Substantial rearrangements, single nucleotide frameshift deletion and low diversity in mitogenome of Wolbachia-infected strepsipteran endoparasitoid in comparison to its tephritid hosts.

Insect mitogenome organisation is highly conserved, yet, some insects, especially with parasitic life cycles, have rearranged mitogenomes. Furthermore, intraspecific mitochondrial diversity can be reduced by fitness-affecting bacterial endosymbionts like Wolbachia due to their maternal coinheritance with mitochondria. We have sequenced mitogenomes of the Wolbachia-infected endoparasitoid Dipterophagus daci (Strepsiptera: Halictophagidae) and four of its 22 known tephritid fruit fly host species using total genomic extracts of parasitised flies collected across > 700 km in Australia. This halictophagid mitogenome revealed extensive rearrangements relative to the four fly mitogenomes which exhibited the ancestral insect mitogenome pattern. Compared to the only four available other strepsipteran mitogenomes, the D. daci mitogenome had additional transpositions of one rRNA and two tRNA genes, and a single nucleotide frameshift deletion in nad5 requiring translational frameshifting or, alternatively, resulting in a large protein truncation. Dipterophagus daci displays an almost completely endoparasitic life cycle when compared to Strepsiptera that have maintained the ancestral state of free-living adults. Our results support the hypothesis that the transition to extreme endoparasitism evolved together with increased levels of mitogenome changes. Furthermore, intraspecific mitogenome diversity was substantially smaller in D. daci than the parasitised flies suggesting Wolbachia reduced mitochondrial diversity because of a role in D. daci fitness.

Occurrence of fruit flies (Diptera: Tephritidae) in the municipality of Alvorada do Gurguéia in South Central Piauí, Brazil

The state of Piauí is in a Cerrado/Caatinga transition area, which is known the richness of its fauna and flora. Therefore, this work aims to know the diversity of fruit fly species in cashew orchards and native forest fragments in the south-central region of Piauí. Fruit flies were collected in McPhail traps and by sampling fruits. The material collected from the traps and the fruits in the two areas were taken to the Laboratory of Phytotechnics from Campus Prof. Cinobelina Elvas at the Universidade Federal do Piauí (UFPI/CPCE), municipality of Bom Jesus, Piauí, Brazil. In McPhail traps, six species of the genus Anastrepha: A. alveata, A. dissimilis, A. fraterculus, A. manihoti, A. obliqua, and A. zenildae were captured, of which A. fraterculus, A. obliqua, and A. zenildae were present in both areas. Anastrepha alveata, A. dissimilis and A. manihoti occurred only in the cashew orchard. In fruit sampling, A. fraterculus, A. obliqua, and A. zenildae infested wild-guava fruits, Myrcia tomentosa (Myrtaceae), in native forest. Anastrepha fraterculus, A. obliqua and A. zenildae were obtained directly from M. tomentosa fruits and from traps. The availability of host fruits is the factor that most influences the population fluctuation of fruit flies, mainly in the native forest area. Anastrepha manihoti is reported for the first time in the state of Piauí. This is the first record of A. fraterculus, A. obliqua and A. zenildae, infesting wildguava fruits, M. tomentosa.

Phylogenomic approach reveals strong signatures of introgression in the rapid diversification of neotropical true fruit flies (Anastrepha: Tephritidae).

New sequencing techniques have allowed us to explore the variation on thousands of genes and elucidate evolutionary relationships of lineages even in complex scenarios, such as when there is rapid diversification. That seems to be the case of species in the genus Anastrepha, which shows great species diversity that has been divided into 21 species groups, several of which show wide geographical distribution. The fraterculus group has several economically important species and it is also an outstanding model for speciation studies, since it includes several lineages that have diverged recently possibly in the presence of interspecific gene flow. Our main goal is to test whether we can infer phylogenetic relationships of recently diverged taxa with gene flow, such as what is expected for the fraterculus group and determine whether certain genes remain informative even in this complex scenario. An analysis of thousands of orthologous genes derived from transcriptome datasets of 10 different lineages across the genus, including some of the economically most important pests, revealed signals of incomplete lineage sorting, vestiges of ancestral introgression between more distant lineages and ongoing gene flow between closely related lineages. Though these patterns affect the phylogenetic signal, the phylogenomic inferences consistently show that the morphologically identified species here investigated are in different evolutionary lineages, with the sole exception involving Brazilian lineages of A. fraterculus, which has been suggested to be a complex assembly of cryptic species. A tree space analysis suggested that genes with greater phylogenetic resolution have evolved under similar selection pressures and are more resilient to intraspecific gene flow, which would make it more likely that these genomic regions may be useful for identifying fraterculus group lineages. Our findings help establish relationships among the most important Anastrepha species groups, as well as bring further data to indicate that the diversification of fraterculus group lineages, and even other lineages in the genus Anastrepha, has been strongly influenced by interspecific gene flow.

A new species of emDoryctobracon/em Enderlein (Hymenoptera, Braconidae) parasitizing larvae of emAnastrepha/em Schiner (Diptera, Tephritidae), with illustrated key to species of emDoryctobracon/em that parasitize fruit-infesting tephritids in Brazil.

Doryctobracon maculatus Marinho, a new species of Braconidae (Opiinae) collected in the municipalities of Piracicaba and São Roque, state of São Paulo, Brazil, is described and illustrated. This new species is placed in group of species with areolate propodeum, but is easily distinguished from other species of this group, and other members of the genus by the noticeable black to dark-brown spots on the head, mesosoma and metasoma. This new species was reared in larvae of Anastrepha pseudoparallela (Diptera, Tephritidae) in passion fruits, Passiflora alata Curtis (Passifloraceae). An illustrated key to species of Doryctobracon recorded in Brazil is presented.

Hybridization, Behavioral Patterns, and Pre- and Postzygotic Isolation Between Two Recently Derived Species of Walnut-Infesting Rhagoletis Fruit Flies in the Highlands of Mexico.

Episodes of isolation and secondary contact among populations of insects of Nearctic origin during Pleistocene glacial/postglacial climatic cycles had a strong evolutionary influence on the diversity of flies in the genus Rhagoletis in mountainous areas of Mexico. As a series of experiments undertaken to gather support for phylogenetic hypotheses on the origin of three walnut-infesting species in the suavis group, we examined pre- and postzygotic isolation between Rhagoletis completa Cresson, 1929 and R. ramosae Hernández-Ortiz, 1985. Despite morphological, biological, and behavioral differences, these two species were found to be capable of hybridization. Mating experiments in large enclosures revealed asymmetric sexual isolation. There were notable differences in male sexual behavior. While R. ramosae males mated exclusively on host fruit, R. completa males used fruit and alternative mating locations. During fruit-guarding and male-male contests, R. completa and R. ramosae males adopted markedly different wing postures. R. completa females were more reluctant to copulate with heterospecific males than R. ramosae females. During no choice crosses in small enclosures, there was a reduction of egg hatch for the hybrid cross of R. completa males × R. ramosae females. Our results and previous studies on reproductive isolation between other species pairs in the suavis group support a clade in which R. ramosae, R. zoqui Bush, 1966, and R. completa are close relatives all still capable of hybridizing.

White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp- mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp- mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

Separating two tightly linked species-defining phenotypes in Bactrocera with hybrid recombinant analysis.

BACKGROUND: Bactrocera tryoni and Bactrocera neohumeralis mate asynchronously; the former mates exclusively around dusk while the latter mates during the day. The two species also differ in the colour of the post-pronotal lobe (callus), which is predominantly yellow in B. tryoni and brown in B. neohumeralis. We have examined the genetic relationship between the two characters in hybrids, backcrosses and multigeneration hybrid progeny. RESULTS: Our analysis of the mating time of the parental species revealed that while B. tryoni mate exclusively at dusk, B. neohumeralis females pair with B. neohumeralis males during the day and with B. tryoni males at dusk. We found considerable variance in mating time and callus colour among hybrid backcross individuals of both sexes but there was a strong although not invariant trend for callus colour to co-segregate with mating time in both sexes. To genetically separate these two phenotypes we allowed the interspecific F1 hybrids to propagate for 25 generations (F25) without selection for mating time or callus colour, finding that the advanced hybrid population had moved towards B. tryoni phenotypes for both traits. Selection for day mating in replicate lines at F25 resulted in significant phenotypic shifts in both traits towards B. neohumeralis phenotypes in F26. However, we were unable to completely recover the mating time profile of B. neohumeralis and relaxation of selection for day mating led to a shift back towards dusk mating, but not yellow callus colour, by F35. CONCLUSION: We conclude that the inheritance of the two major species-defining traits is separable but tightly linked and involves more than one gene in each case. It also appears that laboratory conditions select for the B. tryoni phenotypes for mating time. We discuss our findings in relation to speciation theory and the likely effects of domestication during the generation of mass release strains for sterile insect control programmes.

Host range and species diversity of Tephritidae of three plant formations in Western Burkina Faso.

In Western Burkina Faso, the host range of fruit flies was evaluated in three plant formations between May 2017 and April 2019. Samples of 61 potential hosts were collected and incubated for fruit fly emergence. Twenty-seven hosts including cultivated and wild fruit were identified. Among cultivated fruit species, mango, and guava were the most infested while high infestation incidences were observed in the fruit of the indigenous plants Vitellaria paradoxa, Annona senegalensis, Sarcocephalus latifolius, and Saba senegalensis. Low infestation rates were observed in Anacardium occidentale, Citrus species, Opilia celtidifolia, and Cissus populnea. The highest infestation index (1648.57 flies kg-1) was observed from V. paradoxa. Eleven new host fruit infested with many fruit fly species are reported in Burkina Faso. A total of 18 fruit fly species were reared; Bactrocera dorsalis (42.94%), Ceratitis cosyra (29.93%), and Ceratitis silvestrii (22.33%) dominated those that emerged. Four fruit fly species have been detected for the first time in Burkina Faso. The main suitable fruit hosts are abundant and available from May through August during the rainy season and become rare and have low infestation from November to April during the dry season. This is the first study of its kind in the region. This study shows that the three plant formations had an impact on population dynamics of the three tephritid species of economic importance in Western Burkina Faso. This information should be integrated into the development of a fruit fly pests management strategy.

Towards the implementation of a DNA barcode library for the identification of Peruvian species of Anastrepha (Diptera: Tephritidae).

The genus Anastrepha is a diverse lineage of fruit-damaging tephritid flies widespread across the Neotropical Region. Accurate taxonomic identification of these flies is therefore of paramount importance in agricultural contexts. DNA barcoding libraries are molecular-based tools based on a short sequence of the mitochondrial COI gene enabling rapid taxonomic identification of biological species. In this study, we evaluate the utility of this method for species identification of Peruvian species of Anastrepha and assemble a preliminary barcode profile for the group. We obtained 73 individual sequences representing the 15 most common species, 13 of which were either assigned to previously recognized or newly established BINs. Intraspecific genetic divergence between sampled species averaged 1.01% (range 0-3.3%), whereas maximum interspecific values averaged 8.67 (range 8.26-17.12%). DNA barcoding was found to be an effective method to discriminate between many Peruvian species of Anastrepha that were tested, except for most species of the fraterculus species group, which were all assigned to the same BIN as they shared similar and, in some cases, identical barcodes. We complemented this newly produced dataset with 86 published sequences to build a DNA barcoding library of 159 sequences representing 56 Peruvian species of Anastrepha (approx. 58% of species reported from that country). We conclude that DNA barcoding is an effective method to distinguish among Peruvian species of Anastrepha outside the fraterculus group, and that complementary methods (e.g., morphometrics, additional genetic markers) would be desirable to assist sensu stricto species identification for phytosanitary surveillance and management practices of this important group of pestiferous flies.

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini).

Accurate species-level identifications underpin many aspects of basic and applied biology; however, identifications can be hampered by a lack of discriminating morphological characters, taxonomic expertise or time. Molecular approaches, such as DNA "barcoding" of the cytochrome c oxidase (COI) gene, are argued to overcome these issues. However, nuclear encoding of mitochondrial genes (numts) and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications. One insect group for which these molecular and morphological problems are significant are the dacine fruit flies (Diptera: Tephritidae: Dacini). We addressed these issues associated with COI barcoding in the dacines by first assessing several "universal" COI primers against public mitochondrial genome and numt sequences for dacine taxa. We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region. Our new primers were tested alongside universal primers on a selection of dacine species, including both fresh preserved and decades-old dry specimens. Additionally, Bactrocera tryoni mitochondrial and nuclear genomes were compared to identify putative numts. Four numt clades were identified, three of which were amplified using existing universal primers. In contrast, our new primers preferentially amplified the "true" mitochondrial COI barcode in all dacine species tested. The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable. Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.

New occurrences of Anastrepha Schiner (Diptera: Tephritidae) in the state of Minas Gerais, Brazil / Novas ocorrências de Anastrepha Schiner (Diptera: Tephritidae) no estado de Minas Gerais, Brasil

Occurrences of Anastrepha barbiellinii Lima, Anastrepha connexa Lima, Anastrepha consobrina (Loew), Anastrepha kuhlmanni Lima, Anastrepha leptozona Hendel, and Anastrepha xanthochaeta Hendel are recorded for the first time in the state of Minas Gerais, Brazil. The species were captured in McPhail traps baited with hydrolyzed protein (5%) in orchards of cultivated species and in a remnant of the Atlantic Forest in the municipality of Viçosa, Minas Gerais, or obtained from the collection of species deposited in the Regional Museum of Entomology (UFVB) of the Universidade Federal de Viçosa, Campus Viçosa, in the state of Minas Gerais.(AU)

Ocorrências de Anastrepha barbiellinii Lima, Anastrepha connexa Lima, Anastrepha consobrina (Loew), Anastrepha kuhlmanni Lima, Anastrepha leptozona Hendel e Anastrepha xanthochaeta Hendel são registradas pela primeira vez no estado de Minas Gerais, Brasil. As espécies foram capturadas em armadilhas do tipo McPhail com um atrativo alimentar de proteína hidrolisada (5%) em três pomares de espécies cultivadas e um remanescente de Mata Atlântica no município de Viçosa, Minas Gerais, ou obtidas por meio de levantamento de espécies de Tephritidae depositadas no Museu Regional de Entomologia (UFVB) da Universidade Federal de Viçosa, Campus Viçosa, estado de Minas Gerais.(AU)

Fruit fly identification, population dynamics and fruit damage during fruiting seasons of sweet oranges in Rusitu Valley, Zimbabwe.

In 2003, the pest species Bactrocera dorsalis (Hendel) was reported for the first time in Kenya, Africa, and subsequently on many other African countries. In this work, 20 locations along the Rusitu Valley (Zimbabwe) were sampled in 2014 during the sweet oranges fruiting seasons, to verify the fruit fly taxonomy, invasion source, population dynamics, and fruit damage. The trapped fruit flies were identified using morphological traits and molecular techniques, as B. dorsalis. The haplotype network analysis revealed that Zimbabwe COI sequences were identical to other African B. dorsalis sequences. Fruit fly trappings per day varied during the year, although it remained always high. The same applies to fruit damage, most likely due to the permanent availability of cultivated and wild fruit varieties during the year. Rusitu Valley was invaded by B. dorsalis, most likely from neighbouring countries. Ten years after the first report in Kenya, the complete or near complete invasion of Africa has been achieved by B. dorsalis. In northern Africa the distribution is clearly limited by the Sahara desert. The large population size, the polyphagous nature of the species, and the continuous availability of suitable host fruit species during the year complicates the eradication of this species.

Population genetics of the solanum fruit fly, Bactrocera latifrons (Hendel) (Diptera: Tephritidae).

The solanum fruit fly, Bactrocera latifrons (Hendel), is an important pest species of commercial plants in the family Solanaceae. In this study, the population genetic structure of B. latifrons was investigated using mitochondrial cytochrome c oxidase I sequences. A mitochondrial DNA haplotype network revealed no major genetic break, but haplotypes from recently invaded areas in Japan, Tanzania, and Kenya were genetically divergent. The overall haplotype network is approximately star-shaped, characteristic of recent demographic expansion of populations. This is also supported by large negative values of neutrality tests. Despite the overall pattern of recent population history, genetic structure analysis revealed considerable genetic structuring with 33% of pairwise comparisons being significantly different. Populations that were genetically different from the others usually possess low genetic diversity, suggesting that genetic drift is potentially a factor driving genetic differentiation. Local extinction and recolonization processes related to the availability of host plants are most likely responsible for a founder effect and subsequent genetic drift in a population.

Host plant-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi.

Elucidating the mechanisms and conditions facilitating the formation of biodiversity are central topics in evolutionary biology. A growing number of studies imply that divergent ecological selection may often play a critical role in speciation by counteracting the homogenising effects of gene flow. Several examples involve phytophagous insects, where divergent selection pressures associated with host plant shifts may generate reproductive isolation, promoting speciation. Here, we use ddRADseq to assess the population structure and to test for host-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi (L., 1758) (Diptera: Tephritidae). This tephritid is distributed throughout Europe and western Asia, and has adapted to two different genera of host plants, Prunus spp. (cherries) and Lonicera spp. (honeysuckle). Our data imply that geographic distance and geomorphic barriers serve as the primary factors shaping genetic population structure across the species range. Locally, however, flies genetically cluster according to host plant, with consistent allele frequency differences displayed by a subset of loci between Prunus and Lonicera flies across four sites surveyed in Germany and Norway. These 17 loci display significantly higher FST values between host plants than others. They also showed high levels of linkage disequilibrium within and between Prunus and Lonicera flies, supporting host-related selection and reduced gene flow. Our findings support the existence of sympatric host races in R. cerasi embedded within broader patterns of geographic variation in the fly, similar to the related apple maggot, Rhagoletis pomonella, in North America.

Faunistic analyses of fruit fly species (Diptera: Tephritidae) in orchards surrounded by Atlantic Forest fragments in the metropolitan region of Curitiba, Paraná state, Brazil / Análise faunística de moscas-das-frutas (Diptera: Tephritidae) em pomares rodeados por fragmentos da Mata Atlântica na região metropolitana de Curitiba, Paraná, Brasil

Abstract The objective of this trial was to characterize the assemblage structure of fruit flies (Diptera: Tephritidae) by determining the diversity and faunal indices. Fruit flies were collected for ten years between 2003 and 2015 in six municipalities of the Metropolitan Region of Curitiba, Paraná. The orchards were divided into three crop protection strategies: organic, conventional, and integrated pest management. The characteristic of each community was determined using the Shannon-Wiener, Margalef, and Pielou's indexes. The frequency, constancy, and dominance indexes were also determined. Biological material was analyzed using the explanatory variables: site, crop, plant protection strategy, and susceptibility period. A total of 8,089 fruit flies were collected, of which 4,681 were females. The species Anastrepha daciformis Bezzi, A. dissimilis Stone, A. distincta Greene and A. pickeli Lima were recorded for the first time in Paraná State, the occurrence of three other species was recorded for the first time in the Metropolitan Region of Curitiba (A. montei Lima, A. obliqua Macquart, and A. sororcula Zucchi), in addition A. fraterculus (Wiedemann), A. grandis (Macquart) and Ceratitis capitata (Wiedemann). A. fraterculus accounted for 98.5% of the collected species. Only A. fraterculus and A. sororcula were found in fruits. Fruit flies occurred predominantly in peach trees cultivated under organic conditions. The Shannon index indicated that the municipality of Cerro Azul had the highest diversity and richness whereas apple orchards had the lowest diversity. Crops cultivated under organic conditions exhibited the highest diversity whereas those cultivated under conventional conditions had the highest evenness index. The largest specimens number was collected during the period of crop susceptibility.

Resumo O objetivo deste estudo foi caracterizar agrupamento de espécies de moscas-das-frutas (Diptera: Tephritidae) pela determinação da diversidade e dos índices faunísticos. As moscas-das-frutas foram coletadas por dez anos entre 2003 e 2015 em seis municípios da Região Metropolitana de Curitiba, no Paraná. Os pomares foram divididos em três estratégias de proteção de culturas: orgânica, convencional e integrada. A característica de cada comunidade foi determinada usando os índices de Shannon-Wiener, Margalef e Pielou. Os índices de freqüência, constância e dominância também foram determinados. O material biológico foi analisado utilizando as variáveis explicativas: local, cultura, estratégia de proteção e período de susceptibilidade. Foram coletadas 8.089 moscas-das-frutas, sendo 4.681 fêmeas. As espécies Anastrepha daciformis Bezzi, A. dissimilis Stone, A. distincta Greene e A. pickeli Lima foram registradas pela primeira vez no Estado do Paraná, e pela primeira vez, na Região Metropolitana de Curitiba, as espécies A. montei Lima, A. obliqua Macquart e A. sororcula Zucchi), além de A. fraterculus (Wiedemann), A. grandis (Macquart) e Ceratitis capitata (Wiedemann). A. fraterculus foi responsável por 98,5% das espécies coletadas. Apenas A. fraterculus e A. sororcula foram encontrados em frutos. As moscas-das-frutas ocorreram predominantemente em pessegueiros cultivados sob condições orgânicas. O índice de Shannon indicou que o município de Cerro Azul teve a maior diversidade e riqueza, enquanto que a cultura macieira teve a menor diversidade. As culturas sob condição orgânica exibiram a maior diversidade, enquanto as cultivadas em condições convencionais apresentaram o maior índice de uniformidade. O maior número de espécimes foi coletado durante o período de suscetibilidade à cultura.

Old residents and new arrivals of Rhagoletis species in Europe.

The genus Rhagoletis (Diptera: Tephritidae) comprises more than 65 species distributed throughout Europe, Asia and America, including many species of high economic importance. Currently, there are three Rhagoletis species that infest fruits and nuts in Europe. The European cherry fruit fly, Rhagoletis cerasi (may have invaded Europe a long time ago from the Caucasian area of West Asia), and two invasive species (recently introduced from North America): the eastern American cherry fruit fly, R. cingulata, and the walnut husk fly, R. completa. The presence of different Rhagoletis species may enhance population dynamics and establish an unpredictable economic risk for several fruit and nut crops in Europe. Despite their excessive economic importance, little is known on population dynamics, genetics and symbiotic associations for making sound pest control decisions in terms of species-specific, environmental friendly pest control methods. To this end, the current paper (a) summarizes recently accumulated genetic and population data for the European Rhagoletis species and their association with the endosymbiont Wolbachia pipientis, and (b) explores the possibility of using the current knowledge for implementing the innovative biological control methods of sterile insect technique and incompatible insect technique.

Pheromone emission patterns and courtship sequences across distinct populations within Anastrepha fraterculus (Diptera-Tephritidae) cryptic species complex.

Sexual behavioural isolation can result from sexual selection and represents a relevant factor associated with the speciation process. We analysed the pheromone emission pattern and the courtship of males of five different populations of the Anastrepha fraterculus cryptic complex: Brazil (Vacaria, Tucumán and Piracicaba), Colombia and Peru. The time of pheromone emission was recorded in each population every 30 min during the day. The behavioural sequences of courting were video recorded and analysed using EthoSeq software. Males from different populations have showed different period of pheromone emission - Vacaria, Piracicaba and Tucumán executed calling only during the morning, Colombia only in the afternoon and Peru during both periods. The general frequencies of the courtship units of the males were distinct among the populations. Three groups were formed in the classification from the function of 14 behavioural routines: Vacaria, Piracicaba and Tucumán formed a single group (Brazil-1), while Colombia and Peru formed two distinct groups. In the probabilistic trees generated, the behavioural units that most contributed to the occurrence of copulation were distinct among the three groups formed: Brazil-1 (Contact, Alignment and Arrowhead-1); Colombia (Flying, Mobile, Contact and Alignment); Peru (Flying, Arrowhead-1 and Calling). Our results indicated differences in sexual behaviour that may explain the behavioural isolation found between the distinct groups in addition with the temporal isolation found between the Brazil-1 and Colombia populations. The evolutionary implications for the A. fraterculus cryptic species complex are discussed.

Faunistic analyses of fruit fly species (Diptera: Tephritidae) in orchards surrounded by Atlantic Forest fragments in the metropolitan region of Curitiba, Paraná state, Brazil.

The objective of this trial was to characterize the assemblage structure of fruit flies (Diptera: Tephritidae) by determining the diversity and faunal indices. Fruit flies were collected for ten years between 2003 and 2015 in six municipalities of the Metropolitan Region of Curitiba, Paraná. The orchards were divided into three crop protection strategies: organic, conventional, and integrated pest management. The characteristic of each community was determined using the Shannon-Wiener, Margalef, and Pielou's indexes. The frequency, constancy, and dominance indexes were also determined. Biological material was analyzed using the explanatory variables: site, crop, plant protection strategy, and susceptibility period. A total of 8,089 fruit flies were collected, of which 4,681 were females. The species Anastrepha daciformis Bezzi, A. dissimilis Stone, A. distincta Greene and A. pickeli Lima were recorded for the first time in Paraná State, the occurrence of three other species was recorded for the first time in the Metropolitan Region of Curitiba (A. montei Lima, A. obliqua Macquart, and A. sororcula Zucchi), in addition A. fraterculus (Wiedemann), A. grandis (Macquart) and Ceratitis capitata (Wiedemann). A. fraterculus accounted for 98.5% of the collected species. Only A. fraterculus and A. sororcula were found in fruits. Fruit flies occurred predominantly in peach trees cultivated under organic conditions. The Shannon index indicated that the municipality of Cerro Azul had the highest diversity and richness whereas apple orchards had the lowest diversity. Crops cultivated under organic conditions exhibited the highest diversity whereas those cultivated under conventional conditions had the highest evenness index. The largest specimens number was collected during the period of crop susceptibility.

An integrated diagnostic setup for the morphological and molecular identification of the Ceratitis FAR complex (C. anonae, C. fasciventris, C. rosa, C. quilicii, Diptera, Tephritidae).

The Ceratitis FAR complex (Diptera, Tephritidae) includes four economically important frugivorous flies (Ceratitis anonae, Ceratitis fasciventris, Ceratitis quilicii, Ceratitis rosa) whose immature stages and adult females cannot be properly resolved through morphological identification. In order to develop a simplified molecular tool for the identification of two of these species (C. rosa, C. quilicii), we selected a subset of six microsatellite markers out of a panel of 16 loci that were previously developed for the molecular differentiation of the taxa within the complex. These six markers were first tested in silico and then used for the actual genotyping of C. quilicii and C. rosa, resulting in the correct identification of all male reference specimens. Here, we propose an integrated morphological and molecular setup for the identification of the four species of the FAR complex. The decision map relies on preliminary DNA barcoding or morphological identification (when possible) to exclude species not belonging to the complex followed by (a) morphological identification of all adult male specimens and female C. anonae, (b) molecular identification via a panel of 16 microsatellite markers for immature stages, damaged vouchers and samples potentially including adult female C. fasciventris/C. quilicii/C. rosa and (c) molecular identification via a reduced panel of six microsatellite markers for samples including only C. quilicii and C. rosa. This simplified diagnostic setup was profitably implemented in the framework of the ERAfrica fruit fly project and will help correctly identify species within the FAR complex for their early detection and monitoring.

The complete mitochondrial genome of Bactrocera biguttula (Bezzi) (Diptera: Tephritidae) and phylogenetic relationships with other Dacini.

Bactrocera biguttula is an African olive fruit fly that does not attack cultivated olives but rather develops in the fruits of wild species of Olea and Noronhia. The complete mitochondrial genome of an individual specimen was characterized in comparison to other Bactrocera. The phylogenetic relationships of B. biguttula with other Dacini were investigated, with special focus on B. oleae, an agricultural pest known to attack cultivated and wild olives. The sequence had a total length of 15,829 bp, and included the typical features of insect mitogenomes, similarly to the other Bactrocera analysed. Start codons included ATG, ATC, ATT, and TCG (in COI). The majority of stop codons (TAA) were fully encoded, whereas in some cases only TA or T were present. The complete sequence was biased towards A + T, with a positive AT-skew and a negative GC-skew. The predicted cloverleaf structure of tRNASer1 showed absence of the DHU arm, a common feature in insects and other Metazoans. Phylogenetic reconstruction showed that B. biguttula and B. oleae are sister species, having diverged from a common ancestor < 10 Myr ago. This result warrants future genomic comparisons between these two closely related species for investigating the specific adaptations to the different hosts.