fly, Stomoxys calcitrans feeds on blood from practically any warm-blooded
animal, including humans, pets and livestock. It is also called the biting housefly since it is
distinguishable from the common housefly only by experts - until it bites. During periods of
high activity, humans can be severely pestered. Serious biting problems that affect tourists are
common and breeding in both e.g. dairies and seaweed contribute. Individual flies may feed
more than once per day. Peaks of feeding activity commonly occur during the early morning
and again in the late afternoon. Stable flies prefer feeding on lower parts of the hosts such as
the legs. Both males and females feed on blood, the females for producing viable eggs, the
males for survival. Stomoxys calcitrans are nuisance flies which inflict irritating bites. They
can, when numerous, weaken livestock by their blood sucking activity, interrupt cattle's
normal feeding and resting activities, which in turn reduces weight and milk production.
74
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Besides being vicious biters, Stomoxys calcitrans may transmit animal diseases such as hog
cholera.
General description
Adult Stomoxys calcitrans appear almost identical to houseflies. They are 7 to 8 mm long and
have four distinct, dark longitudinal stripes on the thorax and several dark spots on the
abdomen with sharp mouthparts protruding from the head, which distinguishes it from the
harmless House fly (Musca domestica). The eggs are about 1 mm long and have an off-white
colour. Larvae have a typical maggot-like shape. There are three larval stages; the last stage
larva is about 10 mm long and has a cream white colour. The chestnut brown pupa is 6 to
7 mm long. Stable fly pupae are very similar in appearance to housefly pupae and are difficult
to distinguish since, in their natural habitat, they are usually mixed with houseflies.
Life cycle
Females deposit their eggs in a variety of decaying animal and plant wastes, but are rarely
found in fresh manure. The eggs hatch after 1 to 3 days, and the young larvae immediately
begin to feed, completing development in 14 to 26 days. Large hay or straw bales that are in
contact with moist soil may serve as larval development sites. As in the housefly life cycle,
the third-stage larvae seek drier environments for pupation, which lasts 5 to 26 days
depending on temperature. The entire life cycle from egg to adult is generally completed in
three to six weeks. Stable flies usually overwinter as larvae and pupae, but in the southern
areas the adult flies are often active throughout the winter.
Habitat and climate
Stable flies are common around confined animal rearing facilities, but can also be pests in
open pastures (Talley, 2008). This fly prefers excrement mixed with straw, soil, silage or
grain but is also found in wet straw, hay, grass clippings, other post harvest refuse and poorly
managed compost piles. Stable flies often become abundant around feedlots, dairy cattle
loafing areas, and horse stables. They prefer sunny, outdoor conditions, although a few will
enter buildings and breed there. Since Stable flies can successfully overwinter in central and
northern parts of Europe, they survive in most European climates.
Global distribution
Originally from Europe, Stomoxys calcitrans is now a worldwide pest of livestock and man.
EU distribution
Stomoxys calcitrans is widely distributed in Europe and principally found everywhere where
food is available (e.g. warm-blooded animals as pets, livestock and humans). The anticipated
maximum area of release is more or less identical with Europe and excludes only arctic parts.
Genetic modification
The only genetic modification that has been demonstrated had no effector gene beside the
fluorescent marker (see table 4).
Purpose of development
The genetic modification was developed for the purpose of male production for SIT.
Masculinisation of females has not been demonstrated. Therefore, it is likely that all
applications will be female elimination or sexual sterilisation.
75
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
8.8.
Cydia pomonella (Codling moth)
Importance
The Codling moth, Cydia pomonella, is the most serious pest on apple and pear worldwide.
Especially under climatically suitable conditions, it also attacks walnut, and other tree fruits.
Most nourishment is obtained by feeding on the proteinaceous seeds.
General description
Adult Cydia pomonella is greyish with light grey and copper stripes on its wings, and has an
average wingspan of 17 mm. In resting position, it is around 10 mm long. Eggs are white,
transparent and around 1 mm in size. Newly hatched larvae are 2 mm and the last instar is 15
to 20 mm long. They are yellowish with a black head and get more reddish over time. The
pupa is brown and around 10 mm long.
Life cycle
Two to three generations per year are possible when the climate is warm enough, but under
less suitable conditions, there is only one generation. In spring, each female lays 60 to 100
eggs individually close to fruits or directly on the fruit, the larvae attacking the fruit
immediately after hatching. Each larva burrows into the fruit, eats for around three weeks and
then leaves the fruit to overwinter and pupate elsewhere. Around mid July, the moths hatch
out of the pupae. These only lay around 30 to 60 eggs. The larvae feed until autumn,
overwinter in a cocoon outside of the fruit and pupate in spring.
Habitat and climate
The species lives in orchards, private gardens and wherever host trees are cultivated. Adults
become active when evening temperatures exceed 13 °C. Below 10 °C it is not possible for
the eggs to develop and therefore egg laying ceases with 15 °C. Development to adult stage
starts in spring when temperatures are about 10 °C.
Global distribution
Cydia pomonella is native to Europe and was introduced to North America, where it has
become one of the regular pests in apple orchards. It is found almost worldwide, generally
wherever apples and other tree fruits are cultivated.
EU distribution
Cydia pomonella is found all over Europe, wherever food for the larvae is present, i.e. in
orchards, private gardens and wherever host trees grow.
Genetic modification
Transgenic modifications are planned to facilitate SIT (see table 4). Based on the fact that
(unlike most insects discussed in this report) female sex is determined by a dominant W
chromosome, insertion of a conditional dominant lethal on that chromosome would create a
female elimination strain. Prior to release, the dominant lethal would be induced to kill
females. A likely effector for this trait is a Notch gene allele (N60g11) which permits normal
development at temperatures around 20 °C but disallows it at temperatures in excess of
approximately 30 °C. The fluorescent transgene marker would be used in conjunction with
this trait or independently.
76
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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Purpose of development
The purpose of the development of GM-Codling moth is population suppression via SIT.
8.9.
Pectinophora gossypiella (Cotton pink bollworm)
Importance
Pectionophora gossypiella, the Cotton pink bollworm is a major cotton pest causing failure of
buds to open, fruit shedding, lint damage and seed loss. When developing fruits are infested,
weight of the bolls and yield are reduced.
General description
The wingspan of the adult is about 13 mm. It has grey palps with three dark bands and darker
patches on the forewings. Hind wings are smoky with a deep fringe of hair like scales. The
egg is light green, round, flattened, minutely ornamented and becomes dark before hatching.
Larvae are pallid at first, and become pinkish later on with a small dark spot at the bases of
the setae.
Life cycle
The Cotton pink bollworm can produce 4-6 generations per year, each lasting about 30 days.
The female lays up to 450 eggs (average 125) with the emerging caterpillars developing either
on buds and flowers or on the bolls. Depending on climatic conditions the larval stage varies
from 8 to 14 days. The pupa develops on the ground (6-20 days) with larvae that are fullgrown in November entering hibernation. Depending on the climate two types of generations
can be distinguished. Short cycle generations where larvae pupate soon after becoming fullfed and long cycle generations (in cold winter seasons) where larvae enter a resting stage for 8
to 10 month before pupation (The Caribbean Pest Information Network, 2010b).
Habitat and climate
Pectinophora gossypiella feeds not only on cotton but also on other plants like Okra, Hibiscus
and Lucerne. As stated above the climatic conditions influence the development time of larva
and pupa as well as the generation type.
Global distribution
Being native to Asia Pectinophora gossypiella has become an invasive alien species in most
of the world’s cotton growing regions. It is now distributed throughout tropical South
America, Africa, Asia, Australia, including subtropical regions of Pakistan, Egypt, USA and
Mexico, wherever cotton is grown. It is also widespread in the Lesser and Greater Antilles
(University of California, 2010).
EU distribution
Today the Cotton pink bollworm is distributed almost worldwide in cotton producing regions.
For Europe the presence is reported e.g. in Greece (Stavridis et al., 2009). It was also recorded
in Denmark where the species most likely was introduced through human activities (Karsholt,
1994).
77
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Genetic modification
Two traits are available: pink bollworm modified with a fluorescence marker gene and pink
bollworm being genetically sterile without radiation exposure. Using these techniques, males
are produced that are more competitive against wild-type males than radiation-sterilised
specimens.
Purpose of development
Pink bollworm modified to carry a marker gene helps to distinguish between the GM- and the
non-GM-pink bollworm in the field, allowing tests of field performance. Genetically sterile
males are used for the same purpose as the conventional SIT species, with the exception, that
the negative effects of the irradiation are avoided.
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Risk assessment of GM-arthropods
Based on the previous chapters that provide baseline information on GM-arthropods, the
following sections address potential adverse effects, their likelihood and consequences as well
as methods suitable of assessing these effects. In addition, key parameters of the GMarthropods are presented as well as baseline information necessary. Surrogate and modelling
approaches are discussed as well as implications for the implementation of an ERA of GMarthropods.
9.
Specific areas of potential risk to be addressed in the ERA of GM-arthropods
The following chapters provide considerations on risk issues to be taken into account when
conducting an ERA of GM-arthropod applications. The structure of the analysis follows the
approach described by EFSA (2006b) and EFSA (2010b) and takes into account potential
direct, indirect, immediate, delayed and cumulative long-term adverse effects on the biotic
and abiotic environment. Possible adverse effects on the environment may concern species
level (population size), species-species interactions (gene flow, hybridisation, predation), or
multi-species aspects (biodiversity). They may also affect ecosystem services such as
pollination or ecosystem compartments such as soil or water-bodies. Besides environmental
effects possible adverse effects of the GM-arthropod on human health need to be considered.
Other possible effects of the environmental release of GM-arthropods with regard to socioeconomic, ethic, or legal aspects (e.g. as reviewed by Macer (2005)) are outside the scope of
this report.
The analysis is primarily focused on RA requirements of GM-arthropod applications for
large-scale environmental release, e.g. commercial releases of GM-arthropods. However, this
analysis also takes into consideration possible adverse effects connected to the accidental
release of GM-arthropods. Those could happen e.g. caused by incidents at mass rearing
facilities for GM-arthropods which lead to local exposure of the environment. Another route
could be the release of GM-arthropods during transport, e.g. from mass rearing facilities to
the location of intended application (field trial or unconfined release). One of the specifics of
these situations is that the scale of the accidental releases might be different from large scale
unconfined application of GM-arthropods, usually with fewer individuals of GM-arthropods
released. Another relevant difference is that the area of release and thus the receiving
environment might be different to the areas considered for intended release of these GMarthropods. For accidental releases of GM-arthropods the associated occupational risks for
workers and handlers, which are unintentionally exposed to the GM-arthropods, need to be
considered.
For the presented analysis potential adverse effects concerning possible environmental
releases of relevant GM-arthropods in mainland Europe and the respective receiving
environments are discussed in detail. The EU additionally incorporates a number of overseas
territories of different size and location. These geographically range from Greenland in the
Northern hemisphere to Antarctic territories in the very South, and comprise, at different
latitudes between these extremes, a considerable number of different, mostly tropical
environments with their associated fauna and flora. These territories differ in size and human
population (different population densities or without permanent population like e.g. the
British Antarctic Territory and the British Indian Ocean Territory). Additionally, they usually
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
border non-EU countries. To present a comprehensive treatment of all such overseas
territories would encompass nearly all tropical ecosystems of the world, which are highly
diverse and different to the European habitats. Thus conducting an exposure assessment for
each of these receiving environments is difficult to present within the scope of this report.
Therefore, relevant GM-arthropods from the overseas territories were considered only for
identification and characterisation of hazards (see table 5).
The analysis is presented according to the sequential approach outlined in EFSA (2006b) and
detailed in the current document on ERA (EFSA, 2010b). In the outlined structure for RA, a
number of steps are conducted, starting with a hazard identification step (leading to problem
formulation), characterisation of hazards and evaluation of exposure of the potential receiving
environment, followed by a characterisation of the overall risk. Similar approaches were put
forward in other national and international fora, e.g. recently in a guidance for RA of living
modified organisms (LMOs) in the framework of the Cartagena Protocol (CBD, 2010a). The
analysis is taking into account the general principles for RA of GMOs, specifically the
principles of comparative safety assessment as outlined by EFSA, FAO/WHO and OECD.
Possible adverse effects of GM-arthropods have already been discussed by the IAEA (2006)
and the USDA (2008) and a number of other authors or institutions (e.g. CBD, (2010a)).
Furthermore, guidance documents for RA of certain GM-arthropod species (e.g. on GMmosquitoes by CBD, (2010a)) and specific types of applications (e.g. for field trials of GMmosquitoes by Benedict et al. (2008)). In the following, the potential adverse effects of GMarthropods of possible relevance for future applications in the EU as outlined in previous
chapters of this report (see chapter 8) are discussed. Also species x trait combinations, which
are hypothetical with regard to application in the EU, but which are associated with an
increased potential for specific adverse effects are considered. Such applications which would
present challenges for RA and may be regarded as worst case for RA are discussed to increase
the robustness of the analysis.
Potential adverse environmental effects can result from several sources (Handler and
Atkinson, 2006). They can either be due to the nature of inserted transgenes and their
products, which might exert specific adverse effects e.g. toxicity. Furthermore, the
characteristics of the transformation system used for the design of a specific GM-arthropod
application might result in adverse effects, e.g. possibility for transfer of GM-traits to other
organisms. Thirdly, the characteristics of the different GM-arthropods itself can result in
adverse effects on the environment and human and animal health. For an analysis of adverse
effects associated with the GM-arthropod organism, the characteristics of the parental species
need to be considered as well as any changes of these characteristics due to the genetic
modification.
In general, similar overarching categories for adverse effects like discussed for other types of
GMOs need to be considered for the RA of GM-arthropods. In this discussion, the specific
potential of GM-arthropods for certain adverse effects related to the different indicated
sources have to be taken into account. In the following, types of resulting potential adverse
effects relevant for GM-arthropods are described. The discussion focuses specifically on:
−
Adverse effects associated with gene flow, e.g. transmission of GM-traits, which
confer altered characteristics in receiving individuals, e.g. increased fitness and
increased potential for persistence and spread of modified organisms.
80
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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−
Adverse effects of the GM-arthropods on other organisms (target and non-target
organisms) present in the receiving environments, e.g. changes resulting from
selective adaptation of populations of interacting organisms or effects on species
interacting with the GM-arthropods in a specific receiving environment.
−
Adverse effects on characteristics of the receiving environment and its functions, e.g.
on biodiversity in general, ecosystem services like pollination or biogeochemical
processes.
−
Adverse effects on agricultural management techniques or management practices with
regard to control of vectors for human and animal diseases.
−
Adverse effects of GM-arthropods on human health via different exposure pathways
(direct contact, vector characteristics, and food related effects). With GM-arthropods,
however, the focus of this assessment will be different from other GM-applications,
e.g. GM-plants, since GM-arthropod applications are currently not developed for food
or feed use and most likely will not be developed for such purposes in the near future,
as concluded from ongoing R&D activities. Therefore, only incidential exposure of
humans and livestock is considered and exposure to GM-arthropods from species that
include humans and livestock as hosts, and through this association may act as disease
vectors.
9.1.
Adverse effects associated with gene flow
Gene flow is usually not considered an adverse effect in itself, but an intrinsic characteristic
of the biotic environment. However, when considering the environmental release of GMarthropods a number of aspects of gene flow may be linked to adverse effects, which need to
be taken into account when conducting a RA.
Gene flow aspects are discussed separately for the following mechanisms:
−
Transmission of the inserted transgenic constructs by vertical gene flow to populations
of the same or other species.
−
Transmission of the inserted transgenic constructs by horizontal gene flow.
9.1.1.
Vertical gene flow to populations of the same or sexually compatible species
Hazard identification
The persistence of released GM-arthropods by successful sexual reproduction can lead to
adverse effects if the species in question is a pest species (i.e. an agricultural pest or an
arthropod species) that can spread diseases. In case the GM-arthropod also exerts similar pest
characteristics and the application is increasing the number of viable and reproducing
individuals in the receiving environment, harm can be done to agronomic cultures or the
health of humans or livestock.
With GM-applications developed for suppression or elimination of a certain target species,
the GM-arthropods are usually employed in a way that is self-limiting, i.e. the GM-arthropod
individuals are eliminated from the target population due to the characteristic of the
application itself: application of a sterilisation technique (SIT), a dramatic fitness decrease on
81
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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offspring due to a transmitted dominant lethal gene (RIDL), or some milder fitness
disadvantages with other systems (Beech et al., 2009b).
The production of viable offspring responsible for unintended persistence can be due to a
failure of the employed sexing and sterilisation system for SIT applications or the transgenic
function responsible for exerting lethality in offspring for RIDL applications. In case of a less
dramatic fitness penalty of the genetic modification, some reproduction can be expected and
the GM-arthropods may persist in the release area for a number of generations, before they
are eliminated from the target population (Beech et al., 2009b). If released in large numbers in
an area where this species is already occuring, the problems posed by the species can be
aggravated. Failure of a self-limiting mechanism in a GM-arthropod can be considered an
adverse effect (Andow, 2010).
GM-applications aimed at population replacement (see e.g. Marshall and Taylor (2009)) are
designed to intentionally spread through insect populations that vector diseases, e.g.
mosquitoes. This is facilitated by a transgenic genetic drive system, e.g. a hyper-active
transposable element (see chapter 5.2). In inital experiments, Medea-like gene drive-systems
were shown to be spreading through target populations under laboratory conditions, without
conferring a direct fitness advantage to the modified test species, i.e. Drosophila (Chen et al.,
2007a). This category of GM-modifications would in effect be self-sustaining in the target
population after release, and therefore decribed as associated with a greater potential for risk
(Beech et al., 2009a).
Besides propagation of the introduced transgenic constructs in populations of the parental
arthropod species, the possibility for vertical gene flow to syntopic cross-compatible wild
relatives of the parental species needs to be considered. According to Andow (2010), the
interspecific transmission of GM-constructs should be assessed for all GM-applications
against the background of reproductive isolating mechanisms present in a certain species.
GM-applications which are designed for population replacement strategies, like described
above for GM-mosquito species, could also promote spread of GM-traits to related species
and therefore should be scrutinised specifically.
An important factor for persistence and spread of either a GM-arthropod or of hybrids
harbouring a certain GM-construct in the environment is the fitness effect associated with the
respective genetic modification. Dependent on the effects of the specific GM-construct in the
respective genetic background the modified arthropod could demonstrate either increased
fitness favouring persistence and spread or exert a fitness load on the organism. Hybrids
formed between the released GM-arthropods and cross-compatible wild relatives could also
suffer from outbreeding depression.
Assessment of fitness is specifically relevant with GM-applications developed for other
purposes than to suppress populations by lowering the reproductive rate and which are not
sterilised by other means before release. In this respect, Hoy (2006b) hypothesised on the
possibility of developing GM-Metaseiulus occidentalis for use in agricultural pest control,
based on experiences with the selection of pesticide resistant Metaseiulus occidentalis and the
successful transformation of this species with a transgenic marker construct. Hypothetical
GM-traits leading to increased temperature or drought tolerance may enable the GMarthropods to expand its geographical range, and to reach and colonise new areas close to wild
relatives from which it was previously isolated. Finally, enhanced fitness or the ability to
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occupy new niches would allow such populations to increase and invade new communities.
This may cause a population decline or extinction of wild relatives through hybridisation or
through competition, which may trigger a cascade of environmental consequences.
Additionally, increased fitness of the GM-arthropod itself, or of hybrid progeny, due to
vertical gene flow to sexually compatible relatives, via geographical range alterations may
influence the host range of the species considered (compare chapter 9.2.2).
The spread to other habitats can be due to movement of the released GM-arthropods itself. In
case of accidental releases, GM-arthropods could be released directly in receiving
environments other than currently inhabited by the species of interest, provided that the
environmental conditions permit survival and reproduction.
Hazard characterisation
With the GM-arthropods, which might be of possible relevance for the EU within the next
decade (see table 5), mostly SIT and possibly RIDL applications could be notified for release.
Within the European context applications may be notified for GM-mosquitoes and GMfruitflies.
In case of failure of this SIT, it might happen that populations with a sterility of less than
100% or also females are released. Experience with SIT applications based on non-GMarthropods however would suggest a very low incidence of vertical gene flow. Additionally
the respective genetic modifications are not known to confer any intrinsic fitness advantage,
which of course needs to be further assessed under (semi-) field conditions comparable to a
release scenario. The fitness impacts for GM-applications for SIT may thus be similar to that
experienced by non-GM-arthropods due to the sterilisation treatment involved. The reduction
in fitness is compensated for in SIT campaigns by releasing large numbers of steriles,
normally in a flooding ratio of 10:1 (10 steriles for 1 wild male) (Marrelli et al., 2006).
RIDL applications could potentially fail to exert a lethal effect upon transmission of the
construct (Handler et al., 2004). This would facilitate further propagation of transgenic
elements and raise the question whether the inserted transgenic constructs might have any
impact on the fitness of the GM-arthropods. The current evidence available for
characterisation of RIDL applications is from testing in contained facilities, therefore some
uncertainty exists whether this system will be fully efficient in open field environments.
With applications employing genetic drive systems, further propagation of the genetic
construct in the environment is expected. Such an approach could be used to spread different
transgenes, which induce refractoriness against infection by disease-promoting parasites or
viruses (see table 4 for reference). The impact on persistence and invasiveness would then
depend on the overall effects of these modifications on fitness and reproduction.
The frequency for vertical gene flow from the GM-arthropod to closely related species is an
important indicator in assessment of potential effects from transmission of GM-traits. As
mentioned by Benedict et al. (2008) an extensive list of open questions needs to be addressed
even before field trials with such applications should be conducted. Further data from field
testing would then be required with regard to evidence-based hazard assessment of
unconfined releases.
There are indications of fitness loss in GM-arthropods compared to their wild types.
Catteruccia et al. (2003) interpreted based on results from in cage experiments with several
83
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lines of GM-Anopheles stephensi, containing phenotypic marker genes, the direct costs of the
introduced transgene as reduced fitness. Transgene frequencies were lower than expected in
the first generation and tended to decline over time. The analysed transgenic constructs were
lost within 4 to 16 generations. Other data for GM-mosquitoes suggest that a fitness cost is
generally associated with most of the tested transgenic constructs, which is different from one
GM-line to another (Marrelli et al., 2006). Their conclusion is that a modest fitness load can
be expected. Several factors are described to affect the fitness, e.g. leaky basal expression of
toxic effector proteins with RIDL applications and effects of insertional mutation during
transformation. However, there are also studies which do not support a general fitness loss in
GM-arthropods. With a different approach on the basis of life-table parameters, a study on
Anopheles stephensi transgenic lines containing a fluorescent marker gene showed no
significant differences in fitness between the GM-and non-GM-mosquitoes (Amenya et al.,
2010). Comparable observations concern Aedes albopictus which were engineered to aquire
cytoplasmic incompatibility, which effectively sterilizes females upon mating with GM-males
did not affect immature and adult survivorship (Calvitti et al., 2010).
For GM-malaria-resistant mosquitoes a fitness advantage was described when they were fed
with Plasmodium-infected blood (Marelli et al., 2007). In a laboratory situation, a
considerable advantage could be found, which could increase the spread of the transgene in
mosquito populations. When fed on Plasmodium infected mice, the GM-mosquitoes displayed
a higher fecundity and lower mortality than sibling non-GM-mosquitoes and gradually
replaced non-GM-mosquitoes in a cage experiment. These findings suggest that when feeding
on Plasmodium-infected blood, GM-malaria-resistant mosquitoes have a selective advantage
over non-GM-mosquitoes. However, the authors concluded that due to the actual low
infection rate of host species in the receiving environments the fitness advantage could be
very small and not sufficient to promote establishment of the transgenic construct in field
populations (Marelli et al., 2007).
In GM-arthropods with more than a single transgene, it should be considered whether the
combination of transgenes may lead to enhanced persistence or invasiveness.
Exposure characterisation
Relevant for conducting an ERA is adequate knowledge of the areas inhabited by the parent
species ahead of the release. Some basic information on habitats of the species considered
relevant for GM-applications are summarised in the respective sections of chapter 8. While
some species are quite restricted to specific habitats (e.g. areas of olive cultivation for
Bactrocera oleae), habitats for others like Ceratitis capitata and mosquitoe species are less
well defined. Furthermore, it needs to be considered whether the receiving environment(s) of
the GM-arthropod are populated by wild relatives with which are able to hybridise. This
situation differs from species to species and differs substantially between Europe and overseas
territories.
Fauna Europaea (2004) mentions no further congeners for Europe in the case of Ceratitis,
Bactrocera and Stomoxys. On the other side, there are 13 Aedes species, 32 Anopheles
species, 14 Athalia species and 57 Cydia species recorded for Europe, which indicates some
hybridisation risk. Thus, the potential for hybridisation and gene flow should therefore be
assessed.
84
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If hybridisation is plausible, then it is important to assess if GM-traits will enhance the fitness
of hybrid arthropods present in the respective environment. It is specifically important to
assess whether the GM-arthropod has any different capacity for gene transfer than its
conventional counterpart. The gene(s) inserted may modify the potential for hybridisation due
to e.g. altered behaviour of the GM-species, fertility, or changed sperm viability and
compatibility.
As suggested by Andow (2010) the size and frequency of releases of GM-arthropods should
be considered for assessment of spread of GM-traits. Recurrent releases in the same area can
lead to extended exposure of the receiving environment even for applications which are not
self-sustaining and thus should be also considered for the assessment of release programs for
GM-arthropods.
Another relevant factor with regard to the ability to spread is the mobility of the released GMarthropods (deduced from and compared with the mobility of the parental species). Whenever
possible this assessment needs to be based on data from the respective receiving environment
to avoid misestimations. Like shown in a study on the dispersal rate of two mosquito species
Aedes aegypti and Aedes albopictus the dispersal distances in semi-rural and urban parts of
Singapore were substantially larger than assumed (Liew and Curtis, 2004).
9.1.2.
Horizontal gene transfer
Hazard identification
Horizontal gene transfer needs to be considered as another mechanism for dispersal of
transgenic elements. Different mechanisms are possible for horizontal gene transfer to occur,
eg. uptake of transgene DNA released from from GM-arthropods upon decay or ingestion and
integration into a microbial host (soil microorganisms or gut microflora), transposition into
new host species (other arthropod or other animal species) by transfer through host
parasite/parasitoid interactions (Loreto et al., 2008; Gilbert et al., 2010) and transfer by insect
viruses (e.g. Jehle et al. (1998)).
Microorganisms, especially bacteria, are capable of exchanging genetic material directly
between each other and even across species boundaries using different mechanisms i.e.
conjugation, transduction or transformation. Horizontal gene transfer can be initiated by
uptake of cell free DNA from the environment, which may also include DNA derived from
GM-arthropods. Such a mechanism could potentially result in transfer of transgenic DNA
from GM-arthropods to microorganisms.
Horizontal transfer of TEs is well described for different arthropod species involving different
TEs. Using sequence analysis and comparison a large number of TEs have been found in
different species and their evolutionary relations have been studied. The results provide some
evidence that horizontal gene transfer has taken place (Silva et al., 2004). Horizontal transfer
was found for some genetic elements, which are also used for the construction of
transformation vectors to generate GM-arthropods (see Chapter 6.1). E.g. Mariner-like TE are
widespread among diverse groups of arthropods of interest in this report (e.g. in Apis
mellifera, Ceratitis capitata) and have been shown to be implicated in recent horizontal
transfer events (Robertson and Lampe, 1995; Lampe et al., 2003). Such elements are also
found in other animals and there is evidence that horizontal transfer across wider taxonomic
distances happenes (Ivics et al., 1997; Lampe et al., 2003). A wide body of evidence also
85
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indicates horizontal transfer of Drosophila TE, involving different classes of TE, e.g.
Mariner, Minos, hAT family transposons hobo, hosimary, harrow and SPIN, among others
(Pace et al., 2008; Loreto et al., 2008; Bartolome et al., 2009; Depra et al., 2010; Mota et al.,
2010).
Integration of transgenes or entire transgenic constructs used for generation of GM-arthropods
into a novel host has consequences for the host and accordingly for the potential that any
adverse effect for the environment could result. Consequences could derive from several
mechanisms:
−
From disrupting genetic functions in the novel host species upon stable integration.
−
The integrated transgenes could be expressed in the new host with effects depending
on the nature of the respecting transgens (including the fluorescent marker).
−
The integrated transgenic construct could be re-mobilized, if suitable endogenous
transposases are present in the host. However, this would require that the entire
transgenic construct is integrated with functional inverted repeats, which are necessary
for mobilisation.
Potential adverse effects resulting from horizontal gene transfer thus depend on the functions
mediated by the transferred transgenes or modifications introduced in the genome of new
hosts upon integration and their effect on host organisms and their interactions with the
environment. Propagation of the inserted elements and spread within a host population
however would require that the transgenic modification would result in a fitness advantage for
the GMOs, like resistance to selection pressures or the interaction with a (transposition)
system present in the host, which provides for further spread without an increase in fitness.
Some TE-derived transgenic constructs (e.g. constructs based on the Mariner family of TE)
do not require a lot of species-specific factors (Ivics et al., 1997). For some components this is
unlikely (some of the promoters used are species- and/or tissue specific) while other
components are less specific and could be expressed more widely. If this is the case it has to
be ascertained whether the trait offers some fitness advantage upon which its persistence
through multiple generations could be expected.
Hazard characterisation
Adverse effects by horizontal gene transfer on the environment and any receiving organisms
are dependent on the nature and function of the respective transgenes. With regard to
horizontal gene transfer to bacteria the transgenes which most likely could be used in the
future would probably not give rise to foreseeable adverse effects. However, as noted by
USDA (2008), it would be possible to use markers such as neomycin phosphotransferase
(G418 resistance) or hygromycin B phosphotransferase (hygromycin resistance) genes for
construction of GM-arthropods. These markers were occasionally used in Drosophila, but
little, if any, in pest insect transformation. Such antibotica resistance genes could have the
potential to adverse effects, similar as discussed for antibiotic resistance markers in GMplants. Furthermore, the potential for adverse effects to happen depends on the probablilitiy
that transgenes are taken up by microorganisms and further propagated in these organisms
due to stabilisation by integration into the host genome (e.g. via homologous or nonhomologous recombination processes).
86
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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With regard to transposition events it needs to be considered that the TE-vectors used for
transformation have a different potential to be re-mobilised. Some versions of transformation
vectors can be stabilized by removing the inverted repeats, thereby making re-mobilization
impossible. Furthermore, it should be considered whether trans-mobilization is likely: e.g. the
piggyBac element which is used very often in GM-arthropod construction has been extremely
difficult to mobilise in experimental systems, even when the transposase was provided in
trans. This also can affect the consequences of further horizontal gene transfer.
Another crucial factor is whether the integrated transgenes can be expressed in the respective
hosts (microorganisms or other host arthropods/animals). For some components this is
unlikely (some of the promoters used are species- and/or tissue specific) while others are less
specific and could be expressed more widely. Therefore, the potential for expression of
transgenes in other hosts should be assessed with regard to the possible transfer pathways.
Exposure characterisation
During mass release of GM-arthropods e.g. for pest control, a considerable number of
arthropods will be available for predators ingesting the released GM-animals, or parasitoids
and parasites, some of them are considered to be vectors for TE (Loreto et al., 2008). A
number of pathogens of the arthropod species should be considered such as entomopathogenic
fungi of mosquitoes (Scholte et al., 2004). Environmental microbial communities may include
certain human or animal pathogens (e.g. Pseudomonas aeruginosa or some
Enterobacteriaceae), or non-pathogenic bacteria, which could serve as first recipients of genes
derived from GM-arthropods and the transgenes could be then transferred to other
microorganisms including pathogens and higher organisms.
Dead GM-arthropods will be deposited in the top layer of the soil in terrestrial ecosystems or
in water bodies or in the top layer of the sediment of aquatic ecosystems, where
decomposition processes take place. The fate of transgenic DNA and persistence of
extracellular DNA in the environment was described only for DNA derived from GM-plants,
but apart from the specifics of decomposition of arthropods similar behaviour of arthropod
DNA in the environment can be expected (Nielsen et al., 2007; Pontiroli et al., 2007). By this
exposure pathway gut microflora of predators and parasitoids, as well as microorganisms
from soil and waterbodies get into contact with DNA derived from GM-arthropods. The
amount of exposure to GM-arthropod DNA is influenced by:
−
the number of released individuals, their dispersal or accumulation in specific parts of
the receiving environment, and
−
the stability of the DNA, specifically if DNA fragments of relevant size with regard to
horizontal gene transfer persist in the specific environment.
For horizontal gene transfer by transposition an ecological overlap between donor and
recipient species must exist and a suitable vector (Virus, intracellular symbiontic bacterium
like Wolbachia, parasite or parasitoid) must be present mediating transfer of TE-DNA (Silva
et al., 2004; Loreto et al., 2008). Furthermore, as mentioned above the characteristics of the
transformation system used, with regard to re-mobilisation efficiency affect the likelihood of
further horizontal gene transfer.
87
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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9.2.
Interactions of the GM-arthropod with the target organisms
9.2.1.
Triggering adaptive processes in the target population
Hazard identification
Released GM-arthropods which are designed to impact on a target population present in the
receiving area, e.g. to suppress a population by means of SIT or RIDL, are a powerful
selection factor with regard to adaptive evolutionary processes in the target species. The
possibility for such processes to happen is dependent on the characteristics of the GMapplication and the necessary adaptive changes to overcome a specific effect exerted by the
GM-application. The mode of action of SIT applications which employ radiation-sterilisation
of the arthropods before release thus introducing a multitude of different DNA-lesions is less
prone to the development of adaptive responses in the target population than GM-applications
with a single effector trait as a mode of action, like current applications of the RIDL
technique (see chapter 5.3.2), which are based only a single lethal gene.
With applications designed for lower vector competence for pathogens in GM-arthropods,
some of which are developed e.g. in GM-mosquitos (see chapter 5.2), effectivity of the
system for a longer timespan is necessary to achieve the targeted goal. Failure due to
regaining of competence in the target population or acquisition of new competence for other
disease agents therefore is an important issue in assessment of such applications (Andow,
2010). Introduction of a GM-construct spreading through a pest population without being
effective for lowering vector competence in the target population can thus be considered a
relevant adverse effect.
Hazard characterisation
RIDL with a single lethal transgene as a means to achieving suppression of the target
population may result in development of early resistance when the respective RIDL strain is
used against a target population with a high level of genetic diversity. In the RIDL system the
lethal gene and its control element(s) have to interact with the host genome to be functional
and a number of mechanisms are considered that potentially abolish the lethal effect
(reviewed in Handler et al. (2004)). Development of refractoriness of the target population
against the lethal gene can be seen as an analogy to development of insecticide resistance in
agricultural insect pests against insecticidal substances with a single mode of action. Since
some historical experience exists with development of resistance against insecticides in
different target species; the adaptive behaviour against the insecticide may indicate whether a
rapid adaption process against the GM-application will have to be expected.
Basend on this analogy the following data may characterise the potential for adaptive
development with regard to the GM-trait:
−
Data on biology, life cycle, ecology and/or behaviour of the arthropod. Data on
resistance mechanisms that develop in the arthropod and their genetic control,
heritability and linkages to virulence, fitness and selective advantage. Distribution of
the arthropod and its resistant populations in the European environments;
−
Host range of the arthropod;
−
Information on the population genetics, and epidemiology of susceptible and resistant
arthropods;
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−
Frequency of resistant individuals or resistance alleles (these data need to be obtained
in the laboratory first);
−
Mode of action of the active lethal gene towards the target arthropod;
Most of these baseline data need to be established case-by-case with regard to the
characteristics of a specific GM-arthropod application.
Exposure characterisation
With RIDL applications the aim of such GM-arthropod release programmes is that the
majority (or all) of the population should be exposed to the lethal gene in the receiving
environments. All studies on long-term fitness advantages / disadvantages of a transgene so
far were performed only in contained conditions, thus the fate of a GM-population in much
more complex field conditions is virtually unknown. Such knowledge gaps concern the
baseline frequency of resistant individuals or resistance/virulence alleles but also a variety of
other fitness parameters.
9.2.2.
Host range
Hazard identification
Effects on host range need to be assessed in a specific way for GM-agricultural pests and
GM-arthropod applications in species vectoring diseases. Constriction in host range would
likely be considered a benefit since all arthropods species discussed in this report are
considered pest species, whereas a novel host range is of concern. The creation of a novel host
range of a GM-arthropod could be caused by the newly introduced traits. Mosquitoes or the
Stable fly Stomoxys calcitrans could suck blood from more or different species. Such a larger
or modified host range could include additional livestock, thus causing more economic
damage. For fruit flies or other crop pests such as Athalia rosae or Cydia pomonella extension
of the host range on more crop species would also cause an increased economic damage. Such
extensions of host range could result from spread due to increased fitness of the GMarthropods itself as well as to progeny from hybridisation with sexually compatible species
(see also chapter 9.1.1).
With regard to GM-applications aimed at population suppression, also indirect effects should
be considered. The successful control of a specific targeted species could result in expansion
of the range of another species which occupies the available niche. Such effects should be
considered for mosquitoes, where one species could be replaced by another.
With agricultural pests indirect effects could be happen due to a change in pest management
regimes in conjunction with the application of a specific GM-arthropod for population
suppression. As an analogy expansion in non-target crop pests have been recently described
as indirect effects of control of target pests by selective Bt-toxins expressed in GM-crops (Lu
et al., 2010).
Hazard characterisation
Since a change in host specificity and host range could be directly caused by the transgene, it
should be possible to test for a host range change due to the inserted gene before release of the
GM-arthropod. However, such change could also be induced by environmental factors of the
89
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receiving habitat. A careful post-release monitoring on host range changes could clarify this
situation.
Concerning the population level effects, GM-arthropod pest population densities and densities
of possible other replacing pest species need to be monitored for some time.
Exposure characterisation
The creation of novel host ranges of the GM-insect caused by the newly introduced traits is
unlikely for the following reasons: host preference is a complex interplay of numerous effects
including: olfaction, vision, behaviour, co-evolution and seasonal abundance. The effectors
anticipated for application seem not to affect the systems of olfaction, vision etc in a
sufficiently direct and coordinated way among all systems involved to produce such changes.
Population changes and replacement by other pest clearly should be considered for many
species. Mosquito larvae often play a prominent role as detritophagous organisms in aquatic
ecosystems. Eradicating one species may open an attractive niche for another species, e.g.
another mosquito species which is able to fill this gap. Since only adults are pests, a scenario
is possible where a species replacement does not change the negative impact of adults (e.g. on
humans and or livestock). Among the considered species in this report, the broad host range
of Ceratitis capitata and the high number of Cydia species open the scene for many
replacement scenarios.
9.3.
Interactions of the GM-arthropod with non-target organisms
The analysis of interaction of GM-arthropods with non-target organisms needs to be designed
with a view to a number of different aspects:
−
The interactions of the released GM-species with components of the receiving
ecosystem and its functions needs to be determined (e.g. interaction with other
functional groups of species as part of the food web, specifically predators and
parasitoids; function as a host and vector of pathogens; significance for functions like
control of pest species; delivery of specialised functions like pollination services; etc.).
This way the different main guilds of species from interacting functional groups are
identified which are directly or indirectly affected by the GM-arthropod itself or by
effects of the GM-arthropod on the population of the parental species. It should also be
considered whether the GM-arthropod belongs to an indigenous or non-indigenous
species in a specific receiving environment (Hoy, 2006a).
−
Another important aspect is the mode of presence of the released GM-arthropod in the
environment (i.e. whether the GM-arthropod is only present during a specific
timeframe or continuously, whether it is present in fluctuating numbers or at a specific
population level whether it is present throughout its entire life-cycle or only in specific
life-stages). It should be considered that different life stages of the same species may
have different ecological roles (e.g. different feeding habits, connection to different
predators and parasitoids).
−
Furthermore the characteristics of the GM-application itself are important for
assessment of the effects on non-target organisms in the receiving environment
(characteristics of transgenic components contained in the GM-arthropod, purpose of
the modification and effect on the parental species). This concerns the question
whether direct or indirect effects have to be expected.
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In the following, possible hazards and exposure for predators and other antagonists,
biodiversity in general and pollination in special are addressed.
9.3.1.
Effects on predators and parasitoids
Hazard identification
Ecologically, the most important interaction of released GM-arthropods probably is with
antagonists such as predators and parasitoids. Potential adverse effects on these groups could
be due to direct adverse effects by the incorporated GM-products upon predation. Other
adverse effects that could be associated with the fluctuation of abundance of the target species
are dependent on the timing, frequency and size of releases, which may significantly differ
between various GM-applications.
With respect to SIT applications to suppress or eradicate the target population, the number of
released arthropods is around 10-100 times the number of individuals living naturally in the
target area. So there are many additional individuals which (similar as the non-GM naturally
occurring individuals) usually live only for a few days. In this period, many living and dead
arthropods of the target species are available as food for antagonists, such as predators and
scavengers. Depending on the release characteristics this artificially increased amount of food
is available during the time SIT individuals are present in the environment and will decline
sharply when the target species is successfully eradicated. These changes in abundance
certainly have consequences for potential predators and other antagonists.
Loss of available prey through suppression or disappearance of the target species would be an
adverse effect for predators, especially if the specificity of the predator is high and no
sufficient alternative food sources exist.
By feeding on the GM-arthropods the transgene is ingested by a predator together with the
(living or dead) GM-arthropods. Theoretically, a transgene which exerts toxic effects or make
the prey unwholesome for the predator could affect the antagonist.
Hazard characterisation
Since it will be difficult to estimate the magnitude of species loss or decrease in abundance of
antagonists, a recommendable approach is to define key antagonists on the basis of prerelease habitat analyses and to investigate their abundance during meaningful periods before
release. After release comparable investigations of the abundance of these key antagonist
species are performed to show the effect of the GM-arthropod release. Furthermore, it needs
to be tested by feeding experiments whether transgenic products expressed in the GMarthropod harms the predator in any way. Questions concerning horizontal gene transfer are
addressed in chapter 9.1.2.
Exposure characterisation
Depending on their degree of specialisation, antagonists such as predators, parasitoids and
pathogens will be impacted by changes in the abundance of the target organism or other
potential direct effects. Species with larvae living in an aquatic environment (such as
mosquitoes) are characterised by a longer and rather continuous presence in which more or
less specific predators develop (e.g. some water bug species, predacious mosquitoes of the
cosmopolitan genus Toxorhynchites or other flies like the ephydrid shoreflies of the
worldwide distributed genus Ochthera (Minakawa et al., 2007)). Adult mosquitoes are short91
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
living, usually only available for days or weeks and are preyed upon by dragonflies, spiders,
bats, birds and other generalist predators. Predators of larvae, therefore, could more
continuously rely on mosquito larvae as prey (specialists), whereas predators of adults will be
more opportunistic (generalists). The first group of predators could be more affected than the
latter.
Fruit flies, on the other side, live a much more hidden life since egg and larvae are wellprotected in fruits. Their most-effective antagonists are hymenopteran parasitoids and many
species-specific adaptations can be found. Bactrocera species are mainly parasitized by a few
species of parasitic wasps from the Braconidae, Eulophidae, and Eupelmidae families
(Miranda et al., 2008; Boccaccio and Petacchi, 2009). Mediterranean fruit flies are similarly
predominantly parasitized by a variety of hymenopterans which mostly belong to the braconid
family (Argov and Gazit, 2008).
Furthermore, if the GM-arthropod is an alien species (as e.g. Aedes albopictus) there are no
specialised antagonists of this alien species endemic to European habitats to consider and
eradication would not likely cause any biodiversity loss in the invaded area.
9.3.2.
Biodiversity
Hazard identification
For certain GM-arthropod applications facilitating population suppression strategies effects
on biodiversity by a successful implementation of this strategy have to be considered. The
aim of such applications, which might be developed for notification in the EU (see chapter 8),
is either the elimination of a pest organism in a specific area or at least a reduction of the
abundance of the pest species. One of the consequences of such a programme is that the target
organism disappears or is considerably suppressed in the respective ecosystems. Specialized
antagonists, such as some predators or parasitoids of the target species will thus be affected in
the receiving environment. Also other species, linked to the target species via the food web,
could be affected by its disappearance or its decreasing population size.
Hazard characterisation
The target species and its specialist antagonists will certainly be affected by a GM-application
with the aim of population suppression. If other species will get affected the magnitude of
such an effect is difficult to assess. A recommendable approach is to identify key interacting
species for the released GM-arthropod in the receiving environment on the basis of prerelease habitat analyses and to investigate their abundance during meaningful periods before
release. After release comparable investigations of the abundance of these key species have to
be performed to identify any effect of the GM-arthropod release. During hazard
characterisation it should also be assessed which ecosystem functions could be affected by
impacts of a specific GM-arthropod application on biodiversity (“functional biodiversity”).
Exposure characterisation
Due to permanent immigration from surrounding habitats, the target pest will most likely not
be eradicated completely from a given receiving environment, i.e. biodiversity effects could
be low. In islands or otherwise isolated environments higher biodiversity effects are to be
expected.
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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9.3.3.
Pollination
Hazard identification
For many agricultural crops arthropods are very important for pollen transfer between plants,
thereby enabling fertilization and thus improving quality and quantity of yield. Main
pollinator groups are Hymenoptera (e.g. bees), Diptera (e.g. hover flies and other dipteran
families) and Lepidoptera (butterflies and moths), but in specific pollination systems also
other arthropod groups such as Coleoptera may be important. Pollination can be affected by
variety of anthropogenic impacts causing disturbance (Winfree et al., 2009), causing
pollinator decline (Kluser and Peduzzi, 2007) or in the case of specialised systems by loss of a
specific pollinator species.
Impact on plant pollination by GM-arthropods could be direct, in case they are involved in
pollination or indirectly by ways of impact on other arthropod species which are acting as
pollinators. In the first case the genetic modification would need to interfere with
characteristics of the parental species, which are important for the pollination behaviour, e.g.
abundance at the time of pollination, mobility of the animals, preference for certain plants. In
the latter case pollination loss could be caused by decrease of pollinator abundance in the
environment. For agricultural crops this could lead to yield loss, in case of other plants this
may lead to a decrease in abundance of the considered species, thus to a loss of plant
diversity.
Hazard characterisation
Most of the species discussed as of possible relevance for the EU with regard to GMapplications are not important as pollinators. However, some of the mentioned species (e.g.
Cydia pomonella) could be relevant with regard to pollination. For applications which are
aimed to suppress populations of these species, the impact on pollination should be assessed.
The importance of specific groups or single species for the pollination of a given plant species
can be assessed by observations during the flowering period of target plants. In a comparable,
quantitative approach the pollination habits of GM-arthropod can be tested.
Exposure characterisation
Besides transmitting pollen many arthropods visit flowers and collect nectar. Adult male
mosquitoes and fruitflies visit flowers primarily to take up water. Associated pollen transfer
happens only accidentally, thus they are not particularly relevant for pollination (Nagel and
Peveling, 2005). The same is true for Stomoxys calcitrans but in some genera it may be
meaningful to investigate whether they are essential in pollination and if, which exposure is
given (e.g. Cydia pomonella).
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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9.4.
Impact on specific agricultural management practices and management
measures to control arthropods vectoring diseases
Hazard identification
Impacts of the release of GM-arthropods on management practices need to be considered
differently for either applications targeting agricultural pest species or applications with
regard to arthropod species vectoring diseases. The main focus of this chapter is on potential
adverse effects of applications with regard to agricultural pests.
Management of arthropod pest species during cultivation of crops is a very important issue in
agricultural production. Therefore impacts of the release of GM-arthropods in production
areas need to be considered with a view on the associated effects on the current practice of
pests, e.g. by synthetic pesticides, biocontrol measures or other cultivation practices. Relevant
GM-applications to be considered would be the release of GM-arthropods like Mediterranean
or Olive fruit flies to suppress or eradicate pest populations, e.g. by SIT. The release of GMarthropods for biocontrol purposes, like antagonists of agricultural pests modified to be
resistant to certain insecticides, would constitute a hypothetical example for respective GMapplications, e.g. as described by Hoy (2006b). The hypothesis for a potential GM-application
for these purposes was based on experiments to develop strains of the Western predatory mite
Metaseiulus occidentalis, a natural enemy of spider mites in orchards, that are resistant to
certain insecticides, e.g. carbaryl and permethrin (Hoy, 2006b).
Different impacts on agricultural management practices need to be considered with regard to:
−
different management regimes of crop production (conventional, integrated pest
management, organic);
−
different GM-arthropod applications (for SIT or other purposes) and their
consequences, e.g. for application of pesticides in comparison to the current
management of respective crops;
−
management measures according to the intended use of GM-arthropods and measures,
which are necessary in reaction to unintended release of GM-arthropods or in case of
failure of the GM-application.
The different (conventional, integrated and organic agriculture) management systems are
considerably different e.g. with regard to type and varieties of crops cultivated, crop rotation
schemes, reliance on synthetic pesticides or use of biological pest control measures. For
production of livestock and crops conventional agriculture currently is characterised by a
focus on maximising the production output using crop varieties with high yields and high
management inputs including synthetic fertilizers and pesticides. Integrated production
includes natural resources and regulating mechanisms with the central role of agroecosystems to reduce inputs which may lead to adverse effects and to enable sustainable
farming. The management in organic farming strictly relies on crop rotation, green manure,
compost, biological pest control, and mechanical cultivation measures to maintain soil
productivity and control pests, excluding in general the use of synthetic fertilizers and
pesticides.
Since insecticide use will negatively affect any susceptible GM-arthropods, use of different
pesticides and the timing of application is affected by GM-arthropod releases. As applications
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like SIT are most effective when the number of pest individuals is low, GM-arthropod
applications for SIT in conventional agriculture may require increased insecticide use ahead
of the release, or release of larger numbers of GM-arthropods. In contrast to this, the lower
and more buffered pest abundance in integrated or organic systems will demand less GMarthropod input.
Release of insecticide resistant GM-arthropods as natural enemies would be associated with
the use of the respective insecticides. In such situations the consequences of such applications
should be considered in comparison to pest management without release of GM-arthropods.
Accidental release into the environment of GM-arthropods reared for SIT, but before
irradiation to achieve sterility (which in some species is conducted on-site ahead of releases)
or failure of a GM-application for population suppression could result in the need for applying
additional control measures, e.g. additional applications of pesticides.
As indicated above changes in overall control measures can also result from the application of
GM-arthropods for suppression or eradication of parent species. GM-arthropod applications
in this respect could substitute for control of the target species with pesticides or as an
alternative to conventional SIT. In comparison to control strategies with pesticides GMapplications may result in similar effects as conventional SIT programmes, i.e. reduction in
the pesticide amount necessary for control of the targeted species, once the inital target
population is reduced to a size, which results in high efficiency of SIT approaches. In
comparison to conventional SIT relevant factors for assessment are the specific efficiency of
the GM-application (i.e. timeframe to archieve sufficient reduction in target species numbers
and the potential for failure of the suppression mechanism. Overall reduction of pesticide use
as a result of successful pest control strategies by means of release of GM-arthropods may in
turn lead to indirect effects, e.g.expansion of other pest species (see also chapter 9.2.2).
In case of failure of the intended population suppression increase of numbers of arthropod
individuals competent to transmit diseases could result in the necessity that control with using
pesticides has to be increased, leading to a higher pesticide exposition of the environment.
Hypothetical GM-applications for population replacement would need to be compared to
alternative approaches (population suppression by pesticides, SIT or GM-population
suppression applications) with respect to differences in efficiency (timeframe necessary to
achieve suppression/replacement, requirement for additional measures e.g. for initial
reduction of target population, likelihood for failure of approach and consequences of failure).
In addition the indirect effects of the assessed approaches, i.e. measures necessary to control
other relevant disease vectors, which may experience some advantage from suppression of the
target species, should be considered. This would be specifically relevant for overseas
territories, with a more complex situation regarding number of vector species and diseases
present in a specific area and the necessity for control measures.
Hazard characterisation
With regard to releases of GM-arthropod for purposes of agricultural management, which are
most likely, i.e. SIT applications with GM-fruit fly species, relevant factors for assessing
impacts on agricultural management are:
−
the current management of the respective agricultural pest species according to the
respective production system (conventional, integrated, organic);
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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−
the size and location of the release area (e.g. isolated habitats or agricultural areas
which are prone to reinfestation);
−
duration and timing of release(s) (single releases or recurrent releases);
−
changes to agricultural management due to GM-arthropod application (i.e. specific
differences in agricultural management upon application of GM-arthropods compared
to currently implemented pest control measures).
The main differences between the farming systems are known and the most important
parameters affecting a GM-programme have to be assessed. This includes for example the
frequency of insecticide applications, the sensitivity of the GM-arthropod to such compounds
and possible resistance effects of the species and related species. Also, the probability of a
pest outbreak or the average crop damage will influence the intensity of a GM-arthropod
release programme.
Exposure characterisation
Performing a GM-arthropod programme in differently managed agricultural systems means
that the species encounter very different environments. The numbers and abundances of
species potentially getting into contact with the GM-arthropod will be highest in organic
farming systems and lowest in conventional agriculture. Also the potential areas of
interactions will differ in this sequence. In conventional agriculture the average abundance of
a given pest species is low since any increase will be answered by insecticide applications.
Therefore, the number of naturally occurring antagonists will also be low because they are
also affected by these applications, which may lead to sudden pest outbreaks. A GMarthropod release will have to face such situations with stochastic changes between overall
low and sudden high pest peaks.
As shown for three different olive-orchard management regimes in Spain, corresponding to
the three described management techniques, biodiversity differs considerably between
conventional orchards and integrated and conventional management regimes (Ruano et al.,
2004). Conventional orchards have the lowest numbers of arthropods and orchards with
organic management the highest numbers of arthropods and most arthropod groups. E.g.
spiders are most common in organic orchards, heteropteran bugs and coleopterans (among
them many predacious species) usually show highest abundance in organic farming, next in
integrated managed orchards, and lowest in conventional farming.
9.5.
Effects on biogeochemical processes
Hazard identification
Following mass release of GM-arthropods a considerate amount of arthropod material will be
introduced to the soil layer and into water bodies and eventually decomposed, e.g. by
microorganisms. This way transgenic DNA as well as transgene products enter the soil and
water bodies food web and can accumulate in some compartments of these ecosystems
dependent on the mobility and dispersal capacity of the GM-arthropod and the stability of the
DNA/transgene proteins. Potentially, adverse effects from such an accumulation or from any
impact on soil microorganisms or other organisms living in this ecosystem compartment need
to be identified. This could be due to direct negative effects on exposed organisms, e.g. due to
toxicity of the transgenic material deposited. However, with a view to the transgenic elements
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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present in the GM-arthropods under development and specifically regarding GM-applications
which could possibly be of relevance for application in the EU in the next decade, there are no
clear indications for such effects.
Hazard characterisation
As mentioned above, soil and water functionality (biodiversity) needs to be analysed before
and after being exposed to the transgene and also for a meaningful period after release to test
whether the transgene accumulates and soil or water biodiversity changes on the long term.
Some data are available on the extent of potential negative effects due to other hazards (e.g.
pollutants affecting soil organisms causing changes to ecosystem functions), but no adequate
data exist so far to delimit possible negative effect of the transgenes in question with the GMarthropods considered in this report. Risk hypotheses can be tested by use of microcosmsystems with artificial soil compartments and tested by applying large numbers of dead GMarthropods.
Exposure characterisation
If the transgene is stable in soil, water and the food web, an accumulation in compartments
may happen. However, it needs to be taken into account that the area where GM-arthropods
are released usually is large and the GM-arthropods would be dispersed throughout. Therefore
the amount of transgenic material deposited is small compared to the size of the receiving
compartment, limiting exposure as well as possibilities for accumulation. Chances for
substantial accumulation are therefore possibly low, if no tendency of the GM-arthropod to
accumulate at specific locations can be identified.
Biogeochemical processes and ecosystem functions (soil decomposition, food web structure,
diversity of soil or water ecosystems) should be analysed for any effects by the introduction
of GM-arthropod material. These data need to be provided for the area of intended release
(depending on the mobility of the GM-arthropod) prior to any release. A monitoring system
needs to be established to regularly investigate possible changes in the above mentioned
parameters.
9.6.
Effects on human health
Hazard identification
The release of GM-arthropods could result in effects on human health by different exposure
routes, either during accidental release incidents, or resulting from contacts of humans after
intended releases of large numbers of GM-arthropods into the environment.
For accidental release incidents as well as for intended releases adverse effects on persons
handling the GM-arthropods need to be considered. E.g. during mass rearing, transport or
release staff might come in unintended contact with large numbers of the GM-arthropods.
Skin and sensitive mucous epithelia could be exposed to transgenic products during these
incidents. This could lead to adverse effects if the transgenic products have some potential to
cause allergies or irritation and thus would elicit an adverse physiological response. The same
could happen to other persons that are present in the area where (accidental) releases take
place.
Only female mosquitos actively target and bite humans for blood-feeding, therefore the
presence of viable GM-female mosquitoes in the environment should be addressed
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specifically during the RA. Through such mosquito bites humans might come in contact with
transgenic components, if present in the mosquito saliva. The transmitted transgenic
components thus have to be assessed whether they could elicit any physiological or
immunological response in exposed persons taking into account the amount of the transgenic
substances transmitted to humans. Specific consideration should be given in this respect to
hypothetical applications like recent R&D work on GM-mosquitoes as means of vaccinating
people by transmission of transgenic immunogenic proteins in their saliva (Crampton et al.,
1994). The assessment of such applications needs to consider that with delivery to humans of
proteins of medical relevance would be depending on the behaviour of the GM-mosquitoes,
with all uncertainties regarding predictability involved.
Concerning GM-applications in species able to transmit diseases e.g. mosquitos, additionally
potential changes in vector competence need to be addressed. An assessment is necessary,
whether the released strain may transmit diseases more efficiently than non-modified or
naturally occuring mosquito species. Although vector competence can change, this kind of
data usually is well documented and accessible for most mosquito species.
Another way of exposure of humans to material derived from GM-arthropods is through
ingestion. Although currently no GM-arthropods are developed for direct application for feed
or food use, there are several pathways which could lead to an accidental ingestion of GMarthropod material. Fruit flies and other agricultural pests for instance deposit their eggs in
host crops and developing fruits. GM-applications in these species could lead to
contamination of foods, like fruits with eggs and developing larvae which express transgenic
products if reproduction is possible in the environment after release. Additionally released
GM-arthropods, including GM-mosquitoes, may be swallowed accidentally. Through this
contact expressed transgenes, e.g. marker genes, with an allergenic potential could lead to
adverse effects as well as any accidental contact of transgenic proteins to sensitive human
epithelia (e.g. mucous membranes).
Hazard characterisation
It needs to be tested, whether the transgene could result in harm to humans, e.g. by potential
toxic or immunogenic/allergic effects. An assessment of such effects will be based on
methods established for the assessment of toxicity and allergenicity of GM-traits in the
framework of food safety assessment. If transgenes with a known immunogenic potential are
deliberately used, as in the mentioned experiments with GM-arthropods as vaccinating agents,
the dose-dependent effects of such a transgene need to be evaluated. The vector competence
of the GM-mosquito needs to be characterized or tested with suitable laboratory experiments.
Exposure characterisation
The overall exposure of humans to GM-arthropods through various pathways depends on the
presence of GM-arthropod life stages which are known to interact with humans or which are
present in foodstuffs. As indicated this are either blood-feeding individuals, like adult female
mosquitoes, or eggs and larvae of agricultural pests, which are deposited in and develop in
plant parts like fruit, which are consumed as foods.
If the GM-arthropod applications are designed in a way that no interacting GM-arthropods in
relevant life-stages are present in the environment the expected exposure should be quite low.
This can be achieved in SIT applications for fruit fly species which prevent the production of
developing eggs and larvae in fruits. Releasing only male GM-mosquitoes for SIT
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applications would minimise human exposure, since male mosquitos do not bite or blood-feed
on humans. The crucial aspect with RIDL applications is the development time until the
inherited dominant lethal genes exert their function. If the applied lethal component is active
in eggs, larvae and pupae of the respective GM-mosquitoes, humans would not be exposed
since they usually do not come into contact with these developmental stages. Only if the lethal
transgene acts very late and adult GM-mosquitos develop, exposure of humans through biting
by female GM-mosquitoes could happen. In case vector competence is changed in a certain
GM-mosquito application, exposure of humans to vectored disease agents would also require
contact with female GM-mosquitoes.
However, the likelihood that the respective GM-applications are performing differently as
expected under environmental conditions should be assessed. Instability of transgenes under
environmental conditions which could lead to failures of a GM-application was identified in a
field trial with GM-mites (Metaseiulus occidentails) containing a marker construct derived
from a bacterial lacZ gene (Hoy, 2006b).
For applications like GM-mosquitoes as vaccinating agents characterisation of exposure of
humans to biting GM-female mosquitoes would be a crucial prerequisite for RA.
10.
Methods to investigate adverse effects of GM-arthropods
Several methods are available to investigate adverse effects as described in chapter 9, but any
results demand careful interpretation. Most often, comparisons are needed (for example from
preceding years) to evaluate the obtained data and baseline data are very much needed.
10.1.
Vertical gene flow
Today there are various new molecular genetic tools, which have facilitated the detection of
hybridisation and introgression in cases where hybrid individuals were morphologically
difficult to distinguish from their parental forms. Within this context, particularly important
roles are played by the polymerase chain reaction (PCR) for the in vitro amplification of
specific DNA sequences, and the techniques and genetic markers based on PCR technology,
such as rapid DNA sequencing methods (e.g., cycle sequencing) and microsatellite DNA
markers. The latter allow a genetic resolution at the level of individuals.
The major advantage of PCR-based technologies is their non-destructive and minuscule tissue
requirements, enabling a non-invasive way to study rare or endangered species. Furthermore,
recent statistical developments have facilitated the detection of hybridisation and hybrid
individuals in cases where no taxa-specific markers are available. For example, model-based
Bayesian statistical techniques, which utilize the information of highly polymorphic markers
such as microsatellites (Pritchard et al., 2000; Anderson and Thompson 2002), have already
been widely applied to the study of hybridisation and introgression in natural and maninduced cases (Barilani et al., 2005; Williams et al., 2005; Lecis et al., 2006). The
simultaneous use of cytoplasmic (mitochondrial and chloroplast DNA) and nuclear genetic
markers has become an important standard in studies of introgressive hybridisation. The
combination of these two marker classes allows gaining very detailed insight into the
processes of hybridisation and introgression (Avise, 2000).
These studies take advantage of the fact that these cytoplasmic genomes are usually
maternally inherited, and thus show a pattern of inheritance different to that of recombining
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nuclear markers. A joint use of these marker classes provides information that cannot be
obtained by using either marker class alone. For example, if only one sex of the invaded taxon
hybridises, then the maternally inherited markers will introgress asymmetrically in relation to
nuclear makers, which are inherited by both sexes equally. The direction of the asymmetry
gives information on which combinations of mating occur in the interbreeding of the two
parental taxa. If the interbreeding is restricted to males of the invading taxon with females of
the native taxon, then it would be observed that hybrid individuals always carry a
mitochondrial genotype of the invading taxon, while having a mixture of alleles of the two
parental taxa at the nuclear markers. Thus, measures of association between specific alleles at
nuclear markers and cytoplasmic genotypes can be used to formulate hypotheses of factors
involved in hybrid formation, and the rate and direction of genetic introgression in
hybridisation events. This development of a cytonuclear theory and of statistical models
provided an important framework for hypothesis testing using empirical data in hybridising
taxa (Largiader, 2007).
10.2.
Host range
The experimental confirmation of the host range of herbivorous arthropods bases largely on
the strategy of Wapshere (1974) assuming that the host range of a herbivore is based on the
principle that a plant closer related to the target plant will produce less feeding deterrents to a
specialist arthropod herbivore than a plant that is less closely related. His “centrifugalphylogenetic method” has been the basis for the selection of plants to test ever since. He
added some 'safeguard' criteria for assessing plant species that would not be selected on
phylogenetic grounds alone, such as cultivated plants related to the target plant, or plants for
evolutionary, geographic or climatic reasons not exposed to the candidate agent, or plants
known to be attacked by species related to the candidate agent.
In addition, Briese (2003) suggested three improvements: use true phylogenetic distance
(rather than taxonomic distance) to measure relatedness between plant species; consider the
degree of biogeographic or ecoclimatic overlap and consider the degree of ecological
similarity between potential non-targets. Today, it is generally acknowledged that the
selection of test plant species to analyse the likely host range is well supported by theory
(BIREA, 2010).
Kuhlmann et al. (2005) described and analysed a range of host-range testing programmes and
listed criteria that have been used to select species for testing against arthropod predators and
parasites: ecological similarity between host and test species, overlapping geographic range,
feeding niche, habitat preference, phylogenetic affinity between host and test species,
conservation importance, commercial importance, and role as a beneficial. Biological
similarity between host and test species bases on known host range, phenological overlap with
the target, dispersal capability, morphological similarity, behavioural similarity, overlap of
physiological host range of other agents, and response to host plant, similarity of host plant
structure. Ecological similarities, phylogenetic/taxonomic affinities and safeguard
considerations are applied to ecological host range information to develop an initial test list,
which is then filtered by eliminating those with different spatial, temporal and morphological
attributes and those species that are not readily obtained. The reduced test list is used for the
actual testing but can be revised if new information indicates that more species should be
included.
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10.3.
Symbionts
Many arthropods contain beneficial symbionts, e.g. bacteria of the intestinal flora, and feeding
on a poor diet such as blood, phloem sap or wood are strong predisposing factors for the need
of such beneficial micro-organisms. Most aphids, for example, rely on the bacterium
Buchnera for synthesis of essential amino acids. Cockroaches and termites, many dipterans
and beetles also rely on beneficial symbionts (reviewed in Sanchez-Contreras and Vlisidou
(2008)). Potential adverse effects reducing the diversity of symbionts therefore may threaten
the fitness of the host considerably. The investigation of the symbionts’ diversity is usually
done by molecular tools such as genome sequencing, heterologous hybridisation and
polymerase-chain reaction (PCR) based identification methods and other approaches, for
example denaturing gradient gel electrophoresis fingerprinting (e.g. LaJeunesse et al. (2004)
and Apprill and Gates (2007)). Restriction fragment length polymorphism analysis of the
PCR-amplified 16S rRNA genes is used as rapid approach for determining the phylogeny of
symbionts and mapped restriction site polymorphism (MRSP) proved to be a good
classification method of symbionts (Laguerre et al., 1997).
10.4.
Predation
Adverse effects in the field of predation concern primarily an altered prey composition or
predator spectrum. Useful reviews on methods to quantify predation are given in Sunderland
(1988), Mühlenberg (1993) and Southwood and Henderson (2000). Analyses include direct
observations, identification of killed animals and their remnants but also faeces analyses.
Some predators regurgitate pellets, consisting of indigestible parts of their prey. In aquatic
ecosystems, it is possible to wash out the gut content of some fish species without causing
harm to the fish. The same is possible with the nestlings of some bird species which
regurgitate recently received food. Both approaches allow identifying the prey spectrum in
different predator gilds and habitats quite detailed. Spider webs or ant trails also offer ideal
possibilities for prey analyses since arthropods collected in the last hours can be picked up
(Nentwig, 1983). A semi-natural assessment of the role of predators consists in offering a
known number of prey in natural situations with subsequent analysis (Lys, 1995). More
generally speaking, the role of specific predators can be analysed in field experiments with
exclusion techniques (enclosures).
Several molecular tools for quantification of predation have been developed. Electrophoresis
to separate prey enzymes yield species-specific banding pattern. A variety of immunological
methods has been developed: Serological methods, precipitin methods (including capillary
ring test, double diffusion/Ouchterlony test, and immunoelectrophoresis) and agglutination
methods (including passive haemagglutinin inhibition, fluorescence immunoassay and
enzyme-linked immunosorbent assay ELISA). These methods have been widely used for gut
analyses of sucking species, especially in mosquitoes. They are, to give a few other examples,
useful to identify predators of Lepidoptera and the prey of beetles or spiders.
Though these tools gave quite satisfying results and can still be used, today most of them are
considered to be not state of the art. The availability of PCR-based techniques, their
efficiency, speed and cost-effectiveness to analyse for predation make them a unique tool.
These modern molecular diagnostic approaches include: specific polymerase-chain reaction
(PCR), PCR followed by restriction endonuclease (REN) digestion and gel electrophoretic
analysis of fragment length polymorphisms (PCR-RFLP), random amplified polymorphic
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DNA (RAPD-PCR), DNA barcoding and microsatellite analysis (Gariepy et al., 2007).
Symondson (2002) reviewed the use of these molecular diagnostics for prey identification in
predator diets, and described the techniques used to identify consumed prey. (Behura 2006)
reviewed future directions for the use of molecular techniques in quantifying predation and
concluded that the future developments of these techniques will base on the rapid advances in
genomics, microarray technology, and the miniaturization and automation of processes such
as DNA extraction, PCR and DNA sequencing.
10.5.
Biodiversity
Adverse effects may target biodiversity in a sense that present species disappear or new
species appear and / or that the abundance of species changes. Thus, a biodiversity assessment
demands an assessment of species composition, i.e. collection and identification of species
and their abundance. Technical requirements for such a state of the art assessment vary with
respect to habitat and taxonomic group, so no general recommendations can be given. There
is a vast literature on such biodiversity assessments: with a comprehensive approach. Hill et
al. (2005) address the technical aspects (experimental design, sampling strategy, data analysis
and evaluation), methods for a broad range of habitats, and methods for the major taxonomic
groups. Southwood and Henderson (2000) gives a broad overview and set the frame from a
theoretical and mathematical point of view. Mühlenberg (1993) describes a variety of
methods for specific arthropod groups, while Lampert and Sommer (1999) focus on methods
in the aquatic environment.
10.6.
Pollination
An assessment of adverse effects in the field of pollination can be seen as a subsection of a
biodiversity assessment with the main question if there is any alteration in the change of the
pollinator community of a given plant. Usually this is performed by a survey of pollinators,
which includes observations and collections involving identification of species. A toolkit of
standardised methods to assess pollinator diversity has recently been published by (Potts et
al., 2005). The technique of pollen analysis obtained from pollinators and the conclusion on
visited plant species is demonstrated in Mühlenberg (1993).
10.7.
Water bodies
Methods to detect an accumulation of a transgene in water bodies are the usual molecular
biological methods to analyse this transgene.
10.8.
Soil / decomposition
Methods to detect an accumulation of a transgene in the soil are the usual molecular
biological methods to analyse this transgene. Any potential adverse effect on soil
microorganisms can be detected by common molecular biological and microbiological
methods for the quantification of microbial activity.
Changes in the composition of decomposers (microbial organisms, protozoans or
invertebrates) may influence their degradation potential. This can be quantified with the litter
bag method (Hönemann et al. 2008), originally developed to investigate plant biomass
decomposition, but easily adoptable, e.g., to invertebrate biomass.
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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11.
Keyparameters for the ERA of GM-arthropods
In order to conduct an ERA of GM-arthropods, it is vital to identify crucial parameters for the
different relevant life-history stages. For holometabolous insects those are egg, larva, pupa
and adult. Other GM-arthropods discussed in this report have different development stages
(e.g. crustaceans), but none of them are considered to be of possible relevance for the EU in
the next 10 years and are therefore not further considered in this report. In addition to
arthropod characteristics, it is important to assess environmental variables and genotypeenvironment interactions. Therefore, this chapter discusses key parameters to be considered
when evaluating fitness of GM-arthropods as well as spread, persistence and invasiveness.
Also ecological interactions with other organisms are considered. Table 7 provides an
overview on those parameters indicating the relevance to the respective life-history stage.
Table 7: Relevant arthropod characteristics, environmental variables and genotype environment interactions at different life-history stages
Parameter
Arthropod
characteristics
Environmental
variables
Genotypeenvironment
interaction
Fertility rate
Mating competitiveness
Longevity
Development time
Egg hatching rate2
Larval survival3
Pupal survival4
Adult emergence5
Size / weight
Flight ability
Altered biochemistry
Abiotic stress resistance
Vector competence
Arthropod density
Migration behaviour
Habitat interactions
Climate interactions
Food interactions
Altered host range
Sensitivity to pathogens
Predator interactions
Insecticide resistance
Adult
X
X
X
Life history stage
Pupa1
Larva
X
X
Egg
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
1
if applicable (only present in holometabolous insects)
2
= the frequency of eggs from which larvae can fully free themselves from the egg shell
3
= the proportion of first stage larvae reaching the pupa stage
4
= the frequency of pupae from which adults emerge (in fruit flies the percent of hatched eggs that produce pupae)
5
= in fruit flies the percent of pupae that produce adults.
X
X
X
The definition of some parameters is related to the respective species and the methods
available for different life history stages. Since it is for instance not possible to count larvae in
fruit flies, larval survival is not assessed directly and instead pupal survival can be defined as
percent of hatched eggs that produce pupae. Also the overlap in the definitions of ‘pupal
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survival’ and ‘adult emergence’ reflects those differences between e.g. fruit flies and
mosquitoes. It must also be noted that some of these parameters are not relevant for all
species. For instance, vector competence is only of importance for arthropods capable of
transmitting diseases (e.g. Aedes aegypti, Anopheles gambiae s.s.), thus underlining the
importance of species specificity and a case-by-case approach.
Some parameters are routinely assessed in the laboratory (particularly for species used for
SIT) but not all. It is crucial to keep in mind, that laboratory-generated data may not fully
represent field conditions and that a comprehensive comparison between a GM-arthropod and
the non-GM-species largely depends on scientific knowledge that might not (yet) be available
or is impossible to obtain without actual releases taking place (e.g. dispersal ability).
At the individual level, reproductive fitness and net survival of the assessed GM-arthropod are
key parameters that are highly relevant for the ERA (although in the case of sterile insects the
reproductive fitness should be zero). Reproductive fitness can be assessed by measuring
mating competitiveness (particularly important when using the GM-arthropod for population
suppression) and the fertility rate, e.g. expressed by the number of viable offspring per
female, as the most decisive parameter concerning fertility. Survival can be analysed as the
egg hatching rate, larval survival, pupal survival or adult emergence, as development time for
egg, pupae and larvae, and as longevity of adults. Most of these parameters are common lifetable variables.
The above-mentioned fitness and population parameters are also important to assess possible
spread and migration of a GM-arthropod. In that respect, body size and weight, as well as
flight ability are important parameters to be considered during the ERA. Other important
parameters at the individual level are changes in biochemistry (including toxicity, which is a
parameter relevant for predators) or physiology of the different life-history stages, abiotic
stress resistance (e.g. concerning temperature, humidity), and vector competence in case of
arthropods transmitting diseases. The latter consideration would normally apply to adult
blood-feeding arthropods only, but in view of transovarial (i.e. vertical) transmission of
arboviruses (e.g. dengue) the other life-history stages should also be included in the
assessment.
Environmental variables affect not only individuals but whole populations. Again, one of the
key parameters derives directly from a regular life-table: the number of surviving individuals
determines arthropod density within a given environment and this influences overall
migration behaviour (i.e. the sum of emigration and immigration) as well as spread and
invasiveness. More specific interactions with habitat, climate, and food availability also may
become important and depend on the specific case. In view of climate change, habitat and
climate interactions may become more important in the future, affecting not only spread or
persistence but also trophic interactions.
Genotype - environment interactions refers to the variation in fitness according to
environmental conditions (abiotic as well as biotic). This concerns mainly interactions with
other organisms such as hosts, pathogens, or predators (including parasitoids). With regard to
the RA of GM-arthropods, any changes in these interactions which are specifically due to
genetic modification are of concern. This refers to an altered host range and changes in
pathogen sensitivity, specifically with respect to humans. Also insecticide resistance is an
important parameter, e.g. in SIT programmes insecticides are used at least at the very
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beginning to bring it down the target population to a level where SIT becomes economically
and technically feasible as well as to knock out new infestations. If the target population
would become resistant this would be very detrimental, especially as there are not many
effective insecticides available e.g. for fruit flies. In the case that unanticipated outcome
results from the release of GM-arthropods (e.g. mosquitoes) insecticides would be the firstline response to mitigate the hazard. Resistance to Malathion, DDT and pyrethroids has
already reduced the suite of insecticides that can be used against mosquitoes in numerous
places. Therefore knowledge on the resistance profile of the target species is an important
consideration when considering risk reduction possibilities. Chemical methods used and
allowed in Europoe are based on one of four active ingredients: Bti, methoprene,
diflubenzuron and pyrethroid derivates.
12.
Methods for the assessment of key parameters
All the above mentioned key parameters can be assessed by various methods. These are listed
in table 8 and were also analysed using a SWOT analysis (see chapter 3). In order to enable a
comparison between the different methods for a specific parameter listed in table 7 the
classification follows the same principle. References for the methods are provided in the
respective subchapters. In addition information can be found in Service (1993) and
FAO/IAEA/USDA (2003)
Table 8: Methods for the assessment of key parameters
Parameter
Fertility rate
Mating competitiveness
Longevity
Development time
Hatching rate
Larval survival
Pupal survival
Adult emergence
Size/weight
Flight ability
Altered biochemistry
Abiotic stress resistance
Methods
Life table analysis
Sex ratio test
Cage experiments to observe mating directly
Cage experiments to investigate sterility, sperm marker or marker among
progeny
Field bioassay for egg hatching or sperm marker
Life table analysis
Field estimates
Cages studies
Life table analysis
Field estimates
Direct observations
Life table analysis
Mass collection egg hatching estimates (lab, field)
Life table analysis
Life table analysis
Life table analysis
Nutrient reserves
Direct measurements
Lab test
Lab test flight mill
Field dispersion estimates
Gas chromatography
Enzyme analysis
Bioassay
Proteome profiling
RNA profiling
Bioassay
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Parameter
Vector competence
Arthropod density
Migration behaviour
Habitat interactions
Climate interactions
Food interactions
Altered host range
Sensitivity to insect pathogens
Predator interactions
Insecticide resistance
Methods
Analysis of stress response gene profiles
Gene expression profile
Parasite/agent infection response
Transmission bioassay
Breteau index
Pupal index
House index
Trapping (wild and released arthropod)
Serology
Screening of infested animals
Fruit sampling (infestation rate/fruit damage)
Mark-release-recapture
Population genetic analysis
Rare earth element detection in eggs of treated adults
Reinfestation rate of pest-free areas
Field observations
Analysis of range of habitats occupied
Seasonal abundance
Analysis of range of habitats occupied
Laboratory measures
Host/diet preference
Laboratory bioassays
Larval habitat occupancy
Larval gut content analysis
Field observations
Vertebrate attraction assays
Blood meal source analysis
Survey of host plants
Bioassay
Analysis of immune-response gene expression levels
Predator gut content analysis
Field observations
Allele analysis
Biochemical assay
Bioassay
In the following sections the methods for these parameters are described according to the
outcome of the SWOT analysis, using the SWOT terminology Strenghs, Weaknesses,
Opportunites and Treats. Tables providing an overview can be found in Annex B. It needs to
be noted that some methods can be used to assess different parameters, but in this report
methods are described always in the context of the respective parameter. This way issues that
might be relevant only for a specific parameter can be elaborated allowing the comparison of
the described methods, e.g. life table analysis calculates a given parameter from data obtained
in the laboratory, semi-field or field. Breeding culture was not assessed as a method since this
is the basis for production of materials for the data collection. In that respect it must be noted,
that breeding conditions must be standardised and GM-arthropods that perform well in the
production facility have often been selected for that characteristic, so laboratory and factory
production characteristics do not necessarily correlate with performance in the environment.
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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12.1.1. Fertility rate
To assess the fertility rate, the following methods could be used:
−
Life table analysis
−
Sex ratio test
One possibility to assess the fertility rate is by calculations from a life table analysis (Begon et
al., 1996). The strengths of this method are that data are easy to collect and comparisons to
data collected in other laboratories are possible. Data assessed by life table analysis
presumably accurately reflect underlying biological differences. In most cases males will be
released whose sole function is to inseminate feral females and no progeny are expected to
persist. For this method the opportunities are that the development of GM-production will
lead to the creation of standardised parameters leading to robust comparisons. A possible
threat is that unfavourable life table characteristics may be misinterpreted as indicators of
poor strain performance. While this may be true for the ease of production, it does not
necessarily mean that the efficacy or safety is impaired.
Another method to assess the fertility rate is the sex ratio test (sex ratio being the ratio of
females:males (Begon et al., 1996)). Being also a simple test data can be collected by
unskilled personnel. The test reflects a basic genetic parameter. A weakness of this method is
that some effectors may intentionally skew the sex ratio or sex ratio distortion may be a
natural characteristic of strains of target species (e.g. in mosquitoes). Therefore batch and
temporal consistency is of more importance than the ratio per se (at least in some arthropods).
A general opportunity is that culture methods can be altered in order to change the proportion
of one sex that survives or the development rate to facilitate production or sex separation. On
the other hand one needs to be aware that extensive breeding can distort sex rations but these
effects remain difficult to predict.
12.1.2. Mating competitiveness
For the assessment of mating competitiveness three methods can be used for evaluating GMarthropods:
−
Cage experiments to observe mating directly
−
Cage experiments to investigate sterility, sperm marker or marker among progeny
−
Field bioassay for egg hatching or sperm marker
In cage experiments mating can be observed directly, particularly for species whose
copulation is long. The weakness is that this method is time consuming and, depending on the
species, must also be performed at sunrise or sunset. In mosquitoes mating is often very brief
and difficult to observe. Data collection could be improved by photographic and video
equipment increasing low-light detection of mating behaviour. Indoor cages also can be
constructed that stimulate natural behaviour. Threats are that mating competitiveness is only
one parameter that determines the success of a programme. Poor competitiveness may be
falsely interpreted as meaning a strain is not useful. Reduced longevity but high mating
competitiveness may be offset by increased lifespan and reduced competitiveness. In practice
it is possible to compensate a reduced competitiveness by increasing the number of released
flies.
107
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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Cage experiments (small cages as used for rearing or genetic test crosses) conducted to
investigate sterility, sperm marker or marker among progeny is also a direct measure of
mating frequency and highly relevant to assess GM-arthropod effectiveness (although for
some taxa like fruit flies one or two matings are sufficient to fertilise a female for its entire
life). Obtaining eggs from individual females is often very difficult. Stimulating natural
mating in cages is easy for some arthropods, but difficult for others. Also, by using this
method, insemination with few sperm is difficult to detect and as mentioned before cage data
often do not accurately reflect field performance.
The strength of field bioassays for egg hatching or sperm marker is that it is a highly
meaningful indicator of the performance of the released arthropod. When evaluating data
obtained with this method one must note the fact that obtaining eggs from individual females
can be difficult. Also the ease of collecting adults varies and the numbers that must be
released to obtain useful information makes this assay quite resource intensive. Generally
speaking improved trapping methods that reduce the effort required to collect adults are
needed. Use of sperm markers detectable by PCR will increase throughput. Marking with
stable isotopes during mass rearing may be feasible though analysis of field specimens
remains costly. A threat is that a meaningful assay requires the open release of GMarthropods, probably initially confined in some manner. Locating sites at which this can be
performed may be problematic. Alternatively, semi-field systems (large, outdoor screened
cages) in which the natural ecosystem is simulated may yield more realistic data then
laboratory studies (Ferguson et al. 2008).
12.1.3. Longevity
To assess longevity three different methods are proposed and evaluated:
−
Life table analysis
−
Field estimates
−
Cage studies
Life table analysis is not only a suitable method to calculate the fertility rate but also
longevity (Begon et al., 1996). The strengths, weaknesses, opportunities and threats are the
same as mentioned in the respective subchapter above (see chapter 12.1.1).
Field estimates may provide information that is useful to understand field performance but it
is a very demanding method and in many cases it is difficult to obtain robust data. In fruit
flies released animals are marked with different colours for each consecutive release.
Screening of trapped flies shows how long the flies can be detected. This test includes the
impact of predators on the released flies and shows a combination of effects, e.g. the ability to
find food in alien environments and the agility of the mass reared insect to avoid predation.
Seasonal and yearly values differ. In general, molecular or chemical markers that change
according to age or life stage could improve the data quality. Novel biochemical methods
(e.g. pteridine content of insect head or near-infrared spectroscopy (NIS)) are being developed
for age grading (Perez-Mendoza et al., 2002). But a meaningful assay requires field releases
of GM-arthropods. Locating sites at which this can be performed may be problematic,
although again simulated environments like large screened outdoors cages may be useful.
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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Cage studies are simple to perform and obtain high quality and often precise data. In fruit flies
cage tests in the laboratory means survival without food. It determines the nutritional status of
the produced insect. In field cages food and water is provided to determine longevity under
the climatic conditions in the field. But cage longevity does not accurately reflect field values.
The opportunity of this method in general is that more easily measured surrogates for adult
longevity can be exploited. These include size and energy reserves. For this method no
general threats are described.
12.1.4. Development time
Three methods are described that can assess the parameter development time:
−
Life table analysis
−
Field estimates
−
Direct observations
Data on development time can also be calculated with life table analysis (Begon et al. 1996).
The weakness of this approach is that reproducible values for any life table analysis depend
on highly reproducible environmental, diet and handling conditions. These are currently not
sufficiently standardised between laboratories. In general using outdoor semi-field systems
with simulated natural environments and ambient climate may yield more realistic
information although with a higher degree of variation being an opportunity of this method.
The threats are that favourable life table analysis (i.e. similar to the wild type) can be used to
either support an argument of greater risk or greater efficacy. Conversely, poor strains may be
considered safer but less effective.
The strengths of field estimates are that a truly realistic observation is possible and they can
be useful for planning field releases. On the other hand, the results vary widely by habitat and
season. It is difficult to obtain high quality data and this method is usually based on a model
which requires input data or assumptions that are usually only approximations. Generally
speaking the understanding of the relationship of molecular or morphological markers to
development rate could simplify data collection. The threats are that developmental time will
vary with climate for poikilothermic organisms like insects. Evaluations during different
seasons are therefore necessary and will be time consuming and costly.
The last method proposed for assessing data on the development time is direct observation.
This approach is not open to interpretation, but a very limited amount of data can be obtained
and often insignificant values provide little information relevant to safety or efficacy. In
general, no specific opportunities can be described. The threats are the same as for life table
analysis.
109
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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12.1.5. Egg hatching rate
To assess data on hatching rate the following methods are proposed and evaluated:
−
Life table analysis
−
Mass collection egg hatching estimates
With life table analysis the egg hatching rate can be calculated using data e.g. from the
laboratory (Begon et al., 1996). A full analysis is not necessary to obtain this discrete
measure. No general opportunities and threats are described with respect to hatching rate.
Mass-collection egg hatching estimates either in the laboratory or in the field is the easiest
method to estimate the hatching rate. The weaknesses are that data are confounded if the male
mate chosen affects the number of eggs laid. It is also confounded if sterility differs according
to the number of eggs laid. Obtaining collections of eggs in the laboratory is feasible, but may
not be in the field (for fruit flies e.g. it is in many cases possible to collect eggs in the field,
but it is tedious). In general the opportunities are that laboratory tests can determine whether
the number of eggs laid is affected by the male mate and whether the number of eggs laid is
related to sterility. No threats are described for this method.
12.1.6. Larval survival
To assess larval survival, the following method is proposed:
−
Life table analysis with data from lab culture
Larval survival can be defined as the proportion of first stage larvae that reach the pupa stage.
It can be assessed by life table analysis with data from lab culture (Begon et al., 1996)and be
easily observed but environmental conditions must be standardised. One threat is that data
generated from laboratory conditions may not present field values.
12.1.7. Pupal survival
To assess pupal survival, the following method is proposed:
−
Life table analysis with data from lab culture
Another parameter that can be assessed with life table analysis is pupal survival (Begon et al.
1996). It can either be defined as percent recovery per hatched egg or percent recovery per
first stage larvae (depending on the species and the available methods). It can be easily
observed but results will depend on the handling, larval diet and environmental conditions
meaning that it must be thoroughly standardised. Opportunities are that knowledge of this
parameter can improve production and reduce costs. But pupal survival may be compromised
by transportation so it may not be a good indication of the quality of the material to be
released.
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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12.1.8. Adult emergence
To assess data on adult emergence the following method can be used:
−
Life table analysis with data from lab culture
Adult emergence, e.g. for fruit flies, is a strain-specific characteristic. In the laboratory the
percent recovery from pupae can be calculated by life-table analysis (Begon et al., 1996). In
standard quality control it is also determined how many flies are half emerged and how many
are crippled (FAO/IAEA/USDA, 2003). It is affected, like other life table parameters (egg
hatching, pupal survival) by the genetic or molecular modification that was introduced (e.g.
sexing, marking). Percent recovery from pupae can be easily observed and indicates in part
conditions during the larval stage, so those must be standardised as well as the pupa holding
conditions. One threat is that adult emergence in the laboratory is not a good proxy for
emergence under field conditions with fluctuating environmental conditions.
12.1.9. Size/weight
To assess size and weight respectively two methods are discussed:
−
Nutrient reserves
−
Direct measurements
The strength of the determination of nutrient reserves as a method to assess size and weight is
that measurements of lipid, glycogen and sugar content of laboratory and field specimens are
straightforward and can easily be compared. The weakness on the other hand is that direct
impact of nutrient makeup of laboratory specimens on field performance remains largely
unknown. The opportunities are that tests for comparative performance as a function of
biochemical makeup can be performed and the influence of modifications on makeup studied.
No threats are described.
Direct measurements of size and weight are a simple direct method reflecting general vigour
and indirectly fecundity. No weaknesses are described. In general outside of the production
facility, in which size may reflect the quality of rearing conditions, the effectiveness of
released insects of different sizes is often unknown, thus such knowledge would increase
programme effectiveness. No threats are described.
12.1.10. Flight ability
To assess flight ability three methods are described:
−
Lab tests
−
Lab test flight mill
−
Field dispersion estimates
Laboratory tests are the most readily performed and reproducible data for a meaningful
dispersal characteristic are obtained. Meaningful flight tests do not exist for all species, but
e.g. for Ceratitis capitata, Anastrepha ludens or Bactrocera dorsalis. In general flight
performance assays for various arthropods should be developed. Separate systems might be
useful for either mosquitoes or fruit flies. The threats of this method are that laboratory tests
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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will not provide a credible indicator of flight performance and will not be serve as a basis of
field activity. Realistic tests are expensive and difficult to design (Shirai, 1995; Schumacher
et al., 1997).
The strength of a lab test flight mill is that it is a reasonable surrogate for the measures of
actual flight. But it requires custom equipment and expert technical staff to conduct. Some
arthropods are not stimulated to fly on such mills (e.g. Anopheles arabiensis males) and the
amount of data that can be collected is limited. The general opportunity is that realistic flight
performance devices and procedures that do not require such expertise and technical skill
could be developed. The use of this method is threatened by the fact that flight mills are
custom devices that are not widely available. If no suitable manufacturer can be found this
may prevent widespread use.
Field dispersion estimate (Banks et al., 1988) is the most realistic indicator of field
performance and also important for release strategy/planning (e.g. release density, flight
lines). The weaknesses are that this method is very labour intensive and appropriate sites may
be a limit. Mark-(release)-recapture studies are often time consuming and costly and
dependent on efficient trapping systems. Generally speaking the efforts could be made to
develop laboratory indicators of flight performance and establishing their value as predictors
of field activity. But the threat is that dispersion will be different in varying ecological
settings and under different climate scenarios. For many small arthropods passive drift is most
important.
12.1.11. Altered biochemistry
To assess altered biochemistry five methods can be envisaged:
−
Chromatography
−
Enzyme analysis
−
Bioassay
−
Proteome profiling
−
RNA profiling
Chromatography, e.g. gas chromatography and HPLC, are sensitive methods to determine
numerous compounds including volatiles. They can detect small differences between sibling
species if the natural variation is known. Chromatography is usually a high throughput
method and could be used for establishing baseline data. Specialised equipment and skill is
required and it is firstly necessary to identify what compounds are of specific interest. No
general opportunities can be described. The threats are that evidence of altered gas
chromatography profiles may be misinterpreted as showing some novel characteristic that
may not be biologically meaningful or whose relevance for establishing safety is unknown.
Enzyme analysis is a method that specifically measures changes in activity. The weakness is
that it requires special equipment and knowledge of particular enzymes of interest. It requires
also that there is some understanding of the implications of certain changes for effectiveness
and safety. In general, the opportunities are that a suite of enzymes of special interest for
safety and performance could be determined and recommended for routine baseline data
collection for GM-arthropods. No threats are described.
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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Bioassay is a direct and relevant measure of important performance characteristics, but
biochemical changes that are detectable by bioassay must be known. In general, the suite of
relevant traits that reflect changes in biochemistry should be expanded. These could include
those affecting flight at different temperatures, odour attraction and desiccation tolerance. An
overlap with stress resistance exists and could be exploited. No threats are reported.
The strength of proteome profiling is that extensive baseline data can be collected rapidly. But
the weakness is that the biological significance of changes in the profiles must be established
first. It is expensive and material to be analysed must be generated under highly standardised
conditions. In general, a suite of proteins of particular interest for safety and performance
could be determined and given special attention in these analyses. It could be used for routine
baseline data collection for GM-arthropods. The threat is like for many of the characteristics
of interest above: in the absence of specific knowledge of the significance of changes, they
are hard to interpret.
RNA profiling allows the rapid collection of extensive baseline data. But the biological
significance of changes in the profiles must be established. As for proteome profiling the
method is expensive and the material must be generated under highly standardised conditions.
In general, a suite of RNAs of particular interest for safety and performance could be
determined and given special attention.
12.1.12. Abiotic stress resistance
To assess abiotic stress resistance two methods are described:
−
Bioassay
−
Analysis of stress response gene profiles
Bioassay is a simple method that can be performed repeatedly to determine general vigour
and environmental response. Another strength is that its direct nature makes it much more
credible than indirect measures. Weaknesses on the other hand are that it must be performed
under highly standardised and reproducible conditions with batches of arthropods produced
under similar conditions. In general, developing a suite of standardised assays for routine use
would be valuable for comparing GM- with feral arthropods as well as being useful during
production for quality control purposes. But unless performed under realistic and ambient
conditions it is not suitable as proxy for GM-arthropod performance.
With the analysis of stress response gene profiles (Nesatyy and Suter, 2008), a standard set of
genes can be investigated but it requires expertise and special equipment. In addition the
material needs to be produced under standardised conditions. In general, rapid analysis of a
suit of such genes could provide an indicator of stresses during the production process. A
threat is that interpreting the significance of deviations from standard responses or strains
could be highly speculative.
113
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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12.1.13. Vector competence
To assess vector competence the following three methods can be envisaged:
−
Gene expression profiles
−
Pathogen infection response
−
Transmission bioassays
Gene expression profiles have the strengths that deviations in expression of immune-response
genes could be targeted for study. But changes in gene expression are not definitive indicators
of changes in vector competence. The data are expensive to collect and require highly trained
personnel and special equipment. In general, the development of standardised (micro)arrays
and methods that specifically identify levels of immune response genes would facilitate
exchange of information and relating these to infection outcomes. Threats are that the
significance of aberrant expression will depend also on the accuracy of the sexing.
Pathogen infection response does not require all components of the full transmission cycle. As
infected animals are often needed, special facilities (biosecure, LB2) and highly trained staff
is required. The range of possible diseases to be tested is difficult to determine. In general as
knowledge of specific molecular components of infection is increased, assays can become
more precise and informative. For mosquitoes, membrane feeding assays with pathogeninfected blood may serve as a good proxy for transmission potential. A threat is that the use of
animals is becoming more expensive and socially undesirable. This places pressure on an
evaluation which avoids such experiments yet maintains the credibility of the results.
Transmission bioassays are most informative, accurate and meaningful. The weaknesses are
the same as for the pathogen infection response (infected animals, special facilities, and
trained staff). In general, expanded use of model systems is likely even though they do not
provide the specificity necessary for definitive judgment. The threats are as mentioned before
related to the use of animals and the social pressure on avoiding such experiments.
12.1.14. Arthropod density
To measure arthropod density many methods are available, seven of them are discussed in
this report:
−
Breteau index
−
Pupal index
−
House index
−
Trapping of wild and released arthropods
−
Human serology
−
Screening of infested animals
−
Fruit sampling (infestation rate/fruit damage)
The Breteau index (the number of positive containers, e.g. containing Aedes aegypti larvae
per 100 premises inspected (WHO, 1997; Silver, 2008) is a direct indicator of mosquito
abundance. The weakness is that when similarly appearing species are present, laboratory
114
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determination of which species has been collected is necessary. No specific opportunities and
threats are described.
The strength of the Pupal index (number of pupae per 100 houses (Silver, 2008)) is that it is a
direct indicator of mosquito abundance and a better proxy for adult abundance than indices
based on larvae. The weakness is the same as for the Breteau index. No general opportunities
and threats are described.
The House index (percentage of houses infested with larvae and/or pupae (Silver, 2008)) is a
direct indicator of mosquito abundance and specific for anthropophilic/endophagic species.
The weaknesses are the same as for the Breteau index. In addition this method is very time
consuming and intrusive. In general, the opportunities of this method are that standard indoor
resting boxes that are attractive to numerous species could be devised. A threat of this method
is that permissions are needed for house inspection. If a high number of residents are
unwilling to allow inspection, the value of this method is much diminished.
Trapping either wild or released arthropods is a highly effective standardised method that
indicates adult abundance (Silver, 2008). For fruit flies this is a major component of all
ongoing SIT programmes, in many cases in combination with fruit sampling including
commercial as well as wild hosts. In most cases the seasonal abundance of the target species
is determined prior to the first releases to obtain an estimate for the optimal timing of the
releases and for the required release rates. During a SIT programme large numbers of traps
are used to determine the effectiveness of the releases. After eradication has been achieved,
traps are used as part of an overall quarantine procedure to detect any new infestation. But
effective traps are not available for all target arthropod species, especially at low densities.
Some need batteries and almost all require regular travel by staff for monitoring purposes.
Attractive lures for many target arthropods are not known or not optimised yet. In general,
there are some opportunities: developing new traps appears to be a worthwhile goal, but the
difficulty of doing so should not be underestimated and this is particularly true for traps that
are intended to attract multiple species. Automated data collection and transmission would be
highly desirable. A threat is that the sensitivity of traps for monitoring at very low densities
remains an issue.
Serology is a highly sensitive way to determine the absence of blood feeding insects. The use
of anti-saliva antibodies as markers for presence of target mosquito is another strength. But
specificity for target species may be compromised by the presence of other blood-feeding
insects. In general, the development of highly sensitive and species-specific serological
markers is feasible. Serology tests depend on blood samples of humans, which requires
ethical clearance and may be considered intrusive. No threats are reported.
Screening of infested animals is a direct and sensitive monitoring method for insects that
cause myiasis (Simmons, 2008). The weakness is that it requires travel to or notification of
infested animals and damage has occurred by the time the infestation it detected. In general,
one opportunity is that traps that simulate the odours of infested animals in combination with
shape and colour might provide more reliable monitoring. On the other hand, infestation may
have been caused by closely related species not targeted by the GM-approach.
Fruit sampling to assess the infection rate or direct damage is a method for detecting
problematic levels of infestation, but the damage has occurred by the time the infestation is
detected and as mentioned above, staff travel is required. In general, devices that would
115
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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automatically detect changes in volatiles of fruits and are field deployable could make
detection of infestation more rapid and consistent. Again the threat is that damage may have
been caused by closely related species, meaning that the method lacks specificity.
12.1.15. Migration behaviour
To collect data on migration/dispersal behaviour the following four methods are described:
−
Mark-release -recapture
−
Population genetic analysis
−
Rare element detection in eggs of treated adults
−
Reinfestation rate of pest-free areas
Mark-release-recapture is a reliable and biologically meaningful measure of field performance
but tedious and time consuming (Reisen et al., 1991; Hagler and Jackson, 2001). The recovery
of released arthropods is low for many species making the data highly variable. Marking
methods often cause damage to the released arthropods. The threat of this method is that a
meaningful assay requires the open release of GM-arthropods, probably initially confined in
some manner although this obviously impairs arthropod movement. Also, locating sufficiently
isolated sites at which this can be performed may be problematic.
Population genetic analysis is a sensitive measure of population similarity but requires highly
specialised staff and DNA sequence analysis. The value of the data is doubtful for population
suppression measures since it may not reflect mating compatibility. Moreover, significant
migration can occur between closely related populations that is undetectable by population
genetic analysis. In general, relating the degree of genetic divergence from the target
population that can be tolerated could provide useful indicators of incompatibility that would
interfere with programme success. Another opportunity is that collecting and analysing DNA
sequence data is becoming less expensive and rapid. No threats are described for this method.
The strength of rare element detection (e.g. rubidium) in eggs of treated adults is that it is a
sensitive marking system that causes little harm (Hagler and Jackson, 2001). It can also be
used in natural mosquito larval sites but element abundance declines making inexpensive
detection systems less useful for older insects. In general, the application of high throughput
detection systems would increase the usefulness. No threats for this method are described.
The strength of the reinfestation rate of pest-free areas is that it reflects an invasive character
that could have direct relevance to programme effectiveness, barrier establishment and
containment but so far little data exist. A significant exception is the reinfestation of an area
in Guatemala in which elimination of the mosquito Anopheles albimanus was accomplished
(Lofgren et al., 1974). In general, creating artificially small uninfected zones could provide
information about flight range and migration. For example, placing uninfested fruit trees at
various distances from established infested groves or animal traps for mosquitoes at distances
from villages would demonstrate migration related behaviour, if secondary translocations are
ruled out. The threats are that experiments would have to be conducted taking advantage of
naturally uninfested areas or areas not inhabited by humans.
116
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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12.1.16. Habitat interactions
To assess habitat interactions two methods are recommended:
−
Field observations
−
Analysis of range of habitats occupied
Field observations deliver direct and easily interpretable results but are tedious and must be
carefully conducted with predetermined quantifiable interactions identified before
observation. The subtle differences in behaviour that are relevant may not be known. In
general cataloging relevant behaviours and interactions has not been done so far for many
arthropods. It remains unknown which interactions are likely to be of concern. No threats for
this method are reported.
The strength of the analysis of range of the habitats occupied is that it utilises existing
information without the need for field studies, but specific characteristics that are relevant
with respect to the habitats that are known to be occupied may not have been identified. In
general, changes in habitat occupancy over time may provide information regarding specific
features necessary for persistence, but choice of habitat range may be chosen too narrowly,
particularly when specific biological interactions restrict the range.
12.1.17. Climate interactions
To assess climate interactions three methods can be envisaged:
−
Assessment of seasonal arthropod abundance
−
Analysis of range of habitats occupied
−
Laboratory measurements of life-table changes
Seasonal abundance provides clear relationships between abundance and specific climate
patterns, especially temperature, rainfall and humidity, but data for specific localities are not
always available. Weather data is fairly easy to collect, but arthropods are not and their
abundance is very variable. In general, changes in distribution over time and space (e.g.
altitude changes) may be related to climate change. No threats are reported for this method.
The analysis of range of habitats occupied provides data for predicting the likely distribution,
abundance and potential spread of arthropods, often using existing data. The weakness is, that
this method is model based, thus predictions are somewhat uncertain. In general, modelling
the European distribution of the most important arthropods dealt with in this report would
provide higher resolution of the potentially affected areas. A threat of this method is that
climate change will require reanalysis under various climate change scenarios that are
foreseen over the approaching decades.
Laboratory measurements of life-table changes can be easily conducted and be able to identify
at least some relevant responses (Begon et al., 1996). The weakness of this method is that it
requires the use of arthropods produced under standardised conditions and may not induce or
reflect possible physiological adaptations that increase survival in natural environments. No
general opportunities and threats for this method are reported.
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
12.1.18. Food interactions
To generate data on food interactions the following four methods are described in more detail:
−
Host and diet preference
−
Laboratory bioassays
−
Habitat occupancy
−
Gut content analysis
The strength of host and diet preference is that these constitute the closest measure of the
likelihood of harm when observed in natural settings. On the other hand, it requires field
work, biochemically analysis of specimens and direct observations. Some species are
opportunistic, so changes of hosts may be difficult to detect against a natural background. In
general, knowledge of high value host and diet will improve methods for trapping. The threat
of this method is that a shift in host and diet selection is impossible to predict prior to release.
Laboratory bioassays are easily conducted experimentally with the potential to identify at
least some preferences, but natural food choices may not be known or available for testing in
the laboratory. In general, standard panels of potentially attractive odours could be developed
for standard bioassays of attractiveness to observe and catalogue differences between and
among species. No threats have been identified for this method.
The strength of habitat occupancy is that knowledge of this trait may identify essential
components of the diet that must be present for survival. Data may be existing. The
weaknesses are the insufficient knowledge of specific components of habitats and omnivorous
feeding habits that reduce the usefulness of the information. No opportunities or threats have
been identified for this method.
Gut content analysis is a useful method for identifying dietary components of e.g. mosquito
larvae but many species are opportunistic feeders and the diversity and abundance of
materials in the gut are variable so that changes would be difficult to detect. Also for this
method no opportunities and threats are identified. For a methodological review see King et
al. (2008).
12.1.19. Altered host range
For the assessment of altered host range four methods are described:
−
Field observations
−
Vertebrate attraction assays
−
Blood meal source analysis
−
Survey of host plants
The strength of field observations is that this constitutes the most proximate and direct
measure of the likelihood of harm when observed in natural settings, but while determining
the most likely hosts is straightforward, obtaining quantitative information about changes is
very demanding. No general opportunities and threats have been identified for this method.
118
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Vertebrate attraction assays are a safe and easy to control surrogate for landing catches that
reflect biologically significant characters. The weaknesses are that humans and animals must
be available for the studies. If changes are detected, they must be considered in the light of
other factors that make interactions likely, e.g. the proximities of the vertebrate and the
arthropod. In general, artificial odour baits for blood feeding insects could provide a
standardised procedere against which changes in attraction could be detected more sensitively
and reproducibly without the use of animals. A threat is that changes may be misinterpreted as
hazards unless considerations of other factors that affect feeding are made.
Blood meal source analysis is a good method for establishing the species identity of hosts,
indicating the attractiveness of potential host species in relation to their density (King, 2008).
The weakness is that it requires biochemical assay equipment of a modest sort. Also fieldcollected arthropods are required which may be difficult to obtain. In general, old methods
lack specificity (i.e. do not go beyond groups of hosts, e.g. bovine or equine), new molecular
tools can determine the host species. No threats have been identified for this method.
Survey of host plants is a valuable direct indicator of the extent of plants likely to be affected.
This information would be useful for assessing risk to potential hosts. No opportunities could
be identified for this method, but a threat is that in spite of a negative assessment that changes
would likely be observed. It is also an obvious question that will be asked regarding the safety
of GM-arthropods and will require experimental confirmation.
12.1.20. Sensitivity to insect pathogens
The sensitivity to insect pathogens can be assessed using the following methods:
−
Bioassay
−
Analysis of immune response gene expression levels
Bioassay to assess the sensitivity to insect pathogens is easily performed and quantitative
experiments can be carried out using a few standard agents, but the array of those agents is
limited to those commercially available or easily cultivable. In general, one opportunity is that
a standard array of agents should be chosen that all GM-arthropods and feral counterparts
would be tested against. No threat for this method is identified.
The strength of the analysis of immune response gene expression levels is that deviations in
expression of immune-response genes could be targeted for study thus providing a
standardised set for various GM-products. The weaknesses of this method on the other side
are that changes in gene expression are not definitive indicators of changes in susceptibility.
Also, data are expensive to collect and require highly trained personnel and specific
equipment. In general the development of standardised arrays and methods that specifically
identify levels for immune response genes would facilitate exchanges of information
regarding pathogenicity and sensitivity. No threats are identified for this method.
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
12.1.21. Predator interactions
To assess data on predator interactions two methods are described in detail:
−
Predator gut content analysis
−
Field observations
Predator gut content analysis (King, 2008) provides a direct measure of the diet of predators
and indirectly the dietary importance of the (non) GM-arthropod to that animal but it requires
expertise and sacrificing animals, some of which may be harder to justify (vertebrates) than
others (arthropods). In general, knowledge of key species in the habitats where GMarthropods will be released would provide more robust and focussed attention on the species
that are of greatest environmental concern. A potential threat is that governments and activists
may prohibit or oppose trapping and sacrificing animals (e.g. birds, bats) for this purpose.
The strength of field observations is that it is a reliable method to determine the array of
predators that should be considered. The weakness is that field observations are difficult and
time consuming in many cases. This method may fail to detect significant interactions due to
darkness, occurrence in hidden habitats etc. In general a standard array of predators could be
selected that each GM-arthropod species and its feral counterpart should be tested with. The
threat is that impact on all possible predators cannot be studied and remains partially
unknown.
12.1.22. Insecticide resistance
To assess data on insecticide resistance the following three methods could be envisaged:
−
Allele analysis
−
Biochemical assay
−
Bioassay
Allele analysis is a direct measure of the frequency and nature of insecticide resistance alleles
but not all alleles are known and some resistance mechanisms are polymorphic or, due to
expression levels, not detectable by this method. It requires expert personnel, DNA
sequencing equipment and PCR. In general, an increased amount of genomic data and high
throughput sequencing and SNP analysis will make this method a major tool for strain
comparisons. No threats are identified for this method.
Biochemical assay is a good generic method that can detect changes in enzyme levels. The
weakness is that although methods for most insects have been developed, the relationship of
enzyme levels to resistance must be established for each species. No general opportunities and
threats have been identified.
Bioassay to assess insecticide resistance is the most direct measure of toxicity and likelihood
that a biologically meaningful change has occurred. Data can be collected on large numbers of
individuals over a short period of time. The weakness is that it requires standardised
production of material and testing methods. No general opportunities and threats have been
identified.
120
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
13.
Baseline Information
Performing an ERA requires a considerable amount of qualitative and quantitative
information on the receiving environment into which a GMO is released. Directive
2001/18/EC and its respective annexes (especially Annex IIIA for non-plants) outline which
kind of information has to be provided by applicants in the framework of the submission of a
notification for release of GMOs. This information, related to the GMO, concerns roughly
two parts: the organism and its modifications, and the receiving environment (habitat type)
with its components. In this chapter, specifically the second part with regard to GMarthropods is considered.
13.1.
Habitat type
If appropriate, the receiving habitat(s) should be named according to a habitat classification
system such as the EUNIS habitat type classification (EEA, 2010). This classification is a
pan-European system, which was developed between 1996 and 2001 by the European
Environment Agency (EEA) in collaboration with experts from throughout the EU. It is a
hierarchical system, covering all types of natural and artificial habitats, both aquatic and
terrestrial. Although some habitats are well-defined (e.g. J2.43: Greenhouses), most habitat
types of the species considered in this report can only be defined on habitat level 1, i.e. these
categories are so broad that this classification becomes useless. Mosquitoes, for instance, may
colonise every possible small body of water, irrespective of the habitat type around, thus, their
habitat best can only be described as C (inland surface waters), I (regularly or recently
cultivated agricultural, horticultural and domestic habitats) and J (constructed, industrial and
other artificial habitats). The EUNIS system, however, basically refers to phytosociological
vegetation units and these are not always appropriate in the cases of the discussed GMarthropod species. Also fruit flies such as the Mediterranean fruit fly can occur in a variety of
cultures within the agricultural landscape or in urban areas. In such cases, no specific single
habitat according to a classification can be referred to, but the description of potential habitats
needs to be very general and based on the crucial requirements for occurrence of the
respective species.
A given habitat description further includes its geographic distribution as well as geological
and pedological characteristics in the surrounding area of the intended release if appropriate.
The size of the area for which the ERA needs to be valid depends on the GMO and its
modification, but should be adequate according to the dispersal capacity of the species.
Climatic characteristics including climatic extremes (e.g. temperature, humidity, wind) and
eventually scenarios of climate change are important to understand the possibility of survival,
population establishment, spread and future developments of the GM-arthropod. Climate also
drives the seasonality of the habitat and thus influences the phenology of the GM-arthropod
(including generation time and annual number of generations) and its hibernation, diapause,
or related survival strategies.
Since habitat quality determines population growth of the GM-arthropod, thus dispersal and
the establishment in further areas (by migration and colonization), the habitat-specific
population pressure should be assessed. The ERA should consider the known or supposed
routes of dispersal (such as e.g. water flows, prevailing wind directions or transportation
routes including transport by human activities as trade, etc.), distance to suitable habitats in
the surroundings of the intended release and the maximal distribution range of the considered
121
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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GM-arthropod in Europe. If the target non-GM-pest species is an alien species, a description
of the invasion history and related characteristics should be available.
For agricultural pests, the prevailing management practices in the surroundings of the
intended release (conventional, IP or organic management of crops) should be known as these
have an impact on the quality of the habitat. The impact of agricultural pests may be higher in
conventional management systems, compared to organic farming, which provides a higher
diversity of antagonists and reduces impacts of pest species.
Future changes of the habitat structure due to land-use or climate change have to be discussed
and included into the ERA (e.g. using modelling techniques).
13.2.
Ecology of GM-arthropods within their habitat
To analyse the potential abiotic and biotic interactions of the GM-arthropod in a specific
habitat, a detailed description of these interactions is necessary. Such a description, among
others, will list the most abundant species of flora and fauna, including crops, livestock and
humans. A detailed description of the GM-arthropod ecology with respect to its involvement
in environmental and biogeochemical processes (primary production, nutrient turnover,
decomposition of organic matter, etc.) will allow identifying those species with which
interactions are most likely.
GM-arthropods likely interact with antagonists such as predators, parasitoids, and pathogens
occurring in respective habitats, but also with competitors and symbionts. Adult mosquitoes
additionally interact with their hosts through feeding on their blood. Fruit flies interact with
their host plants by laying eggs into their fruits. Hatched larvae of these flies then develop in
the fruits. The host range may be narrow (Aedes aegypti prefers to take its blood from humans
and the Bactrocera oleae only attacks olive trees) or very broad (Ceratitis capitata is a
generalist feeder and can affect more than 260 plant species (Liquido et al., 1991)). Although
the likelihood of switching to new hosts and their availability in a given territory is difficult to
assess, the ERA should consider the probability of such events (e.g. by demonstrating that this
phenomenon is frequent or rare in the particular arthropod species and its relatives). A
thorough analysis of potentially suitable further host species is considered highly relevant
(e.g. concerning host range switching of mosquitoes).
It is also important to identify at the habitat level species which may influence the survival,
reproduction, vector competence, pathogenicity, toxigenicity or virulence of the considered
GM-arthropod, e.g. by feeding or parasitising them. Further, the identification of non-target
organisms, which may be adversely affected by the release of the GM-arthropod, is
recommended. A thorough analysis of the interactions between the GM-arthropod and other
species in the considered habitat should also detect and identify species with which transfer of
genetic material is possible (e.g. hybridization with related species). Knowledge on the
involvement of the GM-arthropod in environmental and biogeochemical processes is helpful
for estimating which GM-species products could accumulate in the environment and to
determine potential cumulative long-term effects.
122
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
13.3.
Baseline data
Similar to RA on GM-plants, a major obstacle for the conduct of an ERA on GM-arthropods
is the lack of environmental baseline data. This may concern, for example, the number of
predators in a given type of aquatic habitat, the density of an arthropod, or the number of
species preying on a given agricultural pest.
Within an ERA, respective data will probably be obtained during a limited amount of time
only, usually a few weeks up to one year. In addition, field data from different locations (all
relevant geographical areas) will be collected. Such data usually show large variation and it
may be difficult to interpret these data and draw sound conclusions and recommendations.
Data obtained during a second census will certainly be different, which further complicates
interpretations. To detect small differences on a sound statistical basis, large sample sizes are
necessary. The best solution, given such a dilemma, might be the set-up of a targeted
collection of data relevant for an ERA several years before the intended release of a GMarthropod species. There is no consensus on the minimal number of years, but several years
should be considered for such a baseline assessment due to e.g. variability in arthropod
abundance. Ideally, after the GM-species release, this data collection should be continued or
(in case of release activities over several years) should parallel the release activities in areas
with and without GM-arthropods.
These baseline data are difficult to assess, have high variability (e.g. population density),
depend on anthropogenic factors (e.g. coffee prize influences Mediterranen fruit fly densities:
when the market price is low, less beans are harvested, which increases the infection rate by
the pest), and some may even be impossible to obtain in due time (e.g. maximum area of
spread). Therefore, much of this information on receiving environments may also be
considered as a knowledge gap.
123
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
14.
Surrogate Approach
In case the GM-arthropod or the parental species cannot be used in the assessment of a certain
parameter, the assessment of surrogate species was suggested to establish data also relevant
for the original species.
Reasons for proposing a surrogate approach are manifold, e.g. the species under investigation
could be too dangerous because the release may be associated with unwanted adverse effects
like it would be the case for pest species, or the legal framework does not allow such field
experiments like it is the case for many European countries with respect to the release of
biocontrol agents.
A surrogate species needs to be as similar as possible to the GM-arthropod under
investigation. This does not only concern the biological properties of the species itself, but
also the GM-trait. The approach outlined for the RA of GM-fish modified with growth
hormone genes may illustrate a suitable application of the surrogate concept: fish from the
same species treated with injections of growth hormones could possibly be used as a surrogate
for the growth modified GM-fish for the assessment of certain characteristics, taking into
consideration the limitations of such approaches (e.g. the transient effects of such treatments).
The requirements for selecting suitable surrogate species are high: the chosen species has to
be as similar as possible to the GM-arthropod species of concern because data established for
the surrogate species need to be transferable to the original species. Important key
characteristics in this respect include demographic parameters, such as survival and
reproduction, which define population dynamics such as spread. A surplus reproduction is a
prerequisite for unaided natural spread into previously uninhabited areas. However, some
species like mosquitoes may also be translocated unintentionally by human activities (e.g.
translocation of mosquito larvae via shipments of used tyres). Thus, candidate surrogate
species may also be invasive. Also basic ecological interactions with other organisms
(microorganisms, plants, animals, humans) will have to be analysed in the selection process.
Usually the criterion of high similarity is only fulfilled by the closest relatives of a species.
However, even such related species, e.g. in the case of mosquitoes, may differ with respect to
development parameters, habitats, feeding niches (hosts), phenology, biogeography, and
vector competence. Even if a particular trait of a surrogate species is comparable to a GMspecies, and this is tested comprehensively in the laboratory, post-release or post-invasion
changes due to natural selection or other environmental factors cannot be assessed.
Additionally, climate change may change phenology of insect species, e.g. increased number
of generations, or the spread of invasive alien species which often is associated with a change
of their occurrence in certain habitats, usually shifting from urban or artificial habitats to
natural or near-natural habitats. Therefore, the use of closely related species as surrogates
needs to be evaluated very carefully in order to minimise the uncertainties associated with the
assessment.
This difficulty resembles the situation encountered with the RA of introduction of alien
species. The question whether an alien species newly introduced into Europe will become
invasive can also not be answered with certainty by analysing closely related species. The
“invasive elsewhere” criterion, where the impact of an alien species in a comparable region is
assigned to the region under consideration, is a useful proxy for possible effects, but due to
124
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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unexpected changes in the behaviour, morphology or genetic changes of the species in the
considered area, predictions are inherently uncertain.
The use of other strains of the same species as a surrogate may be more promising, but needs
to be assessed on a case-by-case basis as well and may be feasible only for few applications
of GM-arthropods. Suitable surrogates of a given GM-arthropod species could be an
authorised GM-strain of the same species, which is currently not available in the EU, or a
non-GM-strain of the same species which is used for a comparable application with respect to
the modified trait and/or produced for the same purpose.
The latter type of surrogate approach could e.g. be implemented for the assessment of GMsexing strains for SIT-applications. Non-GM-sexing strains of the same species developed for
conventional SIT applications, which are available and implemented for certain species, like
several fruit fly and mosquito species can serve as a surrogate for a SIT application using a
GM-sexing strain. Another example for the use of surrogate applications for RA of GMarthropods could be the comparison of GM-arthropods modified with a fluorescent marker
and a non-GM-arthropod of the same species treated with fluorescent dye.
Both approaches are limited, however, and imply that the genetic modification introduced to
obtain a sexing strain or a fluorescent marker has no other influence on characteristics of the
resulting GMO (like the lethality introduced by RIDL). On the other hand, it assumes that
conventional SIT or the fluorescent dye also have no affect on the set of key characteristics
mentioned above.
A final possibility is to use unmodified non-GM-conspecifics as surrogate species of GMarthropods. Thus, an unmodified non-GM-arthropod could be released as surrogate in
moderate to large numbers to investigate the resulting environmental effects. The data
obtained from a subsequent monitoring programme should be attributable to the conspecific
GM-arthropod, if the genetic modification does not influence the assessed key characteristics
of the GM-species. However, it has to be considered whether it is acceptable to release large
numbers of fertile individuals of the respective species in question. With regard to the GMarthropod species considered in this report (table 5) it is obvious that such a release would not
be feasible, because all of them are pests with respect to human health or agricultural
production or alien (non-indigenous) to Europe.
In conclusion, the surrogate species concept, using either closely related species, varieties
within the same species or non-GM-populations of the same species is tempting to obtain
empirical information on the environmental impact of the GM-arthropod. However, the
central assumption that GM-arthropod and non-GM-surrogate are identical or at least
comparable is questionable. Therefore, and because of the associated environmental risks the
option of releasing modified or alien species for testing needs to be carefully assessed.
Analyses of selected traits of non-GM free-living populations of the same species under
consideration may reveal some insights, but the limitations of such data should be clear and
their interpretation needs to be carried out very carefully.
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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15.
Modelling Approach
Models are commonly used in natural sciences to describe and understand natural systems
and processes or to make predictions on future developments (e.g. habitats of a species under
changed climatic conditions, potential habitats of a GM-species where it could immigrate
after release). Various types of models can be distinguished depending on the amount of data
available or the kind of data used. The modelling approach is also accommodated by EFSA
(2010b) e.g. stating that the use of scientifically sound modelling approaches could provide
further information useful for ERA. For long-term effects it is suggested that models could be
used to explore or test alternative scenarios. EFSA also mentions population dynamic models,
e.g. to test the influence of certain factors on population levels (for GM-arthropods this could
be models where predicted changes of specific biotic or abiotic factors are tested to see, if
they influence the GM-arthropods, e.g. resulting in less abundance or higher fitness).
The quality of a model depends on the use of the correct parameters, the accuracy of the data
used and the significance of the result. The more a priori information is available the more
accurate the model predictions will be. A crucial part of the modelling process is the
evaluation of whether or not a given model describes a system accurately. Another important
issue to consider is the scale and resolution of the data and how these are translated into
recommendations. Downscaling of low resolution environmental input data to a finer
resolution increases variance and thus reduces significance of the model. Any model,
however, must be carefully interpreted and the underlying biological concepts and processes
need to be taken into account. Data and information obtained must be validated and assessed
against meaningful, comparative data.
A tool to assess, map and predict suitable habitats or ecological niches is using statistical
habitat models (HSMs – habitat suitability models) or bioclimatic models. Several statistical
techniques are available for these models (e.g. GLMs – general linear models, GAMs –
general additive models, climate envelope models, classification and regression trees, neural
networks (Watts and Worner, 2008)). The basic idea of most HSMs is to predict the
probability of occupancy in a new range from a set of explanatory variables (e.g. geology,
altitude) and known occurrences or presence/absence data in a native range. With increasing
computer power more realistic variables are included, such as spatially-explicit dispersal to
assess connectivity of habitat patches.
There are several other modelling techniques available (e.g. metapopulation models, cellular
automatons, species distribution models), which need to be thoroughly investigated for their
practicability for an ERA on GM-arthropods. These methods are increasingly used (and
further developed), particularly for predictions of range changes due to climate change or
biological invasions e.g. Guisan and Thuiller (2005), Jeschke and Strayer (2008) and Gallien
et al. (2010). Modeling approaches for non GM-arthropods from species which are of possible
relevance for GMO applications in the EU in the next 10 years are reported in the scientific
literature, especially for mosquitoes (Maguire et al., 1999; Ayala et al., 2009; ECDC, 2009;
Kulkarni et al., 2010; Medley, 2010). Predictions for the future expansion and establishment
of e.g. Aedes albopictus have been made using four different approaches (Genetic Algorithm
for Rule Set Production; GARP model, Random Forest Technique, Multi Criteria Decision
Analysis (MCDA), and impact modelling using IPCC maximum and minimal climate change
scenarios). The GARP model was designed to predict and model the ecological niches of
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
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species (Stockwell and Peters, 1999) and is a simulation of genetic processes of mutation,
recombination, and selection operating as a nonrandom search for a solution consisting of an
optimised rule set describing the niche. This analysis also factored in the risk of tyre imports
from infested countries and the proximity to countries that have already been invaded in order
to develop a list of countries most at risk for future introductions and establishments. The
Random Forest model uses existing presence/absence data of species occurrence and links
these to a variety of climatological parameters. The third method (MCDA) standardises
rainfall and temperature data into specific intervals prior to modelling.
If survival and reproductive fitness, spread, migration, persistence and invasiveness of a GMarthropod and its interactions with other organisms will be assessed using for instance a
population dynamic or habitat modelling approach many prerequisites must be met.
A GM-arthropod differs to a certain degree from the non GM-species most obviously by the
GM-trait; however there could also be some unintended (and unexpected) differences due to
the modification that need to be considered in the model. The quality of a model, however,
must reflect those specific characteristics of the GM-arthropod (resulting from the differences
assessed) and is dependent on the quality of the input data. Therefore accurate data on the
GM-arthropod application in question must be available, especially when used in the context
of an ERA. To run e.g. a population dynamic or habitat model of a GM-arthropod species, the
following questions must be answered:
−
What are the differences between the GM-arthropod and the non-GM-species?
−
Are data available on the GM-arthropod (laboratory or field data)?
−
If not, are data available on the non-GM-species?
−
Is it possible to use data on the non-GM-species for specific parameters in the model?
Many parameters will most likely be assessed in the development process of a GM-arthropod
e.g. for strain evaluation, as it is done currently for SIT species (FAO/IAEA/USDA, 2003).
But there may be a difference if the purpose is the evaluation with respect to an ERA (e.g. the
focus in development process is the quality and effectiveness of the strain, the focus of the
ERA are possible risks) and therefore other parameters have to be assessed additionally and in
some cases other methods may be used. As mentioned in chapter 12 laboratory generated data
are not always representative for field data and the generation of those may be difficult. Data
on the wild relative may also be limited and it is questionable if they can be used as
alternative data for the models (see also chapter 14). The discussion on the RA of GMarthropods revealed that more aspects are unknown than predictable and that there are
scientific gaps in many fields.
In addition relevant anthropogenic factors must be reflected in the model, since the species for
which GM-traits are being developed are subject to human influence and in turn may be
influenced by human behaviour. For example, fruit flies are often introduced along with
international trade routes. There are also some parameters that are very difficult to predict,
e.g. the influence of wind on distribution of the GM-species or the abundance of fruit flies in
relation to the world market price for coffee: if the price is too low, coffee fruits are not
harvested, thus leading to increased species abundance.
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Extrapolating risk estimates for phenotypes tested in laboratory and/or semi-natural
environments to other phenotypes and more complex and multiple environments with the
modelling approach is very difficult. Firstly, as stated above, many data generated in the
laboratory do not represent field settings accurately. Secondly, each phenotype must be
assessed case-by-case, thus assessing differences between the GM-arthropod and other traits
or the non-GM-species may require specific data collection. Thirdly, the GM-arthropod needs
to be assessed in connection to the receiving environment (e.g. a potential spread will be more
rapid in a climatically optimal environment). Since behaviour and other species traits
potentially vary with climate conditions, input data needs to be generated in representative
European habitats, but parameters can be assessed for their significance (e.g. for changing
climatic conditions). Therefore multiple European environments must be taken into account
when modelling possible effects, e.g. by using a biogeographic approach in the ERA.
Useful information for informing models and the ERA may be obtained by some
understanding of natural variation that exists in traits of potential concern. Model predictions
should be consistent with the natural prevalence of e.g. variations in host preference, variation
in size and fecundity. Traits of concern in the GM-arthropod should be compared not only
with the average values but also the range of variation that exists naturally. This will indicate
the likelihood of a GM-trait that differs from the mean to become widespread. Moreover, such
an analysis will determine whether a deviant trait of the GM could have already been subject
to selection and whether it is in fact novel.
Additional insight for the validity of the model can also be obtained by assessing whether it
accurately predicts known species invasions and spread of novel phenotypes. As an example,
the historical spread of Aedes albopictus in Europe should be accurately reflected by any
model that will be used to predict its future behaviour. Data available on insects that carry
insecticide resistance alleles (Du et al., 2005) and their historical spread may help inform
predictions of the potential spread of phenotypes with similar fitness advantages.
In conclusion, when applying a modelling approach as parts of an ERA of GM-arthropods,
high quality input data are needed of the strain and the non-GM-species. Existing differences
and their consequences as well as those of possible differences need to be known and
reflected in the models. Then, population dynamic or habitat models can be useful, but care
has to be taken in the interpretation of the results.
Since a model is a reductionist attempt to interpret complex causal relationships, it does not
comprise all attributes but only those that seem the most relevant for the modeller. The
outcome of modelling has been shown to differ, depending on the input variables, the
characteristics of the model’s domain and the questions asked. Therefore it is crucial to be
very cautious when model based data are used and to be aware which purpose they serve. A
model cannot predict the future and should not replace a thorough evaluation but can be used
as a supportive tool.
128
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16.
Implications for the implementation of an ERA of GM-arthropods
In the following the results obtained in this report and presented in chapters 9 till 15 are
summarised. Important aspects depend among others on the species modified, the method
used for modification and the sort of problem the species pose (agricultural pest or disease
vector). Therefore case study species, comprising these areas were selected (see chapter 2)
taking into account the status of development and therefore the likelihood of an application
for notification in the next 10 years. Table 9 shows the result of this selection process.
Table 9.
Selection of Case study species
GM-arthropods of possible relevance for the EU
during the next 10 years
Case study species
high quality data available
Species only relevant for
overseas territories
Public health application
agricultural application
Common name
high likelihood of notification
for application
Species name
Conclusion
advanced status of
development (GM)
Selection criteria
Y
Y
Y
Y
Y
Y
N
N
Y
Y
N
N
Y
Y
Y
Y
N
N
Y
Y
Y
Y
N
N
Culicidae, Diptera (mosquitoes)
Aedes aegypti
Aedes albopictus
Yellow fever mosquito
Asian tiger mosquito
Anopheles arabiensis
Anopheles gambiae s.s.
African malaria mosquito
Tephritidae, Diptera (true flies)
Bactrocera oleae
Olive fruit fly
Y
Y
Y
N
N
Y
Ceratitis capitata
Mediterranean fruit fly
Y
Y
Y
N
N
Y
Green bottle fly
Stable fly
Y
N
N
N
Y
N
Y
N
N
N
N
N
Other Diptera (flies)
Lucilia cuprina
Stomoxys calcitrans
Lepidoptera (moths and butterflies)
Cydia pomonella
Codling moth
N
N
N
N
N
N
Pectinophora gossypiella
Cotton pink bollworm
Y
Y
Y
Y
N
N
Three species, the Stable fly (Stomoxys calcitrans), the Turnip sawfly (Athalia rosae) and the
Codling moth (Cydia pomonella) were not selected as case study examples. An attempt to
develop classical SIT for Stomoxys calcitrans was undertaken in the 1970s (Patterson et al.,
1980) but was not further developed thereafter. Considering its pest status at present, it is
considered of marginal veterinary importance within the EU, and although an SIT programme
could be re-developed, this is not likely to receive priority within the EU over the next
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decade. For Atalia rosae similar arguments apply, with an additional note that this species has
never been mass-reared and/or released on any significant scale.
16.1.
Summarised analysis of ERA aspects
Many issues to be considered in an ERA of GM-arthropods were presented in chapter 9.
Some of these issues are relevant for all applications but for some there is a difference
according to the target species, i.e. if the species is an agricultural pest or a disease vector
depending also on the intended use (e.g. population suppression or eradication). It is also
important to differentiate between the purpose of the applications, e.g. SIT or RIDL
applications.
In that respect table 10 shows the most important aspects for consideration in an ERA
concerning the four case study species Aedes aegypti, Aedes albopictus, Ceratitis capitata and
Bactrocera oleae. For the analysis it is assumed that neither 100 % sterility (SIT) is
accomplished, nor only males are released (RIDL).
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Table 10: ERA aspects relevant for Aedes aegypti, Aedes albopictus, Ceratitis capitata and Bactrocera oleae
Adverse effect
• Persistence of released
arthropods (due to failure of
SIT or RIDL system)
Consequences
Exposure
mitigation
• Harm to agronomic cultures • Depending on the number • Control measures to
(olive in case of Bactrocera of viable and reproducing
reduce risk of failure of
oleae, various fruits in case
individuals in the
sexing and sterilisation
of Ceratits capitata)
receiving environment, on system (SIT) and to
the density of host
reduce risk of failure of
• Spread into new areas
plants/fruits and on the
the transgenic function
(either by persistence or
duration of the
responsible for exerting
directly by accidental
programme
lethality (RIDL)
release)
• Standard quality control
has to be implemented
• Removal of females by
physical methods size
differences (mosquitoes),
pupa colour using
automated sorting
(Ceratitis capitata)
• Post release monitoring
• Persistence of released
• Harm to human health
• Depending on the number • Control measures to
arthropod with similar pest
reduce risk of failure of
• Spread into new areas either of viable and reproducing
characteristic (due to failure
sexing and sterilisation
by persistence or directly by individuals in the
of SIT or RIDL system)
receiving environment,
system (SIT) and to
accidental release)
habitat conditions and
reduce risk of failure of
density of parasites as
the transgenic function
well as number of released responsible for exerting
arthropods and duration of lethality (RIDL)
the programme
• Post release monitoring
• Adverse effects resulting from • Increased fitness favouring • Number of released flies, • Assessment of species,
vertical gene flow to crosspersistence and spread
number and density of
where cross-mating is
compatible wild relatives
other related species
possible
• In case of the presence of
potential cross-mating
species, appropriate
timing of the release (e.g.
release the GM-arthropods
131
Concerned species
• Bactrocera oleae
• Ceratitis capitata
• Aedes aegypti
• Aedes albopictus
• Bactrocera oleae
• Ceratitis capitata
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Adverse effect
Consequences
Exposure
mitigation
Concerned species
in a period, where the
compatible wild species is
not present, if feasible
• Post release monitoring
• Adverse effects resulting from • Harm to human health if
• Number of released
• Assessment of species,
• Aedes aegypti
vertical gene flow to crossGM-arthropod exerts
mosquitoes and density of where cross-mating is
• Aedes albopictus
compatible wild relatives
similar pest characteristics
the compatible wild
possible
as the application is
relative
• In case of the presence of
increasing the number of
potential cross-mating
viable and reproducing
species, appropriate
timing of the release (e.g.
release the GM-arthropods
in a period, where the
compatible wild species is
not present, if feasible
• Post release monitoring
• Adverse effects resulting from • Persistence and invasiveness • Amount of cell free DNA • Assessment of pathogens • all
horizontal gene transfer to or
of the receiving organism
in the environment (soil
present in the receiving
via microorganisms
and water harbouring
environment prior to
decaying GM-arthropods) release
• Stability of the transgene • Assessment of transgene
in the respective
stability under the
environment
conditions of the receiving
environment
• Presence of pathogens of
the respective arthropod
• Post release monitoring
species
• Resistance of target organism • Persistence and spread
• Depending on the number • Post release monitoring
• Ceratitis capitata
to RIDL strain
• Harm to agronomic cultures of viable and reproducing • Development of suitable • Bactrocera oleae
individuals in the
backup-measures (e.g.
receiving environment and effective insecticides)
habitat conditions
• Resistance of target organism • Persistence and spread
• Depending on the number • Post release monitoring
• Aedes aegypti
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Adverse effect
to RIDL strain
Consequences
• Harm to human health
• Broadened host range due to
genetic modification
• Blood sucking from other
animals, include livestock,
thus causing higher
economic damage
• Broadend host range due to
genetic modification
• Fruit flies affecting other
crops would also cause
increased economic damage
• Broadened host range of non- • Introduction of new and/or
target species (occupying
more serious pest leading to
empty niche after success of
increased economic harm in
GM programme)
agriculture and new diseases
in human health.
• Loss of prey for
predators/parasitoids
• Decrease in predator
abundance
• e.g. parasitic wasps from the
Braconidae, Eulophidae and
Eupelmidae families for
Bactrocera oleae
• Hymenopterans from the
braconid family for the
Exposure
of viable and reproducing
individuals in the
receiving environment,
habitat conditions and
density of parasites
• Depending on the
potential new hosts in the
receiving environment and
the number of released
GM-arthropods
• Depending on the
potential new hosts in the
receiving environment and
the number of released
GM-arthropods
• Availability of species
with similar functional
niches at all life stages.
E.g. mosquitoes are able
of replacing one another,
Ceratitis has a broad host
range that could be
covered by more
specialised species or
other generalistic feeders.
• Depending on predators
species, I.e. if it’s a
specialised or generalistic
feeder
• Abundance of the predator
before the release time
• Time the species is
present for the predator
mitigation
• Development of suitable
backup-measures (e.g.
effective insecticides)
Concerned species
• Post release monitoring
• all
• Post release monitoring
• all
• Post release monitoring
• all
Post release monitoring
• all
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Adverse effect
• Unintended expression of
harmful products in the GMspecies
Consequences
Mediterranean fruit fly
• Decrease in
predator/parasitoid
abundance
Exposure
• Presence of harmful/toxic
products, portion of GMspecies compared to wild
non GM-species
• Changes in
• Decrease of the target pest • Dependent on the
biodiversity/species
and other species linked via receiving environment and
abundance by changes in food the food web (predators,
numbers of individuals
web resulting from adverse
parasitoids). Increase of
released as well as
effects on
species occupying the
duration of the
predators/parasitoids or from
empty/reduced niche.
programme
the disappearance/reduction of • Especially on island or
the target species
isolated areas.
• For Ceratitis and
Bactrocera also depending
on the cultivation system
(conventional, integrated,
organic)
• Adverse effect on soil
• Changes in soil
• Number and density of
(micro)organisms
decomposition, soil food
released GM-species, rate
web structure, diversity of
of accumulation in soil,
soil and water ecosystems
water or respective
organisms, stability of the
transgene in soil or water
mitigation
Concerned species
• Feeding experiments with • all
key predators of the
receiving environments
• Post release monitoring
• Baseline assessment of
• all
key antagonists and their
abundance and monitoring
after release
• Post release monitoring
• Assessment of baseline
data prior to release and
monitoring after release
• Tests by use of
microcosm-systems with
artificial soil
compartments
• Post release monitoring
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Adverse effect
Consequences
• Changes in vector competence • Released strain may
transmit diseases more
efficiently or new diseases
• Allergenic or toxic effects due • Allergenic reaction
to accidental uptake of GMspecies (swallowing of
mosquitoes, uptake of fruits or
olives infested with GMlarvae. The transgenic
components could be present
in mosquito saliva or changed
composition as unintended
effect of modification
• Adverse effects to sensitve
• Allergenic effects or
human epithelia
physiological response to
mucous membranes
Exposure
mitigation
Concerned species
• Depending on the number • Development of suitable • Aedes aegypti
of viable and reproducing
backup-measures (e.g.
• Aedes albopictus
individuals in the
effective insecticides)
receiving environment,
• Post release monitoring
habitat conditions and
density of parasites as
well as number of released
arthropods and duration of
the programme
• Depending on the GM• Tests for allergenic
• all
trait, the number of
potential of transgenic
released individuals, the
components before release
density of humans and the
frequency humans get in
contact with the GMspecies
• Depending on the GM• Tests for allergenic
• all
trait, the number of
potential of transgenic
released individuals, the
components before release
density of humans and the
frequency humans get in
contact with the GMspecies
135
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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Possible adverse effects presented could be the result of the application itself or from failures
of the application, e.g. if not 100 % of the released individuals are sterile (in case of SIT) or
not 100 % of the released individuals are males (RIDL). There is a possibility that this could
happen since e.g. numerous effects are known that abolish the lethal effect and currend GSS
are only 30-50 % sterile. Those failures could lead amongst others to increased persistence
and invasiveness of the target species, due to the increased population. The increased
population of the target species could also increase the likelihood of other adverse effects
discussed. Possible adverse effects as a result of SIT or RIDL failure need to be addressed for
all GM-arthropods developed with this technique and therefore a set of respective basic
information could be demanded from the notifier as well as information on the quality
standards developed. In addition backup-measures should be available that could be applied
in case of failures (e.g. insecticide use, trapping methods).
The species modified can influence the likelihood and effect of a number of possible risks
resulting from its unique biology, the niche it occupies and the role it plays in the
environment. Therefore issues for the respective species need to be addressed not only on a
case-by-case basis but also relating to the specific receiving environment. Therefore an ERA
needs detailed information e.g. on the respective species, the receiving environment
(including a description of predators and parasitoids present as well as species where crossmating could occur).
In addition the genetic modification used can lead to specific risks and adverse effects. The
use of HEGs is e.g. more problematic than other techniques described in this report. In that
respect detailed information should be demanded and respective backup-measures need to be
in place.
Depending on the problem a target species pose consequences could be quite different.
Persistence and invasiveness of agricultural pests (Bactrocera oleae, Ceratitis capitata) would
have e.g. mostly economic consequences whereas the spread of species serving as vectors for
human diseases (Aedes aegypti, Aedes albopictus) would also have health consequences.
16.2.
Issues to be considered in an ERA of GM-arthropods
Based on the outcome of this study the following issues are proposed to be considered in an
ERA of GM-arthropods are summarised which are derived from the considerations on ERA
aspects presented in chapter 9. In addition issues referred to in Hoy (2006b), IAEA (2006),
Benedict et al. (2008), CBD (2010a) and Andow (2010) are included. These proposals do not
pre-empt any further discussion by the EFSA GMO-Panel or EFSA working groups dealing
with this subject.
The issues proposed relate to:
−
The parental species which is modified
−
The nature of the genetic modification present in the GM-arthropod
−
The characteristics of the GM-arthropod
−
Specifics of the receiving environment and the conditions of the environmental release
Issues with regard to the parental organism are suggested to encompass:
1.
Is the parental arthropod species an indigenous or alien species?
2.
What is the habitat range of the parental organism?
136
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3.
What is the host range with known pest species or the prey range with species for
biocontrol purposes?
4.
Is the parental arthropod species a species of conservation concern or interacting in a
significant way with species of conservation concern?
5.
How mobile is the parental species?
6.
Are applicable control and monitoring measures available for the parental species?
The following issues with regard to the genetic modification are suggested:
1.
What is the nature and function of the inserted transgenes?
2.
Is the genetic modification stably inherited in the GM-arthropod?
3.
Which transformation system was used?
Regarding the GM-arthropod following issues are suggested:
1.
Is there any change in reproductive success compared to the parental species?
2.
Is there any change in survival and fitness compared to the parental species?
3.
Is there any change in mobility compared to the parental species?
4.
Is there any significant change of composition and of phenotypic characteristics?
5.
Is there a change in behaviour or with regard to ecological services provided by the
GM-arthropod in relation to the parental species?
6.
With regard to disease transmitting arthropods: Is there a change in vector
competence?
7.
Is there any change in reaction to abiotic or biotic stresses?
8.
How effectively is the modification functioning in the environment?
9.
Is there any change regarding the possibility to control the GM-arthropod compared to
the parental species?
10. In which organs are the transgenes expressed and at what level are they expressed?
Issues with regard to the environment and release characteristics are suggested to encompass:
1.
Does the receiving environment contain cross-compatible wild relatives of the parental
species?
2.
Is the receiving environment isolated from other ecosystems?
3.
Are there environmental factors which can influence the dispersal of the GMarthropod?
4.
Is the release conducted in a single introduction or is there a continuous release of
GM-arthropods?
5.
Is the release conducted at a single site or at different locations?
6.
Which number of GM-arthropods is released in comparison to the wild living
population of the parental species?
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Expertise for ERA of GM-arthropods
The following chapters provide information on scientific disciplines and fields of expertise
that might feed an ERA of GM-arthropods as well as on the two databases enclosed to this
report. The databases contain relevant literature and information on research institutes and
academics for an ERA of GM-arthropods respectively.
17.
Scientific Disciplines and fields of expertise
Scientific disciplines and fields of expertise that might feed an ERA of GM-arthropods were
collected by the project team (see table 11). Fields of expertise listed are the result of the
expert interviews as described in detail in chapter 1. The recommended fields where cleared
of synonyms (e.g. population biology and population ecology where proposed, but since those
are more or less the same, only population ecology was included), but it is noted that the term
for one discipline is not necessarily the same everywhere. Besides that those recommended
fields of expertise that are not within the scope of this project (e.g. economics, ethics) were
excluded.
In the future the identification of possible hazards for a specific GM-arthropod application
may lead to the need of specific expertise not covered in this chapter.
Table 11. Scientific disciplines and fields of expertise
Scientific discipline
Field of expertise
Biology
Developmental biology
Microbiology
Physiology
Parasitology
Taxonomy
Invasion biology
Quarantine biology
Applied biology (incl. applied ecology and entomology)
Molecular biology
Proteomics/ transcriptomics/ genomics
Insect molecular biology
Biotechnology/transgenics
Genetics
Evolutionary genetics
Molecular genetics
Population genetics
Entomology
Insect immunity
Medical entomology/vector control
Regulatory entomology
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Scientific discipline
Ecology
Field of expertise
Evolutionary ecology
Molecular ecology
Microbial ecology
Insect ecology
Community ecology (incl. symbiosis, insect-pathogen
interactions,
Population ecology
Behavioural ecology
Landscape ecology
Disease ecology
Chemistry
Biochemistry
Medicine
Evolutionary medicine
Human medicine
Veterinarian medicine
Immunology
Epidemiology
Agricultural Science
Phytopathology
Agricultural pest control/biological control
Toxicology
Ecotoxicology
Statistics/Informatics
Mathematical modelling
Bioinformatics
Geographic information science
Biosafety
(Environmental) risk assessment
Other
Sterile insect technique
Monitoring
Mass rearing
All scientific disciplines and fields of expertise as listed in table 11 could contribute to an
ERA of GM-arthropods. They might feed an ERA for various reasons e.g. to understand basic
issues of the respective modification and the respective species biology, possible
consequences in relation to the modification and/or the respective species as well as on the
ecological interactions and behaviour of the GM-arthropod with its environment. Also fields
of expertise that provide important methods (e.g. informatics, statistics) need to be
considered. Issues of concern are e.g. insect transformation and transgenesis, gene drive
systems, stable and site-specific genomic introgression of transgenes, (non-) autonomous
139
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
transposable elements and medea, HEG, RIDL, horizontal gene transfer, gene drive systems
for endosymbionts as well as trait stability and gene mobility assessments, mutagenesis
quantitative genetics, transposon/virus behaviour, assesement of the vector stability (remobilisation potential) and transgene integrity (mutation, reversion).
Other important aspects are the genetic structure of the respective populations (insect
populations are genetically very divers and can rapidly respond to selection), compatibility
between released and endemic population, fitness factors/costs associated with GMarthropods, mating competitiveness and reproductive behaviour (at the individual as well as
on the population level), life-table studies and dispersal studies, host-parasitoid or predatorprey interactions, size and structure of the target population, adaption and spread of GMpopulations in the wild, host/species relationships and host range, gene flow and its
consequences, the persistence of the transgene in the environment and the effect on target and
non-target organisms, resistance of the GM-trait, effects on larger spatial scales in food webs
and (improved) modelling approaches (populations, genetic or age structure of populations).
Important is also the knowledge of the development of contained semi-field sytems for the
study of GM-vector ecology as well as on studies of the fitness of the GM-arthropds.
The understanding of the pathology of diseases and their spread, the understanding of the
interaction of the insect vector with humans and the effectiveness of the GM-arthropod should
also be covered. Knowledge on the assessment of tests of susceptibility to other non-target
pathogens might be needed as well as knowledge on the distribution/evolution of the target
disease.
Important issues are also the risks of GM-arthropods, the transgene or the recombinant DNA
in the environment; trait spread factors, the hazards of gene expression, the quantification of
risks as well as the likelihood of failure or the release programme and knowledge on
monitoring systems (that could help in both assessing the potential impact of GM-arthropods
and in identifiying invading species at an early stage). Also expertise on confirming that the
GM-line is performing as expected, its distribution and long term effects on non target
populations is important.
Crucial issues in the production process are strain evaluation at various levels of mass rearing,
quality rearing diets, and maintenance of desired field attributes and of the genetic
modification.
A short description of each scientific discipline and field of expertise listed is provided below.
17.1.
Biology
Biology is a natural science concerned with the study of life and living organisms, including
their structure, function, growth, origin, evolution, distribution and taxonomy. Biology is a
vast subject containing many subdivisions, topics, and disciplines.
Developmental biology
Developmental biology is the study of the process by which organisms grow and develop.
Modern developmental biology studies the genetic control of cell growth, differentiation and
"morphogenesis," which is the process that gives rise to tissues, organs and anatomy.
140
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Microbiology
Microbiology is the study of microorganisms, which are unicellular or cell-cluster
microscopic organisms. This includes eukaryotes such as fungi and protists, and prokaryotes.
Viruses, though not strictly classed as living organisms, are also studied. In short
microbiology refers to the study of life and organisms that are too small to be seen with the
naked eye. Microbiology is a broad term which includes virology, mycology, parasitology,
bacteriology and other branches.
Physiology
Physiology, a subcategory of biology, is the science of the functioning of living systems. In
physiology, the scientific method is applied to determine how organisms, organ systems,
organs, cells and biomolecules carry out the chemical or physical function they have in a
living system
Parasitology
Parasitology is the study of parasites, their hosts, and the relationship between them. As a
biological discipline, the scope of parasitology is not determined by the organism or
environment in question, but by their way of life. This means it forms a synthesis of other
disciplines, and draws on techniques from fields such as cell biology, bioinformatics,
biochemistry, molecular biology, immunology, genetics, evolution and ecology.
Taxonomy
Taxonomy is the practice and science of classification.
Invasion biology
Invasion biology deals with invasive species. The term invasive species is subject of debate
but generally it is a subset of plants or animals (alien species) that are introduced to an area
(outside the natural past or distribution), survive and reproduce and cause harm (economically
or environmentally, through threatening biological diversity) within the new area of
introduction (see e.g. CBD, 2010b). This discipline has also developed RA methods.
Quarantine biology
Quarantine biology is a discipline that deals with the limitation on the freedom of movement
of an individual, to prevent spread of a disease to other members of a population (see also
IPPC 2010).
Applied biology
Applied biology is a subfield within biology, considering the application of the science of
biology to real-world e.g. management questions in the fields of agriculture, forestry and
stored product pests. It deals also with an integrated treatment of the ecological, social, and
biotechnological aspects of natural resource conservation and management.
141
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
17.2.
Molecular biology
Molecular biology is the study of biology at a molecular level. The field overlaps with other
areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology
chiefly concerns itself with understanding the interactions between the various systems of a
cell, including the interactions between DNA, RNA and protein biosynthesis as well as
learning how these interactions are regulated.
Proteomics/ Transcriptomics/ Genomics
Proteomics is the research on the proteome, the totality of all proteins in a creature/tissue/cell
under predefined conditions for a specific point in time. Transcriptomics is a branch of
molecular biology dealing with the study of messenger RNA molecules. Genomics is the
study of genomes of organisms.
Insect molecular biology
Insect molecular biology is a subdiscipline of molecular biology dealing with the structure,
function, mapping, organisation, expression and evolution of insect genoms.
Biotechnology/Transgenics
Biotechnology is a field of biology and any technological application that uses biological
systems, living organisms or derivates thereof, to make or modify products or processes for
specific use. Biotechnology draws on the pure biological sciences and in many instances is
also dependent on knowledge and methods from outside the sphere of biology.
Transgenic technology creates GMOs using genetic engineering techniques.
17.3.
Genetics
Genetics, a discipline of biology, is the science of heredity and variation in living organisms.
Evolutionary genetics
Evolutionary genetics is the broad field of studies that attempts to account for evolution in
terms of changes in gene and genotype frequencies within populations and the processes that
convert the variation within populations into more or less permanent variation between
species. It considers the effect of micro-evolutionary changes among populations due to
evolutionary forces, which account for the emergence of macro-evolutionary patterns in the
long term. A focus of evolutionary genetics is to describe how the evolutionary forces shape
the patterns of biodiversity observed in nature.
Molecular genetics
Molecular genetics is a field of biology that studies the structure and function of genes at the
molecular level.
Population genetics
Population genetics is the study of allele frequency distribution and changes under the
influence of the four main evolutionary processes: natural selection, genetic drift, mutation
and gene flow. It also takes into account the factors of population subdivision and population
structure. It attempts to explain such phenomena as adaptation and speciation. Regarding the
ERA of GM-insects some concerns were raised since many of the schemes are based on
142
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
simple hypotheses of population structure of the pest arthropod and there are some doubts, if
the assumptions are correct.
17.4.
Entomology
Entomology is the scientific study of insects. It is a specialty within the field of biology.
Though technically incorrect, the definition is sometimes widened to include the study of
terrestrial animals in other arthropod groups or other phyla, such as arachnids, myriapods,
earthworms, and slugs.
Insect immunity
It is the study of the insect immune system.
Medical entomology/vector control
The discipline of medical entomology, or public health entomology, and also veterinary
entomology is focused upon arthropods that impact human health. Veterinary entomology is
included in this category, because many animal diseases can "jump species" and become a
human health threat, for example, bovine encephalitis (known as "mad cow disease").
Medical entomology also includes scientific research on the behaviour, ecology, and
epidemiology of arthropod disease vectors, and involves a tremendous outreach to the public,
including local and state officials and other stake holders in the interest of public safety.
Vector control is a field of expertise that deals with methods to limit or eradicate mammals,
birds, insects or other arthropods which transmit disease pathogens.
Regulatory entomology
Regulatory entomology deals with the analysis of risks and the meeting of regulatory
requirements.
17.5.
Ecology
Ecology is the interdisciplinary scientific study of the distributions, abundance and relations
of organisms and their interactions with the environment. Ecology is also the study of
ecosystems. Ecology is closely related to the disciplines of physiology, evolution, genetics
and behaviour.
Evolutionary ecology
Evolutionary ecology lies at the intersection of ecology and evolutionary biology. It
approaches the study of ecology in a way that explicitly considers the evolutionary histories
of species and the interactions between them. Conversely, it can be seen as an approach to the
study of evolution that incorporates an understanding of the interactions between the species
under consideration. The main subfields of evolutionary ecology are life history evolution,
sociobiology (the evolution of behaviour), the evolution of interspecific relations
(cooperation, predator-prey interactions, parasitism, mutualism) and the evolution of
biodiversity and of communities.
143
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Molecular ecology
Molecular ecology is a field of evolutionary biology that is concerned with applying
molecular population genetics, molecular phylogenetics and genomics to traditional
ecological questions.
Microbial ecology
Microbial ecology is concerned with the relationships of microorganisms with one another
and with the environment.
Insect ecology
Insect ecology is the scientific study of how insects, individually or as a community, interact
with the surrounding environment/ecosystem.
Community ecology (incl. symbiosis, insect-pathogen interactions)
Community ecology is a subdiscipline of ecology which studies the distribution, abundance,
demography, and interactions between coexisting populations. Interactions between
populations, determined by specific genotypic and phenotypic characteristics, are the primary
focus of community ecology.
Experts in the field of symbiosis are concerned with close and often long-term interactions
between different ecological species.
Experts in the field of insect-pathogen interactions describe and study those interactions as it
is relevant e.g. for studying malaria.
Population ecology
Population ecology is a major sub-field of ecology that deals with the dynamics of species
populations and how these populations interact with the environment.
Behavioural ecology
Behavioural ecology is the study of the ecological and evolutionary basis for animal
behaviour, and the roles of behaviour in enabling an animal to adapt to its environment.
Landscape ecology
Landscape ecology is the science of studying the relationships between spatial patterns and
ecological processes on a multitude of landscape levels and organisational levels.
Disease ecology
Disease ecology deals with vector-borne diseases, the ecological and demographic changes
resulting from the introduction and the habitats of disease vectors.
144
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
17.6.
Chemistry
Chemistry is the science of matter and the changes it undergoes, being concerned with
composition, behaviour, structure and properties of matter as well as the changes it undergoes
during chemical reactions.
Biochemistry
Biochemistry is the study of the chemical processes in living organisms. It deals with the
structures and functions of cellular components such as proteins, carbohydrates, lipids,
nucleic acids and other biomolecules.
17.7.
Medicine
Medicine is the art and science of healing. It encompasses a range of health care practices
evolved to maintain and restore health by the prevention and treatment of illness.
Contemporary medicine applies health science, biomedical research, and medical technology
to diagnose and treat injuries and disease, typically through medication, surgery, or some
other form of therapy.
Evolutionary medicine
Evolutionary medicine is the application of modern evolutionary theory to understand health
and diseases. Issues are e.g. the evolution of pathogens in terms of their virulence and
resistance to antibiotics.
Human medicine
Human medicine is a field of medicine focusing on humans.
Veterinarian medicine
Veterinarian medicine applies the field of medicine to higher animals.
Immunology
Immunology is a broad branch of biomedical science that covers the study of all aspects of the
immune system in all organisms. It deals with the physiological functioning of the immune
system in states of both health and disease; malfunctions of the immune system in
immunological disorders (autoimmune diseases, hypersensitivities, immune deficiency,
transplant rejection); the physical, chemical and physiological characteristics of the
components of the immune system in vitro, in situ, and in vivo. Immunology has applications
in several disciplines of science, and as such is further divided.
Epidemiology
Epidemiology is the study of factors affecting the health and illness of populations, and serves
as the foundation and logic of interventions made in the interest of public health and
preventive medicine. Epidemiologists rely on a number of other scientific disciplines such as
biology (to better understand disease processes), biostatistics (the current raw information
available), Geographic Information Science (to store data and map disease patterns) and social
science disciplines (to better understand proximate and distal risk factors).
145
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
17.8.
Agricultural science
Agricultural science is a broad multidisciplinary field that encompasses the parts of exact,
natural, economic and social sciences that are used in practice and understanding of
agriculture. Veterinary science, but not animal science, is often excluded from the definition.
Phytopathology
Phytopathology or plant pathology is the scientific study of plant diseases caused by
pathogens and environmental conditions.
Agricultural pest control/ Biological control
Pest control refers to the regulation of management of species defined as a pest, usually
because it is perceived to be detrimental to a person`s health, the ecology or the economy.
Biological control is defined as the reduction of pest populations by natural enemies and
typically involves an active human role. Natural enemies of insect pest, also known as
biological control agents, include predators, parasitoids and pathogens.
17.9.
Toxicology
Toxicology is the study of the adverse effects of chemicals on living organisms. It is the study
of symptoms, mechanisms, treatments and detection of poisoning, especially the poisoning of
people.
Ecotoxicology
Ecotoxicology aims to quantify the effects of stressors upon natural populations,
communities, or ecosystems. Ecotoxicology incorporates aspects of ecology, toxicology,
physiology, molecular biology, analytical chemistry and many other disciplines.
17.10. Statistics/Informatics
Statistics is the formal science of making effective use of numerical data relating to groups of
individuals or experiments. It deals not only with the collection, analysis and interpretation of
data but also with the planning of data collection, in terms of the design of surveys and
experiments.
Informatics is the science of information, the practice of information processing, and the
engineering of information systems. Informatics studies the structure, algorithms, behaviour,
and interactions of natural and artificial systems that store, process, access and communicate
information. It also develops its own conceptual and theoretical foundations and utilizes
foundations developed in other fields.
Mathematical modelling
Mathematical modeling is the process of developing a mathematical model. A mathematical
model uses mathematical language to describe a system (see also chapter 15). Mathematical
models are used in many sciences e.g. in physics, biology, meteorology.
Bioinformatics
Bioinformatics is the application of information technology and computer science to the field
of molecular biology.
146
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Geographical information science
Geographic information science is the academic theory behind the development, use, and
application of geographic information systems (GIS).
17.11. Biosafety
The field of biosafety is concerned with the prevention of large-scale loss of biological
integrity, focusing both on ecology and human health.
(Environmental) risk assessment
RA is the determination of quantitative or qualitative value of risk related to a concrete
situation and a recognised hazard. Quantitative RA requires calculation of two components of
risk: the magnitude and the probability of occurence.
17.12. Others
Sterile Insect Technique
The Sterile Insect Technique is a method of biological control, whereby sterile insects are
released reducing the next generation (see also chapter 5.3.1)
Monitoring
Monitoring is the systematically observation and control of certain procedures and processes
e.g. to determine species occurrence and spread.
Mass rearing
Mass rearing is the production of insects competent to achieve programme goals with an
acceptable cost/benefit ratio and in numbers per generation exceeding ten thousand to one
million times the mean productivity of the native population female.
147
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
18.
Research institutes and academics
During this project information on research institutes and academics having experience in
scientific disciplines and fields of expertise that might feed an ERA of GM-arthropods to be
commercially released into the environment were collected. As stated in chapter 1, mainly
renowned experts provided the respective information.
The information collected is contained in two databases enclosed to this report. A MS
Access® database provides the core information on institutes and academics whereas relevant
publications are contained in a Reference Manager® database.
18.1.
Publication Database
This database contains scientific literature relevant for the ERA of GM-arthropods, basic
information on GM-arthropods and information on techniques of genetic modification. Also
the references citied in this report are imported as well as relevant publications of the
academics listed in the enclosed MS-Access database. The Reference Manager® Database
(Reference Manager®, version 11) provided is named GM_arthropods.
The database contains in most cases the full record (including abstracts). In addition two
fields were added.
−
Provided: This field indicates if the respective reference is provided as full text (see
also legal notice). The respective publications will be enclosed to the final report as
pdf-files on CD-ROM.
−
Save_ID: Full text references are ordered by the reference ID. The field Save_ID was
created serving as a backup in order to prevent accidental loosing of the ID when
copying references into another database.
Legal notice
References provided as full text on CD-ROM should only be used for purposes of reviewing.
Any other use will infringe the copyright. Umweltbundesamt GmbH excludes any liability
concerning this matter.
18.2.
Experts and Institutions Database
As stated above information on research institutes and academics are provided in a MS
Access® database named “GM_arthropods”, which is enclosed to this report. The EntityRelationship-Model (see figure 1) demonstrates the content of this database. It shows the
entities (institution, expert, country and field of expertise) as well as their properties and the
relations between the various entities.
148
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
fax
additional
information
address
email
phone
phone
homepage
email
ID
additional
information
ID
unit_name given name
n
institution institute_name
m
expert
surname
fax
homepage
n
m
1
n
cathegory
field of
expertise
country
EU
ID
cathegory
name
ID
name
Figure 1. Entity-Relationship Model of database GM_arthropods
The database contains various information on institutions and experts, e.g. contact details and
their respective homepage. In addition the fields of expertise of the respective experts are
provided as well as the country where the experts work and the institution is placed.
In order to prevent redundancies all experts are connected to an institution. E.g. the address of
an academic is the same as his departments, since the table ‘expert’ is connected with the
table ‘institution’ the address is listed only once. In the few cases where an expert works as a
private consultant or as freelancer, the information contained in the table ‘expert’ and the
table ‘institution’ is in most cases the same. The name of the institution is in that case
‘consultant + expert name’.
6 tables were necessary to realise the entity-relationship model. Figure 2 shows those tables
and the fields contained therein.
The tables ‘expert’ and ‘institution’ are the main tables of the database and contain the most
information. Tables ‘country’ and ‘fields of expertise’ were created in order to prevent
redundancies and the two tables ‘institution_expert’ and ‘expert_expertise’ were necessary to
allow n:m relationships. A short description of each table, their fields, data types and purpose
is given below.
149
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Figure 2. MS Access® database GM_arthropods: tables and relationships
Table ‘expert’
Each dataset in this table represents one expert and provides specific information related to
this person. Table ‘expert’ consists of 9 fields.
−
ID_expert (AutoNumber, key field): ID_expert is a unique number for each dataset in
this table and necessary for its distinct definition.
−
surname_expert (text): expert`s surname
−
given_name_expert (text): This field provides the expert`s given name. The first name
is written out, additional names abbreviated
−
email_expert (text): personal e-mail address
−
phone_expert (text): personal phone number
−
fax_expert (text): personal fax number
−
homepage_expert (text): personal homepage
−
add_information_expert (memo): This field contains additional information relevant to
the scope of this project, e.g. major projects
−
category_expert (text): This field provides information on the type of expertise an
expert can provide. The 4 options are managed in a lookup table and given below;
more than one selection is possible.
• basic science
• applied science
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
• regulatory science
• advisory function
Table ‘institution’
Each dataset in this table contains information about a specific institution. Normally this
information is not specific for one person except for freelancers as described above. Table
‘institution’ contains 10 fields:
−
ID_institution (AutoNumber, key field): ID_institution is a unique number for each
dataset in this table and necessary for its distinct definition.
−
name_institution (text): This field provides the name of an institution. This can be the
name of a person (in case of a freelancer), the name of an enterprise or of an university
department. If the respective institution is hierarchical structured, all levels are listed
(e.g. University of Vienna, Faculty of Life Sciences, Department of Evolutionary
Biology). For this database the lowest level (e.g. the department) is the most important
and referred to in the other fields of the respective dataset.
−
address_institution (text): institution`s address
−
country_institution (number): institution`s place; interconnection with table ‘country’
−
email_institution (text): This field provides a general e-mail address (e.g. secretariat)
of the institution on the lowest level in hierarchy.
−
phone_institution (text): This field provides a general phone number (e.g. secretariat)
of the institution on the lowest level in hierarchy.
−
fax_institution (text): This field provides a general fax number (e.g. secretariat) of the
institution on the lowest level in hierarchy.
−
homepage_institution (text): This field provides the institution`s homepage on the
lowest level in hierarchy.
−
add_information_institution (memo): This field provides additional information of
releavance for the ERA of GM_arthropods, e.g. major project, core capabilities.
−
cathegory_institution (text): This field contains information on the type of institution.
The 3 options are managed in a lookup table and are the following:
• consultant
• public
• private
Table ‘field_of_expertise’
This table contains the fields of expertise of those experts listed in the respective table. The
table consists of the following 2 fields:
−
ID_expertise (AutoNumber, key field): ID_expertise is a unique number for each field
of expertise listed in this table and necessary for the distinct definition of each dataset.
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Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
−
name_expertise (text): This field contains the name of the field of expertise, as stated
on the personal homepages of the respective experts.
Table ‘country’
This table contains those countries where the institutions of the respective table are placed and
consists of the following 3 fields:
−
ID_country (AutoNumber, key field): ID_country is a unique number for each country
listed in this table and necessary for the distinct definition of each dataset.
−
name_country (text): country`s name
−
EU (yes/no): This field indicates whether a country is currently a member state of the
European Union or not.
Table ‘institution_expert’
This table establishes a n:m relationship between institution and expert, since one expert can
work for more than one institution and one institution employs many experts. The table
contains the following 3 fields:
−
ID_institute_expert (AutoNumber, key field): This field gives a unique number for
each dataset of this table and necessary for its distinct definition.
−
surname_expert_IE (number): expert`s surname; interconnection to table ‘expert’
−
name_institution_IE (number): institution`s name; interconnection to table ‘institution’
Table ‘expert_expertise’
This table establishes a n:m relationship between the tables ‘experts’ and ‘field_of_expertise’,
since one expert comprises more than one field of expertise and for each field more than one
database entries are likely. The table consists of the following 3 fields:
−
ID_expert_expertise (AutoNumber, key field): This field gives a unique number for
each dataset of this table and is necessary for its distinct definition.
−
name_expertise_EE (number): name of the field of expertise; interconnection to table
‘field_of_expertise’
−
surname_expert_EE (number): expert`s surname; interconnection to table ‘expert’
The information provided in the database is based on the respective homepages of experts and
institutions and on information retrieved from the experts via a form some filled in. Therefore
the field of expertise as provided in the database is not necessarily defined in the same way as
in chapter 17. Since not all experts returned the form and the quality of the homepages is
variable it was in many cases not possible to provide the full dataset (e.g. fax number of the
expert is missing). Also content of the fields ‘additional information’ is based on those
information provided and therefore variable.
152
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Cross-cutting considerations
This part of the report gives attention to important topics that are not within the scope of this
report but very much related. Those were definded as cross-cutting issues and some
considerations are given below.
19.
Paratransgenesis
As indicated in chapter 4 paratransgenesis, the use of GMMs which are associated with
arthropods is not directly in the remit of this report since the arthropods are not genetically
modified themselves. However, paratransgenesis was proposed as an alternative approach to
the application of GM-arthropods, e.g. to reduce vector competence in certain arthropod
species or to facilitate population suppression of arthropod pests (Coutinho-Abreu et al.,
2010). Paratrangenesis applications are also developed when the arthropod host species for
some reason may not be modified easily.
Paratransgenesis has been explored or is in development for a wide range of applications:
−
Genetic modification of species-specific gut bacteria of tsetse flies to impair the
vectorial capacity for transmission of Trypanosoma parasites by expression of effector
genes with trypanocidal activity, e.g. monoclonal antibody genes (Aksoy et al., 2008).
−
Genetic modification of symbionts of triatomine bugs as a means to reduce the ability
of this reduviid bug to transmit the agent of Chagas disease, Trypanosoma cruzi.
Symbionts like the gut bacterium Rhodococcus rodnii and Corynebacteria, which were
modified to express the antimicrobial protein cecropin or anti-Trypanosoma antibodies
(Aksoy et al., 2008; Durvasula et al., 2008).
−
Genetic modification of bacteria like Alcaligenes which are colonising the gut of the
glassy-winged sharpshooter Homalodisca coagulate, an important vector of the plant
pathogen Xylella fastidosa, which is the agent causing Pierces´s disease in crops like
grapes (Bextine et al., 2004). Again a strategy to modify the gut bacteria to express
transgenes that impair pathogen transmission was explored.
−
Genetic modification of yeast which colonises social insects such as termites. The
GM-yeast was developed to express lytic enzymes that are directed against the
arthropods and against symbiotic protozoa, which are critically important for the
termites as being responsible for the digestion of woodstuffs (Cooper et al., 2009).
−
Genetic modification of viruses infecting e.g. mosquitoes, like symbiontic
densoviruses or alphaviruses, which could be used to express transgenes in mosquitoe
populations, like single chain antibody molecules (Vanlandingham et al., 2005;
Coutinho-Abreu et al., 2010).
−
Genetic modification of symbiotic bacteria to mosquito species, including application
of Wolbachia, Ricksettia-like intracellular bacteria. A specific strain of Wolbachia,
wMelpop reduces the lifespan of infected Aedes mosquitoes, while still showing one
of the characteristic features of Wolbachia bacteria, i.e. being able to spread in the host
population (Brelsfoard and Dobson, 2009). Wolbachia which is only maternally
transmitted is also a potential agent to introduce cytoplasmic male incompatibility
which could be applied in population suppression strategies. Wolbachia bacteria were
153
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also shown to colonise the salivary glands of Aedes mosquitoes which is an important
factor for development of disease control applications (Zouache et al., 2009).
Crucial differences of GM-arthropod applications and paratransgenesis are a different
spectrum of species targeted, which is due to the availability of species-specific symbiotic
organisms with characteristics supporting the envisioned purposes, without being associated
with risks of environmental releases. Additionally the systems used for spread of the GMmicrobes in the targeted arthropod population are different than with respective approaches in
GM-arthropods. Strategies for paragenesis that are explored in this respect are spread through
infection (GM-viruses, GM-microbes efficiently infecting social insects like termites) or the
strong gene drive system occurring in endosymbiontic Wolbachia. Finally the different
transgenic elements used for transformation and the applied transformation systems for
generating GM-microbes and the different biology features of the GM-microbes demand a
different assessment approach.
The RA of paratransgenesis applications need to focus on the characteristics of the GMM and
conducted according to the guidance provided for GMMs (EFSA, 2006a), which does not
cover this type of applications yet. However, like the environmental release of GM-arthropods
applications of paratransgenesis are developed for environmental release and thus similar RA
issues are relevant for the assessment, which differ from the ones usually addressed during
RA of GMMs. Given the similarities in the purposes of GM-arthropod applications on the one
side and paratransgenesis on the other and the fact that the paratransgenic microorganisms
usually are closely associated with their arthropod hosts, some cross-cutting considerations
may be drawn from the assessment of GM-arthropod applications towards assessment of
paratransgenesis.
Considerations with regard to the respective arthropod species, which is used as a host in
paratransgenesis would be relevant for RA of paratransgenesis also, including baseline data
established for these species. Safety considerations regarding the basic strategies relevant to
both types of GM-application (population suppression by SIT or other approaches, population
replacement with the aim to incapacitate vector species) should similarly be applicable for
paratransgenesis.
154
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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Conclusions
In order to provide comprehensive background information in the area of GM-arthropods and
to present scientifically sound and up to date data on this emerging issue for the European
Union various aspects were addressed in this report. Since no GM-arthropod applications are
market-ready, only a few field trials have been conducted and several strategies and
programmes are still under development this report tried to be as comprehensive as possible.
Not only the species for which transgenesis has been accomplished (often for a proof of
concept) were presented and those selected that could possibly be of relevance for the EU
within the next 10 years but also background information was included to build a basis for
ERA. To accommodate the fact that many applications are still under development or only
envisaged more speculative developments were addressed, too, and comparable conventional
approaches (that could also be improved by transgenic techniques) discussed.
Various purposes of GM-arthropods were assessed ranging from the production of products of
interest to population replacement and population suppression, the use of beneficial
arthropods as biocontrol agents and GM-arthropods used as vectors of vaccination. Regarding
population replacement strategies potential risks e.g. the drive mechanism that could transfer
transgenes to other related species (because a high mobility of genes is required for this
application), or the problems concerning the transposable elements where the transgene
inserts at random positons leading to the difficult prediction of the consequences, were
addressed. With respect to population replacement strategies also incapacitating vectors and
the accomplishments needed for the implementation of this technology are discussed.
Regarding strategies and developments to suppress the target population SIT and RIDL were
discussed. SIT is a conventional technique that is applied for many pest species worldwide
and that could be improved in several ways using GM-arthropods (e.g. genetic sterility is
supposed not to reduce the fitness of the released males whereas sterilisation with
conventional measures like radiation does). Since some similar issues need to be addressed
when using conventional SIT and SIT using GM-arthropods (e.g. releases are done
prophylactic and routinely, continuously, entire areas are treated not only the infested sites)
this technique was described in detail. A lot of practical information is available on this issue
that could feed the ERA of GM-arthropods. Those aspects were presented.
Also RIDL is discussed in more detail since strains are available for e.g. Aedes aegypti,
Ceratis capitata and Bactrocera olea although some further developments are needed, e.g.
RIDL has only been proven to work at very small scale in the laboratory and under confinded
conditions. For this approach risks are discussed regarding the failure of the system e.g. if the
target population devolps resistance to the lethal gene. In addition a number of effects are
known that can abolish the lethal effect since the lethal gene and its control element have to
interact with the host genome accurately.
Also the use of GM-arthropod applications employing RNAi based immunity to vectored
disease agents and beneficial arthropods as biological control agents where addressed,
describing the concept behind and the current status of development.
Based on this background information specific areas of potential risks that should be
addressed in an ERA of GM-arthropods were compiled using existing literature on this or
closely related issues and additionally by addressing additional aspects. Areas addressed were
155
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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adverse effects associated with gene flow (vertical and horizontal gene transfer), potential
risks regarding the interactions of the GM-arthropod with the target population (triggering
adaptive processes in the target population as well as changing the host range), and risks
concerning the interactions of the GM-arthropod with non-target organisms (effects on
predators and parasitoids, biodiversity and pollination). Also risks related to impacts on
specific agricultural management practices and management measures to control arthropods
vectoring diseases where addressed as well as effects on biogeochemical processes and on
human health. In the assessment hazards were identified and characterised and an exposure
characterisation provided, discussing the potential adverse effects, their consequences and
likelihood of occurrence.
In order to provide information on assessment endpoints and methodologies important for an
ERA of GM-arthropods not only methods were provided suitable for investigating the
addressed adverse effects of GM-arthropods but also methods for the assessment of important
key parameters. Those parameters were those addressing crucial arthropod characteristics
(e.g. fertility rate, longevity, or mating competitiveness), environment variables (e.g.
arthropod density, migration behaviour, habitat interactions) and genotype-environment
interactions (e.g. sensitivity to pathogens and insecticide resistance) that are important when
evaluating e.g. fitness, spread, persistence and invasiveness. The proposed methods for the
assessment of the various key parameters were evaluated using SWOT analysis in order to
provide important information on advantages and disadvantages of a method as well as on
strengths and weaknesses that enables the validation of data presented in an ERA of GMarthropods.
In addition aspects on the kind of baseline information about receiving environments needed
for conducting an ERA of GM-arthropods was presented. They cover information on the
habitat type, geological and pedological characteristics, climate characteristics and
information on management practices. An additional aspect should be the assessment of
potential interactions of the GM-arthropod with the biotic and abiotic environment in a
specific habitat. Therefore a lot of information is needed e.g. on abundance of flora and fauna,
a detailed description of the GM-arthropod ecology and possible antagonists such as
predators, parasitoids and pathoges. Baseline data are difficult to assess, have a high
variability and also depend on anthropogenic factors.
Since some information for the ERA of a GM-arthropod needs to be gained in semi-field and
field tests the possibility of using the surrogate approach was assessed considering strains of
the same species, SIT-applications of the same species, or non-GM species modified with
conventional methods to possess similar characteristics. It was assumed that it depends on the
data to be assessed, the species tested and the specific modification and characteristics of a
GM-arthropod whether or not surrogates can be used. It was concluded, that there are
limitations when assessing data with surrogate species and their interpretation needs to be
carried out very carefully.
With respect to the difficulties related to the assessment of data for the ERA of GMarthropods also the possible use of modelling approaches was discussed. It was concluded,
that there are some possible application areas, but that it is very important to use sound data in
the model, and to choose the model most suitable for addressing the question to be answerd.
In addition it needs to be considered that a model does not comprise all attributes of reality
156
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a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
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and that the outcomes of modelling have been shown to differ from the real situation.
Therefore it is very important, that model based data are used cautiously.
To summarise the various aspects addressed concerning the ERA of GM-arthropods
implications for the implementation of and ERA were addressed. The ERA aspects were
elaborated using four case study species as more practical examples leading to a list of issues
to be considered in an ERA of GM-arthropods, regarding the parental organism, the genetic
modification, the GM-arthropod and the receiving environment.
Information is presented on the expertise needed for conducting an ERA on GM-arthropods.
This includes important scientific disciplines and fields of expertise as well as scientific
institutions and academic that are either already involved in aspects related to an ERA or that
could feed an ERA with their expertise.
In this report various knowledge gaps are addressed including paratransgenesis, the lack of
baseline information, the surrogate approach and the aspect of overseas territories of the EU.
In paratransgenesis not the arthropod is genetically modified but microorganisms living
within the arthropod, therefore it was concluded that this technique could not be covered by
ERA considerations for arthropods alone, since although some aspects are similar many
different issues need to be considered. Therefore it was concluded, that paratransgenesis need
to be dealt with in guidelines concerning genetically modified microorganisms.
It was also concluded, that a lot of baseline information crucial for an ERA on GM-arthropods
is not available at the moment (e.g. number of predators in a given type of habitat). Another
problem is that there is a lot of natural variation in the environment and species abundance
differs from one year to another Since respective data for an ERA will probably be obtained
during a limited time it may be difficult to interpret these data. In addition large sample sizes
are necessary to detect small differences on a sound statistical basis. The scientific gaps
concerning important baseline information need to be considered further.
Another point for further consideration is the use of surrogate species, since it is clear that at
some stage of the development more realistic data under field conditions need to be assessed,
but that the associated risks with this approach need to be taken into account.
A mayor knowledge gap addressed in this report is the issue of overseas territories. When
including overseas territories in an ERA of GM-arthropods one needs to be aware of the fact,
that this widens the range of receiving environments to almost all climatic zone of the world.
However, it needs also be kept in mind that the application of GM-arthropods may be
especially important for those territories regarding arthropods vectoring diseases.
One can only speculate on the rate of development of GM-arthropods in the next years but the
species to be modified are most likely one of those described in this report and for which a lot
of research has already been going on. However, the likelihood of an application does not
only depend on whether or not the species can be modified but also on the functioning of the
whole system. Since many factors are influencing the success of the application the
development within the next years is difficult to predict.
157
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Appendices
APPENDIX A
183
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
184
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
185
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exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
APPENDIX B
Table 1: Fertility rate
Method
Life table analysis
Sex ratio test
186
Internal factors
Strengths
External factors
Weaknesses
• Reflects biological differences • none
• Comparisons possible
• Easy to collect
• Reflects basic genetic parameter • Some effectors may skew sex ratio
• Sex ratio distortion could be natural
• Routinely collectable
(e.g. mosquitoes)
• Little training necessary
• For some arthropods batch and
temporal consistency is more
important
Opportunities
Threats
• Exchange of standardised
• Result may be biased by
parameters
unfavourable life table
characteristics
• Improvement of robustness
• Sex proportion can be changed by • Distortion of sex ratios possibly
due to extensive breeding
culture methods
• Production or sex separation can be
facilitated
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 2: Mating competitiveness
Method
Internal factors
Strengths
External factors
Weaknesses
• Mating observation difficult
• Difficult to locate in the field
• Natural behaviour in the lab difficult
to stimulate
• Labour intensive
• Sometimes must be performed at
inconvenient day times
Cage experiments to
• Some GM sperm have no visible
• Direct
• Highly relevant to effectiveness marker
investigate sterility,
• Obtaining eggs often difficult
sperm marker or marker
• Ability to stimulate natural mating
among progeny
in cages differs with the species
• Insemination with few sperm
difficult to detect
• Cage data do not always reflect field
values
Field bioassay for egg
• Collection effort varies within the
• Meaningful indicator of
species
performance
hatching or sperm
• Large numbers must be released
marker
• Obtaining eggs often difficult
• Time and personnel consuming
Cage experiments to
• Direct and meaningful
observe mating directly
Opportunities
• Increased low light detection by
photographic/video equipment
• Stimulating natural behaviour by
special cages
• Stimulating natural behaviour by
special cages
• Use of sperm markers detectable
by PCR
• Marking with stable isotopes
187
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Threats
• Poor competitiveness may be
misunderstood as uselessness of
the strain
• Reduced longevity but high
mating competitiveness may be
offset by increased life span and
reduced competitiveness
• Cage observations difficult to
translate in terms of field
efficacy
• Open releases required
• Improved trapping methods
needed
• Location of sites may be
problematic
• Analysis of field specimens
costly
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 3: Longevity
Internal factors
Method
Life table analysis
Field estimates
Cages studies
Strengths
External factors
Weaknesses
Opportunities
Threats
• Reflects biological differences • Comparable results difficult to
obtain
• Comparisons possible
• Easy to collect
• Seasonal and yearly differences
• Information useful for
understanding field performance confound usefulness
• High quality and precise data
• Simple to perform
• Exchange of standardised
parameters
• Improvement of robustness
• Improvement of data quality by
molecular and chemical markers
• Novel biochemical methods
• Cage data do not reflect field values • Surrogates exploitable
• Recommendations may be
biased by unfavourable life table
characteristics
• Field release needed
• Locating sites could be
problematic
• None
Table 4: Development time
Method
Internal factors
Strengths
Life table analysis
• Simple to collect
Field estimates
• Realistic data
• Useful for field releases
planning
Direct observations
• Direct
• Not open to interpretation
External factors
Weaknesses
Opportunities
• Only controlled experiments useful • More realistic information by
semi-field systems with simulated
• Data not meaningful due to lacking
environments
standards
• High quality data difficult to obtain • Simplified data collection by
understanding relationship of
• Data variations by habitat and
markers to development rate
season
• Model may be based on unsound
input data and assumptions
• Limited data obtainable
• None
• Little information relevant to safety
or efficacy
188
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Threats
• End point of analysis
informational rather than strain
evaluation
• Development time climate
dependent
• Evaluations during different
seasons necessary
• Time consuming and costly
• Development time climate
dependent
• Evaluations during different
seasons necessary
• Time consuming and costly
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 5: Hatching rate
Internal factors
Method
Life table analysis
Mass collection egg
hatching estimates (lab,
field)
Strengths
External factors
Weaknesses
• Comprehensive accounting of
potential rate of increase
• Simplest method
Opportunities
Threats
• Full life table analysis not necessary • None
• None
• Confounded if male affects number
of eggs
• Confounded if sterility differs
according to eggs laid
• Data collection in the field may not
be feasible
• None
• Laboratory tests can determine
influence by male and sterility
Table 6: Larval survival
Internal factors
Method
Strengths
Life table analysis with • Easily observed
data from lab culture:
percent of larvae
reaching the pupal stage
External factors
Weaknesses
• Data depend on handling, diet and
environmental conditions
• Standardisation needed
Opportunities
Threats
• Production can be improved and
costs reduced
• Not a good proxy for quality of
released material
Table 7: Pupal survival
Method
Life table analysis with
data from lab culture:
percent recovery from
eggs
Internal factors
Strengths
• Easily observed
External factors
Weaknesses
• Data depend on handling, diet and
environmental conditions
• Standardisation needed
Opportunities
• Production can be improved and
costs reduced
189
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Threats
• May be compromised by
transportation
• Not a good proxy for quality of
released material
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 8: Adult emergence
Method
Life table analysis with
data from lab culture:
percent recovery from
pupae
Internal factors
Strengths
External factors
Weaknesses
• Easily observed
• Less depended upon precise
conditions
• Standardised conditions needed
Opportunities
• None
Threats
• Laboratory data do not reflect
field conditions
Table 9: Size/weight
Method
Internal factors
Strengths
External factors
Weaknesses
Nutrient reserves
• Straightforward
• Comparable
• Impact on field performance
unknown
Direct measurements
• Simple and direct
• Reflects general vigour and
indirectly fecundity
• None
Opportunities
• Comparative studies possible
• None
• Influence of modifications on
biochemical makeup can be
studied
• Increased programme effectiveness • None
if knowledge on size effects can be
obtained
190
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Threats
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 10: Flight ability
Method
Lab test
Lab test flight mill
Field dispersion
estimates
Internal factors
Strengths
• Readily obtained
• Reproducible
• Meaningful dispersal
characteristic
• Reasonable surrogate
• Most realistic indicator
External factors
Weaknesses
Opportunities
Threats
• Meaningful flight tests not available • Flight performance assays for
• Laboratory data may not reflect
for all species
various insects could be developed field activity
• Separate systems might be useful • More realistic tests expensive
and difficult
• Equipment and expert staff needed • More realistic devices could be
• Not widely available
developed that require less
• Not all insects can be stimulated to
expertise
fly on the mill
• Small amount of data collectable
• Labour intensive
• Depended on varying ecological
• Laboratory indictors developable
settings
• Appropriate sites may be limited
• Time consuming and costly
• Efficient trapping systems needed
191
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 11: Altered biochemistry
Internal factors
Method
Strengths
Gas chromatography
• Sensitive measure
• Very small differences
detectable
Enzyme analysis
• Specific changes in activity
measurable
Bioassay
• Direct
Proteome profiling
RNA profiling
External factors
Weaknesses
• Low throughput
• Specialised equipment and skill
required
• Informal if no specific parameters
identified
• Special equipment and knowledge
of particular enzymes required
• Biochemical changes detectable
must be known
• Baseline date rapidly collectable • Biological significance of changes
must be established
• Expensive
• Standardised conditions required
• Expensive
• Baseline date
• Standardised culture and stage
• Rapid collection
conditions needed
• Biological significance of changes
must be established
Opportunities
Threats
• None
• Altered profiles not necessarily
indicate novel characteristics
• Suite of enzymes for routine
baseline data collection
determinable
• Suite of relevant traits that reflect
changes should be expanded
• Suite of proteins should be
determined for routine collection
• None
• A suite of RNAs should be
determined for routine collection
• Specific knowledge on
significance of changes needed
192
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. • None
• Specific knowledge on
significance of changes needed
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 12: Abiotic stress resistance
Method
Bioassay
Analysis of stress
response gene profiles
Internal factors
Strengths
External factors
Weaknesses
• Standardised conditions needed
• Simple and direct
• Insects must be produced under the
• Repeatedly performable
same conditions
• Highly credible
• Standard sets of genes assayable • Expertise and special equipment
needed
• Standardised condition for
production of material
Opportunities
Threats
• Suite of standardised assays should • Not suitable as proxy for
be developed
performance if not performed
under realistic conditions
• Could indicate stresses during
• Interpretation of deviations
production
could be speculative
193
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 13: Vector competence
Method
Internal factors
Strengths
Gene expression
profiles
• Deviations in expressions of
immune-response genes
targetable
Pathogen infection
response
• Not all components of the full
transmission cycle required
Transmission bioassay
• Accurate and meaningful
External factors
Weaknesses
• Changes in gene expression not
definitive indicator of changes in
vector competence
• Expensive
• Highly trained personnel and
equipment needed
• Infected animals required
• Range of possible diseases difficult
to determine
• Special facilities and highly trained
staff needed
• Infected animals required
• Range of possible diseases difficult
to determine
• Special facilities and highly trained
staff needed
Opportunities
Threats
• Standardised arrays and methods
developable
• If only males are released,
changes that are relevant in
females may be misconstrued
• Can become more precise and
informative if molecular
components of infection
determined
• Membrane feeding assays with
infected blood may serve as proxy
for transmission potential
• Expanded use of model systems
likely
• Use of animals expensive and
socially undesirable
194
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. • Use of animals expensive and
socially undesirable
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 14: Arthropod density
Internal factors
Method
Strengths
External factors
Weaknesses
• Laboratory examination necessary if • None
similar-appearing species present
• Laboratory examination necessary if • None
similar-appearing species present
Breteau index
• Direct indicator
Pupal index
• Direct indicator
• Better than indices based on
larvae
• Laboratory examination necessary if
• Direct indicator
similar-appearing species present
• Specific for
anthrophilic/endophagic species • Time consuming and intrusive
• Effective traps not available for all
• Highly effective
species
• Standardised
• Travelling required
• Attractive lures often not known or
not optimised
• Specificity compromised by
• Sensitive
presence of other blood-feeding
• Anti-salvia antibodies as
insects
markers
• Travelling required
• Direct and sensitive
• For myiasis causing insects
House index
Trapping of wild and
released arthropods
Human serology
Screening of infested
animals
Fruit sampling
(infestation rate/fruit
damage)
• Direct
Opportunities
• Travelling required
Threats
• None
• None
• Standard indoor resting boxed
could be devised
• Permissions for inspection
needed
• New traps developable
• Automated data collection and
transmission desirable
• Low sensitivity by low
arthropod density
• Development of serological
markers feasible
• None
• Advanced traps (odour, shape,
colour)
• No differentiation between
target species and related
• Lack of specificity
• No differentiation between
target species and related
• Lack of specificity
• Devices detecting changes
automatically would make
detection more rapid
195
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 15: Migration behaviour
Internal factors
Method
Strengths
Opportunities
Threats
• Time consuming
• Transgenic markers when available
• Data highly variable
• Marking often damage released
insects
• Specialised staff and DNA sequence • DNA sequence data becoming less
expensive and rapid
analysis required
• For population suppression data are • Indicators of incompatibility
doubtful
interfering with programme
success
• Less useful for older arthropods
• High throughput detection systems
would increase usefulness
Mark-release -recapture • Reliable
• Biological meaningful
Population genetic
analysis
External factors
Weaknesses
• Sensitive
Rare element detection • Sensitive
in eggs of treated adults • Causes little harm
• Can be used in natural mosquito
larval sites
Reinfestation rate of
• Few data exists
• Reflects invasive character
pest-free areas
• Data collection by creating small
uninfested zones artificially
• Requires open field release
• Locating sites may be
problematic
• None
• None
• Requires naturally uninfested
areas or areas uninhabited by
humans
Table 16: Habitat interactions
Method
Internal factors
Strengths
Field observations
• Direct
• Easily understood
Analysis of range of
habitats occupied
• No need for field studies
• Utilises existing information
External factors
Weaknesses
• Must be carefully conducted
• Quantifiable interactions must be
predetermined
• Specific characteristics relevant
among occupied habitats may not
have been identified
Opportunities
Threats
• Cataloguing relevant behaviours
and interaction
• None
• Information about changes in
habitat occupancy over time
• Habitat range chosen may be
too narrow to reflect actual
potential distribution.
196
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 17: Climate interactions
Internal factors
Method
Assessment of seasonal
arthropod abundance
Analysis of range of
habitats occupied
Laboratory measures of
life table changes
Strengths
External factors
Weaknesses
• Data for specific locales often not
• Provides clear relationships
between abundance and climate available
• Provides data for potential
distribution, abundance, spread
• Easily conducted
• Identifies some relevant
responses
• Model based
• Predictions uncertain
• Production standards necessary
• May not reflect or induce
physiological adaptations
Opportunities
Threats
• Data on climate change
• None
• Higher resolution data by
modelling European distributions
• Greater uncertainties due to
climate change
• None
• None
Table 18: Food interactions
Method
Internal factors
Strengths
External factors
Weaknesses
Host and diet preference • Most proximate measures when • Changes in opportunistic species
difficult to detect
observed in natural settings
Laboratory bioassays
Habitat occupancy
Gut content analysis
• Easily conducted
• Identification of some
preferences
• Essential components of diet
identifiable
• Data may be pre-existing
• Identification of dietary
components
• Natural food choices may not be
known or available
• Reduced usefulness due to
insufficient knowledge of habitat
and nature
• Changes difficult to detect in
opportunistic feeders
Opportunities
Threats
• Improved trapping by knowledge
of high value diet
• Shift in host/diet choice
impossible to detect prior to
release
• Development of standard bioassays • None
• Differences between and among
species could be catalogued
• None
• None
• None
197
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. • None
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 19: Altered host range
Method
Field observations
Vertebrate attraction
assays
Blood meal source
analysis
Survey of host plants
Internal factors
Strengths
External factors
Weaknesses
• Obtaining quantitative information
• Most proximate and direct
very demanding
measure
• Safe and controllable surrogate • Humans and animals must be
• Reflects biologically significant available
characters
• Changes must be considered also in
the light of other factors
• Biochemical assay equipment
• Good method of high
required
significance
• Identification of attractancy and • Field collected mosquitoes may be
difficult to obtain
availability of hosts
• Doubtful usefulness for changes in
• Valuable direct indicator
GM arthropods
Opportunities
Threats
• None
• None
• Standardised catalogue could be
developed
• Results more sensitively and
reproducibly
• Old methods lack specificity
• Changes could be misinterpreted
if not considered in context of
natural hosts
• None
• Experimental confirmation may
be required
• None
Table 20: Sensitivity to insect pathogens
Method
Bioassay
Analysis of immuneresponse gene
expression levels
Internal factors
Strengths
• Easily performed
• Quantitatively
• Few standard agents required
• Deviations in expression could
be targeted
• Provides standardisable set
External factors
Weaknesses
• Array of agents limited
Opportunities
Threats
• Standardised array of agents should • None
be chosen
• Expensive
• Standardised array and methods
developable
• Trained personnel and equipment
required
• Changes in gene expression no
definitive indicator of susceptibility
changes
198
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. • None
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Table 21: Predator interactions
Internal factors
Method
Predator gut content
analysis
Field observations
Strengths
External factors
Weaknesses
• Requires expertise
• Direct measure
• Indicates importance of specific • Animals are sacrificed
prey species
• Difficult and time consuming
• Reliable
• Data may be incomplete
Opportunities
Threats
• Knowledge of key species
important
• Trapping and sacrificing
animals may be prohibited
• None
• Impact on predators remains
partially unknown
Table 22: Insecticide resistance
Method
Internal factors
Strengths
Allele analysis
• Direct measure
Biochemical assay
• General method
• Can detect changes in enzyme
levels
• Direct measure
• Quick data collection on large
numbers of individuals
Bioassay
External factors
Weaknesses
Opportunities
• Not all alleles known
• Mainstay for strain comparisons
• Some resistance mechanisms not
detectable
• Expert personnel and DNA
sequencing equipment / PCR needed
• Relationship enzyme level to
• None
resistance must be established for
each species
• Standardised production required
• None
199
The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out
exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following
a tender procedure. The present document is published complying with the transparency principle to which the European Food Safety
Authority is subject. It may not be considered as an output adopted by EFSA. EFSA reserves its rights, view and position as regards the
issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. Threats
• None
• None
• None
Defining Environmental Risk Assessment Criteria for Genetically Modified Insects to
be placed on the EU Market
Abbreviations
Bti
Bacillus thuringiensis israelensis
ERA
Environmental risk assessment
EU
European Union
GM
Genetically modified
GMO
Genetically modified organism
GSS
Genetic sexing strain
HEG
Homoendonuclease gene
RA
Risk assessment
RIDL
Release of insects carrying a dominant lethal
RNAi
RNA interference
SIT
Sterile insect technique
TE
Transposable element
200