ANNEX XX
ABSTRACTS 3RD RCM TUCUMAN
Evaluation of genetic sexing strain, Bactrocera dorsalis Salaya1 for its species complex
members and molecular characterization of Y-chromosome sequences of Bactrocera
dorsalis
Nidchaya Aketarawong1, Siriwan Isasawin1, Anna R. Malacrida2, Giuliano Gasperi2,
Sujinda Thanaphum1
1
2
Department of Biotechnology, Faculty of Science, Mahidol University, THAILAND
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.
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 phylogenetic-based 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.
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.
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.
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.
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).
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.
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
Royal Museum for Central Africa, Tervuren, BELGIUM
3
CIRAD, UMR PVBMT, St Pierre, La Réunion, FRANCE
2
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.
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
Institute of Organic Chemistry and Bichemistry of the Czech Academy of Prague, CZECH
REPUBLIC
3
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), BRAZIL
2
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.
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.
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 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.
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.
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.
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):206208.
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 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.
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.
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.
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.
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 (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.
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
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:
435-443.
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.
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.
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
M. K. Schutze1,2, 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
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
2
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
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.
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 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.
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.
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.
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.
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-4,5dimethoxyphenol, 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.