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

1Department of Biotechnology, Faculty of Science, Mahidol University, THAILAND2Department 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

1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague, CZECH REPUBLIC..

2Institute of Chemical Technology Prague, CZECH REPUBLIC3Universidade 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

1Department of Agricultural Extension, Ministry of Agriculture and Cooperatives, Chatuchak, Bangkok 10900, THAILAND2Department of Agriculture, Ministry of Agriculture and Cooperatives, Chatuchak, Bangkok 10900, THAILAND3Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkhen Campus, Chatuchak, Bangkok 10900, THAILAND4Department 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

1University of Antwerp, Antwerp, BELGIUM2Royal Museum for Central Africa, Tervuren, BELGIUM3CIRAD, 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.

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

1Universidade Federal de Alagoas, BRAZIL2Institute of Organic Chemistry and Bichemistry of the Czech Academy of Prague, CZECH REPUBLIC3Empresa 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.

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

1Department of Genetics, Development and Molecular Biology, School of Biology, Faculty of Sciences, Aristotle University of Thessaloniki, GREECE2Department of Biology, University of Patras, GREECE3Department of Environmental and Natural Resources, University of Patras, Agrinio, GREECE4Insect 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

1Insect Pest Control Laboratory, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Seibersdorf, AUSTRIA2National Agricultural Research Centre, Park Road, Islamabad, PAKISTAN3USDA-APHIS, 41-650 Ahiki Street, Waimanalo, HI 9795, USA4Insect 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., MEXICO2Universidad del Tolima, COLOMBIA3Escuela Politécnica del Ejército, ECUADOR

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):206-208.

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

1Universidade Federal da Bahia (UFBA), BRAZIL2CENARGEN-EMBRAPA, EMBRAPA–Semiárido, BRAZIL3Medfly Rearing Facility Brazil (Moscamed), BRAZIL

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

1Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, MALAYSIA

2School of Environmental & Natural Resource Sciences, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, MALAYSIA

3Phi-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

1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague, CZECH REPUBLIC2Institute of Chemical Technology Prague, CZECH REPUBLIC3Universidade 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

1Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, ITALY2Department of Biotechnology, Mahidol University, Bangkok 10400, THAILAND3Instituto de Genética “Ewald A. Favret” (INTA), B1712WAA Castelar, Buenos Aires, ARGENTINA4Institute of Organic Chemistry and Biochemistry ASCR, CZ-166 10 Prague 6, CZECH REPUBLIC*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

1Sokoine University of Agriculture, TANZANIA2Royal 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

1Instituto de Ecología, A.C., Xalapa, Veracruz, MEXICO2Cátedra de Terapéutica Vegetal, Departamento de Sanidad Vegetal, Facultad de Agronomía y Zootecnia, UNT, Tucumán, ARGENTINA3Instituto de Genética “E.A. Favret”, INTA Castelar, Buenos Aires, ARGENTINA 4Universidad Federal do Bahia, BRAZIL5SENASA, PERU6Universidad del Tolima, Ibagué, COLOMBIA7Infochemical Group Institute of Organic Chemistry and Biochemistry, CZECH REPUBLIC8Embrapa Uva e Vinho, Estação Experimental de Vacaria, BRAZIL

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

1CRC for National Plant Biosecurity, L.P.O. Box 5012, Bruce, A.C.T. 2617, AUSTRALIA2School of Earth, Environmental and Biological Sciences, Queensland University of Technology, G.P.O. Box 2434, Brisbane 4000, Queensland, AUSTRALIA3Pakistan Museum of Natural History, Garden Avenue, Shakarparian, Islamabad, PAKISTAN4 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 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

1Universidade Estadual De Santa Cruz, Bahia, BRAZIL2United States Department Of Agriculture, USA3Florida Department of Agriculture and Consumer Services, USA4Ohio State University, USA5Universidade de Sao Paulo, BRAZIL

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

1Florida 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

1Universidade Federal de Alagoas, Maceio, BRAZIL2Royal Museum for Central Africa, Leuvensesteenweg 13, B-3080 Tervuren, BELGIUM3Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Prague, CZECH REPUBLIC

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

1School of Environmental & Natural Resource Sciences, Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, MALAYSIA2Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, MALAYSIA3Phi-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.