1
IAEA-D41023-CR-3
LIMITED DISTRIBUTION
WORKING MATERIAL
RESOLUTION OF CRYPTIC SPECIES COMPLEXES OF
TEPHRITID PESTS TO OVERCOME CONSTRAINTS TO SIT
APPLICATION AND INTERNATIONAL TRADE
THIRD RESEARCH COORDINATION MEETING OF A
FAO/IAEA COORDINATED RESEARCH PROJECT HELD IN
TUCUMAN, ARGENTINA FROM 26-31 AUGUST 2013
Reproduced by the IAEA
Vienna, Austria 2014
__________________________________________________________________________
NOTE
Material in this document has been supplied by the authors and has not been edited by the
IAEA. The views expressed remain the responsibility of the named authors and do not
necessarily reflect those of the government of the designating Member State(s). In
particular, neither the IAEA nor any other organization or body sponsoring the meeting
can be held responsible for any material reproduced in this document.
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Table of Contents
A.
B.
C.
D.
E.
F.
G.
Background Situation Analysis ................................................................................... 3
The Co-ordinated Research Project (CRP) .................................................................. 4
Report for the 3rd RCM (Tucuman 2013) ................................................................... 5
Conclusions on Current Status and Recommended Future Activities for the CRP
Participants .................................................................................................... 10
Anastrepha fraterculus Complex............................................................................... 10
Background Situation Analysis ..................................................................... 10
Baseline and Progress on Anastrepha fraterculus ......................................... 12
Specific Progress and Individual Research Plans: Anastrepha fraterculus
Complex......................................................................................................... 19
Bactrocera cucurbitae ............................................................................................... 30
Background Situation Analysis ..................................................................... 30
Baseline and Progress on Bactrocera cucurbitae .......................................... 30
Specific Progress and Individual Research Plans: Bactrocera cucurbitae.... 33
TAXONOMY ................................................................................................ 33
Bactrocera dorsalis Complex .................................................................................... 36
Background Situation Analysis ..................................................................... 36
Baseline Knowledge on Five Priority Species in the Bactrocera dorsalis
Complex......................................................................................................... 37
Specific Progress and Individual Research Plans .......................................... 45
Ceratitis FAR Complex .............................................................................................. 57
Background Situation Analysis ..................................................................... 57
Baseline and Progress on the Ceratitis FAR Complex.................................. 57
Specific Progress and Individual Research Plans: Ceratitis FAR Complex . 62
Logical Framework .................................................................................................... 67
Workshop on “Reviewing Evidence to Resolve Species Complexes of Tephritid
Pests” (31. August 2013) ............................................................................... 77
Appendices................................................................................................................. 96
Appendix 1: 3rd RCM Participants ............................................................................ 97
Appendix 2: Agenda ................................................................................................ 108
Appendix 3: Abstracts 3rd RCM Tucuman ............................................................. 113
Appendix 4: Working Groups ................................................................................. 147
Appendix 5: Protocols for Collection and Shipment of Live and Dead Insects for
Vouchering, Rearing, Morphology, Morphometrics, Chemical Ecology
and Molecular Assays ......................................................................... 148
Appendix 6: Guidelines for Performing Mating Compatibility Field Cage Test ... 151
Appendix 7: Male Sex Pheromonal Components in Bactrocera Species – Extraction
of Rectal (Pheromonal) Gland for Chemical Analyses ...................... 163
Appendix 8: List of References .............................................................................. 169
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RESOLUTION OF CRYPTIC SPECIES COMPLEXES OF TEPHRITID PESTS TO
OVERCOME CONSTRAINTS TO SIT APPLICATION AND INTERNATIONAL
TRADE
A. Background Situation Analysis
Tephritids are among the worst pests in agriculture and are of major economic importance in
nearly all tropical, subtropical and temperate countries worldwide. These pest species cause
enormous devastation to both food production and international trade and are major targets of
insecticide applications. They are among primary causes of poverty, malnutrition and poor
production and trade in fresh horticultural commodities in large areas of tropical developing
countries (Waterhouse 1993; Allwood & Leblanc 1996; Lomborg 2004), where climatic
conditions are favourable for labour-intensive fruit and vegetable-based agroindustries.
The study of the biology and management of tephritids therefore requires significant
international attention to overcome these transboundary hurdles and to assist Member States
in developing and validating environment-friendly suppression systems to support viable
fresh fruit and vegetable production and export industries. Such international attention has
resulted in the successful development and validation of a Sterile Insect Technique (SIT)
package for Mediterranean fruit fly. Demands for R&D support for this pest species are
diminishing due to successful integration into control programmes. There is now an urgent
need to focus on the increasing demands from Member States in Africa, Asia and Latin
America to address other major tephritid species or groups of economic importance. These are
groups of species where the morphology is very similar or identical but biologically they are
distinct. Some major pest fruit fly species occur within complexes, and the uncertainty related
to their questionable technical status results in major constraints to SIT application and
international trade.
Problem to be addressed:
Considerable concern has been expressed by many Member States that some major tephritid
pest species complexes include taxonomically described species that are actually only
geographical variants of the same species. Conversely, some fruit fly populations grouped
taxonomically within the same pest species display different biological and genetic traits and
show reproductive isolation which suggest that they are different species. This uncertain
taxonomic status is having important practical implications on the effective development and
use of the SIT against such complexes with respect to the rearing of the correct species and
significantly affects international movement of fruit and vegetables, resulting in the
establishment of trade barriers to important agricultural commodities which are hosts to pest
tephritid species. Moreover, this problematic situation appears to occur in four of the most
important pest genera within the Tephritidae family: Anastrepha, Bactrocera, Ceratitis and
Rhagoletis.
Regional insect rearing facilities that would produce sterile males for use in different
countries or regions are desirable as this would make the SIT more cost-effective. It is of
prime importance therefore to the success of these regional area-wide integrated pest
management (AW-IPM) programmes that have an SIT component that the mass-reared flies
from such species complexes as Anastrepha and Bactrocera, etc. are behaviourally
compatible with the target native fruit fly pest populations in the various recipient regions. If
4
species complexes remain unresolved then these desirable outcomes will be difficult or
impossible to achieve. The resolution of some of the major cryptic species complexes (i.e.
insects whose specific status is questionable) is therefore critical both for SIT application and
to assist subtropical and tropical Member States to overcome non-tariff trade barriers in order
to export their fresh fruit and vegetable commodities to international high value markets.
A ranking of the major fruit fly pest complexes with respect to the economic damage and
regional impacts indicates that a number of them are of concern to Member States. They are:

Known complexes
o Anastrepha fraterculus
o Bactrocera dorsalis
o Ceratitis anonae, C. fasciventris, C. rosa

Suspected complex
o Bactrocera cucurbitae
A Consultants’ Meeting, held from 6 to 10 July 2009 in Vienna, Austria, discussed the
complexes noted above and related technical issues and prioritised them as to economic
importance and potential for SIT application. The three complexes and the suspected one
were confirmed to be of significant importance that needed to be resolved to facilitate world
agricultural trade and SIT programmes.
B. The Co-ordinated Research Project (CRP)
This Co-ordinated Research Project (CRP) is based on the mentioned Consultants Meeting
that reviewed the background and scientific progress made in this area, assessed the potential
for conducting co-ordinated R&D and formulated a proposal for a CRP on “Resolution of
Cryptic Species Complexes of Tephritid Pests to Overcome Constraints to SIT Application
and International Trade”.
The overall objective of this new CRP D4.10.23, approved for the period 2010-2015, is to
assist Member States in overcoming constraints to SIT application against fruit flies and in
facilitating international trade through the resolution of cryptic species complexes of major
tephritid pests.
The specific objectives are:
a) to define the species limits within the target complexes, and
b) to develop robust species-specific diagnostic tools.
The main targets are the Anastrepha fraterculus (Latin America), Bactrocera dorsalis (Asia &
Pacific, Africa), and Ceratitis anonae, C. fasciventris C. rosa (Africa) species complexes and
with lower priority a possible Bactrocera cucurbitae (Asia & Pacific, Africa) complex. In
each of these groups there are questions concerning either the validity of some of the species
or their capacity to be diagnosed.
This international network research project is operated as part of the IAEA Research Contract
Programme. The new FAO/IAEA CRP D4.10.23 includes 22 research teams on all
continents, of which 15 are research contract holders and 7 are agreement holders. In addition
several other research teams are participating fully funded by their institutions and
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governments. The duration of the CRP is 5 years and Research Co-ordination Meetings
(RCMs) are held every 18 months.
These CRPs and RCMs have the objective to encourage networking and close collaboration
among institutions in developed and developing countries, and to provide a forum for
information exchange between scientists and a focused approach to the development, capacity
building and technology transfer of environment-friendly technologies.
The first RCM for this project was held in Vienna from 2 to 6 August 2010 and the second
RCM was held at the EcoCentre of Griffith University in Brisbane, Australia from 31st
January to 3 February 2012 (reports available).
C.
Report for the 3rd RCM (Tucuman 2013)
Objectives of the Meeting
The objectives of the third RCM were as follows:
a) to present, discuss and review the results of the research carried out by participating
researchers since the 2nd RCM;
b) to co-ordinate and harmonize the processing and analysis of the experimental results;
c) to discuss the next activities and agree on protocols for the final phase of the CRP;
d) to prepare work plans for each participating institute for the final phase of the CRP;
e) to co-ordinate the training and other needs and provision of material over the next 18
months; and
f) to strengthen research and administrative linkages between research groups and
institutions.
The meeting was attended by 52 scientists and observers from 18 countries. The list of 3rd
RCM participants is given in Appendix 1. The meeting was formally opened by the host,
Teresa Vera of Universidad Nacional de Tucumán, who welcomed the participants and
described the logistics and organisational aspects. Jorge Hendrichs, as Scientific Secretary of
the CRP, explaines the objectives of the meeting, the importance of resolving the issues that
surround the existence of cryptic Tephritid species complexes and reviewed the agenda of the
3rd RCM and of the workshop on the final day with an external panel of experts on
“Reviewing Evidence to Resolve Species Complexes of Tephritid Pests” (see Section F).
The agenda that was prepared for the RCM (Appendix 2) was followed with only minor
changes. The progress reports of the participants covered the results they obtained during the
18 month since the 2nd RCM of the CRP. The abstracts of these presentations are given in
Appendix 3.
Between the 3rd and 5th days of the RCM participants were divided into three separate
working groups (Appendix 4) to review overall achievements based on original coordinated
research plans, to assess results and scientific/technical conclusions for the different genera,
thematic areas/approaches and techniques, and to review research needs to be addressed and
other activities to be implemented. Also the progress made by the individual research teams
was evaluated and research proposals/plans for the final 18 months discussed for the different
genera and approaches/thematic areas. In addition, cross-cutting research and the scientific
and administrative linkages between participating institutes were detailed.
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Protocols for collection and shipment of live and dead insects for rearing, morphology,
chemical biology and molecular assays and procedures for field cage tests (Appendices 5, 6,
and 7) were updated. Relevant references are presented in Appendix 8.
Working Groups
The three working groups that were established based on pest fruit fly groups being worked
on were:
Group 1: Anastrepha fraterculus (possibly five to seven distinct pest species)
Group 2: Bactrocera dorsalis pest species complex (B. dorsalis s.s., B. carambolae, B.
papayae, B. philippinensis and B. invadens, some of which are not distinct species)
Group 3: Bactrocera cucurbitae (possibly different populations) and the Ceratitis FAR pest
species complex (comprising at least three species: C. fasciventris, C. anonae and C. rosa).
Group participants firstly reviewed the progress made since the 2nd RCM in addressing the
identified gaps in each thematic area in relation to the baseline knowledge determined at the
start of the CRP. In addition to this, participants in each group also provided each research
group’s specific progress made since the 2nd RCM and the specific work plans for the final 18
months of the CRP.
Conclusions of the three Working Groups are reported in Section D. A summary of thematic
areas in relation to the species complexes being addressed by researchers is presented in
Table 1. Given that a number of researchers and institutions are involved in various subprojects within the CRP, Table 2 was updated to facilitate identifying these project linkages
and interactions.
Working Groups then reviewed their inputs into the Logistic Framework (Table 3) to ensure
that targets previously set were reasonable and feasible both technically and with respect to
the duration of the CRP. This is reported in Section E.
After these tasks were completed all Working Groups rejoined to present and discuss any
changes and new inputs and agreed on the content on a draft Working Material for the 3rd
RCM of this CRP.
During the final day (August 31st 2013) a workshop was held with an external panel of
independent experts to review all the evidence collected so far during the CRP to resolve
species complexes of tephritid pests. The resulting summaries and character matrices for each
of the species complexes are presented in Section F.
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Table 1. Thematic areas in relation to Tephritid species complexes and suspected complexes
being addressed by researchers
Thematic area
Complex
Molecular
Anastrepha Canal
fraterculus Castañeda
Cladera
De Lima
Drosopoulou
Malacrida
Passos Roriz
Selivon
Silva
Steck
Vilardi
Zacharopoulou
Bactrocera Aketarawong
Armstrong
dorsalis
Augustinos
Barr
Drosopoulou
Hasanuzzaman
Haymer
Khamis
Malacrida
Thanapum
Yesmin
Zacharopoulou
Ceratitis
FAR
complex
Bactrocera
cucurbitae
Delatte
Haymer
Khamis
Quilici
Virgilio
Delatte
Malacrida
Virgilio
Zacharopoulou
Behaviour
Chemical
Ecology
Taxonomy /
Morphology
Abraham
Dias
Joachim-Bravo
Kovaleski
Mendonça
Nascimento
Paranhos
Rull
Vera
Brizova
Mendonça
Nascimento
Vanickova
Segura
Teal
Bartolucci
Canal
Hernández-Ortíz
Selivon
Steck
Caceres
Amornsak
Chinvinijkul
Clarke
Ekesi
Haq
Hendrichs
Hee
Ji
Schutze
Shelly
Srikacher
Tan
Chinvinijkul
Hee
Kalinova
Tan
Teal
Wee
Chen
Clarke
Drew
Jessup
Ji
Leblanc
Romig
Steck
Schutze
Tsuruta
de Meyer
Ekesi
Mwatawala
Quilici
Kalinova
de Meyer
Steck
Delatte
Ekesi
Haq
Mwatawala
Quilici
-
de Meyer
Leblanc
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Table 2. Project linkages
Thematic area
Participant
Country
Aketarawong/Sujinda
Thailand
Armstrong
New
Zealand
Bravo/Diaz/Beatriz
Canal/Castañeda
Clarke/Schutze
Brazil
Colombia
Australia
de Meyer/Virgilio
Belgium
Delatte/Quilici
Reunion
do Nascimento /
Dias/Adriana/Beatriz
Drew/Romig
Brazil
Australia
Ekesi / Khamis /
Mohamed
Kenya
Ekesi / Khamis /
Mohamed
Kenya
Hee/Wee/Nishida/Tan
Malaysia
Malacrida, Gasperi
Mwatawala
Mwatawala
Hernandez
Qinge
Silva
Italy
Tanzania
Tanzania
Mexico
China
Brazil
Steck
USA
Suksom/Sunynee
Thailand
Tan/Hee/Wee
Malaysia
DNA
Cytology
Genomics
Taxonomy,
Morphology/metrics
Behaviour
(pre,post
Zygotic)
Pheromones/Hydrocarbons
Host
range
Distribution
9
Vanickova/Kalinova
Czech
Vanickova/Kalinova
Vera
Czech
Argentina
Zacharopolou
Barr
Barr
Greece
USA
USA
Seibersdorf
FAO/IAEA
Bactrocera cucurbitae
Ceratitis FAR complex
Anastrepha fraterculus
Bactrocera dorsalis complex
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D.
Conclusions on Current Status and Recommended Future Activities for the CRP
Participants
Anastrepha fraterculus Complex
Background Situation Analysis
The South American fruit fly, Anastrepha fraterculus (Wiedemann) s.l. is present in most
countries of the Americas from the USA to Argentina (Hernández & Aluja 1993, Steck 1999,
Zucchi 2000, 2007). On the South American subcontinent, this species is found in two broad,
possibly unconnected bands: one along the western edge, including both highland and
lowland areas of the Andean range, and the other along the east coast. However, there are
recent data of its presence in parts of the Brazilian Amazon basin (Zucchi et al. 2011). Its
centre of diversity is South America and it has been reported to infest about 80 host plants
including major fruit crops (Norrbom & Kim 1988, Norrbom 2004, Zucchi 2008). This highly
destructive pest imposes quarantine restrictions for fruit export to many countries (Steck
1999).
The high levels of variability found among different populations throughout the geographical
range of A. fraterculus have led to the conclusion that it is a complex of cryptic species rather
than a single biological entity (Stone 1942, Morgante et al. 1980, Solferini & Morgante 1987,
Malavasi & Morgante 1982, Steck 1991, Steck & Sheppard 1993, Selivon 1996, HernándezOrtíz et al. 2004, 2012). Differences have been reported based mainly on morphology, pest
status and genetics (including karyotype, isozyme and molecular analyses); these are
reviewed in Steck (1999) and some aspects discussed in subsequent studies (McPheron et al.
1999, Norrbom et al. 1999, Silva 2000, Smith-Caldas et al. 2001, Hernández-Ortíz et al.
2004, Selivon et al. 2005, Goday et al. 2006, Prezotto 2008, Cáceres et al. 2009, Barr et al.
2005, Silva & Barr 2008, Selivon & Perondini 2007, Ludeña et al. 2010). However, in order
to establish the existence of different species, it is necessary to systematically correlate these
genetic and morphological differences with the existence of reproductive isolation and other
life history related traits (hosts, demography, etc).
Reproductive incompatibility has been reported both at pre- and post-zygotic levels (Selivon
1996, Vera et al. 2006, Cáceres et al. 2009) among some populations. At the pre-zygotic
level, mating compatibility was evaluated among six different populations from Mexico,
South America, involving lowland (Peru) and highland (Colombia) areas from the Andean
region, and the south-eastern part of the continent (Brazil and Argentina). Most of the
populations were shown to have some level of incompatibility with each other and thus
sexually isolated. Flies were sexually active at different times of the day suggesting different
sexual behaviour. Hybrid females tended to mate with hybrid males and as a result Segura et
al. (2011) suggested that hybridization is a possible speciation mechanism.
Post-zygotic studies between two populations from Brazil (Selivon et al. 1999) and between
one Argentine population and one Peruvian population (Cáceres et al. 2009) found partial
hybrid inviability and sex ratio distortion confirming the existence of post-zygotic barriers. In
the former case, cytological, isozyme and molecular studies revealed differences among
groups (Malavasi & Morgante 1982, Selivon 1996, Selivon et al. 2005, Goday et al. 2006);
while for the latter case, differences between groups were also found in terms of male sex
pheromones and karyotypes (Cáceres et al. 2009).
11
The combined results of these studies (see incomplete table below to be further developed and
refined with increasing evidence) suggest the existence of at least seven different biological
entities which in this document are referred to as: A. sp. 1 aff. fraterculus (Yamada & Selivon
2001), A. sp. 2 aff. fraterculus (Yamada & Selivon 2001), A. sp. 3 aff. fraterculus (Selivon et
al. 2004), A. sp. 4 aff. fraterculus (Selivon et al. 2004), A. fraterculus Mexican morphotype
(Hernández-Ortíz et al. 2004) and A. fraterculus from Andean highlands and Venezuela
coastal lowlands (Steck 1991). A recent comprehensive morphological study supports the
existence of these seven morphotypes (Hernández-Ortíz et al. 2012).
Morphotype
(according to
Hernández-Ortiz)
Brazilian 1
Brazilian 2
Brazilian 3
Peruvian
Andean
Venezuelan
Mexican
Population/s
Tucuman, Posadas,
Concordia (all from
Argentina), Vacaria,
Sao
Joaquim,
Pelotas, Piracicaba
(all from Brazil)
Brazil
Reference
Comments
Yamada & Selivon A.
sp.
1
aff.
2001
fraterculus
sensu
Hernández-Ortiz
Selivon
2012
Yamada & Selivon A.
sp.
2
aff.
2001
fraterculus
sensu
Selivon
Sympatric
morphotypes?
Brazil
Selivon et al. 2004
A.
sp.
3
aff.
fraterculus
sensu
Selivon
Sympatric
morphotypes?
Peru, Ecuador
Selivon et al. 2004
A.
sp.
4
aff.
fraterculus
sensu
Selivon
Sympatric
morphotypes?
Colombia highlands
Steck 1991
Sympatric
Hernández-Ortíz et morphotypes?
al. 2012
Venezuela lowlands
Steck 1991
Hernández-Ortíz et
al. 2012
Mexico and Central Hernández-Ortíz et
America
al. 2004
Hernández-Ortiz et
al. 2012
Despite previous studies, which have provided strong evidence supporting the existence of
several species, there are still major knowledge gaps (see below). Moreover, the described
studies have used different methodologies, have not used the same identified biological
material, and most importantly, they did not include all of the morphotypes. In order to be
able to formally describe and name these putative species, it is necessary to apply a complete
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set of methodologies to all of them in a comprehensive study involving populations from the
entire geographic distribution range. This will allow the characterization of each putative
species and will provide sound diagnostic tools for addressing the related management and
trade issues.
Definition of species limits and formal naming of these putative species will be critical for
importing authorities in determining which of them may or may not be quarantine pests. This
will immediately allow some countries to gain access to international fresh fruit markets for
those countries and commodities which can be determined to be outside the geographic and
host range of correctly delimited A fraterculus s.s. Implementation of the SIT also requires a
thorough understanding of species boundaries and sexual compatibility.
Baseline and Progress on Anastrepha fraterculus
DNA
- Knowledge at the start of the CRP:
 Only three gene regions have been studied for representatives of A. fraterculus s.l.
populations. These are the COI, 16S, and period genes. COI and period genes are most
useful in terms of inter-population variation. Data is available for only 24 specimens
from only 16 populations representing five of the above mentioned seven morphotypes
(see Table below):
Number of specimens examined for each morphotype and gene region
Morphotype / biological
entities from:
COI
16S
Period
SC - 2
ES - 1
BA -1
SP - 2
MG -1
ARG - 1
-
-
SP - 1
-
-
3
-
2
Andean
3
1
4
Venezuelan lowland
1
-
1
Brazil, Argentina, other
countries?*
Peruvian (=A. sp. 4 aff
fraterculus of Selivon)
Mexican
* For some populations there are still no data that confirm the morphotype. Therefore it is advisable to confirm
all those populations that are used for behavior, pheromones or other studies.
- Gaps identified at the start of the CRP:
 There are no data yet for several of the morphotypes.
 The total number of specimens analyzed so far is small.
 Microsatellite data are not yet available.
 Other molecular markers have not been applied.
13
- Progress up to the 3rd RCM in addressing identified gaps:
 Extensive sampling was performed in Colombia (9 populations), Peru (2 population),
Bolivia (1 population), Panama (1 population) and Brazil (34 populations) which
resulted in the collection and preservation of more than 350 individuals.
 Samples from the colonies established at Seibersdorf were also preserved for DNA
extraction.
 DNA was extracted and voucher specimens preserved. CO I data was obtained from
Brazil (34 populations), Mexico (one population) and Argentina (one population); CO
I, CO II and ITS I were screened in Colombia (9 populations); ITS I was screened in
Bolivia (1 population), Peru (2 populations), Venezuela (2 populations) and Brazil (1
population).
 Mitochondrial markers (CO I, CO II, ITS-I) were used to study 9 Colombian
populations (5 individuals/pop, 45 total) and compared with individuals from Ecuador,
Brazil, Venezuela, Mexico. Eight of the Colombian populations appeared highly
related to each other, but individuals from Putumayo (south near to Ecuador) appeared
to be the most differentiated and related with an Ecuadorian population.
 Sequencing of Y chromosome is under progress.
 Populations from Ecuador were also analysed (Ludeña et al. 2010).
 Eighteen microsatellite loci sequences were isolated.
 15 SSR easy to score were validated and characterized for their PIC (polymorphic
information content) in two strains (Vienna, Tucuman) and two populations from
Argentina (Concordia, Puerto Yerúa).
 Some of these microsatellites are being used for the analysis of six Brazilian
populations (from Northeast to South).
 90 ISSR loci were developed. Fourteen of them were used to detect host
differentiation between synchronic and sympatric samples from peach, guava and
walnut in Tucuman region. Strong differences were observed between flies collected
from peach and walnut.
 Mitochondrial CO I markers have been used to assess the phylogenetic relationships
between 244 individuals collected from 34 Brazilian populations (collected across the
country from Northeast to South) and from Mexico (Xalapa) and Argentina
(Tucuman). A total of 43 haplotypes have been identified which can be clustered in
seven haplogroups. The geographic distribution of haplogroups is not homogeneous.
CYTOLOGY
- Knowledge at the start of the CRP:
 Karyotypes have been described for at least 24 species of Anastrepha.
 These include the following morphotypes of A. fraterculus s.l.: Mexican, A. sp. 1 aff
fraterculus (Argentina, Brazil), A. sp. 2 aff fraterculus (Brazil), A. sp. 3 aff fraterculus
(Brazil), and A. sp. 4 aff fraterculus (Peruvian morphotype from Peru and Ecuador).
 Polytene chromosomes were used to compare Argentine and Peruvian populations.
- Gaps identified:
 Polytene chromosome banding patterns not yet described.
 No karyotype data available for Andean and Venezuela lowland morphotypes.
- Progress up to the 3rd RCM in addressing identified gaps:
 Polytene chromosome banding patterns was described for A. sp. 1 aff fraterculus from
Argentina (Giardini et al. 2009).
 Karyotypes from 8 populations from Colombia (Andean morphotype) were described.
14
ALLOZYMES
- Knowledge at the start of the CRP:
 Extensive data on allozyme variation are available for various populations of the
following morphotypes: Mexican, Andean, Peruvian, Venezuelan lowland, A. sp. 1 aff
fraterculus (Argentina, Brazil) and A. sp. 2 aff fraterculus (Brazil).
- Gaps identified:
 As data are not directly comparable between the various studies performed in different
laboratories, it is not a priority to gather new isozyme data.
- Progress up to the 3rd RCM in addressing identified gaps:
 No work conducted as not priority.
MORPHOLOGY
- Knowledge at the start of the CRP:
 Published morphometric data indicate that at least three morphotypes can be
recognized in continental America (“Mexican”, “Andean” and “A. sp. 1 aff
fraterculus”), based on discriminant function analysis of particular characters from the
aculeus, wing and mesonotum (Hernández-Ortíz et al. 2004).
 Unpublished data (Hernández-Ortíz et al. in prep.) suggest the presence of four
additional morphotypes called “Venezuelan”, “Peruvian” 4 aff fraterculus (Peru and
Ecuador), “A. sp. 2 aff fraterculus” (Brazil) and “A. sp. 3 aff fraterculus”(Brazil).
 Additional morphometric data using aculeus and wing characters show differences
between the types A. sp. 1 aff fraterculus (Brazil) and A. sp. 2 aff fraterculus (Brazil).
 Egg morphological differences have been described between the three Brazilian
morphotyes (Selivon & Perondini 1998, Selivon et al. 2004).
 Only preliminary descriptions of larval stages are available (Steck et al.1990, Frias et
al. 2006).
- Gaps identified:
 Formal taxonomic descriptions of Mexican, Andean, and “A. sp. 1 aff fraterculus”
(Brazil) morphotypes are not yet published.
 None of the larval stages has been thoroughly described and compared among
morphotypes.
 Egg morphology is unknown for four morphotypes (Andean, Venezuelan, Mexican
and Peruvian).
- Progress up to the 3rd RCM in addressing identified gaps:
 A study including the seven adult morphotypes using a multivariate approach was
published (Hernandez-Ortiz et al., 2012).
 A morphometrics analysis involving 18 populations from Ecuador and Colombia were
carried out. An 8th morphotype was recognized in Ecuador. Eggs were sent to Denise
Selivon for description.
 SEM observations on third instar larvae have been made on five populations
representing three morphotypes (Mexican, Peruvian and A. sp. 1 aff fraterculus).
 A morphometric study of the mouthparts of third instar larvae from the Andean
morphotype was carried out.
 Egg morphology of several Anastrepha species from the fraterculus group was
described (Dutra et al. 2011a).
 Second and third instar larval morphology from some species (Anastrepha bahiensis,
Anastrepha coronilli and Anastrepha turpiniae) from the fraterculus group were
described (Dutra et al., 2012).
15
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The morphometric analysis of several Brazilian populations was initiated to identify
the corresponding morphotype.
New collections of A. fraterculus larvae and adults from Peru and Panama were made.
SEM studies on other members of fraterculus group were performed (A. suspensa and
A. obliqua).
PHEROMONE AND CUTICULAR COMPONENTS / CHEMICAL ECOLOGY
- Knowledge at the start of the CRP:
 Pheromone composition has been described so far for only two populations (A. sp. 1
aff fraterculus, as well as probably A. sp. 4 aff fraterculus from Peru) with different
methodologies.
 Chemicals released by calling males were first investigated by Lima et al. (2001)
using a single population from Pelotas, Rio Grande do Sul, Brazil (morphotype to be
defined). These chemicals were identified as alcohols, mono- and sesquiterpenes and
three isomeric lactones. Later, Caceres et al. (2009) showed that a population from
Argentina differed in its composition from a population from Peru (probably A. sp. 4
aff fraterculus). Preliminary studies carried out on the response elicited by the
pheromone produced by A. fraterculus males from an Argentine laboratory population
have shown that female antennae respond to six out of thirteen compounds identified
as volatiles released by calling males of this population, and these chemicals were
identified as: (Z)-3-nonenol, (E,Z)-3,6-nonadienol, geranyl acetone, alfa-farnesene and
(S,S)-epianastrephin.
 There are few studies concerning hydrocarbon composition in pupae and adults of A.
fraterculus.

Preliminary studies performed by Vanickova et al. (2012) have shown that there are
quantitative and qualitative differences in the cuticular hydrocarbon composition
between virgin males and females depending on age (Argentine laboratory
population).
 Qualitative and quantitative differences were observed in the chemical composition of
cuticular hydrocarbons according to the age of the fly.
- Gaps identified:
 The composition of the pheromone is not known for all morphotypes.
 The function of the identified chemicals either alone or combined in eliciting
attraction of conspecific females towards males is still unknown.
 Antennal response has been studied in only one Argentine population.
 Cuticular hydrocarbon composition is known only from virgin male and females from
one Argentine population and one southern Brazil population.
- Progress up to the 3rd RCM in addressing identified gaps:
 The pheromone volatiles from six Brazilian laboratory F2 populations (Bento
Gonçalves, Alagoas, Piracicaba, Vacaria, Pelotas, Sao Joaquim), one Argentinean
population (Tucuman, Brazilian 1 morphotype), one Peruvian population (Piura + La
Molina Peruvian morphotype), and from hybrids of a cross between the Peruvian and
Argentinean population were characterized.
 The chemical characterization of the pheromone volatiles from one population from
Mexico (Mexican morphotype) and one population from Colombia (Tolima, Andean
morphotype) was initiated and is in progress.
 Cuticular hydrocarbons from males and females from one Argentinean (Tucuman,
Brazilian 1 morphotype), males from four Brazilian population (Bento Gonçalves,
Alagoas, Piracicaba and Vacaria), males and females from one Peruvian population
16
(Piura + La Molina, Peruvian morphotype), males from one population from
Colombia (Tolima, Andean morphotype), and males and females from one population
from Mexico (Mexican morphotype) were characterized.
BEHAVIOUR
- Knowledge at the start of the CRP:
 A. fraterculus s.l. has a lek mating system.
 Mating occurs primarily in the early morning, with some populations showing
different mating times (midday for Peruvian population and late afternoon for
Colombian population (respectively A. sp. 4 aff fraterculus and Andean/Venezuela
Highland morphotypes sensu Vicente 2012).
 Pre-zygotic isolation was detected among populations at the continental level.
 Male courtship has been described for one Argentine population.
 There is some evidence suggesting that populations confined together in field cage
trials form segregated leks.
 For one Argentine population, it has been shown that female post-copulatory
behaviour depends on attributes of the first male suggesting that female decision on
remating depends on the conditions of the first male.
- Gaps identified:
 Mating compatibility has not been evaluated among all morphotypes.
 Mating behaviour has been described for only 3 morphotypes.
 Factors involved in pre-zygotic isolation are unknown (temporal, ecological or sexual
isolation, role of pheromones in mate recognition/acceptance).
 Male courtship, male-male interactions and female responses within and among each
putative species is not completely known.
 The factors that determine lek location should be determined (environmental vs.
avoidance of mixed leks).
 Female post-copulatory behaviour after mating with males from other populations is
unknown.
- Progress up to the 3rd RCM in addressing identified gaps:
 Mating compatibility between the Mexican morphotype and the Peruvian morphotype
as well as between the Mexican morphotype and two populations from Brazil (Vacaria
and Pelotas, presumably Brazilian 1 morphotype) and one population from Argentina
(Brazilian 1 morphotype) was evaluated.
 Mating compatibility evaluations among four populations from southern Brazil (Bento
Gonçalves, Pelotas, Vacaria and São Joaquim) showed that southern populations are
compatible. A detailed behavioural analysis including calling and mounting sound
analysis showed similarity between these populations supporting the mating
compatibility tests.
 Mating compatibility tests between a southeast population from Brazil (Piracicaba)
and three southern populations (Vacaria; São Joaquim and Bento Gonçalves) showed
partial incompatibility. Courtship behaviour analysis showed some differences in the
calling and fanning behaviour of Piracicaba compared with some southern strains.
Sounds analysis showed a higher fundamental frequency in calling (long pulses) and
mounting signals of Piracicaba compared to the southern populations.
 Mating compatibility tests between Parnamirim (a Brazilian northeastern population,
morphotype to be confirmed) and Bento Gonçalves (a Brazilian southern population,
morphotype to be confirmed) showed compatibility between them.
17
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Preliminary results of the sequence of the different components from the courtship
behavior suggested a different pattern in Parnamirim strain (northeast Brazil)
compared with other strains from the south (Bento Gonçalves) and the southeast
(Piracicaba).
High levels of sexual isolation were detected between the Mexican morphotype and
the populations from southern Brazil and Argentina, even though they are sexually
active at the same time (i.e. not temporally isolated).
Mating compatibility tests were performed between males and females of the Andean
morphotype (Colombia, Ibague) and males and females of the Brazil 1 morphotype
(Argentina), the Mexican morphotype (Mexico, Xalapa) or the Peruvian morphotype
(Peru, La Molina). Marked temporal differences in calling activity were detected: the
Andean morphotype mated at dusk, the Mexican and Brazil 1 morphotype early in the
morning, and the Peruvian morphotype around midday.
Mating compatibility tests between two recently established laboratory strains from
Colombia revealed sexual compatibility.
Remating propensity and duration of the refractory period of Argentine and Peruvian
females were different yet independent of male origin; however there was a tendency
for Peruvian females mated with Argentine males not to allow sperm transfer.
DISTRIBUTION
- Knowledge at the start of the CRP:
 A. fraterculus s.l. is widely distributed from southern Texas to northern Argentina in
South America, but the detailed regional patterns for the 7 morphotypes are not well
understood.
 Current unpublished information reveals that the Mexican morphotype extends from
Mexico to Central America; the Venezuelan morphotype occurs only in one area of
the Caribbean lowlands of Venezuela; the Andean morphotype is found in the
Venezuelan and Colombian highlands; while the Peruvian morphotype (A. sp. 4 aff
fraterculus) has been found in at least three localities from Ecuador and Peru.
 The distribution of the three Brazilian types is not well known and some of them occur
in sympatry.
- Gaps identified:
 The detailed distributions of all morphotypes in South America are unknown.
 Elevational transects in Andean countries are lacking and needed to determine limits
of highland and lowland morphotypes.
- Progress up to the 3rd RCM in addressing identified gaps:
 Information was published on the distribution of A. fraterculus morphotypes: Mexican
morphotype extends from Mexico to Central America; the Venezuelan morphotype
occurs only in one area of the Caribbean lowlands of Venezuela; the Andean
morphotype is present in the Venezuelan and Colombian highlands; while the
Peruvian morphotype is found in at least three localities from coastal areas of Ecuador
and Peru (Hernandez-Ortiz et al., 2012).
 Distribution of the Andean morphotype in Colombia was published (Castañeda et al.
2010), with no records below 900 m elevation.
 The extensive sampling performed for DNA studies in Brazil will allow updating the
distribution of the morphotypes found in this country.
 Morphometric studies revealed the presence of two morphotypes in Ecuador: the
Peruvian morphotype in the low lands and a new 8th morphotype (Ecuadorian) in the
Andean Valleys.
18
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Surveys in Peru showed that the fraterculus cryptic species complex occurs in mid-tohigh elevations of eastern Andean valleys of central Peru. ITS I sequence data for
these Peruvian Andean populations and a population from Santa Cruz, Bolivia, match
that of the Brazilian 1 morphotype.
HOST RANGE
- Knowledge at the start of the CRP:
 There are numerous host records available for A. fraterculus s.l., but these have not
been associated with the various morphotypes.
 Host lists have been published for several countries, e.g.,Venezuela (Hernández &
Morales, 2004), Brazil (Zucchi 2007, Zucchi 2008).
- Gaps identified:
 Host ranges for most morphotypes are unknown.
 Update and revision of available host lists to correct errors is needed.
- Progress up to the 3rd RCM in addressing identified gaps:
 In those areas where only one morphotype is present (Mexico and Central America,
highland Venezuela and Colombia, Argentina), the host range is being updated based
on existing records.
 Data on two years survey from different hosts was published for Colombia (Castañeda
et al. 2010) and afterwards expanded with data from entomological collections.
 New host plant information has been obtained in Ecuador and Peru.
POST-ZYGOTIC COMPATIBILITY
- Knowledge at the start of the CRP:
 Crosses between A. sp. 1 aff fraterculus and A. sp 2 aff fraterculus resulted in a
reduction of egg hatch and larval viability and also a distorted sex ratio.
 The same has been observed in crosses between populations from Peru and Argentina.
 It is possible to maintain hybrid lines, including those with their initial sex ratio
distortion maintained, under artificial (laboratory) rearing conditions.
 The occurrence of Wolbachia was confirmed for three populations (Brazil, Argentina,
Peru (morphotypes to be defined).
- Gaps identified:
 The occurrence of hybrid sterility has not been evaluated.
 The role of Wolbachia in post-zygotic incompatibility has not been determined.
- Progress up to the 3rd RCM in addressing identified gaps:
 Compatibility among one population from Argentina (Tucuman, Brazilian 1
morphotype) and two populations from Brazil (Vacaria and Pelotas, morphotypes to
be defined) was demostrated.
 Females of the Mexican morphotype caged with Peruvian or Brazil 1 (Tucuman)
morphotype males either did not lay eggs or laid unviable eggs. Brazil 1 morphotype
females mated to Mexican morphotype males laid eggs that hatched in significantly
lower proportion than those of females mated to homotypic males. The other
heterotypic crosses produced eggs equally viable to those laid by females mated with
homotypic males.
 Colombian morphotype females mated with Mexican, Peruvian or (putative) Brazilian
2 morphotype males laid eggs with significantly lower hatch rate than those laid by
females mated with homotypic males. Heterotypic crosses involving Colombian
morphotype males produced numerically lower egg hatch than homotypic crosses but
19
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the difference was not significant. The cross of F1 x F1 males and females resulted in
high fertility levels.
Preliminary results of postzygotic tests with Brazilian populations have suggested no
postzygotic isolation among strains from different regions (Piracicaba vs. Bento
Gonçalves, Bento Gonçalves vs.Parnamirim and Parnamirim vs. Piracicaba).
Postzygotic tests with six Colombian populations showed no isolation among them.
Specific Progress and Individual Research Plans: Anastrepha fraterculus Complex
DNA
Anna Malacrida
- 5 year plan:
 Identify SSR markers in different populations possibly representing specific entities
across the A. fraterculus complex range and evaluating their degree of differentiation.
 Develop a database for population variability / differentiation within the A. fraterculus
complex.
 Assess the presence of post-zygotic isolation within A. fraterculus entities.
 Develop species-specific molecular markers as diagnostic tools for A. fraterculus
entities.
- Plans for the first 18 months:
 Develop SSR markers in A. fraterculus for one morphtype.
 Assess if it’s transferable to other morphotypes.
- Plans for the second 18 months:
 Validate the microsatellite loci sequences isolated.
 Assess intra-morphotype variability.
 Assess if they are transferable to other morphotypes in collaboration with Janisete
Silva.
- Plans for the third 18 months:
 Define and classify the microsatellites alleles for the 15 loci.
 Perform population genetic analysis of Argentinean and Brazilian populations using
15 highly informative loci in cooperation with INTA (Argentina) and UESC (Brazil).
 Characterize the Y chromosome from a strain of A. fraterculus from Argentina
(Brazilian 1 morphotype).
 Assist people to develop ITS 2 markers.
- Progress made up to 3rd RCM:
 Eighteen microsatellite loci sequences from A. sp. 1 aff fraterculus were isolated.
 15 SSR have been validated and characterized for their PIC (polymorphic information
content) in two strains (Vienna, Tucuman) and two populations from Argentina
(Concordia, Puerto Yerua).
 Intrapopulation variability and differentiation between the two populations and the
two strains have been assessed.
Janisete Silva
- 5 year plan:
 Obtain samples from A. fraterculus populations in Brazil, including colonies already
established.
 Evaluate DNA variation using mitochondrial and nuclear gene sequences.
 Compare populations for evidence of population structure and potentially distinct gene
pools.
20

Test the monophyly of the A. fraterculus complex using multiple specimens from each
putative cryptic species.
 Elucidate the number of putative species in the A. fraterculus complex in Brazil.
 Map haplotype distribution in Brazil to guide future control programs.
 Perform a morphometric analysis to some of the Brazilian populations in collaboration
with Vicente Hernández-Ortíz.
 Describe immature stages in collaboration with Gary Steck.
- Plans for the first 18 months:
 Obtain collections of different populations of A. fraterculus in Brazil as well as some
other related Anastrepha species for the phylogenetic analysis.
 Screen populations and already established colonies for one mitochondrial DNA
marker (COI).
- Plans for the second 18 months:
 Continue collections of A. fraterculus populations.
 Incorporate additional DNA markers.
- Plans for the third 18 months:
 Assign a morphotype to each of the populations under study, by means of
morphometric analysis (Top priority).
 Complete the ITS-1 analysis (and ITS-2 if possible), including as many morphotypes
as possible (Top priority).
 Validate the usefulness of the 15 microsatellites already developed for morphotype 1
to the other morphotypes using the same approach as in the FAR complex (in
collaboration with Pavia). This point is given top priority.
 Perform a population genetic analysis with wild populations from as many
morphotypes as possible from Brazil and other countries if available. (Top priority)
 Submit an article for publication with the complete study of populations from south
and south east Brazil (in collaboration with Iara Joachim-Bravo).
 Perform morphometric analysis for at least 10 Brazilian populations in collaboration
with Iara Bravo and Vicente Hernandez-Ortiz.
 Sequence Wolbacchia to determine the lineages present in samples from different
locations in Brazil
- Progress made up to 3rd RCM:
 Collections of 33 populations of A. fraterculus in Brazil as well as some other related
Anastrepha species were done, totaling 74 species and 630 sequences were obtained
for the phylogenetic analysis (COI).
 A total of 27 populations from 8 countries were also sampled and screened.
 Sequences of COI for 244 A. fraterculus females of 34 populations from Brazil, one
population from Argentina (Tucuman) and one population from Mexico (Xalapa).
 Two mitochondrial regions (COI and ND6) and six nuclear regions (28S, ITS2, CAD,
Period, TGO and TPI) were tested for 111 Anastrepha samples in collaboration with
University of Texas-Pan American.
 Five Brazilian populations of A. fraterculus were screened for 10 microsatellite loci in
collaboration with Silvia Lanzavecchia, INTA (Argentina) and Anna Malacrida, Pavia
(Italy).
Nelson Canal
- 5 year plan:
 Analyze ITS-1 from different populations of A. fraterculus and A. obliqua from
Colombia.
21
- Plans for the first 18 months:
 Obtain samples and optimize ITS amplification.
- Plans for the second 18 months:
 Continue collections of A. fraterculus populations.
 Perform ITS-1 analyses.
- Plans for the third 18 months:
 Finish the study of A. fraterculus and A. obliqua with mitochondrial markers CO I,
CO II and ITS-1, for Colombian populations (in collaboration with Denise Selivon
USP, Brazil).
- Progress made up to 3rd RCM:
 Samples were obtained from 5 populations of A. fraterculus and 4 of A. obliqua from
Colombia.
 ITS-1 amplification technique was optimized.
 Nine populations of A. fraterculus across the country were collected.
 ITS-1 analysis for eight populations is in progress (sequences are in analysis).
 11 populations of A. obliqua were collected and analysis of CO I, CO II and ITS-1 is
in advance.
Gary Steck
- Plans for the third 18 months:
 Complete ITS-1 analysis for samples remaining from the isozyme study of Steck 1991
[Venezuela (highland and lowland), Brazil (Bahia, Sao Paulo), Costa Rica]; plus
additional samples in the Florida State Collection of Arthropods from Ecuador, Peru,
Bolivia, Brazil; plus colony samples from Seibersdorf (Brazil, Colombia, Mexico).
(Collaboration with Bruce Sutton).
 Facilitate the collection and establishment of a population from Peruvian highlands
(and other areas if possible, e.g., Bolivia, Venezuela). (Collaboration with Bruce
Sutton, Allen Norrbom, Erick Rodriguez)
- Progress made up to 3rd RCM:
 Specimens from different regions in Peru and Bolivia were collected.
 Preliminary ITS-1 sequence data indicates that A. fraterculus populations in
Departamento Cusco (Andean highland and Amazonian mid-elevations) are clearly
different from the Pacific coastal populations of Peru.
 DNA sequences from the Cusco populations, as well as those from a mid-elevation
population on the eastern slopes of the Andes in Bolivia, match those of the
widespread Brazilian 1 morphotype.
CYTOLOGY
Nelson Canal
- 5 year plan:
 Examine and compare karyotypes from different populations of A. fraterculus and A.
obliqua from Colombia with previously described karyotypes.
- Plans for the first 18 months:
 Perform karyotype analysis of the Andean morphotype.
- Plans for the second 18 months:
 Perform karyotype analysis of the remaining A. fraterculus populations.
- Plans for the third 18 months:
 Describe the karyotype of the Mexican and Ecuadorian morphotypes (in collaboration
with Vicente Hernández-Ortiz).
22
- Progress made up to 3rd RCM:
 Karyotype of the Andean population was described for 4 A. fraterculus and 2 A.
obliqua populations.
 The karyotypes of seven populations of A. fraterculus from Colombia (Andean
Morphotype) and four populations of A. obliqua were described.
MORPHOLOGY
Vicente Hernández-Ortiz / E. Arévalo / G. Palma / J. Martinez / J. Tigrero
- 5 year plan:
 Perform a comparative morphometric analysis of populations of the nominal species
Anastrepha fraterculus (Wiedemann) throughout Mexico and Central America in
order to describe the involved species.
 Study the morphological variability of populations from Colombia, Ecuador and Peru,
comparing samples from lowland Pacific coastal plain with those samples from
highland Andean areas from those countries.
 Describe the inter-population variability and morphotypes established in each
geographic area and identify their distribution patterns.
 Determine the usefulness of morphological characters to recognize and describe the
species of the complex.
- Plans for the first 18 months:
 Collect A. fraterculus field samples.
 Preserve and mount structures.
 Carry out digital imaging.
 Measure structures in 12 populations.
- Plans for the second 18 months:
 Obtain samples from some countries in Central America (Costa Rica and Honduras)
and additional populations from the Colombian highlands.
 Complete the morphometric analysis intitated in the first phase of the CRP.
 Prepare a protocol for sample preparation to be sent to participants of a training course
to be held in Brazil in 2012.
 Give a one to two week training course on morphometrics and morphotype
identification of A. fraterculus.
 Start preliminary analysis on wing shape using geometric morphometry for Mexican,
Andean and Peruvian morphotypes.
- Plans for the third 18 months:
 Provide taxonomic support on morphometrics for groups studying chemical ecology,
DNA sequences, and larval studies.
 Compile host plant lists for Mexican, Andean, and Peruvian morphotypes.
 Prepare a manuscript describing the Mexican, Andean and Peruvian morphotypes as
new species.
- Progress made up to 3rd RCM:
 Samples from 6 populations from Ecuador,4 populations from Colombia and 2
populations from Mexico were obtained.
 Up to 80% of the samples were preserved and mounted.
 Digital imaging were performed on those slides.
 Almost 50% of the samples were measured for morphometric analysis.
 Samples from Colombia (10 populations) and Ecuador (8 populations) were obtained.
 The morphometric analysis of the Colombian and Ecuadorian populations was
initiated and completed.
23
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A protocol for sample preparation for the participants of a morphometrics training
course was elaborated.
A one week training course on morphometrics and morphotype identification of A.
fraterculus was given in Brazil in November 2012.
The analysis of wing shape using geometric morphometry for Mexican, Andean and
Peruvian morphotypes was completed.
Nelson Canal
- 5 year plan:
 Describe egg and larval external morphology of A. fraterculus and A. obliqua from
Colombia.
 Study adult morphology of different populations from A. fraterculus and A. obliqua
from Colombia.
- Plans for the first 18 months:
 Collect A. fraterculus field populations.
 Establish laboratory colonies.
 Perform morphological studies on immature and adult stages.
- Plans for the second 18 months:
 Continue collections of A. fraterculus and A. obliqua from Colombia.
 Start a morphometric analysis in collaboration with Vicente Hernández-Ortíz.
 Describe egg morphology of A. fraterculus and A. obliqua.
- Plans for the third 18 months:
 Complete morphometric analysis of A. obliqua females.
 Descriptive and morphometric studies on 3rd instar larvae of Mexican, Andean, and
Peruvian morphotypes (with G. Steck).
- Progress made up to 3rd RCM:
 Samples were obtained from 5 A. fraterculus and 4 A. obliqua populations from
Colombia.
 Efforts to established colonies from all populations were made and at the moment two
populations from A. obliqua have been colonized.
 Adults from all populations were measured and analysis is in progress.
 The mouthparts of 3rd instar larve from some populations were measured using light
microscope.
 Nine populations of A. fraterculus and 11 populations of A. obliqua from Colombia
were acquired.
 The morphometric analysis of A. fraterculus was completed in collaboration with
Vicente Hernández-Ortíz. The morphometric analysis of A. obliqua is underway
including also 1 population from Mexico and 1 population from Brazil.
 Training was provided to M. Rosario Castaneda to analyze egg morphology of
Colombian populations of A. fraterculus and A. obliqua.
 The morphological study of the 3rd instar larvae of A. fraterculus from Colombia was
completed.
Gary Steck
- 5 year plan:
 Develop a strategy for sampling, acquiring host plant and geographic distribution data,
rearing and breeding specimens for diverse studies of adults and immature stages,
preservation, and vouchering of specimens.
24

Assist in the acquisition of field collections, as needed, based on participation of
various agencies throughout the Americas.
 Examine and describe immature stages of various populations of the A. fraterculus
complex in search of diagnostic characters to separate discrete species, as provided by
cooperating teams.
- Plans for the first 18 months:
 Design a population sampling strategy for the A. fraterculus complex throughout its
geographic range that will maximize the types of data that can be used for description
of discrete species, and determining their geographic distributions, and associations
with host plants.
 Describe immature stages of various populations of the A. fraterculus complex
(contingent on provision of specimens by cooperating teams of the CRP).
- Plans for the second 18 months:
 Continue with the SEM and optical microscope observations on 3rd instar A.
fraterculus larvae.
 Continue collaboration with Dutra et al. on description of immature stages of various
species of Anastrepha.
 Submit a proposal to get fundings from the US Farm Bill to strengthen collection
network.
 Attempt to establish collaboration with people from Universidad Nacional in Cuzco,
Peru in order to obtain A. fraterculus material to be used in the CRP.
- Plans for the third 18 months:
 Complete the larval descriptions for 3rd instar larvae of Mexican, Andean, and
Peruvian morphotypes (in collaboration with Nelson Canal).
 Acquire new collections of Anastrepha spp. and populations of the A. fraterculus
complex in Peru (also other countries if permits can be obtained) (in collaboration
with Bruce Sutton, Allen Norrbom, Erick Rodriguez).
- Progress made up to 3rd RCM:
 Exploratory trip to Peru in search of collaborators for A. fraterculus field collections.
 Samples from 4 A. fraterculus colonies established at Seibersdof and one from Peru
were obtained.
 SEM observations carried out on 3rd instar larvae from 5 A. fraterculus populations
representing 3 morphotypes.
 Description of immature stages of various species within the fraterculus group
resulting in 3 publications.
 Erick Rodriguez was trained in SEM and optical microscope observations on 3rd instar
larvae of Anastrepha spp.
 Collaboration with Dutra et al. continued to describe immature stages of various
species of Anastrepha and results were published (Dutra et al. 2012).
 A proposal was submitted and 2 years of funding from the US Farm Bill was received
to acquire new collections of Anastrepha larvae and adults to improve ability to
identify larvae using morphological and molecular techniques.
 Collaboration with entomologists at the Universidad Nacional San Antonio Abad
Cuzco, Peru was established to obtain samples of Andean populations of Anastrepha
spp.
 Two collecting trips were made to Peru and ca. 37 spp. of Anastrepha were obtained
along with larvae collections from 14 host plants.
 A collecting trip was made to Panama to acquire samples of Anastrepha adults and
larvae for morphological and molecular study.
25
BEHAVIOUR
Iara Joachim-Bravo / Vanessa Dias / Kelly Roriz
- 5 year plan:
 Evaluate the level of reproductive isolation among populations from three Brazilian
morphotypes through demographic parameters (fertility, sex ratio, and larva and pupa
viability), sexual compatibility and competitiveness.
 Describe the pre-mating behaviour pattern from the three Brazilian morphotypes.
 Analyze comparatively the acoustical signals emitted during the courtship in each
morphotype.
- Plans for the first 18 months:
 Collect infested fruit in the field from different regions in Brazil.
 Establish laboratory colonies of two Brazilian morphotypes (A. sp 1 aff fraterculus and
A. sp 2 aff fracterculus) from the flies collected in the field.
 Initiate compatibility tests between the two established colonies of the two Brazilian
morphotypes.
 Initiate laboratory video recordings in order to characterize the courtship behaviour of
the two established colonies of the two Brazilian morphotypes.
 Perform a comparative analysis of the sounds emitted by the flies during the courtship
behaviour from each of the two established colonies of the two Brazilian morphotypes.
- Plans for the second 18 months:
 Preserve specimens from the A. fraterculus colonies studied for vouchering and DNA
extractions.
 Perform morphometric analysis to determine the morphotype to which each A.
fraterculus population belongs in collaboration with Vicente Hernández-Ortíz.
 Continue field sampling with emphasis on obtaining A. sp 3 aff fracterculus.
 Continue mating compatibility among the already established A. fraterculus colonies.
 Continue male sexual behaviour description (lekking behaviour and video recordings).
 Start a comparative analysis of the sounds emitted by A. fraterculus flies during the
courtship behaviour.
- Plans for the third 18 months:
 Continue collections at Una (BA, Brazil northeast region), Parnamirim (RN, Brazil
northeast region), Alagoas (in collaboration with Ruth do Nascimento in the northeast
region).
 Establish new laboratory colonies of A. fraterculus from the flies collected in the field
to be used in compatibility and behavioural tests.
 Publish compatibility and behavioural tests finished among the southern and southeast
populations.
 Continue the compatibility tests comparing populations from northeast, southeast and
southern regions of Brazil established at laboratory as follows: Parnamirim x Bento
Gonçalves (new replicates) and Parnamirim x Piracicaba (to be done once the
morphotype identification is finished).
 Continue laboratory video recordings tests to characterize the courtship behaviour of
different populations (Parnamirim, Bento Gonçalves and Piracicaba).
 Continue the analysis of acoustical communication during mating for the Parnamirim
population.
 Continue post-zygotic compatibility tests with the combinations of populations:
Parnamirim x Bento Gonçalves; Parnamirim x Piracicaba and Bento Gonçalves x
Piracicaba.
26
- Progress made up to 3rd RCM:
 Collections were performed of 6 A. fraterculus populations from south Brazil, 1 from
the Southeast and 7 from the Northeast (morphotypes to be defined).
 Laboratory colonies were established resulting in 6 A. fraterculus colonies from the
south, 1 from southeast and 1 from the northeast.
 Mating compatibility evaluations were performed among 6 of those A. fraterculus
populations (Bento Gonçalves, Pelotas, Vacaria, Sao Joaquim, Piracicaba and Natal
(morphotypes to be defined).
 The lekking behaviour was studied for 3 A. fraterculus populations from the south and
1 from the southeast.
 Video recordings of male courtship were obtained for one A. fraterculus population
from each region.
 Mating compatibility evaluations among four populations from southern of Brazil
(Bento Gonçalves, Pelotas, Vacaria and São Joaquim) were completed.
 Mating compatibility tests between a southeast population from Brazil (Piracicaba)
and southern populations (Vacaria; São Joaquim and Bento Gonçalves) were
completed.
 Mating compatibility tests between Parnamirim (northeast) vs. Piracicaba (southeast)
and Bento Gonçalves (south) populations were initiated.
 The courtship behaviour analysis of Bento Gonçalves, Piracicaba and Parnamirim
populations were initiated.
 The sound analysis of south and southeast populations was completed.
 Kelly Roriz participated in the morphometric course conducted by Vicente
Hernández-Ortiz in 2012.
Teresa Vera / Diego Segura / Solana Abraham / Juan Rull
- 5 year plan:
 Perform mating compatibility tests from as many A. fraterculus morphotypes as
possible.
 Characterize aspects of the sexual behaviour of each population.
 Determine the occurrence of post-zygotic isolation among populations.
 Study the sexual behaviour of the hybrids.
- Plans for the first 18 months:
 Establish / renew A. fraterculus colonies from the putative species at Seibersdorf.
 Perform mating compatibility tests among the colonies.
 Perform post-copulatory behaviour studies.
 Perform post-zygotic isolation studies among the different A. fraterculus colonies.
- Plans for the second 18 months:
 Preserve specimens from the A. fraterculus colonies for vouchering and DNA
extractions.
 Establish new A. fraterculus colonies in Seibersdorf with emphasis on the Andean
morphptype (Colombia), A. sp 2 aff fracterculus (colony from Maceio if Vicente
Hernández-Ortíz confirms its identity), the Mexican morphotype and the Peruvian
morphotype.
 Continue mating compatibility studies with the A. fraterculus colonies established at
Seibersdorf in collaboration with Nelson Canal.
 Perform experiments in field cages in Seibersdorf with artificial leks to determine if
females show different attraction to the pheromones of males from the different
morphotypes.
27
 Continue female post-copulatory behaviour studies.
 Obtain extracts from the male accessory glands in order to characterize the proteins.
- Plans for the third 18 months:
 Perform experiments in field cages in Seibersdorf with artificial leks to determine if
females show different attraction to the pheromones of males from those
morphotypes/populations that exhibit the same time of mating activity but some
degree of sexual isolation (Brazilian 1 and Mexican morphotype and Piracicaba).
 Perform bioessays with pheromone extracts to determine female attraction to the
pheromones of males from other morphotypes (in collaboration with Blanka Kalinova,
Radka Brizova and Peter Teal).
 Obtain extracts from the male accessory glands in order to determine female
receptivity after the injection of the extracts.
 Initiate the description of the protein profile of the male accessory glands’ proteins (in
collaboration with Seibersdorf and INBIOTECA, Mexico).
- Progress made up to 3rd RCM:
 Four new A. fraterculus colonies were established at Seibersdorf (Mexico, Vacaria,
Pelotas and Piricaba).
 Mating compatibility between the Mexican morphotype and the Peruvian morphotype,
as well as between the Mexican morphotype and two populations from Brazil (Vacaria
and Pelotas) and one population from Argentina, was evaluated.
 Female post-copulatory behaviour of the Peruvian morphtype and one population from
Argentina, after mating with males from the same origin or with males from the other
population, were evaluated.
 Post-zygotic incompatibility between one population from Argentina (Tucumán) and
two populations from Brazil (Vacaria and Pelotas) was evaluated.
 Preliminary experiments to evalauate post-zygotic incompatibility between the
Mexican morphotype and the Peruvian morphotype, and between the Mexican
morphotype and a population from Brazil, were performed.
 New A. fraterculus colonies were established at Seibersdorf (Perú, Colombia, Mexico
and Parnamirim).
 Mating compatibility between the Colombian morphotype was evaluated with the
Peruvian, Mexican and A. ff morphotype.
 The evaluation of female post-copulatory behaviour of the Peruvian morphtype and
one population from Argentina after mating with males from the same origin or with
males from the other population was completed and the manuscript was sent for
publication.
 Specimens from the A. fraterculus colonies for vouchering and DNA extractions were
preserved and distributed as agreed (in collaboration with Norma Nolasco and María
del Rosario Castañeda).
CHEMICAL ECOLOGY
Ruth do Nascimento / Blanka Kalinova / Lucie Vanickova / Radka Brizova
- 5 year plan:
 Compare the exact composition of the volatile pheromone profile released by A.
fraterculus males from different morphotypes.
 Compare the exact composition of the cuticular hydrocarbons (CHCs) profile of A.
fraterculus males from different morphotypes.
28

Study the CHCs and cuticular esther differences between males and females from
different ages to improve understanding of pre-zygotic isolation.
 Perform behavioural assays to identify the biological relevance of pheromone
differences between populations.
- Plans for the first 18 months:
 Collect and establish A. fraterculus populations under laboratory conditions from two
morphotypes (A. sp 1 aff. fraterculus and A. sp 2 aff. fraterculus).
 Collect and characterize volatiles from calling males of the two A. fraterculus
morphotypes.
 Extract and characterize cuticular hydrocarbons from males and females of the two A.
fraterculus morphotypes.
 Perform Gas chromatography-Electroantennography (GC-EAG) analyses of male
extracts.
- Plans for the second 18 months:
 Preserve specimens from the A. fraterculus colonies for vouchering and DNA
extractions.
 Perform morphometric analysis to determine the morphotype to which each
population belongs in collaboration with Vicente Hernández-Ortíz.
 Identify chemically the volatiles from the remaining populations by GC-MS and GCGCMS.
 Initiate gas chromatography-electroantennography (GC-EAG) analyses of male
extracts in case that behavioural data provide evidence of differences in female
behaviour.
- Plans for the third 18 months:
 Collect field infested guavas in the states of Alagoas and Parnamirim (Rio Grande do
Norte).
 Establish cultures of these populations under laboratory conditions.
 Maintain the A. fraterculus populations derived from Alagoas, Parnamirim, Bento
Gonçalves, Piracicaba, São Joaquim, Pelotas and Vacaria under laboratory conditions.
 Complete the identification of the morphotypes (in collaboration with other Brazilian
participants).
 Perform the experiments described below in order to obtain enough extracts for GC x
GC/TOF-MS analyses of all the populations collected (Alagoas, Parnamirin, Bento
Gonçalves, Piracicaba, São Joaquim, Pelotas and Vacaria) aiming to: a) identify and
quantify the pheromone components and cuticular hydrocarbons of those populations
which have not yet being studied; b) conduct laboratory bioassays to find out which
role they play in the communication among conspecific and heterospecific males and
females;
 Carry on electrophysiological studies to find out which compounds within the
mixtures of volatiles released by four populations of A. fraterculus, from three
different regions of Brazil: Northeast: Alagoas; South: Bento Gonçalves and São
Joaquim; Southeast: Piracicaba, trigger a response on the antennae of conspecific and
heterospecific females. These analyses will be performed in Brazil.
 Use statistical tools to find out similarities and differences between them.
 Analyse the pheromone of a Colombian population (collected by Nelson Canal) by
GCxGCMS in Prague.
 Perform GC-EAD for the available morphotypes different than Brazilian 1 (to be done
in Prague).
- Progress made up to 3rd RCM:
29






Laboratory A. fraterculus colonies were established resulting in 4 colonies from the
south, 1 from southeast and 2 from the northeast from Brazil (morphotypes to be
defined).
Pheromone volatiles from two of these A. fraterculus populations (Bento Gonçalves
and Maceio belong probably to two morphotypes (morphotypes to be defined) were
collected and identified.
The cuticular hydrocarbons from these two A. fraterculus populations were also
analysed.
The pheromone volatiles from several populations involving at least two morphotypes
were characterized (Peruvian and Brazilian 1 morphotypes).
The chemical characterization of the pheromone volatiles from another two
morphotypes was initiated (Mexican and Andean morphotypes).
The cuticular hydrocarbons from several populations comprising the four above
mentioned morphotypes were characterized.
Teresa Vera / Diego Segura / Solana Abraham / Juan Rull
- 5 year plan:
 Characterize the male sexual pheromone profile of each A. fraterculus morphotype.
- For the first 18 months:
 Collect male sexual pheromone from A. fraterculus colonies established at
Seibersdorf, Austria.
- Plans for the second 18 months:
 Collect pheromone and CHC samples from the Andean and Mexican A. fraterculus
morphotype.
 Characterize the male sexual pheromone and CHC profile of the samples collected in
collaboration with Blanka Kalinova and Peter Teal.
- Plans for the third 18 months:
 Extract pheromones in co-operation between Seibersdorf and Prague to be used in the
cross-attraction bioassays.
 Check pheromone quality (in collaboration with Blanka Kalinova, Radka Brizova and
Peter Teal).
- Progress made up to 3rd RCM:
 Male sexual pheromone from 5 A. fraterculus colonies established at Seibersdorf,
Austria was collected.
 The volatiles as well as the CHCs from several populations from Seibersdorf were
collected and sent to Prague and Gainesville for chemical characterization.
30
Bactrocera cucurbitae
Background Situation Analysis
Bactrocera cucurbitae is a major pest of cucurbit crops and has spread from its area of origin
(South East Asia) across Africa, Hawaii, the Indian Ocean, Papua New Guinea and the
Solomon Islands (Severin et al., 1914; Dhillon et al., 2005). In particular, it causes severe
losses in food crops in poor village communities and restrictions to trade in some cucurbit
crops.
Some populations have been identified in Africa, Indian Ocean, Hawaii and South East Asia
that indicate that very closely related species may be present. This problem needs to be
resolved before species-specific treatments such as the SIT can be effectively applied in all
situations and regions. There are a number of single species that differ in host, morphology,
etc. from region to region and B. cucurbitae may be one of these.
Recent population genetic studies can resolve five major groups worldwide (with
microsatellites): African mainland + Seychelles, Réunion + Mauritius, Central Asia, SE Asia,
Hawaii (Virgilio et al., 2010). However phylogeographic patterns could not be discerned
(using mitochondrial and nuclear gene fragments (total of 2764 bp) Virgilio, unpublished).
Field cage compatibility tests were recently conducted between populations of Mauritius,
Seychelles and genetic sexing strain of Hawaii. No incompatibility was found (Sookar et al.,
2010). Further mating tests could help to resolve their species status.
Baseline and Progress on Bactrocera cucurbitae
DNA
- Knowledge at the start of the CRP:
 General information on phylogeography / population genetics available on a
worldwide basis. Also information available on population genetics with regard to the
African mainland and La Réunion.
- Gaps identified:
 More data on potential host races are needed, both within Cucurbitaceae (cultivated
versus wild) and cucurbits versus non-cucurbits.
- Progress up to 3rd RCM in addressing identified gaps:
 Detailed DNA studies on population genetics in Réunion, and to a lesser extent in
Tanzania, have shown that there is no relation between genetic structure and host
plants (i.e. cultivated versus wild cucurbits).
 Mitochondrial variation in B. cucurbitae populations from Hawaii were studied and
little variation detected. Other (nuclear) markers and populations are currently being
explored.
CYTOLOGY
- Knowledge at the start of the CRP:
 Work has been carried on two B. cucurbitae populations regarding karyotyping and
polytene maps.
- Gaps identified:
 Need to have same information from other populations.
31
- Progress up to 3rd RCM in addressing identified gaps:
 No cytogenetic differences found between two populations of B. cucurbitae (Genetic
Sexing Strain Hawaii and Bangladesh wild type) (Zacharopoulou et al., 2011).
 Karyotyping reveals that B. cucurbitae is significantly different from other Bactrocera
(i.e. position of subgenus Zeugodacus in relation to Bactrocera and Dacus).
ALLOZYMES
- Knowledge at the start of the CRP:
 Previous work done by Malacrida et al. (1996).
- Gaps identified:
 No further studies planned; covered by DNA work.
GENOMICS
- Knowledge at the start of the CRP:
 No studies conducted.
- Gaps identified:
 Need to conduct genomic analysis.
- Progress up to 3rd RCM in addressing identified gaps:
 No further studies conducted.
MORPHOLOGY
- Knowledge at the start of the CRP:
 B. cucurbitae can be morphologically differentiated from other species within the
subgenus Zeugodacus.
- Gaps identified:
 Possible confusion with other valid taxa.
 Some populations appear to indicate that very closely related species may be present.
- Progress up to 3rd RCM in addressing identified gaps:
 No other valid taxa have been identified that could cause possible confusion with B.
cucurbitae. Also no differences identified among populations. Therefore no need for
further studies.
MORPHOMETRICS
- Knowledge at the start of the CRP:
 Unexplored so far.
- Gaps identified:
 Could provide additional information for recognition of potential geographic or host
races. Only preliminary work done at Seibersdorf for testing methodology.
- Progress up to 3rd RCM in addressing identified gaps:
 No studies conducted so far.
MALE LURE RESPONSE
- Knowledge at the start of the CRP:
 Attraction of males to cue lure and melolure, not by methyl-eugenol.
- Gaps identified:
 No need for further study with regard to the CRP.
DEVELOPMENTAL PHYSIOLOGY
- Knowledge at the start of the CRP:
32

Data are available from Japan, Hawaii and La Réunion, but have not yet been
compared.
- Gaps identified:
 Need to compare existing biological data from countries where host range is different.
- Progress up to 3rd RCM in addressing identified gaps:
 No studies conducted so far.
BEHAVIOUR
- Knowledge at the start of the CRP:
 Good knowledge of mating behaviour through studies in Japan, Hawaii and recently in
Seibersdorf.
- Gaps identified:
 Mating compatibility in future if need arises.
- Progress up to 3rd RCM in addressing identified gaps:
 Publication of compatibility studies (Sookar et al. 2010), investigating cross-mating
among three populations of B. cucurbitae (Hawaii, Seychelles and Mauritius). No
sexual isolation was discovered.
PHEROMONE COMPONENTS AND CONCENTRATIONS
- Knowledge at the start of the CRP:
 Previous work done by Japanese researchers.
- Gaps identified:
 No need for further work planned in the immediate future; perhaps at later stage to
relate pheromones with potential host races.
- Progress up to 3rd RCM in addressing identified gaps:
 Pheromone analysis and profile on-going for Genetic Sexing Strain from Hawaii:
effect of age and diet (Haq et al., in prep).
HOST RANGE
- Knowledge at the start of the CRP:
 Well documented as a whole.
- Gaps identified:
 See comment under DNA: possibility of host races.
- Progress up to 3rd RCM in addressing identified gaps:
 Work by Sookar on host preference; host range studied specifically in Tanzania
(Mwatawala et al. 2010). Host range in Réunion and Tanzania was studied in relation
to genetic structure.
 Laboratory experiments on behaviour and preimaginal development of populations
from Hawaii and Mauritius on cucurbit and non-cucurbit hosts showed no differences
in egg laying choice and survival of larvae to adults.
 Kenya: (Ekesi). In the studies involving the host range of B. cucurbitae, the insect was
reared from a total collection of 17 plant species comprising 10 families covering
Cucurbitaceae, Solanaceae, Anacardiaceae, Rutaceae and Myrtaceae from surveys
carried out at the Coast, Eastern and Central Provinces of Kenya.
 Populations of B. cucurbitae vary in their preference to host plants and this preference
is driven also by variation among population of the pest.
 Two populations were established originating from bitter gourd and tomato. In the
bitter gourd population, significantly higher number of puparia was recovered from
33

bitter gourd but other cucurbits were also infested. The tomato population exclusively
preferred tomato compared to the other host plants.
Tanzania: a study on host preference studies under natural infestation and under seminatural conditions (cages) are being conducted, testing three hosts; cucumber,
pumpkin and watermelon. Work is in progress.
CUTICULAR HYDROCARBONS
- Knowledge at the start of the CRP:
 No studies yet specifically with regard to B. cucurbitae cuticular hydrocarbons have
been conducted.
- Gaps identified:
 Many gaps, however, no need for work in the immediate future, unless there is a need
to relate cuticular hydrocarbons with potential host races.
- Progress up to 3rd RCM in addressing identified gaps:
 General methodology has been developed for Tephritidae to analyse cuticular
hydrocarbons.
 The CHCs from males and females of B. cucurbitae - age dependent production (day
5, 15, 20, 30 after emergence) were extracted. The extracts were analysed by
GCxGCMS. The preliminary data demonstrate sex-specific differences.
Specific Progress and Individual Research Plans: Bactrocera cucurbitae
TAXONOMY
Marc de Meyer
- 5 year plan:
 Provide morphometric diagnostic markers to differentiate potential geographic and/or
host races.
- Plans for the last 18 months:
 Considering outcome of genetic studies on potential host races, no further activities
are required at this stage.
- Progress made up to 3rd RCM:
 Not done so far; only in case of identification of host races.
Sunday Ekesi / Fathiya M. Khamis
- 5 year plan:
 Collect and share biological material of B. cucurbitae with other CRP participants as
required.
- Plans for the last 18 months:
 Make material available for other CRP studies when required.
- Progress made up to 3rd RCM:
 Two colonies (one population from bitter gourd and one from tomato) have been
established at ICIPE.
BEHAVIOUR / HOST RANGE
Hélène Delatte and Serge Quilici
- 5 year plan:
 Define the host range of La Réunion populations through behavioural experiments
(choice and non-choice on mango and solanaceous fruits) including interspecific
competition with other fruit flies.
34

If required (based on results from Hélène Delatte, Serge Quilici and Maulid
Mwatawala): test mating incompatibility between geographic and/or potential host
races at Seibersdorf.
- Plans for the last 18 months:
 Continue behavioural studies in olfactometers.
- Progress made up to 3rd RCM:
 Ecological studies in La Réunion allow specifying the host range of B. cucurbitae
among wild and cultivated cucurbits at various sites and altitudes.
 Study of B. cucurbitae intra- and interspecific competition was conducted and
finalized.
 Behavioural studies in olfactometers and field cages to specify the preferences of
female B. cucurbitae for different species of host plants are under progress.
Maulid Mwatawala
- 5 year plan:
 Host plant preferences, incidence and infestation rates of B. cucurbitae in Tanzania.
Verification of host choice and the relationship with intraspecific variation.
- Plans for the last 18 months:
 Continue conducting host preference (cucurbit versus non-cucurbits) tests in seminatural conditions.
 Sending material from non-cucurbit hosts to CIRAD and RMCA for genetic analysis.
- Progress made up to 3rd RCM:
 Surveys were conducted throughout Tanzania along several transects, obtaining
material from cucurbit and non-cucurbit hosts.
 Material from cucurbit hosts (wild and cultivated) were sent to Royal Museum CA for
genetic analysis.
 Tanzania. Host preference of B. cucurbitae under natural and semi-natural infestation
is being tested for three hosts (cucumber, pumpkin and water melon).
Sunday Ekesi
- 5 year plan:
 Host plant preferences, incidence and infestation rates of B. cucurbitae in Kenya.
Verification of host choice and the relationship with intraspecific variation.
- Progress made up to 3rd RCM:
 In the studies involving the host range of B. cucurbitae, the insect was reared from a
total collection of 17 plant species comprising 10 families covering Cucurbitaceae,
Solanaceae, Anarcadiaceae, Rutaceae and Myrtaceae from surveys carried out at the
Coast, Eastern and Central Provinces of Kenya.
 Two populations were established originating from bitter gourd and tomato and
differences in preference were observed.
MOLECULAR / CYTOGENETICS
Antigone Zacharopoulou / Antonios Augustinos / Farzana Yesmin
- 5 year plan:
 Confirmation karyotype and polytene similarity among the different geographic B.
cucurbitae populations.
- Plans for the last 18 months:
 Pending outcome of genetic studies on potential host races, no further activities are
planned at this stage.
35
- Progress made up to 3rd RCM:
 Wild populations of GSS Hawaii and Bangladesh were studied. Karyotyping reveals
that B. cucurbitae is significantly different from other Bactrocera (i.e. position of
subgenus Zeugodacus in relation to Bactrocera and Dacus).
 There are no cytogenetic differences between the populations of B. cucurbitae studied.
Massimiliano Virgilio and Hélène Delatte / Serge Quilici / Dave Haymer
- 5 year plan:
 Analysis of the population structure according to potential host races in Tanzania and
La Réunion.
- Plans for the last 18 months:
 Given earlier results, the use of microsatellites to differentiate potential host races
within B. cucurbitae will not be further investigated.
 Use the B. cucurbitae transcriptome dataset to identify genes and gene products in
biological significant pathways (odorant and pheromone binding proteins;
carbohydrate metabolism). This will be explored by the University of Hawaii at
Manoa to provide complementary information on the existence of host races.
- Progress made up to 3rd RCM:
 Genetic structure for different African populations, including La Réunion was
analysed. Weak genetic structuring was found on a continental scale.
 Detailed studies on population genetics in La Réunion; three groups were recovered
but there was no relation between the genetic structure and host plants (i.e. cultivated
versus wild cucurbits).
 A smaller pilot study in Tanzania showed no obvious relationship between hosts and
genetic structure.
 Mitochondrial variation in Hawaiian populations is very little.
DEVELOPMENTAL PHYSIOLOGY
Marc De Meyer
- 5 year plan:
 Literature review: comparison of biological data on developmental rates between
polyphagous (Hawaii) and oligophagous (Mascarenes) strains of B. cucurbitae.
- Plans for the last 18 months:
 Conduct literature review study on B. cucurbitae developmental physiology.
- Progress made up to 3rd RCM:
 So far no studies conducted.
PHEROMONE COMPONENTS AND CUTICULAR HYDROCARBON
Peter Teal / Lucie Vanickova / Sunday Ekesi / Maulid Mwatawala
- 5 year plan:

Pheromone analysis and profile for Genetic Sexing Strain from Hawaii: effect of age and diet.
- Plans for the last 18 months:
 Assess pheromones and cuticular hydrocarbons released by two populations from
different host plants. Assess chemical host plant preferences from two populations
from different host plants.
- Progress made up to 3rd RCM:
 The CHCs from males and females of B. cucurbitae - age dependent production (day
5, 15, 20, 30 after emergence) were extracted. The extracts were analysed by
GCxGCMS and species specific differences were found.
36
Bactrocera dorsalis Complex
Background Situation Analysis
Across Asia and the Pacific the fruit fly subfamily Dacinae contains some 48 recognised pest
species (Drew, pers. comm.). Of these 48 pest species, eight are currently recognized within
the Bactrocera dorsalis complex with some being the worst of all pest species within the
subfamily (Drew and Hancock, 1994). Known collectively as the Oriental fruit fly complex,
they are also categorized as the fourth worst of all global insect pests. While it is difficult to
put a precise value on the economic losses, these have been estimated by the Griffith
University International Fruit Fly Centre in Australia to be approximately US$1 billion per
annum for the Asian region. These losses include destruction of crops, restriction of
international trade, and the establishment of a range of quarantine and regulatory activities
carried out by various regional governments.
Background research has generated data on diagnostics, field surveillance including
quarantine strategies, field pest control, and some export trade (e.g. Tan and Nishida, 1996,
1998; Muraji and Nakahara, 2002; Naeole and Haymer, 2003; Smith et al., 2003; Armstrong
& Ball, 2005; Vargas et al 2009). This knowledge has also identified key gaps in resolving the
B. dorsalis complex, but there is no consensus on species limits of the major Bactrocera
dorsalis complex pest species, particularly B. dorsalis s.s., B. papayae, B. philippinensis, B.
carambolae and potentially B. invadens (Clarke et al., 2005; Drew et al., 2008; Wee and Tan,
2005; Ebina and Ohto, 2006). Failure to resolve this complex prevents further development
towards SIT implementation in Area-Wide Integrated Pest Management (AW-IPM)
programmes against these pest insects and limits international trade.
The Asian region has been a primary source of invasions of species of the B. dorsalis complex
into many other regions of the world, including Africa, Middle East, northern and southern
Pacific Islands (including Papua New Guinea (PNG) and Australia), Indian Ocean, and South
America. Currently some of these introduced species are causing food security and market
access problems in Africa, PNG, and Pacific Islands. Background research into the taxonomy
of the B. dorsalis complex has been unable to provide definitive identification of some species
(Clarke et al., 2005). This confounds the collection of associated host plant records and
geographic distributions. It is absolutely essential that the species be accurately identified in
order to be able to apply AW-IPM field programmes that include an SIT component.
Because trade implications and response systems to detections and/or incursions are not the
same for all species in the complex, "near-enough" identification is not, unfortunately, goodenough. Consequently Member States have difficulty overcoming the phytosanitary barriers
to export trade that satisfy major importers such as Japan, South Africa, USA, Australia, and
New Zealand. Not least is the severe problem arising if one member of the complex is
detected or becomes established in a Member State but is unable to be differentiated from
others in the complex. In this case that State would then be forced to indicate that any and all
members of the complex may in fact be present, which would result in trade embargoes.
In this component of the CRP, a comprehensive biological, morphological, chemo-ecological
and molecular study on pest species of the B. dorsalis complex will be undertaken so as to:
(i) Resolve species limits by seeking a consensus result from different tests including:
37
(a) Identifying the levels of inter- and intra-specific genetic (e.g. cytology) and
molecular variation,
(b) Carrying out morphological and morphometric analysis,
(c) Carrying out biological tests including cross-mating and host use studies,
(d) Pheromone analyses to compare component ratios across putative species
(ii) Examine congruence between data from above mentioned studies, (a) to (d) which may
support taxonomic revision or retain existing species, and
(iii) Develop robust diagnostic tools for the resolved species.
Baseline Knowledge on Five Priority Species in the Bactrocera dorsalis Complex
DNA
- Knowledge at the start of the CRP:
 Mitochondrial DNA
o COI ‘barcode’ gene region, and other mtDNA markers, show no clear
distinction between currently defined species; data from more populations may
improve this technique for population/species differentiation.
 Nuclear ribosomal DNA
o ITS1+2 diagnostic for separating B. carambolae from remaining four species.
 Microsatellites
o Sequences from B. dorsalis s.s., B. invadens, B. oleae and B. papayae are
available and may be useful for population genetic structure but not sure if
they will work with the B. dorsalis complex for species separation.
 Nuclear coding gene data for 16 loci (Muscle-specific actin, guanylyl cyclase receptor,
vitellogenin 1&2 precursors, 70 kDa heat shock protein, white, cytochrome P450,
guanylyl cyclase receptor beta, beta-2 tubulin gene, acetylcholinesterase, odorant
receptor, oderant binding, chemosensory, doublesex, G6PDH, alcohol
dehydrogenase), all of which for B. dorsalis s.s.; only two of these involve other
species in the complex and include at least B. carambolae, B. papayae and B.
philippinensis, but species differentiation has not been assessed.
 Exon primed intron crossing from actin gene – for B. papayae and B. carambolae and
B. dorsalis s.s. showed distinction but population level analyses have not been
undertaken.
- Gaps identified at the start of the CRP:
 There is no adequate and consistent sample coverage for the five target species.
 The lack of discriminatory characters means that either a) the characters exist but are
yet to be discovered, or b) that such characters do not exist and the species are the
same. This highlights the question as to how many negatives do we accept before
stopping?
 The inferences from current knowledge are based on standard phylogenetic and
phylogeographic analyses which may be a limitation to accurate interpretation.
- Progress up to 3rd RCM in addressing identified gaps:
 Significantly improved sample coverage, including from China, Indian subcontinent,
Myanmar, Indo/Malay Archipelago, and African populations. While geographic
coverage has increased, the numbers of individuals from particular locations may still
be insufficient.
 Additional diagnostic genetic markers (mitochondrial, ribosomal and nuclear genes)
have been developed using a comparative transcriptome approach, but they do not
discriminate between four of the five species; B. carambolae forms a monophyletic
38




group according to COI and ITS1 supporting its current species-level status.
Bactrocera kandiensis is also a well resolved clade.
A full multigene phylogenetic analysis has been completed.
Demography (using microsatellites) of B. invadens has been published (Khamis et al
2008) and that for B. dorsalis s.s. has been completed (indicating origin of the species
is China).
Additional
experiments
have
been
undertaken
to
supplement
the
phylogenetic/phylogeographic analyses towards clarifying species limits, including
mating compatibility, cytological, genomic, pheromone studies, morphometric and
morphological studies.
Markers have been developed for the Y chromosome in B. dorsalis s.s. from Thailand.
Differences have so far been found between B. dorsalis and B. carambolae, but not for
among B. dorsalis, B. papayae and B. philippinensis (but sample sizes to be
increased).
CYTOLOGY
- Knowledge at the start of the CRP:
 Studies on mitotic karyotypes identified several forms within the B. dorsalis complex,
but did not definitively distinguish between putative species (e.g. Baimai et al., 2000 ).
- Gaps identified at the start of the CRP:
 Further polytene maps are needed to allow distinguishing between putative species.
 Polytene mapping for these species could possibly be linked with genomic data.
- Progress made up to 3rd RCM in addressing identified gaps:
 Polytene maps are now available for B. dorsalis s.s. (Zacharopoulou et al., 2011).
 For B. papayae salivary gland polytene chromosome maps, metaphase karyotypes and
their morphometric characteristics have been described. In the case of B. carambolae,
metaphase karyotypes and their morphometric characteristics have been described
with construction of polytene chromosome maps in progress.
 There are no structural differences between all five species regarding polytene
chromosome banding patterns.
 Hybrid analysis (B. dorsalis s.s x B. invadens and B. dorsalis s.s. x B. carambolae
bidirectional hybrid) also shows no chromosomal structural differences between
paternal polytene chromosome banding pattern or their F1 hybrid, since hybrids’
polytene chromosomes are tightly synapsed. Minor asynapses were observed in a few
hybrids between B. dorsalis and B. carambolae.
 Localization of six unique sequences to polytene chromosomes (in situ hybridization)
has started for the three members of the complex and their hybrids (as in previous
point) with localization of the hsp70, GLD, OVO, TRA, SXL, Scarlet genes.
ALLOZYMES
- Knowledge at the start of the CRP:
 Not applicable as a means to discriminate between species as it is too dependant on
climatic factors.
 Thai team tried allozymes several years ago but data are not published.
- Gaps identified at the start of the CRP:
 Considered not as priority technique for this CRP.
- Progress made up to 3rd RCM in addressing identified gaps:
39

Preliminary esterase isozyme studies using polyacrylamide gel and laboratory colonies
of B. papayae and B. carambolae have been completed. This can differentiate between
these species in the adult stage.
GENOMICS
- Knowledge at the start of the CRP:
 piggyBac and other mobile elements (in particular the Hopper element) have been
documented to be present in some B. dorsalis species populations and may be used for
species differentiation. Noted as difficult to apply for defining species limits.
 An unpublished Hawaiian B. dorsalis s.s. genome exists.
 Also an unpublished C. capitata genome which could be used as a reference.
 Transcriptomics – under way for B. dorsalis s.s., B. philippinensis and B. carambolae.
There are potentially some markers for these species within that data set.
- Gaps identified at the start of the CRP:
 Current unavailability of the genome information.
- Progress made up to 3rd RCM in addressing identified gaps:
 piggybac is multiple copy, a characteristic which has been shown to differentiate B.
dorsalis s.s. and B. invadens.
 There is now a public web portal for accessing the current scaffold and contig
structure of the Hawaiian B. dorsalis s.s. genome. Annotation is in progress. Whole
body transcriptome data has been published for B. dorsalis s.s. (Taiwan, base line lab
strain, 5-day old). Whole body transcriptome data developed for B. dorsalis s.s.
(Saraburi, Thailand, F2-3) has been provided to the Hawaiian laboratory (USDAARS-PBARC, Hilo) to assist with annotation and placement of introns that may be
useful for species marker development.
 The genome sequence for C. capitata as a potential reference is under development.
 Transcriptomics for different tissues and the two sexes are available for C. capitata
and deposited in Vectorbase (Genbank). This will be used to help interpret the
genomic data being developed for both C. capitata and B. dorsalis as well as to
interpret the whole body transcriptome data that has been developed for B. dorsalis
s.s., B. papayae, B. philippinensis, B. carambolae and B. invadens. These data have
been used to identify sequences/genes related to odorant- and pheromone-binding
proteins, reproductive behaviour/reproduction, mate and host recognition.
 Comparative transcriptome data for B. dorsalis s.s., B. papayae, B. philippinensis, B.
carambolae and B. invadens have been analysed for ‘species’ specific SNPs.
 Y-chromosome sequence data have been developed for C. capitata and is under
development for B. dorsalis s.s. These data can be used for sexing individuals at any
stage of development.
MORPHOLOGY
- Knowledge at the start of the CRP:
 A revision of the SE Asian Dacini is under way, but is not expected to impact on the
four nominated pest species in this CRP (Drew, pers. comm.).
- Gaps identified at the start of the CRP:
 Egg and immature morphology – not investigated for B. dorsalis complex species.
 Explicit intra-specific population-level variation in both external and internal
morphological characters.
 Insufficient understanding of relative environmental/genetic influences on
morphological phenotype.
40
- Progress made up to 3rd RCM in addressing identified gaps:
 Major revision of SE Asian tephritids is complete. Updated information on all five
species included. The status of B. philippinensis will change.
 B. invadens vs B. dorsalis s.s. colour morphs described.
 Egg and immature material from Seibersdorf lab colonies has been gathered and is
being included as part of a major project describing the immature stages of tephritids.
 The range of morphological variation has been assessed for B. dorsalis s.s and B.
invadens from across their geographic ranges.
 Morphological pattern variation in B. dorsalis s.s (Seibersdorf culture) has been
assessed for flies fed varying quantities of food (standard lab diet). No clear
correlation was observed.
 Crosses have been undertaken between colour morphs (pale brown and black scutum)
of B. dorsalis from Pakistan. Preliminary results suggest colour morph is a simple
qualitative genetic trait.
MORPHOMETRICS
- Knowledge at the start of the CRP:
 A relatively large number of papers on morphometrics for B. dorsalis complex species
exists. In some cases resolution occurs but not always. Often also with overlap
between morphometric traits and often impossible to determine what might be within
and between population variation.
 Geometric morphometrics (shape analysis) shows some promise but needs further
research.
 Morphometric approaches have rarely been linked adequately with other
morphological or genetic approaches.
- Gaps identified at the start of the CRP:
 Explicit intra-specific population-level variation in both external and internal
morphometric characters.
 Insufficient understanding of relative environmental/genetic influences on
morphometric phenotype.
- Progress made up to 3rd RCM in addressing identified gaps:
 Aedeagi have been measured from 200+ specimens of B. dorsalis s.s., B. carambolae,
B. papayae, B. invadens (and B. verbascifoliae) from SE Asia and Africa. The same
samples have been sequenced for the COI gene and microsats and the combined
dataset has been analysed. Bactrocera papayae aedeagi are longer as expected (B.
verbascifoliae is shorter, but they are also smaller flies).
 Aedeagi have been measured from a latitudinal gradient from northern Thailand to
Peninsular Malaysia for B. dorsalis s.s. and B. papayae. Results show a significant and
continuous latitudinal cline from north to south, with northern Thailand flies the
shortest and Malaysian flies possessing the longest aedeagi.
 Morphometric analysis using wing veins and tibia lengths was also used to
discriminate B. invadens from different localities, and with comparison with other
Bactrocera species (B. kandiensis, B. dorsalis s.s., B. zonata, B. correcta, B.
cucurbitae and B. oleae). The results showed no resolution for species within the B.
dorsalis complex (but they did separate from the non-dorsalis flies) and these results
were also reflected in the COI sequence analysis.
 Geometric morphometric wing shape data has been obtained for B. dorsalis s.s., B.
papayae, B. carambolae, B. philippinensis, B. occipitalis, B. cacuminata, B. opiliae,
and B. musae. Results are consistent with B. dorsalis s.s., B. papayae and B.
41



philippinensis representing the same species which displays strong isolation-bydistance patterns within SE Asia. All ‘outgroup’ species (B. occipitalis, B.
cacuminata, B. opiliae and B. musae) resolve strongly from the four target species
studied here.
Bactrocera invadens morphometric data has been analysed for aedeagus length,
postsuttural lateral vittae and wing shape. This has been compared with B. dorsalis
from across its geographic distribution and reveals no evidence of species-level
differences.
Morphometrics of genitalia, head width and wings have been undertaken for B.
dorsalis s.s. (Seibersdorf colony) reared under different larval densities. Eggs of a
range of species have been measured.
Juvenile stages of all target species have been collected into alcohol and passed to G.
Steck for morphological examination.
DEVELOPMENTAL PHYSIOLOGY
- Knowledge at the start of the CRP:
 There are differences between B. carambolae and B. papayae and B. dorsalis s.s. e.g.
in attaining sexual maturity and in age-related development of response to
parapheromones – e.g. work done at Seibersdorf and by Dr Tan on B. carambolae
(Wee & Tan 2000; Wee et al. 2002; Wee & Tan, 2007).
 There are differences in ovariole numbers between some Bactrocera species but has
not been tested on B. dorsalis complex species.
- Gaps identified at the start of the CRP:
 Baseline developmental data for each of the species, e.g. thermal thresholds and daydegree requirements.
 It is noted that there is a huge gap in knowledge on the application of this technique to
species discrimination.
 Unknown how this knowledge can be used on its own for species discrimination.
- Progress made up to 3rd RCM in addressing identified gaps:
 This area was extensively reviewed by A. Clarke (pers. comm.) and while potentially
useful, is logistically challenging and was not pursued.
MALE LURE RESPONSE
- Knowledge at the start of the CRP:
 Each species nominated for this CRP respond to methyl eugenol (ME).
 However some species are more sensitive to ME at low concentrations e.g B. dorsalis
s.s. is more sensitive to low concentrations of ME than B. carambolae (Wee et al.,
2002).
 Initial feeding on high-concentration ME reduced response to subsequent exposure in
B. dorsalis s.s. (Shelly, 1994).
 The metabolites derived from being fed ME can be used to discriminate B.
carambolae.
- Gaps identified at the start of the CRP:
 Lack of specific or detailed knowledge of the response of B. philippinensis and B.
invadens.
 The metabolites derived from flies being fed ME need to be tested in B. philippinensis.
- Progress made up to 3rd RCM in addressing identified gaps:
42




Male sex pheromone components (from rectal glands) of B. invadens and B.
philippinensis following ME consumption have been identified and are the same as for
B. dorsalis s.s.
Male sensitivity to ME in small cages complete and show that B. carambolae has
much lower sensitivity relative to B. dorsalis, B. papayae, B. philippinensis and B.
invadens.
The male mating advantage seen in the species following ME-feeding begins later for
B. carambolae relative to B. dorsalis.
Lure sensitivity in field cage trials are underway.
PHEROMONE COMPONENTS AND RATIOS
- Knowledge at the start of the CRP:
 Pheromone components (both rectal gland and volatiles) following male consumption
of methyl eugenol (ME) are exactly the same in laboratory cultures of each of three
complex species: B. dorsalis s.s. and B. papayae. (Tan & Nishida, 1996; Wee & Tan,
2007).
 Occurrence of wild hybrid flies (e.g. between B. papayae and B. carambolae) in MEbaited traps.
 Endogenous pheromone components are different between B. carambolae and both B.
dorsalis and B. papayae.
- Gaps identified at the start of the CRP:
 Wild populations have not been tested to confirm the ratio of the pheromone systems
between species.
 Pheromone components of B. philippinensis and B. invadens have not been
characterised.
- Progress made up to 3rd RCM in addressing identified gaps:
 Pheromone components post-ME feeding identified for laboratory-reared and wild
males of B. invadens and B. philippinensis. These components are identical to those
recorded for B. dorsalis and B. papayae.
 Endogenous pheromone components for the above four species are different to those
of B. carambolae.
 Pheromone emissions analysed for B. dorsalis, B. papayae, B. philippinensis and B.
carambolae reared under identical laboratory conditions. B. papayae and B.
philippinensis profiles are virtually identical and B. carambolae emerges as clearly
different.
 The issue of pheromone ratios in wild flies has been discussed as a group. The
consensus is that as differences in pheromone components are quantitative rather than
qualitative between wild and lab flies, this issue will not be pursued as it is not
considered critical to the question.
BEHAVIOUR
- Knowledge at the start of the CRP:
 Host preference and performance: knowledge for various individual Bactrocera
populations/species from isolated studies.
 Mating compatibility: knowledge for individual species crosses from isolated studies.
Evidence for mating compatibility between a mix of wild/long-term cultures: B.
papayae/B. carambolae; B. dorsalis/B. invadens; B. dorsalis/B. carambolae; B.
philippinensis/B. dorsalis.
43
- Gaps identified at the start of the CRP:
 Host preference and performance: comparative knowledge across populations/species.
 Mating compatibility: comparative knowledge across populations/species from across
their geographic range acquired under equivalent/simultaneous conditions.
- Progress made up to 3rd RCM in addressing identified gaps:
 An assessment of published host lists has been made in a comparative sense
among the species, but differences in sampling effort make such comparisons
biologically difficult. The extreme logistic difficulties of doing this work in the
field means that it has not been undertaken.
 Comparative sexual compatibility studies have been completed in field cages at
Seibersdorf for among ‘wild-ish’ (within F5) B. dorsalis s.s. (Thai origin), B.
papayae (Malaysian origin), B. philippinensis (Philippine origin), and B.
carambolae (Suriname origin). Pre and post-zygotic (egg-hatch) data demonstrate
complete compatibility among B. dorsalis s.s., B. papayae, and B. philippinensis;
whereas all three of these species are relatively incompatible with B. carambolae.
 Mating compatibility tests have also been undertaken between B. dorsalis s.s.
(Thai origin) and B. invadens (Kenya origin) – results demonstrate compatibility
between these two species, regardless of ME feeding.
 B. dorsalis and B. carambolae remained relatively sexually incompatible
following ME feeding by males.
 Further compatibility tests (prezygotic and postzygotic to F2) have been completed
between B. dorsalis from Wuhan (China) and Tandojam (Pakistan) and a new
colony of B. invadens from Kenya. Full mating compatibility has been
demonstrated between both B. dorsalis populations and B. invadens. Hybrid
offspring obtained between both crosses are also viable to the F2 generation.
 Extensive mating compatibility studies between populations of B. dorsalis s.s.
(from 12 provinces; 6 regions), B. papayae (6 provinces; 4 regions) and B.
carambolae (5 provinces; 3 regions) conducted in Thailand. Current results
completed for intra-specific intra-regional crosses. No evidence of incompatibility
observed so far.
 B. dorsalis and B. carambolae from the north are relatively sexually incompatible
with conspecific populations from the south of Thailand. B. dorsalis from the
north of Thailand is sexually compatible with B. papayae from the south of
Thailand. B. papayae is compatible with B. carambolae in the north of Thailand,
but not in the south of Thailand.
HOST RANGE
- Knowledge at the start of the CRP:
 Well documented for most pest populations/species being tested in this CRP, but not
yet fully for B. philippinensis and B. invadens.
 Host range data are constantly being updated in all regions.
- Gaps identified at the start of the CRP:
 Host range data not fully documented for B. philippinensis and B. invadens.
- Progress made up to 3rd RCM in addressing identified gaps:
 Host range assessment are continuously underway and new host records are being
added for B. invadens in Africa.
 It is recognised that the published host list for B. philippinensis is incomplete, but
members of the CRP have no ability to add to the list.
44
DISTRIBUTION
- Knowledge at the start of the CRP:
 Distribution data provided in the original and revised species descriptions (Drew
& Hancock, 1994).
 Despite not explicitly defined as a ‘species character-state’ in original and revised
descriptions, collection locality is considered in many operational keys to species.
For example, B. dorsalis within Thailand is restricted to central/northern Thailand
and B. papayae to southern Thailand, but new data from Seibersdorf suggests this
is not correct.
 Distributions may change over time.
 New information obtained from this CRP may contribute to revised distribution
lists.
- Gaps identified at the start of the CRP:
 Do the listed distributions provided in the species descriptions and revised
descriptions remain valid? Particularly the hypothesised zone of transition
between B. dorsalis s.s. and B. papayae on the Thai/Malay Peninsula?
 Endemic range of B. invadens is not known.
- Progress made up to 3rd RCM in addressing identified gaps:
 There is no apparent zone of transition between B. dorsalis s.s. and B. papayae on the
Thai/Malay Peninsula based on population genetic data (e.g. Fst values and
demonstrated gene flow) and morphological data that supports that these are not
different species, but rather a continuum.
 Morphological and wing shape analysis suggests that the native range of B. invadens
need not have been restricted to Sri Lanka but may have been more widely distributed
across the Indian subcontinent. B. invadens has been officially documented in Bhutan.
 CRP participants note that B. invadens has continued to spread throughout subSaharan Africa.
 New specimens of B. philippinensis and B. papayae have been collected and examined
from across the Indonesia Archipelago (R. A. I. Drew).
PROTEOMICS
- Knowledge at the start of the CRP:

No proteomic descriptions available, nevertheless, proteomics may be a good
approach to use for species discrimination, especially for proteins encoded by fast
evolving genes.
 Potentially useful if both proteomics and genomics are combined.
 There are some proteins that may be species specific in Tephritidae, e.g. proteins in
the male accessory gland.
- Gaps identified at the start of the CRP:
 No proteomic descriptions exist for B. dorsalis complex species.
 No knowledge of this technique being used for species discrimination.
 No potential species specific proteins identified for target species.
- Progress made up to 3rd RCM in addressing identified gaps:
 Pheromone binding proteins have been identified and characterised in C. capitata (as a
model species) and for some of these proteins the specificity of the pheromone volatile
component has been identified.
 The transcriptome datasets are used to infer the proteomes of these species. While
these can be stage specific, they are now used instead of classic proteomic approaches
45
for identifying classes of proteins critical for biological functions. Classic proteomics
has not been and will not be further pursued to look for species specific markers.
METABOLOMICS
- Knowledge at the start of the CRP:
 None
- Gaps identified at the start of the CRP:
 Nothing has been done on Tephritids, but may have some potential for species
discrimination.
- Progress made up to 3rd RCM in addressing identified gaps:
 Work is in progress in defining the genes and protein products involved in key
metabolic pathways of B. dorsalis.
Specific Progress and Individual Research Plans
TAXONOMY AND MORPHOLOGY
Qinge Ji
- 5 year plan:
 Establish colonies of populations of economic importance of the B. dorsalis complex
from different regions for other work (e.g. behavioural) with other members of the
CRP where possible.
 Confirm the species of B. dorsalis complex in mainland China based on
morphological characters or with supporting evidence where available (e.g.
corresponding pheromone, molecular or behavioural information).
- Plans for the first 18 months:
 Survey the populations of B. dorsalis complex in different regions in mainland China.
 Collect specimens of B. dorsalis complex by ME trapping and collecting damaged
hosts. Specimens dry pinned for morphological screening with corresponding material
alcohol preserved for future genetic screening.
 Describe and illustrate the morphological variations if they occur in collaboration with
R. A. I. Drew.
- Plans for the second 18 months:
 Continue to collect samples in different regions, covering all of mainland China.
 Continue species descriptions with Prof. D. Drew.
 Establish colonies of economically important B. dorsalis complex species for crossmating studies.
 Explore the potential to establish a culture of B. dorsalis s.s. at Seibersdorf (reared
fresh from field).
 Continue to maintain both dry pinned and alcohol collections.
- Plans for the third 18 months:
 Continue collection of fruit flies from locations of mainland China not sampled in
previous years.
 Continue the molecular analysis of B. dorsalis specimen collected.
 Cross-mate the 8 laboratory strains established and assess egg hatch and other
parametersof the offspring into three generations.
- Progress made up to 3rd RCM:
 Samples collected from fruit and through the establishment of trapping networks with
different traps types and lures from 13 provinces in China.
 Over 35,000 specimens collected and sorted.
46





Specimen identified and deposited at Fujian Agriculture and Forestry University.
Described new distribution records of known species and described some new species
(some of which may fall in the B. dorsalis complex).
Seventeen species of Bactrocera (described and undescribed) were identified.
Five new Bactrocera species described.
Genetic structure of B. dorsalis flies was analysed, indicating that the mt COI gene
within populations was greater than that between the different populations.
R.A.I Drew
- 5 year plan:
 Extensive morphometric analyses of aedeagus (and aculeus where available)
measurements of five target species from specimens already housed in existing
collections in Brisbane, in addition to material obtained through the CRP. This data
will be correlated with geographic origin of samples.
 Review existing host fruit database from fruit fly surveys across SE Asia.
- Plans for the first 18 months:
 Collaborate with Qinge Ji on morphological work from mainland China and prepare
illustrations.
 Provide diagnosis, under current taxonomic species definitions, on fly cultures
established for behavioural experiments as undertaken by other members of the CRP:
Seibersdorf, Thailand, and Malaysia research groups. N.b. minimum of 30 whole
specimens of both sexes dry pinned required with 2-3 week turnaround on
identification.
- - Plans for the second 18 months:
 Undertake a thorough morphological examination of B. dorsalis material received
from China. This will include morphometric studies and also made available for
molecular work where possible.
 Provide diagnosis, under current taxonomic species definitions, on fly cultures
established for behavioural experiments as undertaken by other members of the CRP.
N.b. minimum of 30 whole specimens of both sexes dry pinned required with 2-3
week turnaround on identification.
- Plans for the third 18 months:
 Not defined.
- Progress made up to 3rd RCM:
 Collaboration with China, Pakistan Sri Lanka, Indonesia and other locations in
southeast Asia and the Sub-Indian continent on identification of samples.
 Morphological revision undertaken based on good collections of B. invadens from
Africa and compared these with original specimens of B. invadens from Sri Lanka; all
samples examined to date are B. invadens.
 Comprehensive taxonomic monograph on southeast Asian Bactrocera published.
 Synonimization of B. philippinenesis with B. papayae.
Tony Clarke / Mark Schutze
- 5 year plan:
 Morphological and morphometric analyses of adults from all five target species in the
Bactrocera dorsalis complex.
 Morphological and morphometric analyses of juvenile stages (to be done by Gary
Steck).
- Plans for the first 18 months:
47

Continued development of geometric morphometrics as a potential discriminatory and
diagnostic tool.
 Collection of material for dispersal to molecular workers in CRP.
 Collaboration with R.A.I. Drew on morphometric data collation and analyses.
- Plans for the second 18 months:
 Finalise data analysis and publications.
 Pending financial situation, address the B. tryoni complex through a multidisciplinary
approach.
 Construct specimen database for upload to DIR-SIT.
- Plans for the third 18 months:
 Finalise and publish work on B. invadens.
 Contribute to the formal synonymization of B. invadens, B. papayae, and B.
philippinensis with B. dorsalis.
- Progress made up to 3rd RCM:
 Completed collections of material for B. dorsalis s.s., B. papayae, B. philippinensis
and B. carambolae from appropriate locations in Taiwan, Thailand, Malaysia,
Singapore and the Philippines.
 Completed collection of material of B. invadens from five countries in Africa and the
Indian Subcontinent.
 Morphological and morphometric data analysis of B. invadens and B. dorsalis material
collected from across their geographic range completed.
 Geometric morphometrics using a generalised procrustes analysis shows a population
structure that agrees with the single species hypothesis for B. dorsalis s.s., B. papayae,
B. philippinensis, and that there is evidence of isolation by distance structured around
the land-bridges of the South China Sea; B. carambolae is distinct.
 All work on south east Asian target taxa of the B. dorsalis complex completed and
published.
Sunday Ekesi / Fathiya M. Khamis
- 5 year plan:
 Collect and share biological material of B. invadens with other CRP participants as
required.
 Conduct morphometric analyses involving African, Sri Lankan, and Indian
populations of B. invadens in relation to populations of the B. dorsalis complex.
- Plans for the first 18 months:
 Obtain collections of different populations of B. invadens from Africa, Sri Lanka and
India for morphometric analysis and share with workers in CRP.
 Conduct morphometric analyses involving African, Sri Lankan, and Indian
populations of B. invadens.
- Plans for the second 18 months:
 Geometric morphometric and molecular markers to be generated for the collected
species.
- Plans for the third 18 months:
 None.
- Progress made up to 3rd RCM:
 B. invadens collected from Africa (Kenya, Uganda, Nigeria) and Sri Lanka. Other B.
dorsalis complex species (B. dorsalis s.s. and B. kandiensis) collected from Hawaii
and Sri Lanka. Outgroup species (B. correcta,, B. cucurbitae, B. oleae and B. zonata)
collected from Sri Lanka, Kenya and Mauritius.
48

Morphometric analysis completed, carried out in conjunction with COI gene
confirmation.
BEHAVIOUR
Tony Clarke/Mark Schutze/Seibersdorf staff
- 5 year plan:
 Specimens from each established culture confirmed by R.A.I. Drew.
 Post-zygotic compatibility measured to egg-hatch of F1 from crosses.
 Where possible, characterise additional elements of the mating system of B. tryoni
complex as a model system for the target species (e.g. acoustic communication,
pheromone capture).
- Plans for the first 18 months:
 Carry out inter-specific cross-mating compatibility between at least 4
population/species (B. papayae (Serdang*, Malaysia), B. dorsalis s.s. (Saraburi and
Nakhon Si Thammarat, Thailand), B. philippinensis (Guimaras Is., Philippines), and B.
carambolae (Paramaribo, Suriname)). Collect data as per FAO/IAEA protocols (e.g.
compatibility indices).
 Possible inclusion of Todd Shelly in carrying out remaining cross-mating trials at
Seibersdorf.
 Possible R&D for Todd Shelly on studies on ±ME effects in cross-mating trials.
- Plans for the second 18 months:
 Finalise data analysis and publications.
 Complete studies on ME effects in cross-mating trials.
 Pending financial situation, address the B. tryoni complex through a multidisciplinary
approach.
 Construct specimen database for upload to DIR-SIT.
- Plans for the third 18 months:
 Confirm B. kandiensis identity and complete compatibility study.
 Seibersdorf maintaining colonies.
- Progress made up to 3rd RCM:
 Inter-specific cross-mating compatibility and postzygotic studies between
population/species of B. dorsalis, B. papayae, B. philippinensis, B. invadens, and B.
carambolae complete.
 Preliminary compatibility studies between B. kandiensis and B. dorsalis carried out at
Seibersdorf.
 ME male feeding did not reduce relative incompatibility between B. dorsalis and B.
carambolae.
Suksom Chinvinijkul/Nidchaya et al.
- 5 year plan:
 Include B. carambolae as per other species.
 Specimens from each established culture confirmed by R.A.I. Drew.
 Post-zygotic compatibility measured from egg-hatch of F1 to pupation and sex-ratios
from crosses.
- Plans for the first 18 months:
 Confirm that B. dorsalis s.s. is represented by one species in Thailand, by carrying out
intra-specific cross-mating compatibility among six geographic populations of B.
dorsalis s.s. Populations sourced from different hosts.
49

Confirm that B. papayae is represented by one species in Thailand, by carrying out
intra-specific cross-mating compatibility among three geographic populations of B.
papayae. Populations sourced from different hosts.
 Based on objectives 1+2, carry out inter-specific cross-mating compatibility tests
between B. dorsalis s.s. and B. papayae.
- Plans for the second 18 months:
 Based on objectives 1 and 2, carry out inter-specific cross-mating compatibility tests
between B. dorsalis s.s. and B. papayae.
 Prioritise inter-specific cross-mating tests between geographically extreme populations
(North-South, East-West) of B. dorsalis s.s., B. papayae (North-South) and B.
carambolae.
- Plans for the third 18 months:
 Confirmation of identity of populations used in the Thai north-south mating
compatibility studies using molecular markers and the completion of pheromone
analysis.
 Prepare publication for final CRP proceedings.
- Progress made up to 3rd RCM:
 All B. dorsalis s.s. intra-region cross-mating complete.
 All B. papayae and B. carambolae intra-region cross-mating complete.
 All north and south inter-regional cross-mating studies complete.
 B. dorsalis and B. carambolae from the north are relatively sexually incompatible with
conspecific populations from the south of Thailand. B. dorsalis from the north of
Thailand is sexually compatible with B. papayae from the south of Thailand. B.
papayae is compatible with B. carambolae in the north of Thailand, but not in the
south of Thailand.
A.K.W. Hee / S.L. Wee / K.H. Tan
- 5 year plan:
 Will focus on cryptic species response to ME (laboratory reared) and genetic analysis
(in collaboration with Thai laboratory) of wild flies that have responded to ME.
 Male cryptic species (B. invadens, B. philippinensis, B. dorsalis s.s. and B. papayae)
behavioural response to ME.
 Identification of putative antennal polypeptides specific to the above-mentioned
species following exposure to ME, as means for possible use in species discrimination.
 Original plan involving cross-mating studies has been abandoned due to serious
biosecurity and ethical issues in conducting the outdoor field cage studies involving B.
invadens, B. philippinensis and B. dorsalis s.s.
 Molecular analysis outsourced to Thailand group.
- Plans for the first 18 months:
 Establishment of wild/wildish fruit fly culture of B. papayae and cultures of B.
invadens, B. philippinensis and B. dorsalis s.s. (to be obtained from IAEA
Seibersdorf).
- Plans for the second 18 months:
 Males of target species (B. invadens, B. philippinensis, B. dorsalis s.s. and B. dorsalis
s.l.) assessed for behavioural response to ME using quantal response bioassays.
 Identification of putative antennal polypeptides specific to the above-mentioned
species following exposure to ME, as a proteomic technique for possible use in
species discrimination.
- Plans for the third 18 months:
50

Maintain colonies for ongoing chemical ecology.
- Progress made up to 3rd RCM:
 Establishment of wild fly cultures of B. papayae and that of B. invadens, B.
philippinensis and B. dorsalis s.s. successfully obtained from IAEA Seibersdorf under
strict biosecurity containment. Flies used in chemical ecology (see below).
Sunday Ekesi / Fathiya M. Khamis
- 5 year plan:
 Investigate whether heritable life history differences exist between African and Asian
populations of B. invadens.
- Plans for the first 18 months:
 Collection and establishment of colonies of African and Asian population.
- Plans for the second 18 months:
 Investigation of heritable life history differences among populations.
- Plans for the third 18 months:
 None.
- Progress made up to 3rd RCM:
 Collections of B. invadens from Africa and a sample from Sri Lanka and other
Bactrocera species including B. kandiensis (Sri Lanka), B. zonata (Mauritius), B.
dorsalis s.s. (Hawaii), B. cucurbitae (Kenya), B. oleae (Kenya) and B. correcta (Sri
Lanka). Samples from Sri Lanka of B. invadens and B. kandiensis were too few to be
shared with members of the CRP.
CHEMICAL ECOLOGY
Suksom Chinvinijkul et al.
- 5 year plan:
 Rectal-gland pheromone collection and analysis for B. dorsalis s.s. (from all six
regions) and B. papayae (from all three regions) – from year 3 of CRP. Analysis
outsourced to Malaysian group (Tan et al).
- Plans for the first 18 months:
Collection and forwarding of materials to Malaysia for rectal-gland pheromone
collection and analysis.
- Plans for the second 18 months:
 Collection and forwarding of materials to Malaysia for rectal-gland pheromone
collection and analysis.
- Plans for the third 18 months:
 Identification of the material provided for chemical ecology analysis to be confirmed
by further independent evidence. Material from all crossing colonies to be sent to
Mahidol University, and outstanding material to be sent to Alvin for remaining
pheromone analysis.
- Progress made up to 3rd RCM:
 Material has been collected and sent to Malaysia for rectal-gland pheromone
collection and analysis.
S.L. Wee / A.K.W. Hee / Ritsuo Nishida / K.H. Tan
- 5 year plan:
 Rectal gland analysis on material collected and supplied by Thai group (see above).
51

Pheromone collection (pheromone gland (+/-ME fed) and headspace) and analysis for
wild/wildish individuals of all five target species.
- Plans for the first 18 months:
 Pheromone collection (pheromone gland (+/-ME fed) and headspace) and analysis for
wild/wildish individuals of B. carambolae and B. papayae.
 Components and ratios measured.
- Plans for the second 18 months:
 Pheromone collection (pheromone gland (+/-ME fed) and headspace) and analysis for
wild/wildish individuals of the five target species.
 Determination of the pheromone system of the field morphohybrids.
- Progress made up to 3rd RCM:
 Sex pheromones of B. invadens and B. philippinensis following ME consumption have
been identified.
 Headspace sampling for B. invadens, B. philippinensis, B. dorsalis and B. papayae
male pheromone emissions with and without ME consumption has been analysed.
Quantitative and qualitative analysis of ME related pheromone components has been
completed. The ratio of the two components (E)-coniferyl alcohol and 2-allyl-4,5dimethoxy phenol were found to be identical across the four species.
 Analysis of material supplied by Thai collaborators showed inconsistency with
morphological identifications of species used in mating compatibility studies in
Thailand.
- Plans for the third 18 months:
 Complete headspace analysis of male pheromone emissions of ME fed and unfed flies.
 Following the confirmation of identifications of Thai material, resolve inconsistency
previously detected in Thailand.
 Complete the probit bioassay of males of the four species to ME to measure relative
dose response.
 Complete analysis of the ME exposed male antenna protein profiles of the four
species.
P. Teal/I ul Haq/Seibersdorf
- Plans for the third 18 months:
- Complete work on the sensitivity of B. dorsalis, B. carambolae and B. invadens to
different doses of ME in field cages.
- Confirm qualitative and quantitative profiles of male released volatiles of B. invadens,
B. philippinensis, B. papayae, B. dorsalis and B. carambolae to assess species limits
within the target taxa.
- Analyse male pheromone emissions collected from male flies exposed to different
amounts of ME.
- Prepare flies for male antennal protein extracts.
- Progress made up to 3rd RCM:
- Initial work on the sensitivity of B. dorsalis, B. carambolae and B. invadens to
different doses of ME has been completed in field cages.
- Initial qualitative and quantitative profiles of male released volatiles of B.
philippinensis, B. papayae, B. dorsalis and B. carambolae to assess species limits
within the target taxa.
MOLECULAR AND GENETIC
52
Karen Armstrong/QUT
- 5 year plan:
 Undertake phylogenetic and other genetic analyses of the sequence data.
 Based on the inferred species delineations from molecular and behavioural data,
retrospective identifications will be sought from R.A.I. Drew.
 Based on the inferred species delineations from molecular and behavioural data,
molecular diagnostic markers will be developed.
- Plans for the first 18 months:
 Screening mtDNA and nDNA genes for geographic populations of at least the
following four species: B. dorsalis s.s. (Thailand, Taiwan), B. papayae (Malaysia,
Indonesia) B. carambolae (Suriname, Malaysia, Indonesia) and B. philippinensis
(Philippines). Specimens provided by TC/MS through Australia.
 Collaboration with Norman Barr in genetic studies.
- Plans for the second 18 months:
 Finalise data analysis and publications.
 Pending financial situation, address the B. tryoni complex through a multidisciplinary
approach.
 PCR population analysis of selected SNP regions identified through comparative
transcriptome analysis.
- Plans for the third 18 months:
 Publish comparative transcriptome dataset and related SNP and phylogenetic analysis.
 Provide transcriptome dataset from several species/populations to Anna Malacrida to
identify the specific genes related to reproduction and behaviour.
 Confirmation of identity of B. carambolae, B. papayae and B. dorsalis populations
used in Thai north-south mating compatibility studies using molecular markers.
- Progress made up to 3rd RCM:
 All four target taxa plus B. invadens and outgroups (B. occipitalis, B. cacuminata, B.
opiliae, B. kandiensis, B. musae, B. tryoni) have been screened for two mtDNA and
two nDNA and two rDNA regions.
 Data has been incorporated into a single-gene and combined-gene phylogenetic
analysis. The COI and ITS1 gene regions show B. carambolae and B. occipitalis as
distinct relative to the other four species.
 Collaboration with Norman Barr on the barcoding project is being maintained.
 Comparative transcriptome data for B. dorsalis s.s., B. papayae, B. philippinensis, B.
carambolae and B. invadens have been analysed for ‘species’ specific SNPs.
 Using wild populations, new species specific SNP markers have been found for B.
carambolae and B. occipitalis.
 Phylogenetic analysis using loci with polymorphic SNPs has been carried out for B.
dorsalis s.s., B. papayae, B. philippinensis and B. carambolae.
Anna Malacrida
- 5 year plan:
 Microsatellite analysis of B. dorsalis s.s. (Thailand, China, and other SE Asian
locations) and B. invadens (Sub-Saharan Africa and Sri Lanka).
 For at least one species, potential EST library development for specific relevant tissues
associated with behavioural or reproductive biology (from year 3).
- Plans for the first 18 months:
 Extension of microsatellite analysis to specimens provided by Karen Armstrong and
Nidchaya Aketarawong.
53
- Plans for the second 18 months:
 Publish the work on B. dorsalis s.s.
 Screen other samples from China if they become available.
 Derive specific marker for B. dorsalis s.s. from y chromosomes.
 Using the C. capitata transcriptomes, odor and pheromone binding genes that have
been identified, compare B. dorsalis s.s. transcriptomes to identify homologous genes.
- Plans for the third 18 months:
Complete molecular and functional characterisation of the Y-chromosome of B.
dorsalis s.s. (Saraburi) and to extend this characterisation to wild populations and to
other target taxa of the B. dorsalis complex. This will be done in collaboration with
Mahidol University (Thailand).
 Undertake bioinformatics analysis of the transcriptome dataset from several
species/populations provided by Karen Armstrong to identify the specific genes
related to reproduction and behaviour.
 Provide sequences to Antigone Zacharopoulou to be used for in situ hybridisation for
chromosome mapping.
- Progress made up to 3rd RCM:
 The genotyping of six Chinese populations has been performed for B. dorsalis s.s.
 Database of SSR fingerprint of 20 populations of B. dorsalis s.s. across the species
range has been constructed. This allows us to potentially identify the origin of new
outbreaks of B. dorsalis s.s.
 Specific Y-chromosome sequences have been identified in B. dorsalis s.s. (Saraburi
lab strain, Seibersdorf). SNPs within one of them have been identified. Transcribed
sequences have been identified.
Antigone Zacharopoulou / Antonios Augustinos / Elena Drosopoulou / Kostas Bourtzis
- 5 year plan:
 Polytene chromosome analysis of the five target species (as listed previously) from
colonies in Seibersdorf.
 Same for F1 progeny or subsequent generations if available.
 Upon variability of the strains assess the need to analyse more fly strains at the
chromosome level (beyond 18 months).
 Depending on the chromosomal maps obtained, diagnostic characters for each species
will be developed accordingly.
 Given the opportunity, genomic information developed elsewhere (from within or
outside from the CRP) will be mapped onto the chromosomes.
- Plans for the first 18 months:
 Screening using mitotic polytene chromosomes from established colonies.
 Chromosome distribution of the two elements: piggybac and Hopper elements in the
same materials from point 1 through in situ hybridisation.
- Plans for the second 18 months:
 Analysis of polytene chromosomes of more F1 hybrids depending on availability of
material at Seibersdorf.
 As a priority and where possible, in situ hybridisation of uniquely localised markers
for the five target members of the complex, for example genome project derived data,
ESTs – in collaboration with genomics groups.
 Possibly more extended in situ hybridisation of piggyBac and Hopper for more
samples from more populations depending on availability of material at Seibersdorf.
54

Polytene chromosome analysis of closely related outgroup species, depending on
availability of material at Seibersdorf.
- Plans for the third 18 months:
 Publish the results from comparative chromosome analysis.
 Enrich cytogenetic maps using new sequences including those provided by Anna
Malacrida.
 Analyse and compare the complete mitochondrial genomes of the five target taxa of
the B. dorsalis complex.
 Include outgroup (B. kandiensis or B. occipitalis) from target taxa for comparison of
chromosome analysis.
- Progress made up to 3rd RCM:
 Comparison of B. papayae, B. philippinensis, B. invadens and B. carambolae to the
reference map of B. dorsalis s.s. has been done, showing no striking differences.
 Polytene chromosome analysis of bidirectional F1 hybrids of B. dorsalis s.s. and B.
invadens, and B. dorsalis s.s. and B. carambolae has been completed and no
detectable differences have been found except for few minor asynapses in the hybrids
of B. dorsalis and B. carambolae.
 Preliminary in-situ hybridisation of piggyBac and Hopper in the five target members
of the complex has been performed. Six unique genes have been mapped on the
polytene chromosomes of B. dorsalis and the above hybrids.
Nidchaya Aketarawong et al.
- 5 year plan:
 Complete the genetic analysis of B. dorsalis species complex and examine congruence
with other types of evidence and diagnostic characters.
- Plans for the first 18 months:
 Complete screening, start applying microsatellite DNA typing and carry out genetic
analysis of B. dorsalis species population samples from the following theme:
o B. dorsalis species complex colonies from Seibersdorf.
o Wild B. dorsalis species complex population samples across geographical
range.
o Wild B. dorsalis species complex population samples from Malaysian
Peninsula.
 Begin providing genotyping and genetic analyses for the other group sample:
o Behaviour: samples from Suksom et al.
o Chemical ecology: samples from Suk Ling Wee.
o Taxonomy.
 Increase potential to develop and evaluate novel molecular markers, diagnostic tools.
- Plans for the second 18 months:
 Collect B. carambolae from Thai/Malay Peninsula and Indo/Malay Archipelago.
 Collect B. carambolae from collaborators.
 Develop and evaluate molecular markers for B. carambolae populations.
- Plans for the third 18 months:
 Characterize Y-chromosome in different populations/species of B. dorsalis s.s. and B.
carambolae
 Confirmation of identity of populations used in north-south mating compatibility
studies using molecular markers.
 Try to collect samples of B. carambolae from wider georgraphic distribution and
subject to genetic tests.
55
- Progress made up to 3rd RCM:
 Completed collection and genotyping of B. dorsalis s.s. and B. papayae populations
across southern Thailand and the Malay Peninsula using microsatellite markers.
 Analysis of population genetics based on these microsatellites inferred significant
gene flow between sympatric and allopatric populations. Also isolation-by-distance
was not observed across southern Thailand and the Malay Peninsula.
 Microsatellite DNA markers tested on B. dorsalis s.s. and B. papayae didn’t show
fixed alleles consistent with taxa-specific identification.
 Four specific Y-chromosome sequences (contig 1, 2, 3, and 4) have been identified in
B. dorsalis s.s. (Saraburi lab strain, Seibersdorf). These sequences have been tested in
four populations of B. dorsalis s.s. (three lab strains and one wild). No differences
were found for male specific amplification.
 Two populations of B. carambolae from Thailand (north and south), one from
Malaysia and three from Indonesia (Java, Sumatra, Borneo) were received. Of these,
only one sample from Thailand (south) and one from Sumatra were tested for male
specificity of these Y-chromosome sequences. In the wild sample from Indonesia,
only contig 2 display a male specific pattern. In the sample from Thailand, the
amplification patterns indicate no evidence of male specificity for the four contigs.
These preliminary data seem to indicate the differences between the Thailand and
Indonesia samples.
Sunday Ekesi / Fathiya M. Khamis
- 5 year plan:
 Obtain samples of B. invadens populations from Africa, and Sri Lanka including
colonies already established and contribute to the development of a population
genotype database (in collaboration with A. Malacrida).
 Evaluate DNA variation using mitochondrial and nuclear gene sequences and compare
populations for evidence of population structure and potentially distinct gene pools.
 Collect and share biological material of B. invadens with other CRP participants as
required.
- Plans for the first 18 months:
 Obtain collections of different populations of B. invadens from Africa and Sri Lanka
for DNA variation analysis, population genotype database and sharing with workers in
CRP.
 Screen different populations for one mitochondrial (COI) and nuclear (period) gene.
- Plans for the second 18 months:
 Other molecular markers will be used for confirmation (e.g. period gene).
- Plans for the third 18 months:
 None.
- Progress made up to 3rd RCM:
 Collections of B. invadens from Africa and a sample from Sri Lanka and other
Bactrocera species including B. kandiensis (Sri Lanka), B. zonata (Mauritius), B.
dorsalis s.s. (Hawaii), B. cucurbitae (Kenya), B. oleae (Kenya) and B. correcta (Sri
Lanka).
 The morphometric analyses were confirmed with COI gene.
56
57
Ceratitis FAR Complex
Background Situation Analysis
The Afro-tropical group of fruit flies, Ceratitis fasciventris, C. anonae and C. rosa (FAR),
together with C. capitata, are considered to be among the major horticultural pests of that
region (White & Elson-Harris 1992; De Meyer 200la) and these species are of quarantine
significance (EPPO/CABI 1997). They are highly polyphagous and damage a wide range of
unrelated wild and cultivated crops (De Meyer et al. 2002a), resulting in enormous economic
losses wherever they occur (Barnes 2000; Lux 2000; De Meyer 2001b).
Ceratitis rosa, C. fasciventris and C. anonae are considered the three members of the
Ceratitis FAR species complex (Virgilio et al. 2007a, 2007b). From the taxonomic point of
view, C. fasciventris was considered to be a variety of C. rosa (Bezzi 1920). Only recently
has it been recognized as a different entity with species status (De Meyer 2001a).
Unlike C. capitata, which has spread from its home range in East Africa and attained an
almost world-wide distribution over the last century (Fletcher 1989; White & Elson-Harris
1992), C. rosa, C. fasciventris and C. anonae have so far not been reported outside the
African continent (except La Réunion and Mauritius) but are potentially invasive.
Due to the difficulty in distinguishing some members of the complex morphologically, a
number of molecular approaches for species recognition were used (Baliraine et al., 2004;
Barr et al., 2006; Virgilio et al., 2008). However these remain inadequate for quarantine
purposes and much more robust molecular markers are needed.
Baseline and Progress on the Ceratitis FAR Complex
DNA
- Knowledge at the start of the CRP:
 Attempts to develop specific diagnostic markers were made but they are so far
ineffective.
- Gaps identified:
 There is the need for further exploration for markers, especially regarding
microsatellites.
- Progress up to 3rd RCM in addressing identified gaps:
 Microsatellites were developed for the Ceratitis FAR complex. African populations
for all three species were analysed. Population genetic structure for the complex
revealed five clearly distinguishable clusters: C. fasciventris: F1, F2; C. anonae: A; C.
rosa: R1, R2.
 There is still the need for a diagnostic marker, either as a sequence (explore ITS2) or
banding pattern (microsatellites).
CYTOLOGY
- Knowledge at the start of the CRP:
 No data available. Cytology has the potential to provide a diagnostic tool.
- Gaps identified:
 Need for researcher to conduct cytological studies of this complex.
- Progress up to 3rd RCM in addressing identified gaps:
58

No studies conducted so far.
ALLOZYMES
- Knowledge at the start of the CRP:
 Previous work conducted by Malacrida et al. (1996) only on C. rosa (perhaps also
including C. fasciventris).
- Gaps identified:
 No further work considered under the CRP; covered by DNA work.
GENOMICS
- Knowledge at the start of the CRP:
 Current focus on C. capitata. Y chromosome sequences are available. Transcriptomics
from different sexes and different stages are available. This allows identifying genes
related to odorant binding proteins, pheromone binding proteins and genes correlated
to spermatogenesis and male accessory glands.
- Gaps identified:
 This knowledge is not available for representatives of the Ceratitis FAR complex;
such study can be considered.
- Progress up to 3rd RCM in addressing identified gaps:
 No further studies conducted.
MORPHOLOGY
- Knowledge at the start of the CRP:
 Males can be distinguished to some extent.
- Gaps identified:
 Separation of females is difficult.
 Larval morphology not studied.
- Progress up to 3rd RCM in addressing identified gaps:
 After recognition of five clusters (based on molecular work), adult characters for
males (and to lesser extent for females) were re-examined. Consistent morphological
differences were found to distinguish male C. rosa (R1-R2) and C. fasciventris (F1F2). Male morphological differences were used to re-examine and assign available C.
rosa and C. fasciventris specimens to the different genotypes.
 Larval morphology for representatives of each taxonomically recognized species
within the Ceratitis FAR complex was studied. SEM studies for all immature stages
were conducted. Within C. rosa unusual larval findings contradict earlier observations
based on South African populations. No distinct differences are observed between R1
and R2 but more detailed investigation of larval morphology is required.
MORPHOMETRICS
- Knowledge at the start of the CRP:
 No data available.
- Gaps identified:
 Morphometric study on females might have potential for separation of females.
- Progress up to 3rd RCM in addressing identified gaps:
 Morphometric study was conducted on all five genotypes, using wing landmarking
and wing banding. Wing landmarking provides some separation between the three
FAR species and, to a lesser extent, the five genotypes for both sexes. Wing banding is
providing no clear resolution.
59
MALE LURE RESPONSE
- Knowledge at the start of the CRP:
 All males are attracted to trimedlure.
- Gaps identified:
 Response of representatives of the Ceratitis FAR complex to male lures to be
investigated.
- Progress up to 3rd RCM in addressing identified gaps:
 Response of representatives of the Ceratitis FAR complex to EGOlure was
investigated in Tanzania and La Réunion. EGOlure has been shown to be a stronger
attractant for C. rosa (R1 and R2) than trimedlure in Tanzania. Data of the Réunion
study is under analysis.
DEVELOPMENTAL PHYSIOLOGY
- Knowledge at the start of the CRP:
 Developmental physiology has not been used for diagnostic purposes.
- Gaps identified:
 Given the recent progress in the genetic structure of the complex, there is the need for
additional studies to determine whether there are developmental/ physiological
differences between the entities recognized.
- Progress up to 3rd RCM in addressing identified gaps:
 These studies have started for the two C. rosa types. There is a positive linear
relationship between temperature and development rate. A different upper temperature
threshold was observed but not a lower, with the R1 type being more tolerant to higher
temperature.
BEHAVIOUR
- Knowledge at the start of the CRP:
 Some behavioural work on C. rosa, limited for the other entities.
 Limited experiments under forced laboratory conditions have shown that all
taxonomically recognized species of the FAR complex can interbreed (providing
viable offspring for cross-mating between C. rosa and C. fasciventris).
- Gaps identified:
 Mating compatibility studies under semi-natural field cage conditions can contribute
to resolving the specific status of the taxa within the complex.
 Given the recent progress in the genetic structure of the complex, there is the need for
additional studies to determine whether there are behavioural differences between the
entities recognized.
- Progress up to 3rd RCM in addressing identified gaps:
 Mating compatibility studies have started for the two C. rosa entities and appear to
show some reproductive isolation between the two.
PHEROMONE COMPONENTS
- Knowledge at the start of the CRP:
 Pheromones of entities recognized not studied.
- Gaps identified:
60

Pheromone component studies can contribute to resolving the specific status of the
taxa within the complex.
 Given the recent progress in the genetic structure of the complex, there is the need for
additional studies to determine whether there are pheromone differences between the
entities recognized.
- Progress up to 3rd RCM in addressing identified gaps
 Pheromones were investigated and preliminary identified for all three FAR species.
All species share some components but also demonstrate specific elements.
 The sex pheromones of both species are multicomponent. The composition of
investigated pheromones is different from that of C. capitata, except for some
components like farnesene.
HOST RANGE
- Knowledge at the start of the CRP:
 Host range is well known for representatives of the complex.
- Gaps identified:
 Given the recent progress in the genetic structure of the complex, there is the need for
additional studies to determine whether there are host range differences between the
entities recognized.
- Progress up to 3rd RCM in addressing identified gaps:
 Host range for the two F and two R entities have been re-examined based upon
existing records. Information for the F entities is inconclusive because of lack of data
for F1. R1 and R2 do not show major differences in host range except that the host
from temperate climates (Pyrus, Rubus, Coffea) are predominantly infested by R2.
DISTRIBUTION
- Knowledge at the start of the CRP:
 Distribution patterns are well known for representatives of the complex.
- Gaps identified:
 Given the recent progress in the genetic structure of the complex, there is the need for
additional studies to determine whether there are distributional differences between
the entities recognized.
- Progress up to 3rd RCM in addressing identified gaps:
Distribution range has been re-examined based upon existing records. R1 distribution is
limited by latitude (absent southern part of the African continent) and altitude (absent from
higher altitudes). F1-F2 largely geographically separated (western-central versus eastern
Africa). However, isolated populations of ‘western’ type found in Angola, Malawi, Tanzania,
Zambia. New altitudinal transect study has been started in Tanzania to reveal seasonal
changes and altitudinal limitations.
CUTICULAR HYDROCARBONS
- Knowledge at the start of the CRP:
 Cuticular hydrocarbons of the Ceratitis FAR complex not studied.
- Gaps identified:
 Cuticular hydrocarbon studies can contribute to resolving the specific status of the
taxa within the complex.
 Given the recent progress in the genetic structure of the complex, there is the need for
cuticular hydrocarbon comparisons to determine whether there are differences
between the entities recognized.
61
- Progress up to 3rd RCM in addressing identified gaps:
 Cuticular hydrocarbons of both sexes (virgin, 20 days old) were analyzed. Specific
differences were detected between the three taxonomically recognized species of the
complexes. Sexual differences were also found in each species. The CHC from the
two C. rosa (R1, R2) – from Kenya, are obtained and the CHC of males and females
of C. rosa R1 will be analysed by the means of GCxGCMS during September and
October 2013.
62
Specific Progress and Individual Research Plans: Ceratitis FAR Complex
General observation: Given the difficulties in establishing a colony of C. fasciventris F1 type,
it is deemed not feasible to gather all information from the different approaches to
differentiate between F1 and F2. Therefore, the focus is on R1-R2 differentiation, and further
F1-F2 differentiation is not considered. Should a F1 colony be forthcoming in the near future
of the last 18 months, inclusion of F1-F2 will be re-considered.
TAXONOMY
Marc de Meyer
- 5 year plan:
 Provide diagnostic markers to differentiate adults of both sexes for species within the
complex (except below).
 In collaboration with Fellow from Seibersdorf laboratory: provide morphometric
diagnostic markers to differentiate females within the complex
- Plans for the last 18 months:
 Explore the possibility of using the most significant differences in wing landmarking,
as defined by the multivariate analysis, into a suitable identification tool to
differentiate the females of the three FAR species and five genotypes.
 Continue re-examination of taxonomic collections for further assignment of collection
specimens to the different entities.
- Progress made up to 3rd RCM:
 After recognition of five clusters (based on molecular work), adult characters for
males (and to lesser extent for females) were re-examined. Consistent morphological
differences were found to distinguish male R1-R2 and F1-F2. Male specimen holdings
in taxonomic collections (Royal Museum for Central Africa Tervuren, Natural History
Museum London, Stellenbosch University Stellenbosch, South African Collections
Pretoria) were re-examined and assigned to one of the five genotypes.
 Morphometric study, based on wing landmarking and wing banding was conducted by
a MSc student of the University of Antwerp (J. Van Cann) for specimens used in
genotypic study. Results show clear sexual dimorphism in all five genotypes. Wing
landmarking allow separation to a certain extent of all three FAR species and, less so,
of the five genotypes.
Sunday Ekesi / Fathiya M. Khamis
- 5 year plan:
 Collect and share biological material of the FAR group with other CRP participants as
required.
- Plans for the last 18 months:
 Establishment of R2 in Tanzania and further sharing of material with participants as
required (pheromones, endosymbiosis) will be done.
 Attempts will be made to establish a C. fasciventris colony from western Africa.
 Material of South African collections will also be shared with partners on larval
morphology to clarify earlier observed differences.
- Progress made up to 3rd RCM:
 Colonies for the three taxonomically recognized species were established at ICIPE
(Kenya)
and
the
material
was
shared
with
other
teams
for
pheromones/hydrocarbons/larval taxonomy/DNA research.
63



Furthermore colonies for R1-R2 were established at ICIPE (Kenya): lowland
population of Ceratitis rosa from guava fruits collected from Mwanjamba,
Msambweni, Kenya and the highland populations was reared from mango fruits
collected from Kithoka, Meru, Kenya and material shared with partners working on
larval morphology and cuticular hydrocarbons.
Colony of R2 from Tanzania at SUA is progressing well but difficulties are
encountered with the colony from R1 and we have resorted to use environmental
chamber because of the low ambient temperatures.
A third partner (Citrus Research International, South Africa) is also in the process of
establishing colonies of both R1 and R2 from South Africa to conduct studies on
developmental physiology and mating compatibility in conjunction with the Kenyan
and Tanzanian partners.
Gary Steck / Sunday Ekesi
- 5 year plan:
 To provide indication whether morphological characters of immature stages (larvae)
provide diagnostic character for differentiation.
- Plans for the last 18 months:
 Conduct an in depth study of larval morphology for all entities currently available.
Supplement the R1-R2 with material from South Africa to resolve the issue of
contradictory information of an earlier study. This study will be conducted by a PhD
student from ICIPE in collaboration with Gary Steck.
- Progress made up to 3rd RCM:
 Larval morphology for representatives of each taxonomically recognized species
within the FAR complex was studied.
 SEM studies for all immature stages were conducted.
 Within C. rosa unusual findings contradict earlier observations based on South
African populations.
 A sample of R1 3rd instar larvae, originating from the coastal region of Kenya was
received and examined. Preliminary SEM examination shows them to be similar to the
Kenyan highland population, i.e., oral ridge accessory plates are present also in the
coastal population.
BEHAVIOUR
Maulid Mwatawala / Sunday Ekesi
- 5 year plan:
 If necessary: to provide additional information on the specific status in case of
conflicting data from other work packages using mating compatibility tests.
- Plans for the last 18 months:
 Mating compatibility, both pre and post zygotic will be further conducted in Kenya
and Tanzania (pending establishment of the colonies). Contacts will be made with CRI
(South Africa to enquire whether they will also conduct these tests).
 Behavioural studies (including mating, responses to attractant etc) with regard to the
highland population of C. rosa collected from coffee (Ruiru) has previously been
documented (Pontiano Nemeye’s thesis) but no information is available with regard to
the lowland population.
 In addition to assessing mating compatibility, observation on courtship
repertoire/mating behaviour will be recorded in field cages. Similarly, responses of the
64
two populations to trimed lure, EGOlure and synthetic food attractants will be
documented in field cages.
- Progress made up to 3rd RCM:
 Protocols on mating compatibility tests have been exchanged between Kenya and
Tanzania. Studies have started in Kenya: Only 3 replications have been conducted and
the results obtained so far showed some degree of mating incompatibility between the
2 populations of C. rosa and also between the 2 populations and C. fasciventris. The
ISI values ranged from 0.75 to 0.90 with the Highland C. rosa x the Lowland C. rosa
showing the highest degree of isolation. More replications are planned for September
when the colony population increases. Tanzania: The cages for this study have been
designed and the trials will start as soon as fairly large colonies of the two C. rosa type
have been established.
CHEMICAL ECOLOGY
Blanka Kalinova/ Lucie Vanickova
- 5 year plan:
 To improve the efficiency of new and sustainable control methods by means of use the
sex pheromone and cuticular hydrocarbon profile of Ceratitis spp. of the FAR
complex.
- Plans for the last 18 months:
 Pheromone analysis: obtain material of R1 and R2 at beginning of 2014 to study sex
pheromone patterns. Obtain additional material of F2 and A at mid-2014 to complete
the EAG analysis.
- Progress made up to 3rd RCM:
 Pheromones were investigated and preliminary identified for C. rosa and C. anonae.
The sex pheromones of the species are multicomponent. Both species share some
components but also demonstrate specific elements. The composition of investigated
pheromones is different from that of C. capitata, except for some components like
farnesene.
 Cuticular hydrocarbons of both sexes (virgin, 20 days old) were analysed. Specific
differences were detected between the three taxonomically recognized species of the
complexes. Sexual differences were also found in each species. The CHC from the
two C. rosa (R1, R2) – from Kenya, are obtained and the CHC of males and females
of C. rosa R1 will be analysed by the means of GCxGCMS during September and
October 2013 and the results will be compared with the results of chemical analyses of
C. rosa R2.
MOLECULAR
Hélène Delatte / Massimiliano Virgilio / David Haymer / Anna Malacrida
- 5 year plan:
 Provide discriminant markers that can be used as a diagnostic tool to differentiate the
three species of the complex.
- Plans for the last 18 months:
 David: Exploration of utility of ITS2 as a diagnostic markers using about 50 samples
used for the genotyping (5 individuals x 2 populations x 5 genetic entities). Send
material end of 2013, beginning of 2014.
 Anna: use sequences from C. capitata to derive variable changes to the others for the
FAR complex.
65

Hélène/Massimiliano: To evaluate the use of microsatellites as a diagnostic tool by
looking at specific banding patterns.
 Hélène: To look for endosymbionts (i.e. Wolbachia) in R1 and R2 populations
(specimens from established colonies and specimens used for genotyping) to see
whether any differences in presence/absence/strains are observed. Repeat this study
for F1, F2 and A populations.
- Progress made up to 3rd RCM:
 Microsatellites were developed for the FAR complex.
 African populations for all three species were analysed.
 Population genetic structure for the complex revealed five clearly distinguishable
clusters (F1, F2, A, R1, R2). These results were published.
DEVELOPMENTAL PHYSIOLOGY
Maulid Mwatawala / Sunday Ekesi
- 5 year plan:
 Compare developmental/physiological differences between the five entities
recognized, with emphasis on R1-R2 differentiation.
- Plans for the last 18 months:
 Continue studies on Kenyan populations.
 Start studies on Tanzanian and South African populations.
- Progress made up to 3rd RCM:
 Preliminary studies conducted on R1-R2 differentiation with Kenya populations.
Preliminary studies on thermo-tolerance for the two populations of C. rosa have been
conducted. The linear regression model showed a strong positive linear relationship
between temperature and development rate for all stages. Lower development
threshold for all stages were estimated. Parameter estimates for the Brière-1 nonlinear
model predicted the lower and upper temperature thresholds for all stages. This
experiment will need to be repeated.
 Exchange of protocols between Kenya, Tanzania, South Africa (Ekesi, Manrakhan,
Mwatawala) is in progress.
HOST RANGE
Marc De Meyer / Mwatawala
- 5 year plan:
 Redefine host records with regard to the five recognized entities within the FAR
complex.
- Plans for the last 18 months:
 Carry out a further review of existing host records. No focused research on new host
records is planned.
- Progress made up to 3rd RCM:
 Re-evaluation of known host records based upon specimens study of natural history
collections. Inconclusive results as all genotypes appear to be polyphagous. Tendency
for R2 to be recorded from some temperate climate fruits for which there are no
records for R1 (linked to distribution patterns, Cf below)
DISTRIBUTION
Marc De Meyer/Maulid Mwatawala
- 5 year plan:
66

Redefine distribution records with regard to the five recognized entities within the
Ceratitis FAR complex.
- Plans for the last 18 months:
 Carry out a further review of existing geographical records.
 Continue sampling along altitudinal transect and analysis of data.
- Progress made up to 3rd RCM:
 Re-evaluation was carried out of known distribution records. Available specimens
were re-examined and divided according to the 5 groupings.
 R1 distribution is limited by latitude (absent southern part of the African continent)
and altitude (absent from higher altitudes).
 F1-F2 largely geographically separated (western-central versus eastern Africa).
However, isolated populations of ‘western’ type found in Angola, Malawi, Tanzania,
Zambia.
 New altitudinal transect study Tanzania started to reveal seasonal changes and
altitudinal limitations. This study has been conducted for three months along an
altitudinal transect, at 10 locations, about 100 m apart (altitudinal). Material is
currently sorted, in order to determine the overlap/ cut-off points.
67
E. Logical Framework
Table 3.
Logical Framework
NARRATIVE
SUMMARY
OBJECTIVE
VERIFIABLE
INDICATORS
MEANS OF
VERIFICATION
IMPORTANT
ASSUMPTIONS
N/A
Fruit and vegetable
production in Member
States continue to suffer
major losses due to
endemic and introduced
fruit flies of economic
importance.
OVERALL OBJECTIVE
To assist Member
States in achieving
sustainable fruit and
vegetable production
and in overcoming
constraints to SIT
application and
international trade
through the
resolution of cryptic
species complexes of
major tephritid pests.
N/A
International trade in
fruit and vegetable
commodities continue to
increase and be disrupted
by fruit fly pests,
requiring expensive preand post-harvest
treatments and
quarantine measures.
International trade
continues to suffer from
unnecessary restrictions
due to confusion about
the biological identity,
geographic distribution
and lack of diagnostic
tools for pest fruit fly
species.
In contrast to pesticides,
a more environmentally
friendly pest fruit fly
control requires precise
applications based on the
knowledge of the biology
of species.
68
The area-wide integrated
management of fruit flies
of economic importance,
including where
appropriate SIT as the
non-polluting
suppression/eradication
component, will
increasingly be a costeffective method to deal
with fruit flies of
economic importance.
SPECIFIC RESEARCH OBJECTIVE
Identify and define
the biological limits
of closely related
pest species within
the South American
fruit fly (A.
fraterculus) complex.
Facilitate the
taxonomic revision
and develop
corresponding
diagnostic tools to
distinguish these
species.
Identify and redefine
the biological limits
and species status of
five closely related
species of economic
importance within
the Oriental fruit fly
(B. dorsalis)
complex. Develop
corresponding
diagnostic tools to
distinguish these
species.
Determine the
variability within the
melon fly (B.
cucurbitae) regarding
geographic and/or
host races among
Basic and applied
research that
defines biological
species limits of
species within the
A. fraterculus
complex and
develops tools to
distinguish them
so as to facilitate
trade and SIT
implementation.
Sampling protocols,
diagnostic tools,
species descriptions
and geographic
distributions,
trapping and
ecological
information,
presented in reports
and publications.
There are multiple
species present within
the single A. fraterculus
nominal species. This
constrains trade and SIT
application.
Basic and applied
research that
defines biological
species limits
within the B.
dorsalis complex
and develops tools
to distinguish them
so as to facilitate
trade and SIT
implementation.
Sampling protocols,
diagnostic tools,
species descriptions
and geographic
distributions,
trapping and
ecological
information,
presented in reports
and publications.
There is no universal
agreement on the validity
of the currently described
pest species within the B.
dorsalis complex. This
constrains trade and SIT
application.
Mating
compatibility,
morphological and
genetic variability
assessed so as to
allow efficient
Sampling protocols,
diagnostic tools,
geographic
distributions and
ecological
information,
There are concerns that
there may be intraspecific differences
which would interfere
with efficient SIT
applications and trade.
69
populations from
Africa, Indian Ocean
and Hawaii.
Identify and redefine
the biological limits
and species status of
the members of the
Ceratitis FAR
complex.
Develop more robust
species markers and
identification tools
for the members of
the complex.
application of SIT
to B. cucurbitae in
these regions.
Basic and applied
research that
defines biological
species limits
within the
Ceratitis FAR
complex and
develops tools to
distinguish them
so as to facilitate
trade and
protection of
agriculture through
improved
quarantine
services.
presented in reports
and publications.
Sampling protocols,
diagnostic tools,
species descriptions
and geographic
distributions,
trapping and
ecological
information,
presented in reports
and publications.
Diagnostic tools,
reports and
publications.
Recent evidence
indicates the existence of
different entities within
the recognized species of
the Ceratitis FAR
complex. Existing
diagnostic tools for these
entities are not suitably
robust for quarantine use.
Protocols
developed and
network
established for
structured
collecting,
handling and
preserving,
transport,
recording,
vouchering of
specimens.
Protocol available
to all investigators.
Quality of outcomes
highly dependent on
essential co-ordination
between groups working
on A. fraterculus.
Established protocols can
be adopted for use in
collections. Investigators
apply protocols.
Colonies of the
recognized
morphotypes
established and
available for
studies that
required live
material.
Colony reports.
Materials of all
morphotypes can be
obtained and colonies
established.
Cross-mating and
post-zygotic
Reports of
behaviour and
The procedures to
establish species limits
OUTPUTS A.
fraterculus
1. Establish a
network for
targeted,
structured
sampling of the
complex together
with collection of
ecological data.
Develop protocol
for collecting,
handling and
preserving,
transport,
recording, etc. of
specimens.
2. Establish
colonies of all
morphotypes to
allow
behavioural,
chemical,
cytological, larval
morphology, etc.
studies.
3. Carry out
reproductive
70
compatibility
analyses as
required between
selected
populations of
named species –
including crossmating studies on
caged trees under
semi-natural,
choice situations.
Record
behavioural
aspects.
studies carried out
between
morphotypes of
the A. fraterculus
complex under
semi-natural,
choice situations,
to
confirm/evaluate
presence of
reproductive
isolation.
4. Perform
a) Sex
chemical,
pheromone and
cytological,
cuticular
molecular
hydrocarbons
genetic,
profiles
morphological
determined for
and other relevant
recognized
biological studies
morphotypes.
to help
b) Cytological
characterize/
studies
discriminate
performed and
among
karyotypes
morphotypes.
described for
each of the
recognized
morphotypes.
c) Molecular
genetic
analyses
carried out on
material
collected from
across the
geographical
range of A.
fraterculus s. l.
d) Morphological
studies carried
out on material
collected from
across the
geographical
range of A.
fraterculus s. l.
e) Other
mating
compatibility tests.
International peer
reviewed
publications.
are scientifically
acceptable.
a) Reports of
pheromones
studies and
analyses.
a) The samples are
representative of the
true population and
are processed
according to the
established protocol.
b) Karyotype and
polytene
chromosome
data published.
c) Reports and
published
genetic
analyses.
d) Reports and
published
morphological
studies.
e) Published data
b) New gene regions can
be accessed and used to
screen populations.
c) Sufficient
representative
samples available to
conduct appropriate
analysis.
d) The samples are
representative of the
true population and
that they are
processed according
to the established
protocol.
71
potentially
discriminating
biological
attributes
investigated
for all
morphotypes.
on biology of
flies with
particular
reference to
assisting
discrimination
between populations/species.
Published results on
the taxonomy of the
A. fraterculus
complex focussing
on species of
economic
significance.
5. Describe and
diagnose
morphotypes by
collating,
analysing and
interpreting new
and existing data.
Morphotypes
described and
diagnosed based
on the information
collected using
different
approaches.
6. Clarify the
nomenclature for
A. fraterculus s. l.
(If name changes
are required, the
Inter-national
Commission of
Zoological
Nomen-clature
may need to be
involved.)
-Holotypes examined / synonyms
resurrected as
appropriate.
-Consultant
meeting of
specialists held to
review all
evidence collected
to name/ rename
species.
-Species named/
renamed.
Diagnostic tools
developed based
on morphological,
molecular and
other evidence
collected.
Holotypes.
Meeting reports.
Protocols
developed and
network
established for
structured
collecting,
handling and
preserving,
Published protocol
checklist publicly
available.
7. Develop
improved
diagnostic tools
for each newly
described/rename
d species within
the complex.
Published
diagnostic
protocols.
e) Experimental staff
capable of carrying
out standardised
behavioural
protocols.
Independent avenues of
conducted research will
suggest a consensus
outcome.
The supporting research
as part of CRP (mating,
pheromones, behaviour,
etc. studies) is published
and/or made available.
Funding for Consultants
Meeting.
The previous objective
(defining species limits
in A. fraterculus) has
been achieved.
Having determined
species limits, diagnostic
markers can be found.
OUTPUTS B.
dorsalis
1. Carry out
targeted sampling
for species listed
as Bactrocera
dorsalis s. l.
Establish a
laboratory
network for
Centralised group coordinating collection and
curation of samples.
Local collectors
Central repository
accurately locate and
of collection
collect required samples
material curated and according to defined
live cultures
protocols.
72
structured
sampling (for
rearing, morphology, pheromone
and genetic
work).
2. Quantify field
cage mating
behaviour of two
or more
geographically
distinct B.
dorsalis s.s.
populations as a
basis for
subsequent
comparative
studies within the
complex.
transport,
recording, etc. of
specimens.
established and
maintained.
Specialists available for
specimen identification
prior to live culture
establishment.
Within species
variation in mating
behaviour
documented for B.
dorsalis s.s.
Results reported/
published.
3. Carry out crossspecies matings
under seminatural, choice
situations using
wild / wildish
flies as required.
Record
behavioural
aspects as per QC
Manual.
Cross-matings and
behaviour studies
carried out
between species of
the B. dorsalis
complex to
compute and
record mating
isolation indices.
Published results on
cross-mating
studies.
4. Carry out
multiple
generation
crosses to record
post-zygotic
isolation on at
least two
Bactrocera
dorsalis s. l.
species.
5. Determine sex
pheromone
profiles for each
species with and
without prior
methyl eugenol
consumption.
Multiple
generation crosses
carried out in B.
dorsalis complex
which detail postzygotic mating
effects.
Published results on
multiple postzygotic studies.
Confirming that B.
dorsalis s.s. is a single
species allows the
establishment of
centralised rearing
facilities.
Individuals collected
from targeted collection
localities are B. dorsalis
s.s. based on initial
morphological
identification.
Collaborators can be
found to undertake
collections.
Flies can be maintained
under wildish conditions
over minimum
generations to produce
meaningful results.
There is staff available to
undertake experiments
and maintain cultures.
The timing between field
collections, cultures, and
available staff will
coincide.
Experimental staff
capable of carrying out
standardised protocols.
Sex pheromone
profiles
determined for
each species.
Published results on
pheromone profiles.
The volatiles of
Bactrocera dorsalis s. l.
species can be obtained
and are processed
according to established
protocols.
73
6. Establish relative
attraction of
males of different
Bactrocera
dorsalis s. l.
species to methyl
eugenol.
7. Investigate other
potentially
informative
biological
attributes for
discriminating
between species.
Relative taxon
sensitivity to
methyl eugenol
determined.
9. Species status for
Bactrocera
dorsalis s. l.
species of
economic
importance newly
defined based on
outputs 1 to 8.
Species status
defined for at least
B. dorsalis, B.
papayae, B.
philippinensis, B.
invadens and B.
carambolae.
10. Develop
improved
morphological
and molecular
diagnostic tools
based on the
acquired
knowledge (for
adults and
immatures).
At least one
diagnostic tool
developed for each
species defined in
output 7.
Published results on
relative attraction of
males to methyl
eugenol.
Other potentially
discriminating
biological
attributes
investigated for
species.
Reported protocols
made publicly
available.
Published data on
biology of flies with
particular reference
to assisting
discrimination
between
populations/species.
8. Carry out genetic, Genetic,
Published results of
cytogenetic and
cytogenetic and
genetic analyses.
molecular
molecular analyses Reports with
analyses on
carried out for B.
specific statement
material collected dorsalis, B.
on whether genetic
from across the
papayae and B.
data supports
geographical
philippinensis
separate/single
range of species
populations.
species for B.
of economic
dorsalis s.l.
importance.
OUTPUTS Bactrocera cucurbitae
Published results on
the taxonomy of the
B. dorsalis complex
focussing on
species of economic
significance.
Suitable fly specimens
and specialists (including
specialist equipment) are
available to analyse and
compare information.
Experimental staff
capable of carrying out
standardised biological
and behavioural
protocols.
Quality of outcomes
highly dependent on
essential co-ordination
between groups.
New gene regions can be
accessed and used to
screen populations.
Sufficient representative
samples available to
conduct appropriate
analysis.
Independent avenues of
conducted research will
suggest a consensus
outcome.
The previous research as
part of CRP (mating,
pheromones, behaviour)
is completed and made
available.
Diagnostic
The previous objective
protocols published. (defining species limits
in B. dorsalis s.l.) has
been achieved.
Having determined
species limits, diagnostic
markers can be found.
74
1. Develop protocol
for collecting,
handling,
preserving,
transport,
recording, etc. of
specimens from
Africa, Hawaii
and Indian
Ocean.
2. Establish a
network for
targeted,
structured
sampling of
species together
with ecological
data.
Protocol
developed for
structured
collecting,
handling and
preserving,
transport,
recording, and
vouchering.
Protocol available
to all investigators.
Established protocols can
be adopted for use in
collections. Investigators
apply protocols.
Clarification on
existence or nonexistence of host
races within B.
cucurbitae among
populations from
Africa, Hawaii and
Indian Ocean.
Published results on
mating
compatibility,
ecology and
population genetics.
3. Characterize
potential host
races through
ecological,
morpho-metric,
genetic studies. If
there are positive
differences then
pro-ceed to other
studies.
4. Carry out crossmatings as
required between
selected
populations under
semi-natural,
choice situations
using wild /
wildish flies.
Record
behavioural
aspects.
Potential host
races characterized
and differences
explored.
Reports and
publications of
ecological ,
morphometric and
genetic studies.
Flies can be built up in
culture to sufficient
numbers over minimum
generations to produce
meaningful results.
There is staff available to
undertake experiments
and maintain cultures.
The timing between field
collections, cultures, and
available staff coincides.
Host races obtained and
the presence of host races
is confirmed.
Mating compatiMating test reports
bility assessments and publication.
and behaviour
studies carried out
among populations
from Africa,
Hawaii and Indian
Ocean, under
semi-natural,
choice situations
using wild /
wildish flies.
OUTPUTS C. rosa, C. fasciventris, C. anonae
Extent of cross-mating
tests dependent on results
of characterization of
potential host races
through ecological ,
morphometric and
genetic studies.
75
1. Develop
morphological
and molecular
diagnostics to be
able to
distinguish the
recognized
entities.
2. Evaluate of
specific status of
taxa within the
complex (e.g.
molecular,
pheromone,
cuticular
hydrocarbon,
mating
compatibility,
ecology, etc.)
Robust
discriminating
markers developed
and made
available for the
recognized
entities.
Markers to analyse
extensive field
samples.
More robust markers can
be identified and utilized.
Additional support
to the current or
changed specific
status of taxa
within the FAR
complex.
Reports and
publications of
taxonomic and
related studies.
Confirmation depending
on outcome o all other
studies.
Hold Consultants
Meeting.
Consultants
meeting held 6-10
July 2009.
CRP proposal.
Award research
agreements and
contracts to fruit fly
researchers,
taxonomists and fruit
fly control
programmes and
establish CRP.
Research contracts
agreements.
Signed agreements
and contracts.
CRP approved by
committee
Proposals
submitted/accepted.
Agreements and
contracts approved and
funded.
Organise 1st RCM to
plan, co-ordinate and
review research
activities.
1st RCM held in
August 2010.
Working material
of 1st RCM printed
and distributed.
Consultants’ report
refined by participants.
Carry out R and D.
R and D findings.
Research reports.
Hold 2nd RCM to
review results, begin
analysis of collection
data, draft technical
protocols as required,
2nd RCM held in
February 2012.
Working material
printed and
distributed for 2nd
RCM, research
published in
Continued institutional
support, including
support for establishment
of colonies.
Analytical tools
appropriate and
verifiable.
ACTIVITIES
76
and plan future
activities.
scientific literature
and disseminated to
Member States and
Scientific
community.
Continue R and D.
R and D findings.
Research reports.
Hold training course
on A. fraterculus
morphotype
identification.
Hold 3rd RCM to
review results,
continue analysis of
collections and
colony material and
to plan future
activities.
Training course
held in 2012.
Trained A.
fraterculus
taxonomists in
region.
Working material
printed and
distributed for 3rd
RCM, research
published in
scientific literature
and disseminated to
Member States and
Scientific
community.
Hold Consultants
Meeting of
specialists to review
all evidence collected
to review species
limits and
name/rename
species.
Consultants
Consultants
Meeting held, gasp Meeting report.
identified and
decisions on some
species to be
named/ renamed.
Funding and
participation of relevant
specialists.
Carry out final R and
D.
R and D findings.
Research reports.
Continued institutional
support.
Hold final RCM to
review data, reach
consensus and
prepare final report.
Final RCM held in
January 2015.
Final CRP report
distributed and
research published
in scientific
literature.
Funding available to
complete CRP and to
publish proceedings.
3rd RCM held in
June 2013.
Continued institutional
support.
Availability of lecturer(s)
and of funding.
Reports published and
distributed following
each RCM.
Mid-year review of CRP
approved.
77
F. Workshop on “Reviewing Evidence to Resolve Species
Complexes of Tephritid Pests” (31. August 2013)
Recommendations of External Panel
31 August 2013
(Agenda of Workshop in Annex 4)
1. Anastrepha fraterculus Complex Work Group
(see Current Knowledge of Anastrepha fraterculus Complex in Annex 1a and Character
Matrix for the Anastrepha fraterculus Species Complex in Annex 1b)
Strengths:
 Well-defined morphometric framework for differentiating the 7-8 morphotypes
currently recognized.
 Multiple lines of evidence in support of conclusions for separating various subsets of
morphotypes as significantly different entities (e.g. pre- and post-zygotic isolation,
cytology, cuticular hydrocarbons).
 Extensive sampling of some morphotypes across range.
 Extensive network of collaborators across a diverse range of disciplines.
Weaknesses:
 Number of project participants contributing to morphometric identifications limited.
 Molecular data available for study is insufficient, and would be highly desirable.
 Molecular priorities unclear and how these relate to overall goals.
 Movement of material between participants is highly restricted (legislative).
 Different studies are frequently using only subsets of morphotypes, rather than
comparing all 8 morphotypes.
 Inadequate communications between workers doing molecular work.
 Insufficient funds for sampling, molecular and morphometric work.
Recommendations (CRP):
 Prioritization of morphological identification of Brazilian populations within current
morphometric framework.
 Ensure accurate morphotype identification using morphometric methods is made on
all samples as a first step before use in other studies.
 Prioritize objectives for molecular markers and analytical methods (phylogenetics,
haplotype networks, etc.).
 Work plan should ensure that all morphotypes are included in all studies, rather than
subsets.
78





Attempt to collect specimens from Venezuela, Ecuador, Andean Peru and Bolivia and
establish colonies in Siebersdorf lab.
Trade implications should be considered, including communications strategy.
Develop diagnostic tools for all life stages.
IAEA and other relevant agencies be engaged in facilitating movement of samples
and alleviating legislative restrictions.
Ensure samples are vouchered and availability where possible for future molecular
studies.
Future recommendations:


Where possible maintain IAEA colonies used for studies or voucher specimens
Cryopreserve IAEA colonies, if possible, for use in future studies.
2. Bactrocera dorsalis Complex Work Group
(see Current Knowledge of Bactrocera dorsalis Complex in Annex 2a and Character Matrix
for the Bactrocera dorsalis Species Complex in Annex 2b)
Strengths:
 Extensive sampling over entire distribution
 Ready availability of material to participants throughout project duration
 Coordination of participant roles and alignment of activities with project goals
resulting in direct comparability of data and results
 Clearly defined hypotheses
 Clear presence of outlying groups such as B. occipitalis, and B. cacuminata
 Multiple lines of evidence (i.e. morphometric, DNA sequences, mating behavior,
pheromones, etc.) in support of conclusions of synonomy of B. papayae, B.
philippinensis and B. invadens with B. dorsalis but not B. carambolae
 Well-defined plan for publication of results with inclusive authorship.
Weaknesses:
 Vouchering and likely availability of samples for future molecular studies.
Recommendations (CRP):

 Manuscript on synonymy of various species under B. dorsalis should include revised
diagnosis and redescription of B. dorsalis s.s.
 Trade implications should be considered, including communications strategy.
 Consider longer term storage solutions for voucher specimens.
 Develop diagnostic tools where applicable and available for all life stages.
 Ensure samples are vouchered and availability where possible for future molecular
studies
Future recommendations:
 Further investigation of possible hybridization between B. carambloae and dorsalis
complex.
79

Clarification of potential issues of paraphyly of B. dorsalis relative to B. kandiensis in
Sri Lanka and India.

3. Ceratitis FAR Complex Work Group
(see Current Knowledge of Ceratitis FAR Complex in Annex 3a and Character Matrix for
the Ceratitis FAR Species Complex in Annex 3b)
Strengths:
 Extensive sampling over entire distribution
 Ready availability of material to participants throughout project duration
 Coordination of participant roles and alignment of activities with project goals
resulting in direct comparability of data and results
 Clearly defined hypotheses
 Evidence in support of conclusions is relatively strong for FAR and R1 vs R2
differentiation, and based on adult male morphology, mating behavior [?
Clarification of ISI results], microsat and phys data.
 Prioritizing work-plan and project activities based on relative economic importance of
C. rosa and likely availability of material (i.e. rarity of C. fasciventris (West)).
Weaknesses:
 DNA diagnostic tools for differentiating morphotypes and species are relatively weak
and requires development.
Recommendations (CRP):
 Trade implications should be considered, including communications strategy
including NPPO communications.
 Develop molecular and morphological diagnostic tools for all life stages.
 Ensure samples are vouchered and available where possible for future molecular
studies.
Future recommendations:
 Continue to consider other morphological differences between species and
morphotypes, and relationship to distribution.
 Reanalysis of previously published ecological niche modeling data to predict potential
distributions.
 Further exploration of F1 – F2 differentiation.
80
Annex 1a: CURRENT KNOWLEDGE OF ANASTREPHA FRATERCULUS
COMPLEX (August 2013)
The concept that Anastrepha fraterculus is a complex of cryptic species is not recent (Stone
1942, Morgante et al. 1980, Solferini & Morgante 1987, Malavasi & Morgante 1982, Steck
1991, Steck & Sheppard 1993, Selivon 1996, Hernández-Ortíz et al. 2004, 2012) and is based
on the presence of population differentiation and reproductive isolation.
The combined results of these studies, suggest the existence of at least seven different
biological entities which are referred to as: A. sp. 1 aff. fraterculus (Yamada & Selivon
2001), A. sp. 2 aff. fraterculus (Yamada & Selivon 2001), A. sp. 3 aff. fraterculus (Selivon et
al. 2004), A. sp. 4 aff. fraterculus (Selivon et al. 2004), A. fraterculus Mexican morphotype
(Hernández-Ortíz et al. 2004) and A. fraterculus from Andean highlands and Venezuela
coastal lowlands (Steck 1991). A recent comprehensive morphological study supports the
existence of these seven morphotypes (Hernández-Ortíz et al. 2012).
In order to determine if the biological entities correspond to different species, it was agreed
for this CRP that there was a need to use different methodologies to analyse them, to use
properly identified material, and most importantly, to include all of the morphotypes in the
studies.
This approach was followed since the initiation of the CRP and during the last months,
significant new information appeared (see following summary character matrix for the
Anastrepha fraterculus complex).
At the molecular level, SSR markers were developed and applied to assess intra/interpopulation differentiation (Malacrida). In addition, a portion of the cytochrome oxidase c
subunit I was sequenced for several samples obtained from different populations from Brazil,
Argentina and Mexico (Silva). Results indicate the presence of population structuring.
Studies of COI and COII done with nine populations from Colombia showed the occurrence
of the highland morphotype in eight of the populations with the ninth population, collected in
the south, clustering apart (Canal). Preliminary DNA sequence data from Peru showed
differences between populations from Cusco Department and coastal populations, as well as
from those from eastern slopes of the Andes in Bolivia, which match those of Argentina and
southern Brazil (Steck).
Morphometric studies incorporated the geometric approach and new populations from
Ecuador and results allow preliminary interpretations suggesting the occurrence of an
additional eighth morphotype (Hernández-Ortiz). In addition, samples from adults collected
from traps and larvae obtained from 14 different host plants in Peru allowed obtaining
material for taxonomic analysis from regions in which material was scarce. This study is ongoing.
In terms of behavior, studies keep providing evidence in support of the usefulness of field
cages as a tool to determine mating compatibility among populations. As a result, it was
possible to increase the number of population combinations analyzed and delineate better the
degree of compatibility among them. The presence of full compatibility among populations
from southern Brazil was confirmed, the existence of incompatibility between these
populations and the population from Piracicaba (southeast Brazil), and contrary to
expectations, the population from the northeast was compatible with one from the south
(Joachim-Bravo). At the continental level, prezygotic isolation was revealed even when
sexual activity occurred at the same time such as in the case of Mexico and Brazil 1
morphotypes (Rull). The addition of acoustical analysis allowed extending the tools available
to analyze sexual isolation (Joachim-Bravo).
81
At the chemical level, it was evidenced that pheromones can act as a primary method of
reproductive isolation. This is supported by the differences found in the volatiles analyzed
from the different populations (Brizova, do Nascimiento, Teal) as well as for the cuticular
hydrocarbons (Vanickova). In addition the incorporation of the chemical characterization of
the proteins from the males’ accessory glands can provide another diagnostic tool (Rull).
Conclusions
 All evidence so far points towards the fact that previously reported population
differences are correlated with reproductive isolation and if the biological concept of
species is followed, Anastrepha fraterculus is composed of several species.
 Different tools can be used in combination for diagnosis (DNA, morphology,
cytology, sexual behavior and chemical profile of male-emitted volatiles and cuticle
extracts).
 All reproductively isolated populations revealed prezygotic isolation.
 As more populations/areas are incorporated into the comprehensive approach, the idea
of the existence of morphotypes gains support.
 As a consequence it would be advisable to initiate progressively the process of species
definition and revision.
82
Annex 1b: Character Matrix for the Anastrepha fraterculus Species
Complex
Characters
A. sp. 1
aff.
fratercu
lus
A. sp. 2
aff.
fratercu
lus
Peruvian
Ande
-an
Mexican
Venezuelan
A. sp. 3
aff.
fratercu
lus
Ecu
adori
an
Piracicaba
1. DNACO I
2. DNA ITS1
and
ITS2
3. DNA CO II
If
possible
4. Isozymes
5.
Micro
satellites
validat
ion
Done
6.
Micro
satellites
population
analysis
7.
Cytolo
gy
8. Adult
morpholo
gy
9. Wing
geometric
s
10. Larval
morphology
11. Egg
morphology
12. Prezygotic
isolation
P
P
P
P
P
P
P
P
?
P
P
P
P
P
P
P
P
?
P
Partial
isolatio
n
Bentos
Gonçal
-ves
Parn
amiri
m
83
13. Postzygotic
isolati
on
14.
Phero
mones
15.
Cuticu
lar
hydro
carbon
s
16.
Remat
ing
behavi
our
17.
Sexual
behavi
or
18. Time
sexual
activit
y
P
P
P
P
Bento
Gonçal
ves
P
Piracic
aba
P
P
If
possi
ble
P
If
possibl
e
A. sp. 1
aff.
fratercu
lus /
Mexica
n
A. sp. 1
aff.
fratercu
lus
Piracica
ba /
Mexican
19. Sound
20. Host
range
21. Strain
(on
artificial
diet)
Legend
P
Vacaria
/ Pelotas
/ Sao
Joaquin
/ Tucumán
Piura
+ La
Molina
Different to
all
P
P
Tolima
Xalapa
In
progr
ess
Not a
publicat
ion that
reduces
Norrbom's
host
range
Publi
shed
but
need
s to
be
upda
-ted
Differs
from
some
morpho
-type/s
and
overlaps
with
other/s
1. -2. -3. -4. Criteria to distinguish from other populations based on Roger's Genetic Distance equal to
or greater than distance between other recognized species of A. fraterculus group (A.
distincta and A. obliqua).
5. Work developed in conjunction with INTA Buenos Aires and Pavia.
84
6. Not done yet.
7. Based on Selivón´s work and Cáceres et al. 2009; all morphotypes with different
caryotype.
8. Based on Hernández-Ortiz et al. 2012; the Ecuadorian morphotype is not published.
9. Preliminary results show that geometric analysis can differentiate the eight morphotypes.
10. -11. Eggs show morphological differences.
12. All based on field cage mating compatibility tests. Males and females of two populations
released in the cage. Mating compatibility indices range from high isolation (above 0.8),
partial isolation (between 0.4 and 0.6) and random mating (below 0.2. All populations'
combinations within a given morphotype show random mating (sp. 1, Peru, Colombia and
Mexico).
13. Reduction in egg hatch and sex ratio distortion; published by Selivón and Cáceres.
14. All populations analysed present a different chemical profile, even some belonging to the
same morphotype except Bento Gonzalvez and Piracicaba that showed the same chemical
profile. Hybrids between Peru and sp. 1 present components unique to both populations
(the blend is a mixture). Hybrid females prefer hybrid males.
15. As for pheromones, sensitivity seems higher than morphometrics.
16. Based on the remating rate of the females, the time between 1st and 2nd mating and the
percentage of females without sperm transfer; manuscript submitted.
17. Based on video recordings; one publication for one Argentina population (GomezCendra).
18. Some populations are temporarily isolated as they are early morning, midday and dusk
maters.
19. Preliminary data that shows differences in the frequency.
20. Taking into account Norrbom's host list, Colombia and Mexico morphotypes have been
claimed to have a narrower host range than Af ss.
21. Names of the localities from where populations were derived.
85
Annex 2a: CURRENT KNOWLEDGE OF BACTROCERA DORSALIS COMPLEX
(August 2013)
Background
The Bactrocera dorsalis species complex was first recognised by Hardy (1969), and greatly
expanded by Drew and Hancock (1994). Several other taxa have been placed in the complex
outside of these two revisions, of which the only one of current relevance is B. invadens
(Drew et al. 2005). While the majority of species within the B. dorsalis complex are easily
diagnosed on morphological characters, this is not the case for a small group of cryptic taxa
which have been the focus of the current project: B. dorsalis s.s. (Hendel), B. papayae Drew
& Hancock, B. philippinensis Drew & Hancock, B. carambolae Drew & Hancock and B.
invadens Drew, Tsuruta & White.
Quite quickly after their original descriptions, concerns were raised as to the biological
validity of B. papayae, B. carambolae and B. philippinensis, most notably by Prof Keng
Hong Tan and colleagues (Nishida et al. 1988, Tan & Nishida 1996), but these views were
subsequently reviewed and dismissed (Clarke et al. 2005, Drew et al. 2008). Nevertheless,
repeated failure to find robust and consistent biological markers for these taxa (with the
possible exception of B. carambolae), has meant that the question of whether the taxonomic
species correctly align with the biological species has never been fully resolved. This
component of the CRP was established to (i) accurately delimit the biological species of this
clade; and (ii) having done so develop appropriate diagnostic markers.
Current status
Very significant research has been undertaken, within an integrative taxonomic framework,
to biologically delimit the target taxa (see following summary character matrix). The tools
used have included: i) morphological examination, including traditional morphology,
morphometrics of genitalic characters, and fine-scale wing shape analysis using geometric
morphometrics; ii) molecular-based approaches using both phylogenetic and population
genetic analyses of nuclear DNA sequence and microsatellite data coupled with
mitochondrial DNA sequence analysis and comparative transcriptome analysis; iii) studies of
mating and post-zygotic sexual compatibility; iv) analysis of rectal gland (= pheromone)
constituents; and v) comparative polytene chromosone analysis. Not all work has yet been
published, but much has (Tan et al. 2011, Khamis et al. 2012, Schutze et al. 2012a, Schutze et
al. 2012b, Boykin et al. 2013, Krosch et al. 2013, Schutze et al. 2013, Tan et al. 2013).
With one exception, all data so far presented by CRP participants have failed to find evidence
of any species level biological differentiation between B. dorsalis, B. papayae, B.
philippinensis and B. invadens. Any genetic and morphological differences which have been
found are best explained as intra-specific variation (i.e. population level variation), not interspecific variation. Data (pheromonal, genetic, behavioural) suggests that B. carambolae is a
biologically unique, albeit very closely related species to the other four taxa. It is important to
recognise that some critical data sets for B. invadens (e.g. sexual compatibility trials) have
still to be publically presented, but this ‘missing’ data will be presented in Argentina and is
consistent with the other data sets so far presented. The one exception to the general patterns
seen in the data sets was a verbal paper presented by Prof Drew at the 2nd RCM (Brisbane,
Jan 2012), who analysed thoracic colour patterns of B. invadens from Africa and B. dorsalis
from China and concluded that the frequency of colours (predominantly red-brown in the
African flies and black in the Chinese flies) confirmed these two species as described.
Because such colour variation might also be interpreted as intra-specific variation in one
wide-ranging biological species, more work in this area has been done in the last 18 months
and will be presented in Argentina.
86
Prof Drew has recently formally synonymised B. philippinensis with B. papayae in a soon to
be published monograph, but no other taxonomic changes have been made.
Conclusions
 All evidence so far suggests that B. dorsalis, B. papayae and B. philippinensis are one
biological species, while B. carambolae is a closely related but biologically unique
species, albeit natural hybrids between B. carambolae and B. papayae have been
detected in the Penisular Malaysia (Tan, 2005, Wee and Tan 2005).
 B. invadens should also be likely considered a junior synonym of B. dorsalis, but
some additional data sets need to be publically presented and peer considered before
this can be assumed.
 The strength of the case for considering the synonymisation of B. papayae, B.
philippinensis and B. invadens with B. dorsalis rests not on the strength of any one
approach (i.e. mating, pheromone, genetics), but the highly consistent results of all of
these independent approaches.
References
Boykin, L. M., M. K. Schutze, M. N. Krosch, A. Chomic, T. A. Chapman, A. Englezou, K. F.
Armstrong, A. R. Clarke, D. Hailstones, and S. L. Cameron. 2013. Multi-gene
phylogenetic analysis of the south-east Asian pest members of the Bactrocera
dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy.
Journal of Applied Entomology:DOI: 10.1111/jen.12047.
Clarke, A. R., K. F. Armstrong, A. E. Carmichael, J. R. Milne, S. Raghu, G. K. Roderick, and
D. K. Yeates. 2005. Invasive phytophagous pests arising through a recent tropical
evolutionary radiation: The Bactrocera dorsalis complex of fruit flies. Annual
Review of Entomology 50:293-319.
Drew, R. A. I. and D. L. Hancock. 1994. The Bactrocera dorsalis complex of fruit flies
(Diptera: Tephritidae: Dacinae) in Asia. Bulletin of Entomological Research
Supplement No. 2:i-iii, 1-68.
Drew, R. A. I., S. Raghu, and P. Halcoop. 2008. Bridging the morphological and biological
species concepts: studies on the Bactrocera dorsalis (Hendel) complex (Diptera:
Tephritidae: Dacinae) in South-east Asia. Biological Journal of the Linnean Society
93:217-226.
Drew, R. A. I., K. Tsuruta, and I. M. White. 2005. A new species of pest fruit fly (Diptera:
Tephritidae: Dacinae) from Sri Lanka and Africa. African Entomologist 13:149-154.
Hardy, D. E. 1969. Taxonomy and distribution of the oriental fruit fly and related species
(Tephritidae-Diptera). Proceedings of the Hawaiian Entomological Society 20:395428.
Khamis, F. M., D. K. Masiga, S. A. Mohamed, D. Salifu, M. d. Meyer, and S. Ekesi. 2012.
Taxonomic identity of the Invasive fruit fly pest, Bactrocera invadens: Concordance
in morphometry and DNA barcoding. PLoS ONE:7(9) e44862.
doi:44810.41371/journal.pone.0044862.
Krosch, M. N., M. K. Schutze, K. F. Armstrong, Y. Boontop, L. M. Boykin, T. A. Chapman,
A. Englezou, S. L. Cameron, and A. R. Clarke. 2013. Piecing together an integrative
taxonomic puzzle: microsatellite, wing shape and aedeagus length analysis of
Bactrocera dorsalis s.l. (Diptera: Tephritidae) find no evidence of multiple lineages in
a proposed contact zone along the Thai/Malay Peninsula. Systematic Entomology
38:2-13.
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Nishida, R., K.H. Tan, M. Serit, N.H.Lajis, A.M. Sukari, S. Takahashi and H. Fukami. 1988.
Accumulation of phenylpropanoids in the rectal glands of male Oriental fruit fly,
Dacus dorsalis. Experientia 44:534-536.
Schutze, M., A. Jessup, I. Ul-Haq, M. J. B. Vreysen, V. Wornoayporn, M. T. Vera, and A. R.
Clarke. 2013. Mating compatibility among four pest members of the Bactrocera
dorsalis fruit fly species complex (Diptera: Tephritidae). Journal of Economic
Entomology 106:695-707.
Schutze, M. K., A. Jessup, and A. R. Clarke. 2012a. Wing shape as a potential discriminator
of morphologically similar pest taxa within the Bactrocera dorsalis species complex
(Diptera: Tephritidae). Bulletin of Entomological Research 102:103-111.
Schutze, M. K., M. N. Krosch, K. F. Armstrong, T. A. Chapman, A. Englezou, A. Chomič, S.
L. Cameron, D. L. Hailstones, and A. R. Clarke. 2012b. Population structure of
Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in
southeast Asia: evidence for a single species hypothesis using mitochondrial DNA
and wing-shape data. BMC Evolutionary Biology 12:130. doi:110.1186/1471-21481112-1130.
Tan, K. H. 2005. Interbreeding and DNA analysis of Bactrocera dorsalis sibling species. pp
113 – 122. In: Recent Trends on Sterile Insect Technique and Area-Wide Integrated
Pest Management - Economic Feasibility, Control Projects, Farmer Organization
and Bactrocera dorsalis Complex Control Study. Research Institute for the
Subtropics, Okinawa, Japan.
Tan, K. H. and R. Nishida. 1996. Sex pheromone and mating competition after methyl
eugenol consumption in Bactrocera dorsalis complex. pp. 147-153. In: B.
A.McPheron and G.J. Steck (eds.) Fruit Fly Pests – A World Assessment of their
Biology and Management. St.Lucie Press, Florida.
Tan, K. H., I. Tokushima, H. Ono, and R. Nishida. 2011. Comparison of phenylpropanoid
volatiles in male rectal and pheromone gland after methyl eugenol consumptions, and
molecular phylogenetic relationship of four global pest fruit fly species: Bactrocera
invadens, B. dorsalis, B. correcta and B. zonata. Chemoecology 21:25-33.
Tan, K. H., S.L. Wee, H. Ono, and R. Nishida. 2013. Comparison of methyl eugenol
metabolites, mitochondrial COI, and rDNA sequences of Bactrocera philippinensis
(Diptera: Tephritidae) with those of three other major pest species within the dorsalis
complex. Applied Entomology and Zoology 48:275-282.
Wee, S. L. and K. H. Tan. 2005. Evidence of natural hybridization between two sympatric
sibling species of Bactrocera dorsalis complex based on pheromone analysis. Journal
of Chemical Ecology 31:845-858.
88
Annex 2b: Character Matrix for the Bactrocera dorsalis Complex
Characters
B. dorsalis s.s.
B. papayae
B. philippinensis
B. carambolae
B. invadens
B. kandiensis
B. occipitalis
1. Polytene
chromosomes
2. Rectal gland
extracts
following ME
feeding
3. Mechanics of
processing of
ME
4. Thoracic
colour pattern
5. Aculeus
spicules
6. Pre-zygotic
compatibility
7. Post-zygotic
compatibility
8. Multi-gene
phylogeny
9. CO1
haplotype
(pop genetics)
10.
Microsatellite
(pop genetics)
11. Wing shape
12. Y chromosome
13. Post-suttural
lateral vittae
14. Aedeagus
length
15. Cuticular
hydrocarbons
16. General
morphology
Legend
No differences
to other taxa
Differences found
to the other taxa
1. "The complex is homosequential", but with some minor inversions in B. dorsalis vs. B.
carambolae hybrids.
2. The constitutive pheromone compounds and ME derived metabolites are the same for B.
dorsalis, B. papaya, B. philippinensis and B. invadens, but different not for B. carambolae.
3. The physiological processing of ME seems the same for B. dorsalis, B. papayae, B.
philippinensis and B. invadens, but different for B. carambolae.
4. B. invadens is predominantly red-brown and the others black, but it has been shown that
thoracic colour is a qualitative genetic trait, which exhibits at different population
frequencies in different locations.
89
5. Originally thought to be discriminating between B. papayae and B. philippinensis,
subsequently shown not to be in a publication by Khalid Mahmood.
6. In standardised field cage mating trials, random mating shown between B. dorsalis, B.
papayae, and B. philippinensis, but not B. carambolae. In a separate set of tests, B.
invadens was shown to mate randomly with two B. dorsalis populations (China and
Pakistan).
7. Compatibility shown to F2 generation. Non-compatibility shows up as significantly
reduced egg hatch in the F1 population.
8. Bayesian phylogeny using 2 mitochondrial, 2 nuclear and 2 ribosomal genes.
9. Co1 haplotype networks tightly clustered for studies of B. dorsalis, B. papaya, B.
philippinensis and B. invadens. B. carambolae and B. kandiensis share no haplotypes with
this cluster (or each other).
10. Microsatellites detect no differences in B. dorsalis and B. papayae in the Thai/Malay
Peninsula where these two taxa are thought to abut or overlap their distributions.
11. Wing shape between B. dorsalis, B. papayae and B. philippinensis variable, but showing
very strong isolation by distance effects consistent with a single species hypothesis. B.
invadens wing shape very similar to B. dorsalis populations from northern India/Pakistan.
12. --13. Originally thought to be discriminating between B. dorsalis and B. invadens,
subsequently shown to be a clinal trait. Not different between B. dorsalis and B. papayae.
14. Originally thought to be discriminating between B. dorsalis and B. papayae, subsequently
shown to be a clinal trait. Not different between B. dorsalis and B. invadens.
15. Work done on cuticular hydrocarbons for these flies on-going.
16. B. carambolae, B. kandiensis and B. occipitalis relatively straightforward to separate on
subtle, but relatively consistent morphological characters. B. dorsalis, B. papaya, B.
philippinensis and B. invadens cannot be consistently separated in this way.
90
Annex 3a: CURRENT KNOWLEDGE CERATITIS FAR COMPLEX (August 2013)
The Ceratitis FAR complex comprises three described entities:
C. rosa Karsch, 1887
C. rosa var. fasciventris (Bezzi, 1920)
C. anonae Graham, 1908
C. rosa var. fasciventris was given specific status by De Meyer (2001). All three entities are
thus currently recognized as separate species. Males can be easily differentiated, based on
secondary male characters (pilosity of the mid leg. See De Meyer & Freidberg, 2006).
Females cannot be easily differentiated. There are some minute differences in leg and
anepisternal pilosity between C. anonae and the other two species (cf De Meyer & Freidberg,
2006).
However, no mitochondrial or nuclear markers were found so far to serve as unambiguous
differentiating character between the three entities (Barr & McPheron, 2006; Virgilio et al,
2008; Barr & Wiegmann, 2009). Also in laboratory conditions, C. rosa and C. fasciventris
can interbreed (G. Franz, pers. communication) although the hybrids show a higher
fluctuating asymmetry which might indicate a decrease in developmental stability (Erbout et
al, 2008). Therefore, it is not clear whether the three entities correspond to true biological
species.
Pheromone and cuticular hydrocarbon analyses showed that the three entities are clearly
separable based on these methodologies (2nd RCM meeting report and presentations;
Vacinkova et al, subm.). Morphology of immature stages also showed differences specimens
of C. rosa (2nd RCM meeting report and presentation by G. Steck), while the use of
morphological characters to separate the three entities needs further examination. Recent
morphometric research, based on wing landmarking, also allows separation of the three
entities (3rd RCM meeting presentation De Meyer).
Using microsatellite markers, it was shown that the three entities could be recognized but that
both C. rosa and C. fasciventris each consist of two separate genotypes (R1-R2 and F1-F2)
resulting in five different entities within the complex.
Recent work has focused on re-analyzing existing data and extending previously used
methodologies recognizing the five entities:
C. anonae is a single entity and is not further investigated. Both males and (albeit to a lesser
degree) females can be differentiated from the other four entities. It shows a clear geographic
pattern unlike that of the other entities (although with some considerable overlap).
Research on C. fasciventris is restricted to re-analyzing the adult morphology, host plant and
geographic data. Male adults can be differentiated by characters of the mid tibia. Wing
landmarking gives an indication of separation for both sexes but not significantly. The data
on host plants are too limited to make draw any conclusions on possible differences in host
spectrum. The distributional data show two largely separated geographical occurrences with
one type (F2) restricted to eastern Africa, while the other type (F1) mainly found in western
and central Africa (this separations was also confirmed by the markers used in Virgilio et al.,
2008). However the latter type is also found in some isolated areas in Angola, Malawi,
Tanzania and Zambia. No clear geographical or other pattern could be detected. The lack of
91
an established colony of both types has prevented providing material for some of the other
studies (like larval morphology or pheromones) and did not allow checking on mating
incompatibility and physiological differences. Currently there is an established colony of F2
type (from eastern Africa) at ICIPE (Kenya). Contacts have been made with researchers in
western Africa but establishment of live colony of F1 has been unsuccessful so far.
Most research has been focusing on the two C. rosa types, also because C. rosa is considered
economically the most important species within the complex. Adult males can be
differentiated, based on characters of the male mid tibia. Wing landmarking gives an
indication of separation for both sexes but not significantly. There is conflicting information
in the literature regarding developmental physiology and climatic niche for C. rosa (De
Meyer et al, 2008; Duyck et al., 2002, Grout & Stoltz, 2007). Re-analysis of distributional
data indicates that this might be because of failure to recognize the two types that were
indicated by the microsatellite study. One type, R2, appears to occur at lower latitudes on the
African continent and at higher altitudes. It might be more cold resistant than R1 type that is
absent from the colder parts (lower latitudes, higher altitudes) within the geographic range of
C. rosa. In order to corroborate this with experimental data, physiological development
experiments are set up by different teams throughout the range of the species (South Africa,
Tanzania and Kenya). So far live colonies of both types have be established only in Kenya by
ICIPE. South African and Tanzanian teams are currently setting up live colonies. Preliminary
results on the Kenyan experiments will be presented at the 3rd CRM. In addition, material
from the colonies at ICIPE has been made available to other teams for study of larval
morphology and cuticular hydrocarbons. Preliminary investigation of larval morphology
between R1 and R2 from Kenya has not shown any differences (but there is marked
difference with earlier studied material of C. rosa (type unknown) from South Africa. Further
results will be presented at the 3rd CRM. Mating compatibility experiments have been
conducted between the two C. rosa (R1-R2) and the C. fasciventris (F2) populations at
ICIPE. These results will also be presented at the 3rd CRM.
Conclusions
- All evidence so far points towards the fact that the three recognized species within the
FAR complex are indeed three separate species
- Further evidence indicate that C. rosa and C. fasciventris might each comprise two
different entities, while C. anonae remains a single entity
- For the two C. fasciventris types, there is only the indication of some adult
morphological differences and different biogeographic patterns. The latter are not
completely understood so far. Further investigation in the status of the two entities
will require the establishment of live colonies.
- For the two C. rosa types, there is some evidence that these are indeed two separate
species. Results presented at the 3rd CRM and further research that is planned in the
forthcoming months might further reveal the exact status of these two entities.
92
Annex 3b: Character Matrix for the Ceratitis FAR Complex
Discipline
1. Microsatellite
(pop genetics)
2. ND6/COI
C. rosa 1
(R1) (warm)
C. rosa 2
(R2) (cold)
C. fasciventris 1
(F1) (west)
C. fasciventris 2
(F2) (east)
C. annonae
(A)
Remarks
published
published
2. Per/ITS1
published
2. ITS2/other
markers
3. Adult male
morphology
4. Adult female
morphology
5. Wing
morphometrics
published
published
dissertation
6. Larval
morphology
7. Developmental
physiology
7. Pre-zygotic
isolation
7. Post-zygotic
isolation
8. Pheromones
dissertation
8. Cuticular
hydrocarbons
9. Host range
submitted
10. Distribution
11. Y-chromosome
published
12. Endosymbionts
13. Behaviour
1. Recognition of five genotypes is based upon the microsatellite analysis. This splitting up
was confirmed by male morphology.
2. Based on Virgilio et al. (2008).
3. Five types can be differentiated using secondary sexual characters of male mid femur and
tibia ornamentation and shape.
4. Only female C. anonae can be differentiated from the other four types using pilosity
characters on anepisternum and fore femur.
5. Male morphometrics allow some differentiation but not complete.
6. Preliminary studies on larval morphology do not show any distinct differences but need to
be further examined in detail.
7. Study of C. anonae is deemed unnecessary given other means of differentiation. Current
focus is on R1-R2 differentiation and needs to be elaborated.
8. Study done on three 'morphotypes' prior to splitting in five groups. Work will be extended
to R1-R2.
9. All types are polyphagous and most data are too limited to observe distinct differences in
host range, except for R1-R2 regarding temperate fruits.
93
10. All types show distinct distribution ranges.
11. Already published by Malacrida's team.
12. To be done on all 5 populations of the FAR (Wolbachia presence).
13. Behavioural studies (including mating, responses to attractant etc.) with regard to the
highland population of C. rosa collected from coffee (Ruiru) has previously been
documented (Pontiano Nemeye’s thesis) but no information is available with regard to the
lowland population. In addition to assessing mating compatibility, observation on
courtship repertoire/mating behaviour will be recorded in field cages. Similarly, responses
of the two populations to trimedlure, EGOlure and synthetic food attractants will be
documented in field cages.
94
Annex 4: “Workshop on Reviewing Evidence to Resolve Species Complexes
of Tephritid Pests”
31 August 2013
Tucumán Center Hotel, Tucuman, ARGENTINA
SATURDAY, 31 AUGUST
Chairperson:
David Haymer
Panel of Independent Experts:
Rob Duthie, David Haymer, Allen Norrbom, Shaun Winterton
08:00 - 08:15
David Haymer: Opening statements, goals of the workshop, review of the
agenda and other issues.
08:15 - 08:30
Tony Clarke: Brief summary of CRP findings of the Bactrocera dorsalis
complex group, focusing on conclusions and remaining controversies and
research possible gaps.
08:30 - 09:00
Panellists: Questions and discussion to determine/confirm B. dorsalis
complex:
a. specifically what decisions need to be made by the end of the day;
b. the areas where consensus has been reached and can be put aside.
09:00 - 09:15
Teresa Vera: Brief summary of CRP findings of the Anastrepha fraterculus
complex group, focusing on conclusions and remaining controversies and
possible research gaps.
09:15 - 09:45
Panellists: Questions and discussion to determine/confirm for the A.
fraterculus complex:
a. specifically what decisions need to be made by the end of the day;
b. the areas where consensus has been reached and can be put aside.
09:45 - 10:00
Marc de Meyer: Brief summary of CRP findings of the Ceratitis FAR
group, focusing on conclusions and remaining controversies and research
possible gaps.
10:00 - 10:30
Panellists: Questions and discussion to determine/confirm for the Ceratitis
FAR complex:
a. specifically what decisions need to be made by the end of the day;
b. the areas where consensus has been reached and can be put aside.
95
10:30 - 10:45 BREAK
10:45 - 11:30
Panellists: discussion on the controversies/gaps to debate, following two
approaches:
a. addressing a series of direct questions to the specific research groups; and
b. guiding a semi-formal debate between representatives from those groups
with differing opinions, mediated by the chair of the meeting.
12:00 - 14:00 LUNCH
Panellists discuss among themselves the arguments and formalise decisions
as to whether:
a. Sufficient data have been obtained to answer the remaining questions, in
which case they deliver ‘verdicts’.
b. In cases where remaining data are required to answer the questions, they
state what remains to be done and suggests how this should be achieved.
14:00 - 14:30
Panellists: make a presentation to participants of all the groups on the
outcome of their deliberations.
14:30 - 15:30
Panellists: chair a final discussion and guide reaching a consensus on
follow-up actions by research groups during the final 18 month phase of the
CRP.
15:30 - 15:45 BREAK
15:45 - 17:00
Panellists: finalize recommendation and follow-up actions by research
groups during the final 18 month phase of the CRP.
96
G. Appendices
97
Appendix 1: 3rd RCM Participants
LIST OF PARTICIPANTS
3rd RCM “Resolution of Cryptic Species Complexes of Tephritid Pests to Overcome
Constraints to SIT Application and International Trade”
Tucuman, Argentina
26-31st 2013
ARGENTINA
Mr BACHMANN, Guillermo
Laboratorio de Genética de Insectos
Instituto de Genética, INTA Castelar
Nicolas Repetto y de los Reseros s/n (1686), Hurlingham
Buenos Aires, ARGENTINA
Email: geb_84@yahoo.com.ar
Ms CERIANI CAMDESUS, Adriana
Dirección de Cuarentena Vegetal SENASA
Av. Paseo Colón 315, 4º piso, Oficina 14
Buenos Aires, ARGENTINA
Tel: +54 11 4121-5245/5246
Email: aceriani@senasa.gov.ar
Mr CLADERA, Jorge
Instituto de Genética, INTA Castelar
Nicolás Repetto y de los Reseros s/n
(1686) Hurlingham,
Buenos Aires, ARGENTINA
Tel + 54 11 44501876 and
44598372 EXT 106
EMAIL jcladera@cnia.inta.gov.ar
Ms FEDYSZAK, Paola Yael
Programa Nacional de Control y Erradicación de Mosca de los Frutos
SENASA
Av. Paseo Colón 315, 4º, Depto. A – Oficina 13
Buenos Aires, ARGENTINA
E-mail: procem@senasa.gob.ar
98
Ms GOANE, Lucia
Facultad de Agronomía y Zootecnia
UNT-Estación Experimental Agroindustrial
Obispo Colombres (FAZ-EEAOC)
Av. William Cross 3150-Las TalitasTucumán, ARGENTINA
Tel: 0054 381 4521000 (243)
Fax: 0054 381 4521000 (231)
Email: lugoane@gmail.com
Ms JOFRE BARUD, Flavia
Instituto de Biotecnología
Facultad de Ingeniería
Universidad Nacional de San Juan
Av. Libertador General San Martín 1109(O)
San Juan, ARGENTINA
Tel - Fax: 0264 4211700
Email: fjbarud@unsj.edu.ar
Ms JUAREZ, María Laura
Cátedra Terapéutica Vegetal
Facultad de Agronomía y Zootecnia
Florentino Ameghino s/n. Finca El Manantial,
Universidad Nacional de Tucumán
Tucumán (4000), ARGENTINA
Tel: (54) 381-439-0003
Email: lau_zoo@yahoo.com.ar
Ms LOPEZ, María Liza
Instituto de Biotecnología
Facultad de Ingeniería
Universidad Nacional de San Juan
Av. Libertador General San Martín, 1109 (O)
San Juan, ARGENTINA
Tel - Fax: 0264 4211700
E-mail: mllopez@unsj.edu.ar
Ms MENDOZA, Mariana
Universidad Nacional de San Juan
Av. Libertador General San Martín, 1109(O)
San Juan, ARGENTINA
Email: marianamendoza79@hotmail.com
Mr MURUA, Fernando
Programa de Control y Erradicación de Mosca de los Frutos
Av. N. Benavides 8000 Oeste – Rivadavia
San Juan, ARGENTINA
Tel.-Fax: 0264 - 4231996
E-mail: fmurua80@gmail.com
99
Ms RUIZ, María Josefina
CONICET- Estación Experimental Agroindustrial Obispo Colombres
Av. William Cross 3150 - Las Talitas
Tucumán, ARGENTINA
Telefax 54-381-4521000-int.154
Email: josefinaruiz2802@hotmail.com
Mr VILARDI Juan
Universidad de Buenos Aires
Buenos Aires, ARGENTINA
Email: vilardi10@gmail.com
Ms VERA, María Teresa
Universidad Nacional de Tucumán
Avenida Independencia 1800
4000 San Miguel de Tucumán, ARGENTINA
Tel: 0054 381 439 0003
Fax: 0054 381 4390049
Email: teretina@hotmail.com
Ms VILLAGRAN, María Elvira
Estación Experimental Agroindustrial Obispo Colombres
Av. William Cross 3150 - C.P. T4101XAC - Las Talitas
Tucumán, ARGENTINA
Tel: (54 - 381) 452 1000
Email: melvira_v@yahoo.com.ar
AUSTRALIA
Mr CLARKE, Anthony
Associate Professor
Biogeosciences, FaST
Queensland University of Technology
G.P.O. Box 2434
Brisbane, QLD 4001
AUSTRALIA
Tel: 61 7 3138 5023
Fax: 61 7 3138 1535
Email: a.clarke@qut.edu.au
Mr DUTHIE, Robert
Plant Health Consultant
PO Box 212
Bellingen, New South Wales 2454
AUSTRALIA
Tel: (+61) 66551843
E-Mail: rob.duthie@kalang.com.au
100
Mr SCHUTZE, Mark
School of Earth, Environmental and Biological Sciences & CRC National Plant Biosecurity
Queensland University of Technology,
GPO Box 2434
Brisbane, QLD 4001,
AUSTRALIA
Tel: (Int) 61 7 3138 8351
Fax: (Int) 61 7 3138 1535
Email: m.schutze@qut.edu.au
AUSTRIA
Mr CÁCERES, Carlos
Insect Pest Control Laboratory
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
FAO/IAEA Agriculture and Biotechnology Laboratories
A-2444 Seibersdorf, AUSTRIA
Tel.: +43 1 2600 28413
Email: C.E.Caceres-Barrios@iaea.org
Mr HAQ, Ihsan
Insect Pest Control Laboratory
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
FAO/IAEA Agriculture and Biotechnology Laboratories
A-2444 Seibersdorf, AUSTRIA
Tel.: +43 1 2600 28454
Email: I.Haq@iaea.org
Mr HENDRICHS, Jorge
Insect Pest Control Programme
Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture
Wagrammerstrasse 5, 1400 Vienna, AUSTRIA
Tel.: +43 1 2600 21628
Email: J.Hendrichs@iaea.org
BELGIUM
Mr DE MEYER, Marc
Royal Museum for Central Africa
Leuvensesteenweg 13
3080 Tervuren, BELGIUM
Tel : 32 (0) 2 7695360
Fax : 32 (0) 2 7695695
Email : demeyer@africamuseum.be
101
BRAZIL
Ms DE LIMA, Kátia Manuela
Universidade Estadual de Santa Cruz
Laboratório de Marcadores Moleculares e Citogenética
Rodovia Ilhéus-Itabuna, km 16
BRAZIL
Tel Lab: (0055 73) 3680-5431
Tel cel: (0055 73) 8801-4731 ou 8177-8558
Email : katiamanuela2@hotmail.com
Ms GOMES DA SILVA MILLER, Janisete
Centro de Biotecnologia e Genética
Departamento de Ciências Biológicas
Universidade Estadual de Santa Cruz
Rodovia Ilhéus-Itabuna, km 16
BRAZIL
Tel : 00557 3680 5440
Fax : 005573 3680 5226
Email : janisete.silvamiller@pq.cnpq.br
Jgs10@uol.com.br
Ms RUFINO DO NASCIMIENTO, Ruth
Universidade Federal de Alagoas
Av. Lourival Melo Mota, s/n
Tabuleiro do Martins 57072-970
Maceio, BRAZIL
Tel/Fax : 55 82 3214 1361
Email : ruthviacar@yahoo.com.br
Ms SORDI JOACHIM BRAVO, Iara
Medfly Rearing Facility Brazil
Quadra d-13, Iote 15
Distrito Industrial do Sao Francisco
48.900-000 Juazeiro, Bahia, BRAZIL
Email: ibravo@ufba.br
Ms PASSOS RORIZ, Kelly
Empresa Baiana de Desenvolvimento Agrícola
Laboratório de Ecologia Nuticional de Insetos
Rua Barão de Geremoabo, Campus-Ondina-147, Instituto de Biologia
Salvador, Bahia, BRAZIL
Tel: 32836545
Email: kellyroriz@yahoo.com.br
102
COLOMBIA
Mr CANAL DAZA, Nelson
Universidad del Tolima
Barrio Santa Elena, Apartado Aéreo 546
Ibagué, Tolima, COLOMBIA
Tel/Fax : 57 8 2771212, ext 9261.
Email: nacanal@ut.edu.co
Ms CASTAÑEDA, María del Rosario
Universidad del Tolima
Barrio Santa Elena, Apartado Aéreo 546
Ibagué, Tolima, COLOMBIA
Tel/Fax : 57 8 2771212, ext 9261.
Email: mrcasta@ut.edu.co
CZECH REPUBLIC
Ms BRIZOVA, Radka
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Flemingovo nám. 2, CZ-166 10 Praha 6
CZECH REPUBLIC
Telephone: +420 220 183 240
E-mail: Radka.Brizova@vscht.cz
Ms KALINOVA, Blanka
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Flemingovo nam.2, 166 10 Praha 6
CZECH REPUBLIC
Tel: +420 220 183 339
Email: blanka@uochb.cas.cz
Ms VANICKOVA, Lucie
Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic
Flemingovo nam. 2, 166 10 Praha 6.
CZECH REPUBLIC
Tel: +420 220 183 339
Email: luci.vanickova@gmail.com
103
FRANCE
Ms DELATTE, Hélene
UMR "Peuplements Végétaux et Bio-agresseurs en Milieu Tropical"
CIRAD-3P
7, Chemin de l'IRAT
Ligne Paradis
97410 Saint Pierre
La Réunion, FRANCE
Tel 262 262 49 92 35
Email: helene.delatte@cirad.fr
GREECE
Ms DROSOPOULOU, Elena
Department of Genetics, Development and Molecular Biology
School of Biology, Faculty of Sciences
Aristotle University of Thessaloniki
Thessaloniki, GREECE.
Tel: 0030-2310 998291 (office)
E-mail: edrosopo@auth.gr
Ms ZACHAROPOULOU, Antigone
University of Patras
265 00 Patras, GREECE
Email: zacharop@upatras.gr
ITALY
Ms MALACRIDA, Anna
Dipartimento di Biologia Animale
Via Ferrata 1
Università di Pavia
27100 Pavia, ITALY
Tel/Fax +39 0382 986059
Email: annarodolfa.malacrida@unipv.it
104
JAPAN
Mr TSURUTA, Kenji
Pest Identification & Diagnostics Division,
Yokohama Plant Protection Station, MAFF,
1-16-10, Shinyamashita, Naka-Ku,
Yokohama, 231-0801
JAPAN
TEL: +81-45-622-8940
FAX: +81-45-621-7560
E-mail: tsurutak@pps.maff.go.jp
KENYA
Mr EKESI, Sunday
International Centre of Insect Physiology and Ecology (ICIPE)
P.O. Box 30772
Nairobi, KENYA
Email: sekesi@icipe.org
MALAYSIA
Mr HEE, Alvin
University Putra Malaysia
43400 Serdang, Selangor, MALAYSIA
Tel : 603 8946 6643
Email : alvinhee@putra.upm.edu.my
Mr TAN, Ken Hong
Phi Systems Sdn Bhd
Phi Biotech Sdn Bhd (Research)
18-26-A1, Gurney tower
Persiaran Gurney
10250 Penang, MALAYSIA
Tel : 604 890537
Email : khtan@phi-biotech.com
MEXICO
Mr HERNANDEZ-ORTIZ, Vicente
Instituto de Ecología A.C.
Carretera Antigua a Coatepec No. 35, El Haya
Apartado Postal 63. CP. 91070
Xalapa, Veracruz, MEXICO
Tel: 52 228 842 1800 ext 3303
Fax: 52 228 8421800 ext 4222
105
Email: Vicente.hernandez@inecol.edu.mx
Mr RULL, Juan
Instituto de Ecologia A.C.
Carretera Antigua a Coatepec No. 35, El Haya
Apartado Postal 63. CP. 91070
Xalapa, Veracruz, MEXICO
Email: juan.rull@inecol.edu.mx
NEW ZEALAND
Ms ARMSTRONG, Karen
Bio-Protection Research Centre
Lincoln University
P. O. Box 84
Canterbury 8150, NEW ZEALAND
Tel: 64 3 325 3838
Fax: 64 3 325 3864
Email: Karen.armstrong@lincoln.ac.nz
THAILAND
Ms AKETARAWONG, Nidchaya
Department of Biotechnology
Faculty of Science
Mahidol University
272 Rama VI Road
Ratchathewe, Bangkok 10400
THAILAND
Tel: 668 9493 7036
Fax: 662 441 9820 ext 1124
Email:testn@mahidol.ac.th
Ms CHINVINIJKUL, Suksom
Irradiation for Agricultural Development Section
Dept. of Agricultural Extension
Ministry of Agriculture and Cooperatives (MOAC)
Paholyotin Road, Chatujak, Bangkok 10900
THAILAND
Tel: 662 5773682, 9406187
Fax: 662 5773682, 9406188
Email: chinvinijkuls@gmail.com
106
UNITED REPUBLIC OF TANZANIA
Mr MWATAWALA, Maulid
Sokoine University of Agriculture
P.O. Box 3000. Chuo Kikuu
Morogoro, UNITED REPUBLIC OF TANZANIA
Tel: + 255 23 260 3511
Fax: + 255 23 260 4651
Email: mwatawala@yahoo.com
UNITED STATES OF AMERICA
Ms AGUIRRE HAYMER, Beatriz
454 Opihikao Pl.
Honolulu, HI 96825.
UNITED STATES OF AMERICA
Email: Beya9@yahoo.com
Mr HAYMER, David
Professor of Genetics
Dept. of Cell and Molecular Biology
1960 East-West Rd, Biomed T511
University of Hawaii
Honolulu, HI 96822.
UNITED STATES OF AMERICA
Email: dhaymer@hawaii.edu
Mr NORRBOM, Allen L.
Systematic Entomology Lab., USDA
c/o National Museum of Natural History, MRC-168
P.O. Box 37012
Washington, DC 20013-7012.
UNITED STATES OF AMERICA
Tel. 202 633-4564
Fax. 202 786-9422
Email: Allen.Norrbom@ARS.USDA.GOV
Mr STECK, Gary
Division of Plant Industry
Florida Dept. of Agriculture and Consumer Services
P. O. Box 147100
Gainesville, FL 32614-7100.
UNITED STATES OF AMERICA
Tel: 1 352 372 3505 ext 188
Fax: 1 352 334 0737
Email: Gary.Steck@freshfromflorida.com
107
Mr TEAL, Peter
Chemistry Research Unit
Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS
1700 SW 23 Drive
Gainesville, Fl. 32604.
UNITED STATES OF AMERICA
Email: Peter.Teal@ARS.USDA.GOV
Mr WINTERTON, Shaun
California State Collection of Arthropods
California Department of Food and Agriculture
3294 Meadowview Rd.
Sacramento, CA, 95832-1148.
UNITED STATES OF AMERICA
Email: wintertonshaun@gmail.com
108
Appendix 2: Agenda
3RD FAO/IAEA RESEARCH CO-ORDINATION MEETING ON
"RESOLUTION OF CRYPTIC SPECIES COMPLEXES OF TEPHRITID PESTS TO
OVERCOME CONSTRAINTS TO SIT APPLICATION AND INTERNATIONAL
TRADE"
26 – 30 August 2013
Universidad Nacional de Tucumán, Facultad de Agronomía y Zootecnia
Hotel Tucumán Center, Tucumán, ARGENTINA
MONDAY, 26 AUGUST 2013
08:30 - 08:45
Teresa Vera, Jorge Hendrichs. Opening and welcome statements. Goals of
the meeting, review of agenda, administrative and other issues.
SESSION I : Chairperson: Gary Steck
08:45 - 09:15
Iara Sordi Joachim-Bravo, Raul Laumann, Vanessa Simões Dias, Alzira
Kelly Passos Roriz, Clarissa Petitinga, Beatriz Jordão Paranhos, Aldo
Malavasi. Analysis of sexual compatibility among Anastrepha fraterculus
populations from Brazil.
09:15 - 09:45
J. Rull, S. Abraham, M. Mendoza, M.C. Liendo, F. Devescovi, D.F.
Segura, K. Roriz, N. Nolasco, R. Castañeda, E. Tadeo, R. Brizova, A.
Kovaleski and M.T. Vera. Evolution of reproductive isolation among
different morphotypes of Anastrepha fraterculus.
09:45 - 10:15
Suksom Chinvinijkul, Sunyanee Srikachar, Weerawan Amornsak and
Sujinda Thanaphum..Field cage mating compatibility test of Bactrocera
dorsalis complex of the north and the south of Thailand.
10:15 - 10:45
Ruth R. do Nascimento, Lucie Vanicková, Adriana L. Mendonça, Alana
L. Mendonça, Beatriz Jordão Paranhos, Blanka Kalinová and Antônio
Euzébio Goulart Sant´Ana. Chemical studies of the sex pheromone of five
Brazilian populations of Anastrepha fraterculus (Diptera: Tephritidae).
10:45 - 11:00 BREAK
11:00 - 11:30
Peter Teal. Volatile chemicals released by Tephritid flies as a tool to
understanding species diversity.
11:30 - 12:00
Radka Břízová, Lucie Vaníčková, Blanka Kalinová, Michal Hoskovec,
Ruth Rufino Do Nascimento. Pheromones as an indispensible instruments
109
for distiction of Anastrepha fraterculus by age and geographic origin.
12:00 - 12:30
Suk-Ling Wee, Alvin Kah-Wei Hee and Keng-Hong Tan. Emission of
male sex pheromone by four cryptic species, Bactrocera dorsalis, B.
invadens, B. papayae and B. philippinensis, following Methyl Eugenol
consumption.
12:30 - 13:00
Blanka Kalinová, Radka Břízová, Maria Faťarová, Lucie Vaníčková,
Michal Hoskovec. Pheromones of three species of Ceratitis FAR complex.
13:00 - 14:00 LUNCH
SESSION II : Chairperson: Sunday Ekesi
14:00 - 14:30
Lucie Vaníčková, Massimiliano Virgilio, Aleš Tomčala, Radka Břízová,
Adriana de Lima Mendonça, Blanka Kalinová, Ruth Rufino Do
Nascimento, and Marc De Meyer. Cuticular hydrocarbons as a tool for
resolution of the fruit fly species complexes.
14:30 - 15:00
Ishan ul Haq, Marc J.B. Vreysen, Todd E. Shelly, Jorge Hendrichs.
Methyl eugenol sensitivity and its effects on intraspecific mating of three
sibling species of the Bactrocera dorsalis complex.
15:00 - 15:30
Alvin Kah-Wei Hee, Suk-Ling Wee and Keng-Hong Tan. Comparative
sensitivity of males of four globally important cryptic species in the
Bactrocera dorsalis complex - B. dorsalis, B. invadens, B. papayae and B.
philippinensis, to Methyl Eugenol and subsequent male antennal protein
profiles.
15:30 - 15:45 BREAK
15:45 - 16:15
Elena Drosopoulou, A. Augustinos, A. Gariou-Papalexiou, C. CaceresBarrios, K. Bourtzis, P. Mavragani-Tsipidou, A. Zacharopoulou.
Cytogenetic studies in members of Bactrocera dorsalis species complex: A
comparative analysis.
16:15 - 16:45
Nelson A. Canal, Julio C. Carranza, Daniel Zabala, María del Rosario
Castañeda. Population structure of the Andean morphotype of the
Anastrepha fraterculus complex.
16:45 - 17:15
María del Rosario Castañeda, Daniel Zabala, F. M. Patarroyo, Julio C.
Carranza, Nelson A. Canal. Population studies of Anastrepha obliqua
(MacQuart, 1835).
17:15 - 17:45
General Discussion
110
TUESDAY, 27 AUGUST 2013
08:30 - 08:45
Teresa Vera, Jorge Hendrichs. Announcements, adjustments to the
agenda, administrative and other issues.
SESSION III : Chairperson: Blanka Kalinova
08:45 - 09:15
Janisete G. Silva, Katia M. Lima, Norman B. Barr, Raul Ruíz-Arce,
Gary J. Steck, Bruce. A. McPheron, Roberto A. Zucchi. Use of mtDNA
and nuclear markers to delimit and diagnose Brazilian species within the
Anastrepha fraterculus complex.
09:15 - 09:45
L. Paulin, P. Gómez Cendra, M. Juri, S. Lanzavecchia, M. T. Vera, J.
Cladera, J. C. Vilardi. Landscape genetics approach to study Anastrepha
fraterculus populations.
09:45 - 10:15
Karen F. Armstrong, L. M. Boykin, A. Chomic, M. K. Schutze, A. R.
Clarke. Molecular genetic resolution of pest species within the Bactrocera
dorsalis complex
10:15 - 10:45
Anna R Malacrida, Nidchaya Akeratawong, Silvia Lanzavecchia,
Blanka Kalinova, Giuliano Gasperi. Looking for species-specific
sequences in Bactrocera, Anastrepha and Ceratitis species.
10:45 - 11:00 BREAK
11:00 - 11:30
Qinge Ji. Bactrocera dorsalis complex species in mainland China.
11:30 - 12:00
Nidchaya Aketarawong, Siriwan Isasawin, Anna R. Malacrida,
Giuliano Gasperi, Sujinda Thanaphum. Evaluation of genetic sexing
strain, Bactrocera dorsalis Salaya1 for its species complex members and
molecular characterization of Y-chromosome sequences of Bactrocera
dorsalis.
12:00 - 12:30
Sunday Ekesi, Chrysantus M. Tanga, Fathiya M. Khamis and Samira
A. Mohamed. Host plant relationships, temperature tolerance and mating
compatibility studies in Bactrocera cucurbitae and Ceratitis rosa.
12:30 - 13:00
Maulid W. Mwatawala, Massimo Virgilio, Marc De Meyer. Comparative
studies on behaviour and developmental physiology of Ceratitis rosa entities
(R1 and R2) occurring in Tanzania.
13:00 - 14:00 LUNCH
SESSION IV : Chairperson: Peter Teal
111
14:00 - 14:30
Vicente Hernández-Ortiz, Nelson A. Canal and Juan O. Tigrero.
Morphometrics of wild populations from Ecuador and Colombia of the
Anastrepha fraterculus complex: The eighth passenger.
14:30 - 15:00
Gary Steck. Anastrepha species of the Central Andes of Peru.
15:00 - 15:30
M. K. Schutze and A. R. Clarke. The taxonomic history of the Oriental
and invasive fruit fly: from Musca ferruginea Fabricius to Bactrocera
invadens Drew, Tsuruta & White.
15:30 - 15:45 BREAK
15:45 - 16:15
M. K. Schutze, S.L. Cameron, K. Mahmood, W. Bo, C. Cáceres, M.J.B.
Vreysen, A. Pavasovic, A. R. Clarke. Reproductive compatibility,
morphological and morphometric variation, and molecular genetic analyses
of Bactrocera dorsalis sensu lato (Diptera: Tephritidae) from Africa, the
Indian Subcontinent and East Asia.
16:15 - 16:45
J. Van Cann, M. Virgilio, K. Jordaens and M. De Meyer. The Ceratitis
FAR complex: biogeography, host plant spectrum and morphometrics.
16:45 - 17:30
General Discussion and organization of three Working Groups:
a) Anastrepha fraterculus complex, b) Bactrocera dorsalis complex, and
c) Bactrocera cucurbitae & Ceratitis FAR complex.
19:00 - 22:00
Social Event
WEDNESDAY, 28 AUGUST 2013
SESSION V : Chairpersons Jorge Hendrichs and Group Leaders
08:30 - 10:30
Working Groups. Review of achievements based on original coordinated
research plans and drafting of results and scientific/technical conclusions for
the different genera, approaches and techniques. Review of research needs to
be addressed and other activities to be implemented.
10:30 - 10:45 BREAK
10:30 - 13:00
Working Groups – continued
13:00 - 14:00 LUNCH
14:00 - 15:30
Working Groups – continued
15:30 - 15:45 BREAK
112
15:45 - 17:30
Working Groups Presentations – Review of achievements, conclusions
and recommendations on research needs and other activities for the different
genera, approaches and techniques.
THURSDAY, 29 AUGUST 2013
SESSION VI : Chairpersons Jorge Hendrichs and Group Leaders
08:30 - 10:30
Working Groups – Drafting of individual research proposals for the
different genera and techniques, taking into account the proposed R&D of
each research team.
10:30 - 10:45 BREAK
10:45 - 13:00
Working Groups – continued
13:00 - 14:00 LUNCH
14:00 - 15:30
Working Group Presentations – Review of recommendations for the
individual research proposals for the different applications of each research
team.
15:30 - 15:45 BREAK
15:45 - 17:00
General Discussion – Organization of the preparation of final CRP
proceedings in a special issue and potential journals to be contacted.
FRIDAY, 30 AUGUST 2013
SESSION VII : Chairpersons Jorge Hendrichs and Group Leaders
08:30 - 10:30
Working Groups – Drafting and compiling of RCM report.
10:30 - 10:45 BREAK
10:45 - 13:00
Working Group – continued
13:00 - 14:00 LUNCH
14:00 - 16:30
Working Groups – General Discussion: Agreement on content of RCM
report, on information exchange mechanisms, on location of Final RCM and
closure of the RCM.
113
Appendix 3: Abstracts 3rd RCM Tucuman
ABSTRACTS 3RD RCM TUCUMAN
114
Evaluation of genetic sexing strain, Bactrocera dorsalis Salaya1 for its species complex
members and molecular characterization of Y-chromosome sequences of Bactrocera
dorsalis
1
Nidchaya Aketarawong , Siriwan Isasawin1, Anna R. Malacrida2, Giuliano Gasperi2,
Sujinda Thanaphum1
1
Department of Biotechnology, Faculty of Science, Mahidol University, THAILAND
2
Department of Biology and Biotechnology, University of Pavia, ITALY
Abstract:
Bactrocera dorsalis strain Salaya 1 is a translocation-based genetic sexing character based on
a brown-white pupal color dimorphism. This strain showed successful mating
competitiveness with wild B. dorsalis s.s. under semi-natural condition (Fried
competitiveness value (C) = 1.33). A mass release of sterile Salaya 1 males in the field
resulted in suppression of an indigenous pest population (from 23 flies/trap/day to less than 1
fly/trap/day). Consequently, fruit damage was reduced from 30% to 5%. According to results,
the Salaya 1 strain has been applied to wild B. carambolae, a member of B. dorsalis species
complex, in order to test mating competitiveness under the semi-natural condition. However,
we found that C-value was low, suggesting that incompatible mating occurs between the
genetic sexing Salaya 1 strain and wild B. carambolae. The new genetic sexing strain is
therefore being further developed for pest management using the sterile insect technique.
In the meantime, we have a collaborative research project between University of Pavia and
Mahidol University. This project aims to study Y-chromosome sequences of B. dorsalis s.s.
We focus on the Y-chromosome because it plays a key role in determining the development
of male individuals and may contain genes involving male fertility. Five DNA fragments
were isolated from the genome of B. dorsalis s.s. (Seiberdorf strain). After amplification of
several B. dorsalis s.s. strains, we found that four fragments may be a male-specific sequence
and another one may be a male-enriched sequence. More experiments such as in situ
hybridization will be performed to identify the location of those sequences.
115
Molecular genetic resolution of pest species within the Bactrocera dorsalis complex
Karen F. Armstrong1, L. M. Boykin1, A. Chomic1, M. K. Schutze2, A. R. Clarke2
1Lincoln University, NEW ZEALAND
2School of Earth, Environmental and Biological Sciences, Queensland University of
Technology, G.P.O. Box 2434, Brisbane 4000, Queensland, AUSTRALIA
Abstract:
As part of a comprehensive genetic analysis a comparative transcriptome approach was taken
to identify genetic differences amongst the pest taxa B. dorsalis ss, B. papayae, B.
philippinensis, B. invadens and B. carambolae. The aim was to identify novel, potentially
discriminatory gene regions based on the presence of group-specific SNPs (single nucleotide
polymorphisms) which might support the species status of these groups or otherwise.
After filtering out non-insect EST’s (9% other organisms; 35% not annotated) ~37,000 EST’s
were screened for private SNPs, each of which then manually checked for authenticity. Of
many candidate markers identified 22 primer pairs have been designed and further explored
to date; B. occipitalis was included as a “good species” out group. Ten loci produced useful
data. These together with data from three of the previous loci using the same specimens,
excluding B. invadens, were examined for character-based (private SNPs) and phylogeneticbased evidence of differences between groups. Diagnostic differences were observed in
several loci for B. carambolae (11 loci), B. occipitalis (8 loci), B. philippinensis (5 loci) and
B. dorsalis/B. papayae (5 loci). No loci suggested differences between B. dorsalis and B.
papayae. Clusters in maximum likelihood analysis reflected these separations, although the
inter-group relationships were not consistent between loci. Species tree analysis has yet to be
completed.
In conclusion, while a species tree may clarify these relationships as might the inclusion of
further loci, based on the data to date used in isolation in the context of a phylogenetic
species concept, there is still no evidence to suggest that B. dorsalis and B. papayae and
possibly B. philippinensis are discrete taxa. Bactrocera philippinensis does appear to have
some characters that differentiate it from the others. However, this information on its own,
with such small genetic distances and sampling from a narrow geographic base, would be
hard to interpret as anything more than a population variant emphasised by geographic
distance from the interbreeding populations in mainland Asia.
116
Pheromones as an indispensible instruments for distiction of Anastrepha fraterculus by
age and geographic origin
Radka Břízová1,2*, Lucie Vaníčková3, Blanka Kalinová1, Michal Hoskovec1, Ruth
Rufino Do Nascimento3
1
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague,
CZECH REPUBLIC..
2
Institute of Chemical Technology Prague, CZECH REPUBLIC
3
Universidade Federal de Alagoas, Maceio, BRAZIL
Abstract:
Males of Anastrepha fraterculus form leks and release sex pheromone to attract females
during the reproductive behavior. 20 substances were identified in mature male emanations.
Of these substances, 6 compounds were antennally active. In young males, the active
compounds are present only in trace quantities. Their concentration increases with age and
reaches a maximum at the time of sexual maturity. It is very likely that only these substances
are part of the male sex pheromone signal.
In order to estimate whether the variability in pheromone communication correlates with
reproductive isolation observed between some A. fraterculus populations, the emanations of
mature males of “Piracicaba”, “Tucuman”, “Vacaria” and “Peru” populations were
compared. Semi-quantitative analyses showed both qualitative and qualitative differences in
signal quality. The biggest difference was observed in the “Peru” population.
117
Population structure of the Andean morphotype of the Anastrepha fraterculus complex
Nelson A. Canal, Julio C. Carranza, Daniel Zabala, María del Rosario Castañeda
Universidad del Tolima, COLOMBIA
Abstract:
The South American Fruit Fly is an important pest of fruits in Latin America, however, seven
morphotypes have been found in the nominal species Anastrepha fraterculus. Specimens
from Colombia belong to the Andean morphotype, together with specimens from the
Venezuelan highlands. Data on morphology, biology, ecology or genetics of this biological
entity are lacking.
Specimens from nine localities in Colombia were collected, from two different hosts and a
wide range of altitudinal distribution of the species. The main objective was to study the
morphological, cytogenetic and genetic structure of the population of the biological entity
through its geographical distribution. There were not statistical differences among
populations based on adult morphometry, however, the range of the measurements of the
taxonomic characters are wider than previously reported. The karyotype of the species was
described; it differs from the other known karyotypes of the complex and it is variable among
Colombian populations.
Studies of the mitochondrial genes COI and COII show eight populations in a clade differing
from other populations from Latin America; the ninth population, from the South of the
country, near Ecuador, was grouped apart from the other eight and it appear to be related to
Ecuadorian populations, however, its karyotype and morphometry are related to other
populations from Colombia and not from Ecuador.
For quarantine purposes, A. fraterculus from Colombia belongs to a single species along the
Highland Mountains, from the South-West to North- East and the specimens from the South
have to be studied more deeply. Aspects of biology, ecology, quarantine status, chemical
ecology, among other aspects, have to be studied for this widely unknown morphotype.
118
Population studies of Anastrepha obliqua (MacQuart, 1835)
María del Rosario Castañeda, Daniel Zabala, F. M. Patarroyo, Julio C. Carranza,
Nelson A. Canal
University of Tolima, COLOMBIA
Abstract:
The West Indian Fruit Fly Anastrepha obliqua is distributed from Mexico to South Peru and
North-East Brazil, and it is an important pest of mango and other fruits, with quarantine
restrictions. The species is close to A. fraterculus and like this last species, and due to a wide
distribution, some studies have shown population variability and it has been suggested that a
complex of cryptic species may be present in the biological entities. It is important to clear
this fact to support future SIT activities and/or quarantines at international markets.
Morphological, cytogenetic and genetic studies were performed, based on Colombian
populations and including specimens from Brazil and Mexico or data from previous studies.
Genetic variability was found, supported by 100 bootstrap. Cytogenetic variability was found
especially in the size of sexual chromosomes and shape of C-band. Morphometric analyses
show some divergent populations. Results obtained show population variability; however
results are not clear at this moment because differences found are not as remarkable as the
variability found within A. fraterculus.
119
Field cage mating compatibility test of Bactrocera dorsalis complex of the north and the
south of Thailand
Suksom Chinvinijkul1, Sunyanee Srikachar2, Weerawan Amornsak3, Sujinda
Thanaphum4
1
Department of Agricultural Extension, Ministry of Agriculture and Cooperatives,
Chatuchak, Bangkok 10900, THAILAND
2
Department of Agriculture, Ministry of Agriculture and Cooperatives, Chatuchak, Bangkok
10900, THAILAND
3
Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkhen
Campus, Chatuchak, Bangkok 10900, THAILAND
4
Department of Biotechnology, Faculty of Science, Mahidol University, Salaya Campus,
Salaya, Nakhon Pathom, THAILAND
Abstract:
The sexual compatibility among Bactrocera dorsalis complex (Diptera: Tephritidae)
populations from the northern and southern extremes of geographical distribution of Thailand
was assessed in field cages.
Six wild colonies of Bactrocera dorsalis, Bactrocera papayae and Bactrocera carambolae
from Chiang Mai (north) and Nakhon Si Thammarat (south) were collected from infested
fruits and maintained at the fruit fly mass rearing facility in Pathumthani province. Identified
flies of different populations were genetically studied, including a pheromone analysis. Intra–
specific and inter-specific mating compatibility of the 2nd - 4th generation flies was
investigated at the intra-regional and inter-regional level. Bactrocera dorsalis, B. papayae
and B. carambolae males 23, 28 and 28 day old and females 21, 28 and 28 day old,
respectively, were used in these studies. Twenty individuals of each sex of two different
colour marked populations were released into the field cages. A potted mango tree
approximately 180 cm height in each octagonal field cage was set up for the experiment. Five
mated pairs in each replicate were randomized and individually assessed for fecundity.
Sexual compatible mating was quantified with the following indices: the Relative Isolation
Index (RII), Isolation Index (ISI), Male Relative Performance Index (MRPI), and Female
Relative Performance Index (FRPI).
Results of the intra-species mating compatibility tests showed that B. dorsalis from the north
and the south preferred to mate within region (RII=1.73), higher potential to mate appeared in
males from the north than the south (MRPI=0.09), but females from the south showed higher
mating (FRPI=-0.15). Bactrocera papayae showed inter-regional mating compatibility
between north and south (RII=1.04), with males from the south and females from the north
showing higher propensity to mate (MRPI=-0.13, FRPI=0.03). Bactrocera carambolae
showed the same trend in mating as B. dorsalis that preferred to mate within region
(RII=2.41), with both males and females from the south showing higher potential to mate
(MRPI=-0.22, FRPI=-0.27).
The inter-species mating tests between B. dorsalis and B. papayae from the north showed
non-selective mating (RII= 0.98), with both males and females of B. dorsalis from the north
showing higher potential (MRPI=0.19, FRPI=0.38), while in the south (RII=1.94) both males
and females of B. papayae showed higher potential in mating (MRPI=-0.51, FRPI=-0.48).
Inter-regional mating between B. dorsalis and B. papayae from the south showed selectivity
by both males and females of B. dorsalis (RII=2.50, MRPI=0.58, FRPI=0.66).
Bactrocera dorsalis showed high potential species selectivity in the north with B. carambolae
(RII=2.04, MRPI=0.18, FRPI=0.32) and high inter-regional selectivity by B. dorsalis from
the south (RII=5.50, MRPI=0.48, FRPI=0.90).
120
Bactrocera papayae and B. carambolae showed non-selectivity in the north (RII=0.77), with
males of B. carambolae and females of B. papayae showing higher potential (MRPI=-0.37,
FRPI=0.55), but selectivity in the south with males of B. papayae and females of B.
carambolae showing higher potential (RII=1.92, MRPI=0.28, FRPI=-0.17).
Meanwhile infested star apple fruits from Chantaburi province (east), which was recorded as
the host and place that B. carambolae was presented in Thailand, were collected for
reference.
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The Ceratitis FAR complex: biogeography, host plant spectrum and morphometrics
J. Van Cann1, M. Virgilio2, H. Delatte3, K. Jordaens1, 2, M. De Meyer2
1
University of Antwerp, Antwerp, BELGIUM
2
Royal Museum for Central Africa, Tervuren, BELGIUM
3
CIRAD, UMR PVBMT, St Pierre, La Réunion, FRANCE
Abstract:
Earlier studies on morphological and genetic differentiation of the FAR complex are shortly
reviewed. They revealed the existence of five genotypes within the Ceratitis FAR complex,
comprising two groupings within Ceratitis rosa (‘cold’ and ‘hot’), two within Ceratitis
fasciventris (‘dark’ and ‘pale’) and Ceratitis anonae. These different groupings were
confirmed by morphological differences in the male secondary characters, in particular the
leg ornamentation.
Distributional data for C. rosa and C. fasciventris were re-examined. For C. fasciventris the
two types are largely separated in a West-Central group (‘dark’ type) and an eastern group
(‘pale’). However, isolated occurrences of the ‘dark’ type are found in Angola, Malawi and
Tanzania. For C. rosa the two types seem to be separated along temperature clines with the
‘cold’ type restricted to lower latitudes (most part of South Africa) and higher altitudes. For
the ‘cold’ type, precipitation also seems to have an impact on the distribution.
Host plant information is inconclusive. All types are polyphagous with a large overlap of host
plants, especially regarding those of economic significance. In C. rosa there is a tendency for
the ‘cold’ type to be found more in fruits grown in temperate climates, compared to the ‘hot’
type.
Morphometric studies, based on wing landmarking, indicate a clear sexual dimorphism.
There is also an indication of separation between the three established taxa (C. fasciventris,
C. anonae, C. rosa) and to a lesser extent, between the five genotypes.
122
Chemical studies of the sex pheromone of five Brazilian populations of Anastrepha
fraterculus (Diptera: Tephritidae)
Ruth R. do Nascimento1, Lucie Vanicková1,2, Adriana L. Mendonça1, Alana L.
Mendonça1, Beatriz Jordão Paranhos3, Blanka Kalinová2, Antônio Euzébio Goulart
Sant´Ana1
1
Universidade Federal de Alagoas, BRAZIL
2
Institute of Organic Chemistry and Bichemistry of the Czech Academy of Prague, CZECH
REPUBLIC
3
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), BRAZIL
Abstract:
The sex pheromone mixtures released by five Brazilian laboratory populations of A.
fraterculus from two different geographical regions: four from the South and one from the
Southwest of Brazil were studied and the obtained results were compared with those found
for the populations collected in the estate of Maceió, Alagoas (Northeast of Brazil) and
Tucumán, Argentina.
Prior chemical studies, the populations were established under laboratory conditions and as
soon as the males reached sexual maturity, experiments aiming to collect the volatiles
released by calling males of all populations were performed in order to obtain extracts
containing the volatile molecules used by calling males as sex pheromone. These extracts
were analyzed by GC x GC/TOF-MS to determine its chemical composition as well to
quantify the amount of each compound within the volatile mixtures.
These analyses showed that 14 compounds were common to all studied populations, but their
relative proportions vary among the populations. It was also observed that six out of the ten
compounds found to elicit electrophysiological response on A. fraterculus females from a
laboratory population derived from Tucumán, Argentina and maintained in Seibersdorf,
Austria were also present in the volatile mixtures of compounds released by males of the
studied Brazilian populations.
From the obtained results we can reach the following conclusions: (i) Significant quantitative
differences were found in the volatile mixtures released by A. fraterculus males from the five
Brazilian populations studied; (ii) The mixtures of compounds from all studied populations
are similar, but they do differ in the proportions of components; (iii) The five populations
form five distinct groups, but a degree of overlapping is clearly observed among two
populations from the south of Brazil: Bento Gonçalves and São Joaquim and one from the
Southeast: Piracicaba, and (iv) The populations from Vacaria, Pelotas and Alagoas form three
distinct groups.
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Cytogenetic studies in members of Bactrocera dorsalis species complex: A comparative
analysis
Elena Drosopoulou1, A. Augustinos2,3,4, A. Gariou-Papalexiou2, C. Cáceres-Barrios4, K.
Bourtzis4, P. Mavragani-Tsipidou1, A. Zacharopoulou2
1
Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of
Sciences, Aristotle University of Thessaloniki, GREECE
2
Department of Biology, University of Patras, GREECE
3
Department of Environmental and Natural Resources, University of Patras, Agrinio,
GREECE
4
Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in
Food and Agriculture, Vienna, AUSTRIA
Abstract:
In the previous meeting we reported on the polytene chromosome maps of the five members
of the B. dorsalis complex under study (dorsalis s.s., philippinensis, papayae, carambolae
and invadens), pointing to the non-detection of chromosomal rearrangements (mainly
inversions) that could act as diagnostic characters and as barriers to gene flow among the
different members of the complex. However, the above analysis cannot rule out the presence
of small rearrangements, undetectable by microscopic observation.
In respect to this, we focused on two directions: a) the cytogenetic analysis of bidirectional F1
hybrids of B. dorsalis s.s with B. invadens and B. carambolae, and b) the in situ hybridization
of several DNA markers including unique genes on the polytene chromosomes of different
members of the complex, as well as of the aforementioned hybrids. Cytogenetic analysis and
in situ localization of unique genes in the hybrids could reveal the presence of smaller
rearrangements among the parental taxa.
Although ongoing, our main findings can be summarized as follows:
a) no extended asynapses or rearrangements were observed in the two F1 hybrids,
b) a specific inversion was observed in both hybrids always as heterozygous, which derives
from one of the polymorphic inversions observed in the B. dorsalis s.s genome,
c) there seem to be some minor asynapses especially in the B. dorsalis s.s X B. carambolae
F1 hybrids, probably attributed to differential expression (through differential puffing
activity) of the parental chromosomes, and
d) preliminary in situ hybridization results of six unique genes give no evidence of small
chromosomal rearrangements and verify the accuracy of the proposed polytene chromosome
maps of the dorsalis complex as well as the proposed homologies with other Tephritid
polytene chromosome maps.
To summarize, up to now we have no evidence of chromosomal differences that could have
enhanced the limitation of gene flow among the different entities of the complex. Therefore,
it is unclear how these entities could have evolved and maintained as different species,
especially in sympatry. The existence of workable polytene chromosome maps, along with
the localization of more unique genes and markers derived from genome sequencing and EST
projects of B. dorsalis s.s. could facilitate direct synteny comparisons among the members of
the complex. In addition, they could support the finishing of ongoing genome projects, which
in turn can shed light to the actual species boundaries in the complex.
124
Host plant relationships, temperature tolerance and mating compatibility studies in
Bactrocera cucurbitae and Ceratitis rosa
Sunday Ekesi, Chrysantus M. Tanga, Fathiya M. Khamis, Samira A. Mohamed
International Centre of Insect Physiology & Ecology (ICIPE), Nairobi, KENYA
Abstract:
During the period under review, the following program of work was carried out (1) establish
lowland and highland populations of Ceratitis rosa and study their temperature tolerance,
assess mating compatibility between the two populations and share materials with CRP
participants; and (2) establish cultures of Bactrocera cucurbitae from different hosts and
habitats, catalogue their host plants and carry out host preference studies, preferably under
field cage conditions. We were able to successfully rear the lowland population of Ceratitis
rosa from guava fruits collected from Mwanjamba, Msambweni, Kenya, while the highland
populations was reared from mango fruits collected from Kithoka, Meru, Kenya.
Across generations, larval duration of the highland population was longer (20.1-22.4 d) when
compared with the lowland population (16.2-18.7 d). Similarly, across generations,
percentage pupal recovery of the highland population was higher (30.5 to 66.4%) compared
to the lowland population (15.8 to 42.6%). In both populations, pupal recovery increased
from parent generation to the fourth generation. Pupal weight was not affected by population
or generation, but became heavier with increasing generations. Significantly more adults
emerged from the highland population (70.6 to 78.2%) compared with the lowland population
(46.2 to 56.2%). Ten day fecundity was not affected by population, but increased with
increasing generations of rearing. Across generations, egg fertility in the highland population
was significantly higher (50.4 to 70.2%) than that of the lowland population (34.5 to 48.9%).
Temperature had a significant effect on development and survival of both populations of C.
rosa.
Egg development for highland population ranged from 1.8 d at 330C to 7.8 d at 150C, while
that of the lowland population ranged from 1.6 d at 330C to 7.5 d at 150C. At the larval stage,
development ranged from 9.5 d at 330C to 24.9 d at 150C in the highland population while in
the lowland population, it took 7.7 d at 350C to 22.9 d at 150C. Pupal development ranged
from 10.9 d at 300C to 35.4 d at 150C in the highland population. The developmental time of
the pupal stage from the lowland population ranged from 9.1 d at 300C to 34.3 d at 150C.
Regression analysis showed a strong positive linear relationship between temperature and
developmental rate for all stages. Lower developmental thresholds for all stages were
estimated and will be presented. Parameter estimates for the Brière-1 nonlinear model
predicted the lower and upper temperature thresholds for all stages. At the egg stage, survival
rates ranged between 43.5% at 330C to 63.5% at 250C in the highland population and 72.0%
at 350C to 91.5% at 250C in the lowland population. In the highland population, survivorship
at the larval stage ranged between 35.8% at 330C to 77.0 % at 250C while in the lowland
population survival rate ranged between 48.25% at 350C to 89.25% at 250C. At the pupal
stage, survival rate ranged from 61.3% at 300C to 94.0% at 250C in the highland population.
In the lowland population, survival rate ranged from 75.5% at 300C to 95.5% at 250C.
Results obtained from mating compatibility studies showed a high degree of mating
incompatibility between the 2 populations of C. rosa and also between the 2 populations and
C. fasciventris. The ISI values ranged from 0.75 to 0.90 with the highland C. rosa x the
lowland C. rosa showing the highest degree of isolation. All matings were achieved by males
125
of the lowland C. rosa population. No significant difference was observed in latency to mate
among the mating combinations. Mating duration was also similar for all mating
combinations. Generally, over 80% of the mating occurred on the tree canopy for all the
combinations. Overall, this preliminary observation provides some indication of the existence
of variability among populations of C. rosa in Kenya.
In the studies involving the host range of B. cucurbitae, the insect was reared from a total
collection of 17 plant species comprising 10 families covering Cucurbitaceae, Solanaceae,
Anarcadiaceae, Rutaceae and Myrtaceae from surveys carried out at the Coast, Eastern and
Central Provinces of Kenya. Across the localities, highest infestation was recorded from
Momordica charantia (9.8 to 16.2 flies/kg fruits), Citrullus lanatus (8.5 to 11.2 flies/kg
fruits) and Lycopersicum esculentum (5.7 to 12.1 flies/kg fruits). The next most important
group of fruits included Cucumis sativus, Cucurbita maxima and C. moschaata with
infestation indices ranging from 2.6 to 4.4 flies/kg fruits. In general, there was a tendency for
higher fruit infestation in Taveta (lowland) than in Muranga and Nthagaiya (highland).
Bactrocera cucurbitae was also recorded from non-traditional host plants such as Mangifera
indica, Citrus sinensis, C. reticulata and Psidium guajava, but infestation did not exceed 0.5
flies/kg fruits. In some fruit species, B. cucurbitae frequently shared host with B. invadens
and a variety of Dacus spp. Future research activities should also address competitive
interactions among the different fruit fly species especially on cucurbits.
Colonies of two populations of B. cucurbitae (from tomato and bitter gourd) have been
established using the whole fruit rearing technique. In host preference studies conducted in
field cages, results showed that significantly more puparia were recovered from M. charantia
(134.2) compared to the other host fruit species in experiments in which bitter gourd
populations of B. cucurbitae were used. The next preferred lots of fruits were C. lanatus, C.
sativus, C. maxima and C. mosschata with infestation ranging from 74.3-76.5. In experiment
involving the tomato population of B. cucurbitae, the insect exclusively preferred tomato
(117.2 puparia) compared to the other host plants (10.6-18.4 puparia). In this particular case,
no puparia were recovered from guava and mango. In both populations of B. cucurbitae and
on all host plants, adult emergence ranged from 78-82% and did not differ significantly
across treatments. We conclude that populations of B. cucurbitae vary in their preference to
host plants and this preference is driven also by variation among population of the pest.
During the period under review, fruit fly specimens (specifically C. rosa) of different
developmental stages and quantities were shipped to Dr. Gary Steck (USA), Dr. Marc De
Meyer (Belgium) and Dr. Lucie Vanickova (Czech Republic) for different activities related to
the CRP.
126
Methyl eugenol sensitivity and its effects on intraspecific mating of three sibling species
of the Bactrocera dorsalis complex
Ihsan ul Haq 1,2*, Marc J.B. Vreysen1, Todd E. Shelly3, Jorge Hendrichs4
1
Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food
and Agriculture, Seibersdorf, AUSTRIA
2
National Agricultural Research Centre, Park Road, Islamabad, PAKISTAN
3
USDA-APHIS, 41-650 Ahiki Street, Waimanalo, HI 9795, USA
4
Insect Pest Control Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and
Agriculture Vienna, AUSTRIA
Abstract:
Bactrocera dorsalis Hendel, Bactrocera carambolae Drew & Hancock, and Bactrocera
invadens Drew, Tsuruta & White are included in the Bactrocera dorsalis species complex
(Diptera: Tephritidae). They are methy eugenol (ME) responsive and the effects of ME in
enhancing the male mating success are uniform across the B. dorsalis species complex, but
the sensitivity of males within the complex differs.
Effects of ME (feeding or no feeding to either or both types of males) on mating
compatibility between B. dorsalis and B. carambolae, and B. dorsalis and B. invadens were
studied. The sensitivity of all three males to different doses of ME (0.05 ml, 0.1 ml, 0.5 ml)
was also measured by recording the numbers of males arriving at a ME source over the
duration of 1 h. All experiments were run under field cage conditions.
ME had no effect on reducing the mating incompatibility between B. dorsalis and B.
carambolae; they remained relatively incompatible with each other regardless of the
treatment. On the other hand B. dorsalis and B. invadens were compatible without and with
ME exposure (no isolation).
ME seems to increase the total number of matings achieved when given to both types of
males and when given to either B. carambolae or B. dorsalis males only. ME feeding to B.
invadens males only did not increase the total number of their matings. Sensitivity to ME
across the three taxa was inversely proportional to the dose of ME, indicating that further ME
sensitivity testing needs to be carried out at a lower range of doses.
127
Morphometrics of wild populations from Ecuador and Colombia of the Anastrepha
fraterculus complex: The eighth passenger
Vicente Hernández-Ortiz1, Nelson A. Canal2, Juan O. Tigrero3
1Instituto
de Ecología A.C., MEXICO
del Tolima, COLOMBIA
3Escuela Politécnica del Ejército, ECUADOR
2Universidad
Abstract:
In previous studies we made a comprehensive morphometric analysis including a total of 32
populations of the Anastrepha fraterculus complex, originating from Mexico to Argentina.
The most outstanding achievement of this investigation was the development of linear
morphometric techniques for the recognition of 7 morphotypes within the AF species
complex (sensu Hernandez-Ortiz et al. 2012).
Under the approach of linear and geometric morphometrics, 10 Colombian and 7 Ecuadorian
populations were examined, all coming from different locations and altitudinal strata along
both countries, which were compared with other populations along the Neotropics.
Our results showed that all of the Colombian populations belong to a single taxon, and those
are consistent with the Andean morphotype previously described. In contrast, populations
coming from Ecuador exhibited clear differences among them, so that, preliminary
interpretations suggested that they belong to two distinct morphotypes; the Peruvian
morphotype distributed in the lowlands, and a new Ecuadorian morphotype which occurs in
the highlands, that proved to be different from all others studied before. Therefore, these
findings support the presence of the eight morphotype within the Anastrepha fraterculus
complex.
128
Bactrocera dorsalis complex species in mainland China
Qing Ji
Institute of Beneficial Insects, Fujian Agriculture and Forestry University, Fuzhou, Fujian
350002, CHINA
Abastract:
Based on the trapping network established last year, Methyl eugenol (ME) and Cuelure (Cue)
traps were still used for trapping fruit flies in mainland China. Totally more than 35,000
specimens of the Subfamily Dacinae were collected from Henan (Luoyang), Jiangsu (Jintan,
Nanjing), Chongqing, Jiangxi, Fujian (Xiamen, Fuzhou, Zhangzhou, Gutian and Putian),
Guangxi (Nanning, Guilin), Guangdong (Dongguan, Guangzhou), Guizhou (Guiyang),
Yunnan (Jinghong, Yuanjiang and Jianshui) and Hainnan (Wenchang, Danzhou and Sanya),
and then studied and identified.
17 species of Bactrocera Macquart were identified and they were B. bivittata Lin et Wang, B.
philippinensis Drew et Hancock, B. correcta (Bezzi), B. tuberculata (Bezzi), B.dorsalis
(Hendel), B. flavoscutellata Lin et Wang, B. wuzhishana Lin et Yang, B. thailandica Drew et
Hancock, B. rubiginus Wang et Zhao, B. latifrons (Hendel), B. cheni, sp. nov., B.
guiyangensis, sp. nov., B. hardyi, sp. nov., B. jinghongensis, sp. nov., B. nanningensis, sp.
nov., B. nigrifacia, sp. nov., and B. zonata (Saunders) All specimens are deposited in Fujian
Agriculture and Forestry University.
In the meantime, we cooperated with local counterparts to collect damaged fruits in Yunnan,
Guangdong, Guizhou, Hainan, Jiangxi, Hubei, Hunan, Jiangsu, Guangxi and Fujian for
rearing in the laboratory. Laboratory strains from Hangzhou, Shanghai, Hunan, Guangdong,
Yunnan, Hainan, Hubei and Guangxi were established.
In addition theree papers were published:
Zhang Nan-Nan, Ji Qing-E, Chen Jia-Hua. 2011. Three new species and one new record of
the Genus Bactrocera Macquart (Diptera, Tephritidae) from Yunnan, China. Acta
Zootaxonomica Sinica 36 (3):598-603.
Zhang Nan-nan, Ji Qing-e, Yang Jian-quan, Chen Jia-hua. 2011. A new species of the
Genus Bactrocera Macquart (Diptera, Tephritidae: Dacinae) from China. Entomotaxomomia
33 (4):262-266.
Zhang Nan-Nan, Ji Qing-E, Chen Jia-Hua. 2012. A new species of the genus Bactrocera
Macquart from China (Diptera, Tephritidae). Acta Zootaxonomica Sinica 37 (1):206-208.
129
Analysis of sexual compatibility among Anastrepha fraterculus populations from Brazil
Iara Sordi Joachim-Bravo1, Raul Laumann2, Vanessa Simões Dias3, Alzira Kelly Passos
Roriz3, Clarissa Petitinga3, Beatriz Jordão Paranhos3, Aldo Malavasi3
1
Universidade Federal da Bahia (UFBA), BRAZIL
CENARGEN-EMBRAPA, EMBRAPA–Semiárido, BRAZIL
3
Medfly Rearing Facility Brazil (Moscamed), BRAZIL
2
Abstract:
Anastrepha fraterculus, known as South American fruit fly, has been considered as the most
important pest of native fruits in South America and it is a major pest in apple orchards in
south of Brazil. The high level of variability among natural population of A. fraterculus
suggests that is a complex of sibling species and not a single biological entity.
In Brazil genetic and behavioral studies on mating choice support the hypothesis of at least
three different species group in the country. The definition of their taxonomic status will
support the implementation of a SIT program for A. fraterculus in Brazil.
In this paper we finished the evaluation of pre-zygotic sexual compatibility between 4
southern populations, 1 southeast population and 1 northeast population of Brazil. We also
evaluated some aspects of courtship behavior and acoustic communication during courtship.
The populations from southern Brazil (Vacaria, Pelotas, Bento Gonçalves and São Joaquim)
showed full mating compatibility, supporting preliminary data presented in the last report.
These populations seem to belong to the same biological entity.
Comparisons among southern populations (Vacaria, Bento Gonçalves and São Joaquim) and
southeast population (Piracicaba) showed a greater degree of mating incompatibility, also
corroborating last report’s data. Contrary to initial expectations, results of tests between a
southern population (Bento Gonçalves) and a northeast population (Parnamirim) showed
mating compatibility.
The analysis of comparative frequency of behavioral units of southern and southeastern
populations and of Parnamirim (northeast region) showed differences concerning the
behavioral units’ frequency related to copula success (Arrowhead, Attempt, Calling, Fanning,
Graceful + Spin). The data showed a distinction between wild populations (Vacaria and
Bento Gonçalves) and laboratory-reared populations for several years (São Joaquim and
Piracicaba). Additional experiments must be done to elucidate the relevance of these data and
complete the comparisons between populations.
Concerning acoustical communication, data permitted to compare pulse duration, pulse
period (repetition time) and dominant frequency (or fundamental frequency) of 4 populations
(São Joaquim, Vacaria, Bento Gonçalves and Piracicaba) for short pulses and of 3
populations (São Joaquim, Vacaria and Bento Gonçalves) for long pulses. In most of the
cases, there was a significant variation in the acoustic parameters between individuals inside
each population, but when differences in parameters of each population were tested no
significance were found. Significant differences were found in dominant frequency for short
pulses (population of Piracicaba show the higher frequencies in short pulses) and in pulse
130
duration and pulse period for long pulses (São Joaquim population showed the longest pulses
and pulses periods). Cluster analyses did not show any specific group formation between
individual of different populations suggesting that the parameters of the acoustic signals of
males of A. fraterculus from different geographic populations are not involved in sexual
isolation.
131
Comparative sensitivity of males of four globally important cryptic species in the Bactrocera
dorsalis complex - B. dorsalis, B. invadens, B. papayae and B. philippinensis, to methyl eugenol
and subsequent male antennal protein profiles
Alvin Kah-Wei Hee1, Suk-Ling Wee2, Keng-Hong Tan3
1
Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, MALAYSIA
2
School of Environmental & Natural Resource Sciences, Faculty of Science & Technology, Universiti
Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, MALAYSIA
3
Phi-Biotech Sdn. Bhd., 20, Jalan Tan Jit Seng, 11200 Tanjong Bungah, Penang, MALAYSIA
Abstract:
Males of certain putative species belonging to the Bactrocera dorsalis complex are strongly
attracted to, and readily feed on methyl eugenol that is found in over 480 plant species
worldwide. Amongst those species in the complex are some of the world’s most severe fruit
pests such as the Oriental fruit fly, B. dorsalis, along with its very close sibling members, B.
papayae, B. philippinensis and B. invadens. Whilst the first three species continue to threaten
the fruit industry in the Asia-Pacific countries, the rapid establishment of B. invadens in
Africa within the last decade had raised an alarm on this pest that can potentially devastate
the entire fruit industry there.
The identification of these four cryptic species has been the subject of great controversy for a
number of years due to their very similar morphology, mating compatibility, production of
viable offspring and sex pheromone system following pharmacophagy of methyl eugenol, an
extremely potent male fly attractant.
In the present study, the comparative sensitivity of B. dorsalis s.s., B. papayae, B.
philippinensis and B. invadens to methyl eugenol is being investigated. To date, laboratory
trials have demonstrated that the ED50 (effective median dose required to elicit 50% of male
fruit flies tested) of the four species showed no significant difference.
In addition, gel electrophoretic analyses of the male antennal proteins extracted from the four
male sibling species following exposure to methyl eugenol did not show differences.
However, efforts are currently made to evaluate all the male antennal proteins on a finer scale
using the high performance microfluidic capillary electrophoresis. Results of this work as
well as the current behavioural response of the males to methyl eugenol will be discussed in
the meeting.
132
Pheromones of three species of Ceratitis FAR complex
Blanka Kalinová1, Radka Břízová1,2, Maria Faťarová2, Lucie Vaníčková3, Michal
Hoskovec1
1
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague,
CZECH REPUBLIC
2
Institute of Chemical Technology Prague, CZECH REPUBLIC
3
Universidade Federal de Alagoas, Maceio, BRAZIL
Abstract:
Fruit fly males of many species form leks and release sex pheromone to attract females
during the reproductive behavior. Male sex pheromones are highly species-specific and can
therefore help in taxonomic categorization or in species recognition.
Previous research has demonstrated the multi-component nature of the pheromones and
showed that Ceratitis rosa and Ceratitis anonae share some pheromone components, but are
also characterized by certain species-specific substances.
At the upcoming meeting we will report on the composition of the male pheromone in
Ceratitis fasciventris. Like in C. rosa and C. anonae, male sex pheromone in C. fasciventris
is multi-component with shared and specific substances.
133
Looking for species-specific sequences in Bactrocera, Anastrepha and Ceratitis species
Anna R Malacrida1*, Nidchaya Akeratawong2, Silvia Lanzavecchia3, Blanka Kalinova4,
Giuliano Gasperi1
1
Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, ITALY
Department of Biotechnology, Mahidol University, Bangkok 10400, THAILAND
3
Instituto de Genética “Ewald A. Favret” (INTA), B1712WAA Castelar, Buenos Aires,
ARGENTINA
4
Institute of Organic Chemistry and Biochemistry ASCR, CZ-166 10 Prague 6, CZECH
REPUBLIC
2
*The Pavia laboratory actively contributed to this work: Ludvik M. Gomulski, Francesca Scolari,
Marco Falchetto, Mosè Manni.
Abstract:
Our aim is the identification and characterization of species and sex-specific sequences:
- to determine the intra/inter-population diversity, the species range/shift, and the indigenous
or invasive status of newly observed outbreaks.
- to provide insides into genes/expressions that could be associated with barriers to gene flow.
Our attention has been addressed to Bactrocera dorsalis ss, to Anastrepha fraterculus and to
Ceratitis capitata:
a) for B dorsalis ss we have developed an integrated database of SSR genotypes from
populations across the species range for traceability assays. Based on genetic data we
have tried to disentangle the possible major factors that affected the invasion processes.
We have identified sequences that allow to mark the Y chromosome;
b) for Anastrepha fraterculus we have developed SSR markers which allow to assess the
intra/inter-population and strain differentiation, and the mating/reproductive behaviour;
c) for Ceratitis capitata, using genomic/functional genomic approaches, we have identified sequences
correlated to the reproduction and chemoreception that may explain some traits of its behaviour and
provide species-specific markers.
134
Comparative studies on behaviour and developmental physiology of Ceratitis rosa
entities (R1 and R2) occurring in Tanzania
Maulid W. Mwatawala1, M. Virgilio2, M. De Meyer2
1
2
Sokoine University of Agriculture, TANZANIA
Royal Museum for Central Africa, BELGIUM
Abstract:
Determining spatial distribution of C. rosa entities along an altitudinal transect
Studies were conducted along an altitudinal transect in the Morogoro Region of Tanzania.
The transect lies in an embranchment of the Uluguru mountains, which is part of the Eastern
Arc Mountains. The vegetation consists of cultivated land (maize, sugar cane, beans) fruit
orchards (mango, citrus, peach, apple, jambolan, avocado, papaya, feijoa, guava) and fallow
land overgrown with grasses and shrubs. Peach, apple and pear are temperate fruits and
according to their climate demands they can be grown at high altitude. Most tropical fruits do
not occur above a certain altitudinal limit for the same reason. The crops are cultivated by
terracing and are grown in polycultures.
Modified McPhail® traps (Scentry Cie, Bilings, MT, VS) were set at ten stations along the
transect (500-1650 m) approximately at 100 m altitudinal intervals. Three sets of traps, each
baited with either trimedlure, EGO lure or terpinyl acetate were set on the same tree,
replicated three times at each station. Traps were serviced every two weeks, for three months.
The captured flies were brought to the lab where the specimens were counted and identified
and finally preserved in 70% ethanol. At each site a datalogger (iButton® Maxim Integrated
Products, Dallas) was placed to record temperature and relative humidity.
Testing mating compatibility of C. rosa entities (R1 and R2)
Establishment of fruit fly colonies
The studies are being carried out in laboratories at the Sokoine University of Agriculture
(SUA). Colonies of the two C. rosa entities are being established from areas where they
occur in Morogoro Tanzania. Sampling of host fruits concentrated in the low land (550 –
1000 m asl) and in high altitude areas (1650 – 1800 m asl). General methods and indices used
by Cayol et al. (1999) will be used.
Pupae from both strains are placed in separate ventilated Plexiglas cages (30 by 30 by 40 cm)
until emergence. After emergence, females are kept in separate rooms from males to avoid
contact with the male pheromone before the tests. All flies are kept in plastic containers (40
flies in each 1-liter plastic container) with water and food (protein and sugar). At least 48 h
before the test, healthy flies will be selected and marked with a dot of water-based paint
(Deka) on the notum. Two colors will be used (green and red) to mark alternatively R1 and
R2 flies.
The field cages have been made up of screen (20 by 20 mesh), cylindrical, with flat floor and
ceiling (2.9 m diameter and 2.0 m high). Each cage will be supported by a PVC frame and set
over a citrus tree. Four field cages will be used. To compensate for the greenhouse effect
caused by the field cage, a black shading material will be placed on top of each field cage
135
(Cayol et al. 1999). Field cage testing protocols used by Cayol et al. (1999) will be generally
followed.
Comparing developmental physiologies of C. rosa entities (R1 and R2)
Establishment of fruit fly colonies
The studies are conducted in laboratories at the Sokoine University of Agriculture (SUA).
Colonies of the two C. rosa entities are being established from areas where they occur in
Morogoro Tanzania. Sampling of host fruits concentrated in the low land (550 – 1000 m asl)
and in high altitude areas (1650 – 1800 m asl). General methods and indices used by Cayol et
al. (1999) are being used.
Pupae from both strains are placed in separate ventilated Plexiglas cages (30 by 30 by 40 cm)
until emergence. Adults are fed simultaneously with enzymatic yeast hydrolysate (ICN
Biomedical) and sucrose in a ratio of 1:3 and water on pumice granules in different plastic
containers for each species. Fruit flies rearing protocols used by Vayssières et al. (2008) are
being followed. The protocols to be used in the trial will first be harmonized with those used
by International Centre of Insect Physiology and Ecology (ICIPE) and Citrus Research
International, of South Africa.
136
Evolution of reproductive isolation among different morphotypes of Anastrepha
fraterculus
J. Rull1, S. Abraham2, M. Mendoza2, M. C. Liendo3, F. Devescovi3, D. F. Segura3, K. Roriz4, N.
Nolasco5, R. Castañeda6, E. Tadeo1, R. Břízová7, A. Kovaleski8, M. T. Vera2
1
Instituto de Ecología, A.C., Xalapa, Veracruz, MEXICO
Cátedra de Terapéutica Vegetal, Departamento de Sanidad Vegetal, Facultad de Agronomía
y Zootecnia, UNT, Tucumán, ARGENTINA
3
Instituto de Genética “E.A. Favret”, INTA Castelar, Buenos Aires, ARGENTINA
4
Universidad Federal do Bahia, BRAZIL
5
SENASA, PERU
6
Universidad del Tolima, Ibagué, COLOMBIA
7
Infochemical Group Institute of Organic Chemistry and Biochemistry, CZECH REPUBLIC
8
Embrapa Uva e Vinho, Estação Experimental de Vacaria, BRAZIL
2
Abstract:
During the course of the previous RCM meeting we reported results of prezygotic and
postzygotic reproductive isolation among three populations of the Brazil 1 morphotype (after
Hernández-Ortíz et al. 2012). Experimental results and a review of biological evidence
revealed that populations from Argentina and Southern Brazil belong to a single biological
entity and are fully compatible (Rull et al. 2012).
By contrast, adults from the Mexican morphotype exhibited strong prezygotic isolation in the
form of assortative mating (vs. Brazil 1 morphotype adults), timing of sexual activity (vs.
Peruvian morphotype adults), and resistance to penetration by Mexican females when
mounted by heterotypic males of several origins. Hybrids between Mexican adults and adults
of the Brazil 1 or Peruvian morphotypes were either less viable (fertile) or sterile (Rull et al.
in press).
In the case of the Andean morphotype (Colombia) the strongest form of prezygotic isolation
was related to marked differences in the timing of sexual activity (Colombian adults court
and mate at dusk vs. early morning or mid-day for adults of other morphotypes). We
discovered some degree of postzygotic isolation between adults of the Andean morphotype
and adults of the Mexican, Peruvian, Brazil 1 and Brazil 2 morphotypes. Nevertheless, F1
hybrid fertility was partially restored in all cases (Rull et al. in preparation), supporting
previous hypotheses of hybridization in the fraterculus species group as putative speciation
mechanism (Segura et al. 2011).
In a separate experiment, we discovered that Peruvian females tend to store less sperm than
Brazil 1 females irrespective of the origin of their mate and almost one half of the crosses
involving Brazil 1 males and Peruvian females were unsuccessful. Brazil 1 females were
more willing to remate than Peruvian females, irrespective of male morphotype, but latency
to remating was not affected by male or female morphotype (Abraham et al. 2013 submitted).
Collective evidence suggests that all A. fraterculus morphotypes are reproductively isolated
and that prezygotic isolation is stronger than postzygotic isolation. Such finding, and the
existence of numerous sister and cryptic species, suggest that recent differentiation in the
fraterculus species group may have often occurred in sympatry (Coyne and Orr 1997).
References
Abraham S, Rull J, Mendoza M, Liendo MC, Devescovi F, Roriz AK, Kovaleski A,
Segura DF & Vera MT. Differences in female remating propensity in two morphotypes of
137
the Anastrepha fraterculus (Diptera: Tephritidae) cryptic species complex. Bulletin of
Entomological Research. Submitted
Coyne JA & Orr HA (1997) “Patterns of speciation in Drosophila” revisited. Evolution 51:
295-303.
Hernández-Ortiz V, Bartolluci AF, Morales-Valle P, Frías D & Selivon D. (2012) Cryptic
species of the Anastrepha fraterculus complex (Diptera: Tephritidae): A multivariate
approach for the recognition of South American morphotypes. Annals of the Entomological
Society of America 105: 305-318.
Rull J, Abraham S, Kovaleski A, Segura DF, Islam A, Wornoayporn V, Dammalage T,
Santo Tomas U & Vera MT (2012) Argentinean and Southern Brazilian Populations of
Anastrepha fraterculus (Diptera: Tephritidae). Bulletin of Entomological Research 102: 435443.
Rull J, Abraham S, Kovaleski A, Segura DF, Mendoza M, Liendo M & Vera MT (2013).
Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species
within the Anastrepha fraterculus complex. Entomologia Experimentalis et Applicata. In
press
Rull J, Devescovi F, Abraham S, Roriz K, Nolasco N, Castañeda R, Tadeo E, Brizova, R,
Cáceres C, Segura D & Vera MT. Reproductive isolation among Colombian Anastrepha
fraterculus (Diptera:Tephritidae) and four morphotypes of the Anastrepha fraterculus criptic
species complex. In preparation.
Segura DF, Vera MT, Rull J, Wornoayporn V, Islam A & Robinson AS (2011)
Assortative mating among Anastrepha fraterculus (Diptera: Tephritidae) hybrids from two
distinct populations as a possible route to radiation of the fraterculus cryptic species group.
Biol. J. Linn. Soc. 102: 346-354.
138
The taxonomic history of the Oriental and Invasive Fruit Fly: from Musca ferruginea
Fabricius to Bactrocera invadens Drew, Tsuruta & White
M. K. Schutze, A. R. Clarke
School of Earth, Environmental and Biological Sciences, GPO Box 2434, Queensland
University of Technology, Brisbane 4001, AUSTRALIA.
Abstract:
A fly similar to the widely distributed pest tephritid Bactrocera dorsalis, the Oriental Fruit
Fly, was first detected in Africa in 2003. Two years later this fly was deemed new to science
and given the name B. invadens, the Invasive Fruit Fly. Bactrocera invadens is considered
taxonomically different from its Asian cousin due largely to its thoracic colour patterns:
predominantly red-brown in B. invadens and black in B. dorsalis.
However, questions are now being asked as to whether B. invadens is truly a new species, or
rather represents a morphologically variable population of the Oriental Fruit Fly (as was
initially suspected by local researchers).
As a component of species delimitation research within the larger B. dorsalis species
complex, a review of the taxonomic history of B. dorsalis sensu stricto was undertaken and
findings suggest that there are sound taxonomic reasons as to why B. invadens should be
regarded as a junior synonym of B. dorsalis. Specifically, morphological characteristics
described for Musca/Dacus ferrugineus – a species described in 1794 by Fabricius and now a
junior synonym of B. dorsalis – are largely consistent with modern-day B. invadens.
The situation is not straight forward though, with a highly complex taxonomic history that
has fuelled debate for almost two centuries. As a ‘scene setter’ for the week’s discussion, we
detail the history of these species and reveal that the current confusion is nothing new.
139
Reproductive compatibility, morphological and morphometric variation, and molecular
genetic analyses of Bactrocera dorsalis sensu lato (Diptera: Tephritidae) from Africa, the
Indian Subcontinent and East Asia
1,2
M. K. Schutze , S.L. Cameron2, K. Mahmood3, W. Bo4, C. Cáceres4, M. J. B. Vreysen4,
A. Pavasovic2, A. R. Clarke1,2
1
CRC for National Plant Biosecurity, L.P.O. Box 5012, Bruce, A.C.T. 2617, AUSTRALIA
2
School of Earth, Environmental and Biological Sciences, Queensland University of
Technology, G.P.O. Box 2434, Brisbane 4000, Queensland, AUSTRALIA
3
Pakistan Museum of Natural History, Garden Avenue, Shakarparian, Islamabad,
PAKISTAN
4
Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food
and Agriculture, International Atomic Energy Agency, Vienna, AUSTRIA
Abstract:
Bactrocera dorsalis, B. papayae, B. philippinensis, B. carambolae and B. invadens are
sibling pest members within the B. dorsalis species complex of tropical fruit flies. The
species status of these taxa is unclear and this confounds quarantine, pest management and
general research.
Bactrocera dorsalis is distributed from Pakistan to the Pacific with the Thai/Malay peninsula
its southern limit. Bactrocera papayae and B. carambolae occur in the south-east Asian
archipelago, B. philippinensis in the Philippines, and B. invadens in Africa and Sri Lanka.
Since the 2nd RCM in Brisbane, the QUT group has focused on two main activities. The first
was the completion and write-up of previously presented work on the species delimitation of
the South-east Asian members of the complex; the second the initiation (and largely
completion) of new work on the delimitation of B. dorsalis and B. invadens. The latter
activity was undertaken jointly with the FAO/IAEA Insect Pest Control Laboratory,
Seibersdorf, and Dr Khalid Mahmood of the Pakistan Natural History Museum.
Our work on the South-east Asian members of the complex has now been published in a
series of five papers covering a multi-gene phylogenetic analysis, a COI population study, a
microsatellite study, mating behaviour, wing shape analysis and male aedeagus
measurements.
Our conclusions, as previously presented, are that B. papayae and B. philippinensis should be
regarded junior synonyms of B. dorsalis: there is no evidence for these flies being distinct
biological species. In contrast, there is sufficient evidence available to consider B.
carambolae a distinct, albeit very closely related, biological species.
The new work on comparing B. dorsalis and B. invadens has followed the same integrative
taxonomic approach. Pre- and post-zygotic compatibility studies conducted at the FAO/IAEA
Seibersdorf laboratories have shown random mating between a Kenyan population of B.
invadens and B. dorsalis populations from Pakistan and China. There is also no evidence of
post-zygotic incompatibility in these crosses. In contrast, attempted crosses between the
Kenyan population and a population of B. kandiensis from Sri Lanka showed high levels of
mating isolation and post-zygotic incompatibility. These latter results, as well as the earlier
results showing non-random mating of B. carambolae, provide confidence that field cage
mating trials provide accurate measures of mating isolation between very closely related
member of the B. dorsalis complex.
A COI haplotype network including B. dorsalis individuals from across its entire range from
Pakistan to South-east Asia and B. invadens individuals from Africa and Sri Lanka showed
no evidence of any haplotype divergence between B. dorsalis and B. invadens. In stark
contrast, B. kandiensis shares no haplotypes with either B. dorsalis or B. invadens. A
140
multigene phylogenetic study has also been undertaken and we hope to present the analysis of
the results in Argentina.
Morphometric and geometric morphometric analysis has been conducted on similarly widely
distributed samples of B. dorsalis and B. invadens. Attributes studied were wing shape,
thoracic colour pattern, width of the post-sutural lateral vittae and length of male genitalia.
Clinal and/or geographic variation in vittae width was detected; however there was no
evidence of character variation being in any way consistent with purported morphological
discontinuities between B. dorsalis and B. invadens.
We conclude from this work that B. dorsalis is a morphologically highly variable and widely
distributed species, and that ‘B. invadens’ represents only an incursive population of this
wide-ranging species. Wing shape and body colour pattern are consistent with the invasive
African population of B. dorsalis having originated from the northern subcontinent, i.e.
Pakistan or similar.
141
Use of mtDNA and nuclear markers to delimit and diagnose Brazilian species within the
Anastrepha fraterculus complex
Janisete G. Silva1, Katia M. Lima1, Norman B. Barr2, Raul Ruíz-Arce2, Gary J. Steck3,
Bruce. A. McPheron4, Roberto A. Zucchi5
1
Universidade Estadual De Santa Cruz, Bahia, BRAZIL
United States Department Of Agriculture, USA
3
Florida Department of Agriculture and Consumer Services, USA
4
Ohio State University, USA
5
Universidade de Sao Paulo, BRAZIL
2
Abstract:
The South American fruit fly, Anastrepha fraterculus (Wiedemann), is among the most
serious agricultural pests in South America. In Brazil, A. fraterculus has been reported to
infest 81 host species in 20 plant families. A series of morphological and genetic studies have
revealed that A. fraterculus actually comprises a complex of multiple species. The actual
number of putative species within the A. fraterculus complex and their associated
biogeography is yet uncertain.
Differences among cryptic species could have significant consequences for pest quarantine,
management, and eradication. Therefore, it is paramount to study populations from a wide
geographic range in Brazil using mtDNA and nuclear markers in order to capture genetic
diversity of this complex. Analysis of con-specific variation among Anastrepha fraterculus
samples and con-generic variation among fraterculus species group samples is critical to
resolving evolutionary relationships of cryptic species.
The cooperative research program has obtained over 630 expertly identified Anastrepha
samples representing nearly 70 species. This includes 244 A. fraterculus specimens gathered
from several states in Brazil, Argentina, and Mexico plus an additional set of 175 specimens
representing 20 taxa within the fraterculus species group as well as from other Anastrepha
species groups. To date, DNA has been isolated from 630 adult specimens.
We sequenced a portion of the cytochrome oxidase c subunit I from approximately 90% of
the samples. Genetic diversity estimates and Neighbor Joining Tree analysis of the aligned
sequences suggest structuring among populations and species. A subset of the DNA samples
have been screened for variation at additional mitochondrial (ND6) and nuclear (period,
CAD, ITS, 18S, TPI) loci. Based on variation within species and among species, several of
these loci have also been identified as informative on a portion of collections for the study of
cryptic species. Protocols for these genetic markers have been developed and are being used
to generate a multi-gene sequence data set for A. fraterculus collections.
Additional accomplishments include analysis of a subset of populations using 10
microsatellite markers developed by Drs. Silvia Lanzavecchia and Anna Malacrida, who lead
another group in this CRP. Results indicate genetic structuring between some populations.
Our collaborative efforts include morphometric analyses of populations of A. fraterculus
from Brazil with Dr. Vicente Hernández-Ortíz and Dr. Iara Bravo, respectively, who lead two
other groups in this CRP. Brazilian populations that are being genotyped using mitochondrial
and nuclear markers will also be examined for morphometric differences. This is being done
142
through collaboration with taxonomic experts at Universidade de São Paulo, Brazil (Drs.
Keiko Uramoto and Roberto Zucchi) and Instituto de Ecología A.C., Mexico (Dr. Vicente
Hernández-Ortíz). The morphometric and genetic data sets will be available for comparison
to other data sets generated for Anastrepha species.
143
Anastrepha species of the Central Andes of Peru
Gary Steck1
1
Florida Department of Agriculture, Division of Plant Industry, USA
Abstract:
Recently obtained funding for a U.S. Farm Bill project entitled "Enhancement of fruit fly
immature stage identification and taxonomy" has enabled the principal investigators to
undertake extensive field work in South and Central America to acquire larval and adult
stages of numerous Anastrepha species for morphological and molecular studies.
We made two trips to Peru (November 2012 and January-February 2013) and made
collections in Departmento Cusco where the Anastrepha fauna is poorly known. Collection
efforts were successful at elevations of about 3000 m (Sacred Valley), 1100m (Echarate) and
600-800m (Pilcopata). Identification is still on-going, but at least 37 species and 3,000
specimens of adult Anastrepha were collected from traps, larvae were reared to adult stage
from 14 different host plants, and a total of approximately 800 larvae have been preserved.
Additional collection activities are underway in Panama to extend the geographic range of
Anastrepha spp. samples for taxonomic analysis.
In Departamento Cusco, Anastrepha fraterculus s.s. is absent or rare at the lower elevations,
although at least one undescribed species of the fraterculus group is present; fraterculus is
present, but not dominant at mid-elevations; and it seems to be the only species present at
high elevations where it is a serious pest of stone fruits.
Our preliminary round of collecting yielded too few fraterculus specimens to establish a
research colony for more general studies, but we have laid the groundwork for establishing a
colony in the near future - hopefully in Seibersdorf.
Preliminary DNA sequence data indicates that A. fraterculus populations in Departamento
Cusco (Andean highland and Amazonian mid-elevations) are clearly different from the
Pacific coastal populations of Peru. DNA sequences from the Cusco populations, as well as
those from a mid-elevation population on the eastern slopes of the Andes in Bolivia, match
those of widespread fraterculus populations in Argentina and southern Brazil.
144
Volatile chemicals released by Tephritid flies as a tool to understanding species
diversity
Peter E. A. Teal
Chemistry Research Unit, Center for Medical, Agricultural and Veterinary Entomology,
USDA-ARS, 1700 SW 23 Dr., Gainesville, FL. 32604, USA
Abstract:
It is clear that the Tephritids are a wonderfully diverse group of flies. However, as is evident
from the current Coordinated Research Project many times clear species identifications are
next to impossible using common systematic methods. Excellent examples of cryptic species
are documented among members of the Bactrocera dorsalis complex and the Anastrepha
fraterculus complex.
In collaboration with the IAEA/FAO and research groups from around the world we have
analyzed volatile chemicals released by males of several groups from both complexes.
Chemical data show a good correlation between the blends of chemicals males release when
signaling for mates and data supporting the complexes being composed of several
reproductively isolated species. Indeed, there is good evidence that pheromones released by
males are a primary method of reproductive isolation for these species.
There is a not only a need to identify male produced volatiles from these cryptic species but
also to determine the impact of pheromones on reproductive isolation among these groups.
145
Cuticular hydrocarbons as a tool for resolution of the fruit fly species complexes
Lucie Vaníčková1*, Massimiliano Virgilio2, Aleš Tomčala3, Radka Břízová3, Adriana de
Lima Mendonça1, Blanka Kalinová3, Ruth Rufino Do Nascimento1, Marc De Meyer2
1
Universidade Federal de Alagoas, Maceio, BRAZIL
Royal Museum for Central Africa, Leuvensesteenweg 13, B-3080 Tervuren, BELGIUM
3
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague,
CZECH REPUBLIC
2
Abstract:
Resolution of the species complexes in Tephritidae is a very challenging task. Recently, in
fruit fly family (Diptera:Tephritidae), there have been reported several cases on species
complexes including the so called Ceratitis FAR complex and Anastrepha fraterculus
complex. Cuticular hydrocarbons appear to be an excellent tool for chemotaxonomical
resolution of these cryptic species.
In the present study, we report the use of cuticular hydrocarbon profiles of Ceratitis anonae,
C. fasciventris, C. rosa, C. capitata and A. fraterculus for distinguishing the mentioned
species complexes. Quantitative as well as qualitative differences between species,
populations and genders are reported. The CHC profiles consist of mixture of alkanes,
internally methyl-branched alkanes, alkenes and alkadienes. The species and gender-specific
cuticular hydrocarbons were identified and they can be used as chemotaxonomic markers for
recognizing between the species and further between the species complexes.
146
Emission of male sex pheromone by four cryptic species, Bactrocera dorsalis, B. invadens, B.
papayae and B. philippinensis, following Methyl Eugenol consumption
Suk-Ling Wee1, Alvin Kah-Wei Hee2, Keng-Hong Tan3
1
School of Environmental & Natural Resource Sciences, Faculty of Science & Technology, Universiti
Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, MALAYSIA
2
Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, MALAYSIA
3
Phi-Biotech Sdn. Bhd., 20, Jalan Tan Jit Seng, 11200 Tanjong Bungah, Penang, MALAYSIA
Abstract:
Four global cryptic pest species of the Bactrocera dorsalis complex- Bactrocera dorsalis sensu
stricto, B. papayae, B. philippinensis and B. invadens, are known to be strongly attracted to, and
consume methyl eugenol, a phenylpropanoid and very potent male attractant, detected in at least 480
plant species from 80 families across 46 botanical orders.
Whilst chemical and behavioural analyses have led to elucidation of the explicit and implicit roles of
methyl eugenol in the fruit fly-plant interaction, evidence on the sex pheromone production by males
of these cryptic species following ingestion of methyl eugenol has complemented efforts to resolve
the current controversy related to the taxonomic status of the four Bactrocera species.
Following methyl eugenol feeding, males of the four cryptic species are known to produce two major
phenylpropanoid compounds - (E)-coniferyl alcohol and 2-allyl-4,5-dimethoxyphenol, that are
temporarily stored in the rectal gland and ultimately emitted as sex pheromone during dusk prior to
mating. The pheromone emission during courtship period of B. dorsalis s.s, B. invadens, B. papayae,
and B. philippinensis are being investigated using SPME-GCMS analyses and results of this work will
be presented and discussed in the meeting.
147
Appendix 4: Working Groups
Anastrepha fraterculus
complex
Bactrocera dorsalis complex
Bactrocera cucurbitae
Ceratitis FAR complex
M. Teresa Vera (Leader)
Tony Clarke (Leader)
Marc De Meyer (Leader)
Andrea Bartolucci
Karen Armstrong
David Haymer
Radka Brizova
Nidchaya Aketarawong
Helene Detalatte
Carlos Caceres
Suksom Chinvinijkul
Sunday Ekesi
Nelson Canal Daza
Elena Drosopoulou
Blanka Kalinova
Maria del Rosario
Castañeda
Alvin Kah-Wei Hee
Ana Malacrida
Mark Schutze
Maulid Mwatawala
Keng Hong Tan
Massimiliano Virgilio
Jorge Cladera
Vanessa Dias
Peter Teal
Paola Fedyszak
Kenji Tsuruta
Lucia Goane
Ishan ul-Haq
Vicente Hernandez Ortiz
Antogoni Zacharopoulo
Iara Sordi Joachim Bravo
Laura Juarez
Katia Manuela de Lima
Ruth Rufino do
Nascimento
Beatriz Jordao Paranhos
Kelly Passos Roriz
Juan Rull
Janisete G. Silva
Gary Steck
Lucie Vanickova
148
Appendix 5: Protocols for Collection and Shipment of Live and Dead Insects for
Vouchering, Rearing, Morphology, Morphometrics, Chemical Ecology and Molecular
Assays
1. Labelling
1.1. Place of origin (GPS / latitude:longitude, town, country)
1.2. Host fruit, what plant insect was collected from
1.3. Date and time of collection
1.4. Person who collected the insect
Note: strict rules of how to refer to the relationship between the fly and the plants e.g.
trapped in tree, found as larvae in fruit, clearly differentiate between on fruit, in fruit,
ex fruit, etc.
Consider pre-designed forms that should be attached to any specimen, forms attached
as an appendix
2. Live insects
2.1. Collection
2.1.1.
2.1.1.1.
2.1.1.2.
2.1.1.3.
2.1.1.4.
2.1.2.
2.1.3.
2.1.4.
2.1.5.
2.1.6.
From wild infested fruit
Fruit type described, photo
Number of fruit needed to collect
How many trees, orchards, regions were collections made
from? How wide a population should the sample be collected
from?
Base number of insects required for R&D
From harvested fruit placed in the field
From non-toxic traps
From netting, aspiration, etc
From laboratory cultures
Shipment of wild-infested fruit from the field to local laboratory –
packaging to reduce package-induced mortality, temperature
management, etc
2.2. Shipment
2.2.1.
Common considerations
2.2.1.1.
Shipping by:
2.2.1.1.1.
Hand luggage for air travel
2.2.1.1.2.
Booked-on package
2.2.1.1.3.
Via courier
2.2.1.1.4.
Via Post
2.2.1.1.5.
Personally by car
2.2.1.1.6.
Packaging type, size, weight
2.2.1.2.
Quarantine security – FORMS!!!
2.2.1.2.1.
Packaging type, size, weight
2.2.1.2.2.
Description of contents
2.2.1.2.3.
Liquids present
2.2.1.2.4.
Contact names, addresses, phone numbers, e-mail
addresses – both sender and receiver
2.2.1.2.5.
Exporting country requirements
149
2.2.1.2.6.
Importing country requirements
2.2.1.2.7.
Courier company requirements
2.2.1.2.8.
Airline company requirements
2.2.2.
As pupae
2.2.2.1.
Strong packaging material
2.2.2.2.
Reduce desiccation
2.2.2.3.
Vibration, movement of pupae causing physical damage
2.2.2.4.
Access of pupae to air
2.2.2.5.
Permissible transit temperature range
2.2.2.6.
Quarantine security, escape of pupae, adults
2.2.3.
As eggs
2.2.3.1.
Transport of liquids
2.2.3.2.
Reduce leakage, maintain package integrity
2.2.3.3.
Eggs in water, or in agar agar solution
2.2.4.
As infested fruit
2.2.4.1.
Distance to travel
2.2.4.2.
Time to travel
2.2.4.3.
Temperature management to reduce adult eclosion
2.3. Receipt of live insects
2.3.1.
Customs clearance – FORMS!!!
2.3.2.
Retrieving eggs, pupae, adults from package
2.3.3.
Identification and separation of species in a mixed culture, other
insects, parasitoids, etc
2.3.4.
Setting up laboratory culture
2.3.5.
Minimum quantity of insects required to be a valid sized culture –
justification of sample size, recording
3. Dead insects
3.1. Collection
3.1.1.
3.1.2.
3.1.3.
Wild flies reared from fruit, collected from traps, nets, etc
Part/s of insect required for shipment
Retaining remains of insect for trace-back or for study in another
collaborating laboratory
Labelling requirements for receiving agency / laboratory, etc
3.1.4.
3.2. Shipment
3.2.1.
Shipment of insect extracts e.g. DNA extracts, pheromones
3.2.1.1.
Specialist handling and shipment procedures
3.2.2.
Quarantine considerations as above
3.2.3.
Preserved as a dried specimen or in alcohol or alternative preservatives
3.2.3.1.
Alcohol concentration
3.2.4.
Shipment in alcohol and importer, airline, courier requirements
3.2.4.1.
Amount of alcohol
3.2.4.2.
Alcohol concentration
3.2.5.
Alternatives to shipment in alcohol e.g. PEG, etc
3.3. Packaging of specimens
3.4. Receipt of specimens
3.4.1.
Customs clearance
4.
Post receipt handling
4.1. Quarantine security requirements of the receiving institutes
150
4.2. Quarantine security facilities at the receiving institutes
4.3. Quarantine security requirements of the importing country
5.
Trapping
5.1. Type of trap, type of specimen required
5.1.1.
For dry specimen
5.1.2.
Trapped in water
5.1.3.
Trapped in a preserving solution (e.g. PEG)
5.1.4.
With or without toxin
6.
Collection of rectal glands and/or volatiles from live insects – See paper by K.H.
Tan and work by Peter Teal
6.1. Guarding against contamination
6.2. Storage and transport in 100% alcohol
6.3. Other issues described in the paper
6.4. Collection of pheromones (e.g. work done by Peter Teal)
7.
Validation of species by recognised expert
7.1. Reference material
7.1.1.
Validation before experiment start
7.1.2.
Validation after experiment
7.1.3.
Keep Voucher Specimens
7.1.3.1.
How many to keep?
7.1.3.2.
In alcohol and / or
7.1.3.3.
As a dried (pinned?) specimen
7.1.3.4.
Preservation and vouchering these specimens for future
7.1.4.
Sharing these specimens for later molecular or morphology
8.
Other issues to be addressed
8.1.
Feedback on how culture is progressing, return information, courtesy call / email on receipt of goods, etc
8.2.
Agreement reached on terms, agreements and structures for payment
requirements, if necessary, for suppliers, for analysers, etc
8.3.
Consider the possibility of assessing the same individual insects for mating
studies, then morphological studies, then pheromone analysis, then molecular,
etc. What logistics are required if each assessment is to be done in
geographically distant facilities?
8.4.
Share progeny from captured wild females for study
8.5.
Detailed descriptions and documentation of collection methods, dates,
times, vouchering, personnel, experimental designs, etc
8.6.
Collaboration and co-operation
8.7.
Potential / possibility of on-line collaboration, questions, FAQs, problems
and solutions
8.8.
Publication considerations, intellectual property, data sharing rights, legal
requirements
9.
Other reports, manuals, etc that are already published / used? E.g. ICIPE, etc.
9.1. List as many other publications that are already available.
151
Appendix 6: Guidelines for Performing Mating Compatibility Field Cage Test
Note: this document is an adaptation of the guidelines presented in the FAO/IAEA/USDA
Manual for Product Quality Control and Shipping Procedures for Sterile Mass-Reared
Tephritid Fruit Flies (2003). The QC Manual can be downloaded from http://wwwnaweb.iaea.org/nafa/ipc/public/ipc-mass-reared-tephritid.html and a revised version will soon
be available.
Overall Objective
The overall objective of the mating compatibility field-caged test is to determine the
degree of sexual compatibility between two populations/strains under semi-controlled field
conditions by releasing in a confined semi-natural environment (i.e. field cages with trees
inside) sexually mature fertile but yet virgin flies from these two populations to observe their
mating behaviour and interactions during the time of sexual activity.
Specific Objectives
1. To determine the number of mating couples obtained in each of the possible mating
combinations between the two populations.
2. To characterise male calling behaviour (time, location, aggregation in leks, number
and type of males in the lek, aggressive behaviour, etc.).
3. To characterise male – female interactions once the females arrive at the lek.
4. To determine mating duration and any possible correlation with the mating
combination or type of cross (homotypic vs. heterotypic).
5. To determine possible changes in female postcopulatory behaviour.
6. To determine the impact on the above mentioned variables of the origin of the other
population released in the field cage (presence of plasticity in the mating behaviour).
7. Determine sperm transfer.
8. Obtain couples to perform studies of postzygotic compatibility.
Discussion
Mating behaviour field cage tests aimed either to evaluate mating compatibility of male
performance, are the best compromise between laboratory conditions and costly and/or
impractical field observations to assess tephritid fly mating behaviour under semi-natural
conditions.
Sexual compatibility is the degree to which two groups of animals tend to mate randomly
without regard to their group of origin rather than mating selectively with members of their
own group. For tephritid SIT programmes, the sexual compatibility between the target wild
population and the candidate strain or population aimed at being transferred to mass rearing
intended to be used for release should be measured before initiating any large-scale
operations. In some tephritid species, sexual incompatibility could reveal the presence of
sexually isolated populations and, to some further extent, the presence of cryptic species
previously undetected.
The FAO/IAEA/USDA QC Manual offers a standard design for field-cage tests and it is
recommend that within the CRP people follows this protocol with the particular
modifications presented here. Data from these tests can be used to generate simple,
reproducible, meaningful indices of sexual compatibility that can be used for making
comparisons between populations. Given it was concluded in previous CRPs that the
152
nutritional status, sex ratio and density of flies in relation to available canopy surface in the
field cage influences test results, efforts should be made to strictly follow the standards
described thereafter in order to minimize the impact of these variables on the overall results
and thus permit comparisons among different research groups.
Sources and Handling of Flies
Biological material can come from already established laboratory strains or from wild
populations. In both cases pupae should be placed in proper conditions to allow adult
emergence. Within a few hours of adult emergence, select only flying adults and separate the
sexes. Sexing has to be 100% effective. Female cages in which even one male is detected
cannot be used for the tests and will have to be discarded, the same is recommended for male
cages in which one female has been missplaced. Then, hold the flies according to the day of
emergence in laboratory cages (screen or Plexiglas) containing water and a food until
sexually mature. Age of sexual maturity will vary with species or strain so in case no data is
available, preliminary tests may be required to determine the appropriate testing age. The
type of food to be given should be equal to all origins and should resemble the one the flies
consume in nature. Moistened dry fruits such as figs is a good worldwide available option;
however a source of protein is required. Preferably, the sexes should be placed into two
separate rooms at a maximum density of ±40 flies per litre volume for the case of medlfy and
less for bigger flies such as some Anastrepha or Bactrocera spp.
Equipment
 All necessary equipment to maintain the flies in the laboratory from emergence until
release in the field cage: Plexiglas cages with “Flight Ability” devices, aspirators to sex
the flies, 1 l containers, water and food containers, etc.
 All necessary equipment to mark the flies according to the chosen technique (see below):
(1) fluorescent powder; (2) water-based paint, thin soft camel hair brushes and a meshed
bag; (3) cake colorants to dye the diet.
 Outdoor field cage, 2 m tall by 2.9 m in diameter, set up over a plant that fills a large
portion of the volume of the cage (Error! Reference source not found.). The plant should
e a local host plant of the fly species to be tested (citrus, guavas, etc.). Ideally, the plant
should be rooted in the ground, but potted plants may suffice if ground-rooted plants are
unavailable. Artificial trees may also be an option and have the advantage that flies are not
exposed to plant volatiles that alter flies behaviour. Care must be taken in setting up the
cages. The available foliage must provide an abundant substrate for mating behaviour, but
could be lightly pruned (if necessary) so that flies will be visible to the observer. An
average of 20 medium-sized leaves should be available per fly released in the field cage. If
adequate foliage and light are available within the cages and if not more than 150-200 flies
are released per cage (regardless of the type of field cage test done), little if any mating
activity should take place on the screen of the cage.
 Plastic pill vials, scintillation vials, or similar containers of about 10-20 ml preferably
clear, to collect the mating couples (depending on the amount of flies released 50 – 80 per
cage).
 Grease pencils and/or masking tape and pens for marking vials.
 Long-wave ultra-violet lamp (should flies not be marked with paint and identifiable only
by the presence of fluorescent dye) or fluorescent microscope.
153
 A minimum of 4 dental cotton wicks impregnated with water should be placed per tree as
source of water for the flies.
 Data recording forms with pencil.
 Hydro-thermometer and luxmeter.
Procedure
Before releasing the flies in the cages, it would be necessary to mark them in order to
distinguish at which strain/population they belong. Three methodologies are widely used and
can be applied in conjunction:
(1) dyeing with fluorescent powder as in operational programmes
(2) painting the flies in the thorax with water based paint, or
(3) providing the flies with diets dyed with non-toxic food colouring.
Each technique is applied at different times. In the case of fluorescent powder, dying
occurs at the pupal stage before adult emergence with approximately 1.5 gr per kilo of pupae.
In the case of painting the thorax, at least 24 to 48 hours before the test, flies are marked
individually by applying a small dot of paint on the dorsal surface of the thorax. Flies can be
immobilize by chilling them at approximately 5° C for a few minutes or by placing them in a
bag made of mosquito net (18 mesh), placing the bag on a table, and holding the mesh down
gently around each fly, one at a time. Use a thin, soft camel hairbrush to apply a small drop of
paint to the fly. For the case of adding the colorant to the diet, it is advisable to do it since
adult emergence. A small drop of non-toxic food colouring is added to the adult food in order
to obtain the same food with different colours. Flies that ingest the food have their abdomen
coloured with the provided colour. Even though marking of flies with water-based paint or
cake colorants appears to have no effect on mating performance field cage test results,
colours used for marking strains should be randomised among replicates and brands of paints
or colorants could be evaluated ahead of time to ensure that the mark does not affect fly
behaviour or survival.
Immediately after marking, flies are transferred to containers suitable for releasing
them into the field cages with water and food in groups of 25 – 50 flies, depending on the
species, per 1 litre container.
On the day of the test, males are released into a screen cage and given a period of time
(e.g., 15-30 min) to disperse and establish territories. The number of flies to be released
depends on the species; for the case of medfly normally 50 flies of each strain and sex are
released which totals 200 flies per cage while for other species it is recommended to release
25-30 flies of each sex and origin. Time of release should precede the time of peak mating for
that species. Flies should be able to fly out of the container by themselves and should not be
forced to do so by shaking the container or pushing flies out. Flies that are left inside the
container, dead, deformed, or apparently incapable of flying should be replaced. Then,
females are released.
Starting time of mating performance field cage test should be adjusted according to
environmental conditions and fly activity (i.e. early start on hot days) and test duration should
cover most of the known sexual activity period for the species under the local conditions. In
the absence of such information, the test should cover most of the day. Ideally, tests should
be run “blind”; i.e., technicians running the test should not be told which colour of marking
corresponds to wild or sterile. In addition colors should be alternated randomly among
replicates.
154
Data gathering
Collection of mating couples
After release of females, observers should screen the tree for mating couples. This can be
done continuously or a census of mating couples can be done under a certain frequency.
Census frequency should be at least every 10-15 minutes, or even shorter for species that
have shorter mating durations. Capture mating couples individually in the vials, taking care to
get only one pair (and only 2 flies) per vial. Look at the pair carefully in order to confirm that
copulation is taking place; males sometimes mount the female without real genital contact. If
flies are not marked with paint but with fluorescent dye, keep them well identified (number
each vial in accordance to the record is followed in the form) for a later recognition under the
UV lamp or the fluorescent microscope.
Location of couples
Write time the position on the tree (height, substrate, quadrant, and any information
considered relevant) or any other place on the cage from where the couple is collected.
Record all pertinent data, including time and type of male and female for each vial on the
form. Label the vial accordingly, indicating the number assigned to the field cage in which
the mating took place (if more than one cage is run on a given day), day of test and the
number of the mating pair (couples should be numbered in the order they are collected in a
given cage, starting from 1, for the first mating pair). This is extremely critical and attention
should be paid in order not to mix the mating pairs.
Mating duration
Write time when pairs were captured on the form. Once the couple is in the vial, care must be
taken to ensure that the vials are kept in the shade and otherwise handled to minimize thermal
or other stresses to the flies. Observe mating couples in vials on a regular basis (every 5 min
or less) and note the time when uncoupling occurred.
Continue the test until the natural period of peak mating for that species under local
conditions is well over. Once the test is over, with the aid of an aspirator remove the flies that
did not mate from the cage. It is advisable to wash the tree to help removing any chemical
signal left by the flies if the cage is to be used on the following or consecutive days.
In addition to the standard procedure described in the previous paragraphs, it is recommended
to collect the following information:
Male calling time
To determine the extent and timing of the males’ participation in pre-mating
behaviours, a census may be taken at regular intervals (e.g., every 10-15 min or half hour).
During the census, the number, location, and colour of mark are recorded for each male that
is “calling”, or releasing pheromone within the cage or involved in courtship with female.
Calling males are typically (but not always) on the underside of leaves. In medfly and in
some Anastrepha species, they can be identified by the presence of what appears to be a drop
of liquid on the tip of the abdomen (in reality, a sac is extended from the anus) and males also
may inflate pouches on the pleura. Calling males may intermittently vibrate or “fan” their
wings while standing in place; during fanning, anal sac is partially retracted and held under
155
the abdomen, and thus is more difficult to observe. These data can also be used to check diel
periodicity of sexual behaviour among sterile males in comparison with wild males.
Incidence of remating
This requires continuation of the test for a second or even more days. It is recommended then
that this part is held under laboratory conditions to avoid dependency on weather conditions.
Here again, care should be taken when collecting mating pairs during the first day to ensure
that they are not disturbed to the point where they uncouple prematurely. Given that female
behaviour is the core of this part, it is recommended that females are held singly in vials and
two virgin males are offered every certain established period until females remate or the
observation period ends. Remating rate and refractory period are the variables to be analyzed.
The test should be run at leat twice the time of the mean refractory period (if known) and
virgin males of the same population of the females should be offered at regular intervales (i.e.
every day, every two days or three times a week). If possible, an ovisposition substrate can be
provided to the females and the number of eggs laid recorded to be used as a covariable in the
analysis. If female receptivity is very sensitive to fly density, then it is recomended to run the
test under relaxed conditions in order to allow the females reject the males. For meaningful
data, 25 females of each category should be analyzed.
Interpretation
Measures of sexual activity:
For meaningful data, the basic mating performance field cage test (one-day observation)
should be replicated ca. 6 - 10 times. Test replicates with more than ca. 25% of wild flies on
the cage screen during calling periods may reflect inadequate environmental conditions (such
as inadequate light, lack of water, leaves, etc.) and should therefore be repeated. In general it
is suggested that fly activities occurring away from the host tree should not be included in the
data analysis. This statement, however, should be taken with caution in compatibility tests
given that the occurrence of leks and matings in the screen of the cage may also reflect some
kind of spatial (i.e. ecological) isolation. For this reason it is recommended that any
information should be recorded during the test and discarded if considered properly at the
moment of data analysis.
Proportion of mating (PM). The proportion of flies mating measures the suitability of the
flies and the environment for mating and is defined as
PM = No. of pairs collected / No. of females released
Mean percentage of mating provides a useful indication of mating propensity. With C.
capitata, mating propensity is considered adequate when 50% of flies from all combinations
of strain and sex participate in mating. In practice, flies from some wild strains (especially the
females) are more reluctant than sterile females to mate in field cages. At any rate, data from
a cage should be discarded if less than 20% of both males and females from any strain
participate in mating.
Indices of strain sexual compatibility:
Several indices have been developed to quantify the sexual compatibility between
strains/populations. The indices should be computed separately for each cage. Analysis of test
results should involve the use of all of the main indices available (RII, ISI, FRPI, MRPI, see
156
bellow for a detailed description). It is advisable to include χ2 Goodness of Fit analysis to
assess statistically significant departure from random mating. .
Relative Isolation Index (RII). The RII is a measure of mating compatibility between two
strains.
AA  BB
RII 
AB  BA
Values of 1 indicates random mating between strains which is desirable in terms of SIT
control; values greater than one indicate positive assortative mating, i.e., males from
population A tends to mate with females from population A and vice versa (see graph
below). The RII has some advantages over other indices of compatibility being the most
relevant to this type of tests the fact that it is not affected by the overall level of participation
of the different types of flies, but only by whom they chose to mate.
RII 
SS  WW
SW  WS
RII
0
+1
Negative
assortative
mating
Random
mating
+10
+
+100
Positive
assortative
mating
(Sexual
compatibility)
(Sexual isolation)
Graphic representation of the Relative Isolation Index (RII). The value shown represents
the mean value obtained when comparing wild and sterile Ceratitis capitata flies in
field cages.
However, RII also has some disadvantages. First, it is undefined if one of the two heterotypic
crosses is zero. Second, when the number of matings in any one category is small, adding or
subtracting a single mating in that category will cause a large change in the value of the
index. Third, it can be difficult to normalize if the data are to be analysed statistically. Values
of RII larger than 1 indicate that there is some difference in mating behaviour (in a broad
sense) between the two populations, and that one or both strains are tending to mate
assortatively (i.e., like with like). Values of RII that trigger corrective action will probably be
found to vary from species to species, and wild flies from some areas seem to consistently
produce higher RII’s than flies from other areas.
Isolation Index (ISI). The ISI is a measure of mating compatibility.
ISI 
( AA  BB )  ( AB  BA)
AA  BB  AB  BA
Its values range from -1 (complete negative assortative mating; i.e., all matings are with
members of the opposite strain) through 0 (random mating) to +1 (complete positive
assortative mating; total mating isolation of the two strains) (see graph below). The main
advantage of this index is that, by ranging from –1 to +1, it is easier to assess the deviation
157
from the expected value of 0 than it is with index ranging from 0 to infinity. Compared to the
RII, ISI is not as sensitive to a change in a single mating and can always be defined, whatever
are the values of the 4 types of mating. In general, values of ISI consistently larger than 0.5
suggest that some positive assortative mating took place (which should be explained by
analysing the values of MRPI and FRPI).
ISI 
( SS  WW )  ( SW  WS )
SS  WW  SW  WS
ISI
-1
-0.75
-0.5
Negative
assortative
mating
-0.25
0
0.25
0.5
0.75
Random mating
+1
Positive
assortative
mating
(Sexual compatibility)
(Sexual isolation)
FRPI 
( SS  WS )  ( SW  WW )
SS  SW  WS  WW
MRPI 
( SS  SW )  (WS  WW )
SS  SW  WS  WW
FRPI
MRPI
-1
-0.75
-0.5
All matings
by wild
individuals
-0.25
0
0.25
0.5
Equal mating
propensity
0.75
+1
All matings
by sterile
individuals
Graphic representation of the Isolation Index (ISI) and of the Male and Female Relative
Performance indices (MRPI and FRPI). These indices should only be considered
together for a better understanding. The value shown was obtained when
comparing wild and sterile Anastrepha ludens flies in field cages (after Hernandez
et al. 2003).
Male Relative Performance Index (MRPI). The MRPI is a relative measure of mating
propensity of males of one population versus males from the other.
MRPI 
( AA  AB)  ( BA  BB )
AA  AB  BA  BB
A value of 1 indicates all matings in the cage were done by sterile males, and -1 indicates all
mating was done by wild males. Zero indicates that wild and sterile males participated
equally in mating. This index must be used in addition to the mean percentages of different
types of flies participating in mating, and complements the ISI and FRPI (below).
158
Female Relative Performance Index (FRPI). The FRPI is the counter part of the MRPI and
serves as a measure of mating propensity for female flies.
FRPI 
( AA  BA)  ( AB  BB )
AA  AB  BA  BB
The joint analysis of ISI, MRPI and FRPI, should provide a complete and reliable picture of
the sexual compatibility between strains and, should a deviation from the expected standard
be encountered, the reasons why it occurred.
Mating time:
The time at which matings take place is species and/or strain specific, as such in this type of
test it is important to evaluate of this trait is affected or not by the origin of the other
population present.
Copulation duration:
Length of time spent in copula can be an indication of laboratory adaptation of a strain and
can be related to transfer of sperm and accessory gland fluid to the female fly or the
occurrence of some kind of guard posture. Duration of copulation can be compared among
crosses by means of ANOVA or with non parametric statistics. In C. capitata, it has been
noted that an exceptionally long duration of mating (>3 hours) very often resulted in no
sperm being transferred to the female. However, short duration of mating for sterile males,
relative to those of males mating with the same type of female, may be indicative of some
kind of isolation, and may correlate with other related aspects, such as those on the incidence
of remating.
Male calling time:
Calling, or releasing pheromone to attract female flies, is an early and critical step in a
male’s effort to secure a mate. The incidence of calling is a component of mating propensity,
and a low incidence of calling could be indicative of low fly quality or vitality. In addition,
location of calling males is relevant in this type of tests given that they may reflect the
occurrence or not of mixed leks.
Female acceptance:
Estimating the rate at which females accept males from their own origin or from the
other requires more detailed observational studies, quantifying the number of female visits to
the different types of calling and courting males, and recording rejection or acceptance of
males by females. However, these data is of outmost importance in the case of incipient
isolation between two origins.
Incidence of remating:
Remating in wild tephritid females is more common in nature than previously believed.
This behaviour can be interpreted as the outcome from what the female received during the
first mating and Postcopulatory decision affected by her reproductive potential. Failings in
the first election may be reverted in a second mating and in the case of compatibility studies
it may reflect a rejection of the first male.
Modification to the mating performance field cage test
Other variations of the mating performance field cage test may be conducted if deemed
necessary. Examples of possible alternatives include: different ratios; use of various potted
159
host plants; use of higher male:female ratio by releasing females slowly over time (as would
be expected in natural leks in the field); replacement of individuals as the couples are formed;
etc. In the specific case of this cryptic species complexes CRP it is desirable to agree to
which etent changes are appropriate or may impede comparisons between investigations. Use
of tests with alternate designs may be particularly valuable in diagnosing causes of less-thandesirable levels of sexual compatibility but it is recommended to perform first the agreed
protocol.
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163
Appendix 7: Male Sex Pheromonal Components in Bactrocera Species – Extraction of
Rectal (Pheromonal) Gland for Chemical Analyses
Jacentkovski in 1932 first demonstrated the existence of insect sex pheromone by using traps
containing virgin gypsy moth females, which attracted and trapped many males, placed in
gypsy moth infested woods. Only till 1959, Butenandt and coworkers after extracting from
about half a million female silkworms, Bombyx mori, reported the first sex pheromone
'bombykol' was identified using traditional chemistry. We have come a long way since then,
with the help of highly sensitive instrumentation and recording techniques, such as the gas
chromatography-mass spectrophotometry (GC-MS) coupled to an electro-anntenograph
(EAG), a scientist can now extract and identify an insect's sex pheromone from a few
individuals. In the case of fruit flies, particularly those from the genus Bactrocera, (unlike the
lepidopterans) males produce sex pheromone at sexual maturity. For many species, males
make use of external sources of chemical (attractant) to produce sex pheromonal
component(s).Therefore, a single male may be sufficient to confirm identity of the sex
pheromonal component(s) after feeding on the specific attractant, provided proper procedure
is followed.
All Bactrocera species may be categorized into three groups based on their non-response (28
species confirmed and 258 species listed under “lures unknown”) or response to two very
potent attractants – cue-lure (195 species) and methyl eugenol (ME) (ca 84 species) (IAEA
2000, 2005). Approximately a dozen of the ME responsive/sensitive species of Bactrocera
including several putative sibling species of the Bactrocera dorsalis complex, such as B.
carambolae Drew & Hancock, B. dorsalis (Hendel), B. invadens Drew, Tsuruta & White, B.
occipitalis (Bezzi), B. papayae Drew & Hancock, and B. philippinensis Drew & Hancock
form a group of serious polyphagous pests that currently cause high economic losses in the
production of fruits. They are also very important quarantine pests that may interfere or
disrupt international free and fair trade of fresh fruits and vegetables between exporting and
importing countries globally.
Credit must be given to Howlett who first demonstrated that male tephritid flies (Dacus spp.)
were attracted to citronella oil in in 1912. He subsequently showed that the flies were actually
attracted to ME in 1915. Since then, there were much speculations as to the actual role of ME
in tephritid flies.
Males of B. dorsalis and B. papayae are strongly attracted to and compulsively feed on the
ME which acts as a sex pheromone precursor that is converted to two major components - Econiferyl alcohol and 2-allyl-4,5-dimethoxyphenol (Nishida et al. 1988; Tan and Nishida
1996, 1998). Nonetheless, the ME can also act as a booster component to complement
endogenously produced sex pheromone as in B. carambolae in which it is converted only to
E-coniferyl alcohol (Tan and Nishida 1996; Wee and Tan 2007). Table 1 shows the male sex
pheromone components of several Bactrocera species after feeding on an attractant ME or
raspberry ketone in the case of the melon fly B. cucurbitae. In the male B. dorsalis/papayae,
the consumed ME is biotransformed in the crop and its metabolites are then transported via
the haemolymph to the rectal gland (Hee and Tan 2006), where they are sequestered via
rectal papillae and temporarily stored before being released as sex pheromone (Fig. 1) (Khoo
and Tan 2005).
164
Native males seek and ingest ME from natural sources (Tan 2009; Tan et al. 2002, 2006).
There are ca 350 plant species belonging to 61 families that possess ME, in varying
quantities from trace amounts to >90% of essential oils, as a constituent component and/or
release as a component of floral fragrance (Tan, compiled list unpublished). Consumption of
the ME has been shown to improve significantly male mating performance and
competitiveness of B. dorsalis (Shelly and Dewire 1994, 2000; Tan and Nishida 1996), B.
papayae (Tan and Nishida 1996, 1998) B. carambolae (Wee et al. 2007), B. correcta
(Orankanok et al. 2009) and B. zonata (Quilici et al. 2004; Sookar et al. 2009).
Table 1. Sex pheromone after feeding on methyl eugenol or raspberry ketone
B. carambolae
- E-coniferyl alcohol (CF) (endogenously produced - 6-oxo-1-nonanol + N3 methylbutyl acetamide + 1,6-nonanediol + ethyl benzoate)
B. dorsalis
- 2-allyl-4,5-dimethoxyphenol (DMP) + CF + 3,4-dimethoxycinnamyl
alcohol (DCA)
B. papayae
- DMP + CF + DCA
B. umbrosa
- DMP + DCA + 3,4-dimethoxy-hydroxyallyl-benzene
B. cucurbitae
- Raspberry ketone (endogenously produced - 1,3-nonanediol + methyl,
ethyl & propyl 4-hydroxybenzoate + N-3-methylbutyl methoxyacetamide)
Figure 1. Male rectal gland of B. dorsalis showing the presence of auto-fluorescent
compounds during sequestration of sex pheromonal chemicals from the haemolymph at
10 to120 minutes after feeding on methyl eugenol. (Adapted from Khoo and Tan (2005).
White arrows – the chemicals detected within 1 hour.
165
10 min
20 min
40 min
2 hours
Figure 2. Male rectal glands extracted from sexually mature males (15 days old) –
deprived of ME (left) and fed with ME ( right - one day post-feeding)
166
With highly sensitive instrumentation such as the GC-MS that uses fine capillary column to
analyze sex pheromonal components, it is very important that samples injected into the
column must not contain contaminants (especially non-volatile compounds and
macromolecules ) that will clog up the column. It is also important to note that sex
pheromonal compounds are usually volatile and exist in microgram or submicrogram levels.
Therefore, it is pertinent to follow proper rectal gland extraction procedure, before preserving
it for future chemical analyses.
1. Solvent for extraction preferably pure ethanol – redistilled (N.B. This solvent is very
hygroscopic - so do not expose to atmospheric air for long periods, especially in the
humid tropics). If this solvent is not available, then, absolute alcohol (ethanol) is
suitable (N.B. Please provide a 1 ml sample for solvent check for possible impurities).
2. Prepare glass vials (1 or 2 ml with teflon lined screw-caps) for preservation of gland –
fill each vial with 250 microlitre (0.25 ml) of the solvent and tighten the screw cap
until use. (N.B. One vial for preservation of one rectal gland).
3. Fine pointed forceps – Firstly, wash and clean with a detergent; and secondly, rinse
the tips with a few drops of the solvent just before use.
4. Immobilize a male fly, preferably without the use of carbon dioxide, by transferring
the fly into a small clean plastic bag or cleaned container, making sure that the fly
cannot escape, and then place it in either a refrigerator for 20-30 minutes at < 15o C or
a freezer < - 5o C for 1-2 minutes.
5. Rectal gland removal - Place the immobilized fly ventral side up on the stage of a
dissecting microscope (Fig. 3). One may hold the fly in place either with a pair of
forceps or with one's fore finger and thumb. Then, with a pair of cleaned fine pointed
forceps grip firmly the base of the aedaegus (Fig. 3A), and simultaneously pull it
gently away from the fly's abdomen until the rectal gland is completely exposed (Fig.
3B & C) – due care should be taken, by avoiding sudden jerks, to ensure that the
rectal sac does not burst especially when it is bloated. Then cut the hind gut at
approximately 1 mm from the rectal gland (Fig 3 D). The freshly removed gland (Fig.
4) on the forceps tip is immediately introduced into a vial containing pure alcohol.
After that, the teflon lined screw-cap is replaced and tightened (finger tight) to the
vial. To avoid possible leakage during transportation (especially drastic climatic
changes), the narrow gap between rim circumference of the screw-cap and the glass
vial is tightly sealed by simultaneously stretching and wrapping a parafilm strip (ca
1.5 cm x 5 cm) several times around it . After labeling the vial, store the extracted
rectal gland in a refrigerator until dispatch for chemical analyses.
6. For comparison of sexually mature flies fed with and deprived of of an attractant, e.g.
methyl eugenol (ME) – item 5 needs to be repeated for rectal gland removal from the
ME-fed and the ME-deprived (as controls) flies.
167
Figure 3. Removal of rectal (pheromone) gland under magnification. Steps A-D.
A
C
B
D
Figure 4. Freshly removed male rectal gland and associated aedeagal apparatus.
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