On the complementarity of DNA barcoding and morphology to distinguish benign endemic insects from possible pests: the case of Dirioxa pornia and the tribe Acanthonevrini (Diptera: Tephritidae: Phytalmiinae) in Australia.

Fruit flies are considered economically important insects due to some species being agricultural pests. However, morphological identification of fruit fly adults and larvae can be difficult requiring a high level of taxonomic expertise, with misidentifications causing problematic false-positive/negative results. While destructive molecular techniques can assist with the identification process, these often cannot be applied where it is mandatory to retain a voucher reference specimen. In this work, we non-destructively (and partial-destructively) processed larvae and adults mostly belonging to the species Dirioxa pornia (Walker, 1849), of the poorly studied nonpest fruit fly tribe Acanthonevrini (Tephritidae) from Australia, to enable molecular identifications whilst retaining morphological vouchers. By retaining the morphological features of specimens, we confirmed useful characters for genus/species-level identification, contributing to improved accuracy for future diagnostics using both molecular and morphological approaches. We provide DNA barcode information for three species of Acanthonevrini known from Australia, which prior to our study was only available for a single species, D. pornia. Our specimen examinations provide new distribution records for three nonpest species: Acanthonevroides variegatus Permkam and Hancock, 1995 in South Australia, Acanthonevroides basalis (Walker, 1853) and D. pornia in Victoria, Australia; as well as new host plant records for D. pornia, from kangaroo apple, apricot and loquat.

Sensilla on antenna and maxillary palp of Neoceratitis asiatica (Diptera: Tephritidae).

Neoceratitis asiatica (Diptera: Tephritidae) is a disastrous pest for wolfberry production in China. In our preliminary field trapping experiment, both female and male N. asiatica adults were captured by male-produced sex attractant. To provide a solid background for studying mechanism of olfaction, the sensilla of antenna and maxillary palp of N. asiatica adults were studied by means of scanning electron microscopy. Both sexes have 3 antennal segments, including scape, pedicel, and flagellum, while arista is arisen from the proximal dorsal ridge of the flagellum. Two types of sensilla were found on the scape and pedicel, sensilla microtrichia (AnMi) and sensilla chaetica (AnCh). There are five types of sensilla on the flagellum, sensilla basiconica (AnB), clavate sensilla (AnCl), sensilla trichoidea (AnTr), sensilla coeloconica (AnCo), and AnMi. Three types of sensilla were observed on maxillary palp, sensilla basiconica (MpB), sensilla microtrichia (MpMi), and sensilla chaetica (MpCh). AnMi and MpMi are the most abundant sensilla type on antenna and maxillary palp, respectively. With the exception of having longer flagellum in females, the dimension of antennal segments, type and number of sensilla, density of pores on the shaft of AnCl, AnCh, AnB and MpB, and at the base area of AnCh and MpCh didn't show differences between sexes. This lack of inter-sexual differences may be related to chemoreception of male-emitted sex pheromone and of host plant volatiles by both female and male adults.

Thermal effect on the fecundity and longevity of Bactrocera dorsalis adults and their improved oviposition model.

The oriental fruit fly, Bactrocera dorsalis, is a destructive polyphagous pest that causes damage to various fruit crops, and their distribution is currently expanding worldwide. Temperature is an important abiotic factor that influences insect population dynamics and distribution by affecting their survival, development, and reproduction. We examined the fecundity, pre-oviposition and oviposition periods, and longevity of adult B. dorsalis at various constant temperatures ranging from13°C to 35°C. The longevity of female B. dorsalis ranged from 116.8 days (18.8°C) to 22.4 days (34.9°C), and the maximum fecundity per female was 1,684 eggs at 28.1°C. Females were only able to lay eggs at 16.7°C to 34.9°C, and both the pre-oviposition and oviposition periods were different depending on the temperature. We modeled female reproduction in two oviposition models (OMs): 1) the current model developed by Kim and Lee, an OM composed of a fecundity model, age-specific survival model, and age-specific cumulative oviposition rate model, and 2) a two-phase OM modified the logic structure of the current model by separating pre-oviposition, so that oviposition was estimated with the female in oviposition phase who had complete pre-oviposition phase. The results of the two-phase OM provided more realistic outputs at lower and higher temperatures than those of the current model. We discussed the usefulness of the two-phase OM for the reproduction of insects with long pre-oviposition periods.

Influence of wing bands and behavior of Anastrepha fraterculus (Diptera: Tephritidae) by the presence of Megafreya sutrix (Araneae: Salticidae).

The objective of this study was to evaluate the influence of wing bands and the behavior of Anastrepha fraterculus in the presence of Megafreya sutrix. The first experiment used specimens of M. sutrix (n = 40), 20 males and 20 females of A. fraterculus and Musca domestica. The second experiment used 20 individuals of M. sutrix (10 males and 10 females) and 120 of A. fraterculus (20 males and 20 females) for each treatment. Marks were made on the wings of the flies: a group with painted wing bands (n = 40) with their wings fully painted; another with highlighted wing bands (n = 40) and normal bands (n = 40). Recordings were made for 15 min or until the fly was preyed upon. The analyses were performed through observation of the recordings and Chi-square test with Yates correction for continuity. The values found for predation of A. fraterculus were significant when compared to M. domestica when evaluating the predation factor, showing that, A. fraterculus is less predated than M. domestica. It was found that the wing patterns did not influence the predatory behavior of M. sutrix.

Integrated LC-MS and GC-MS-based untargeted metabolomics studies of the effect of azadirachtin on Bactrocera dorsalis larvae.

Azadirachtin exhibits excellent bioactivities against several hundred arthropods. However, current knowlege of its biochemical effect on B. dorsalis larvae is not deep enough. In this study, integrated LC-MS and GC-MS-based untargeted metabolomics were used to analyze the changes of endogenous metabolites and the biochemical effects of azadirachtin on B. dorsalis larvae. Azadirachtin has excellent bioactivities against B. dorsalis larvae in this study, leading to a longer developmental duration, lower survival rate, and low pupa weight. The effect of azadirachtin was investigated on a total of 22 and 13 differentially abundant metabolites in the LC-MS and GC-MS-based metabolomics results, are selected respectively. Pathway analysis indicated that 14 differentially enriched metabolic pathways, including seven influential pathways, are worthy of attention. Further integrated key metabolic pathway analysis showed that histidine metabolism, D-glutamine and D-glutamate metabolism, biotin metabolism, ascorbate and aldarate metabolism, pentose and glucuronate interconversions, and alanine, aspartate and glutamate metabolism in B. dorsalis larvae are significantly relevant pathways affected by azadirachtin. Although extrapolating the bioactivity results in this study to the practical project of B. dorsalis pest management in the field has limitations, it was found that azadirachtin has a significant effect on the primary metabolism of B. dorsalis larvae.

The oesophageal diverticulum of Dirioxa pornia studied through micro-CT scan, dissection and SEM studies.

BACKGROUND: Dirioxa pornia (Diptera, Tephritidae) (Island fly) is an Australian native species related to a number of pestiferous fruit flies but, unlike many of the pest species, has not been studied extensively due to its non-pest status. However, due to D. pornia's apparent reliance on the bacteria for survival it is an ideal species to undertake studies into interaction between Tephritid species and bacteria associated with the intestinal tract. The oesophageal diverticulum, which is a blind-ended protrusion of the oesophagus, has been studied, described and characterised in many other Tephritid species. Unlike many other species where the oesophageal diverticulum has been observed the organ was only observed in male D. pornia. It is speculated that this sexual dimorphism the organ may be the primary location to host beneficial bacteria in the involved in the production of the nuptial gift and the mating success of this Tephritid species. In case of D. pornia, however, no study on any area of the digestive system has been conducted. This study was conducted to locate and characterize the oesophageal diverticulum in D. pornia. A virtual dissection of the alimentary tract was made through micro-computer tomography studies. These studies were followed by dissection and scanning microscopy studies to elucidate the presence of bacteria. RESULTS: The oesophageal diverticulum of D. pornia is part of the foregut and distends from the oesophagus within the head of the fly. The shape of the oesophageal diverticulum corresponds with the Ceratitis type. Scanning microscopy studies of the oesophageal diverticulum show rod-shaped bacterial cells residing along with yeast cells in the lumen. The organ was only observed in male specimens. CONCLUSIONS: This study classifies the oesophageal diverticulum of D. pornia under the "Ceratitis type" of oesophageal diverticula in Tephritid species. The study also proves that micro-CT scanning is possible to locate soft tissues in Tephritid species and the Avizo® Fire software can be successfully used to visualize 3 dimensional (3D) images from x-rays. The methods used in this experiment can be used in future studies for visualising soft tissues of adult Tephritid species through micro tomography. There is sexual dimorphism with the organ only found in males. Finally this study shows that bacteria are present in the oesophageal diverticulum of D. pornia.

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.

De novo transcriptomic analysis of the alimentary tract of the tephritid gall fly, Procecidochares utilis.

The tephritid gall fly, Procecidochares utilis, is an important obligate parasitic insect of the malignant weed Eupatorium adenophorum which biosynthesizes toxic secondary metabolites. Insect alimentary tracts secrete several enzymes that are used for detoxification, including cytochrome P450s, glutathione S-transferases, and carboxylesterases. To explore the adaptation of P. utilis to its toxic host plant, E. adenophorum at molecular level, we sequenced the transcriptome of the alimentary tract of P. utilis using Illumina sequencing. Sequencing and de novo assembly yielded 62,443 high-quality contigs with an average length of 604 bp that were further assembled into 45,985 unigenes with an average length of 674 bp and an N50 of 983 bp. Among the unigenes, 30,430 (66.17%) were annotated by alignment against the NCBI non-redundant protein (Nr) database, while 16,700 (36.32%), 16,267 (35.37%), and 11,530 (25.07%) were assigned functions using the Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) databases, respectively. Using the comprehensive transcriptome data set, we manually identified several important gene families likely to be involved in the detoxification of toxic compounds including 21 unigenes within the glutathione S-transferase (GST) family, 22 unigenes within the cytochrome P450 (P450) family, and 16 unigenes within the carboxylesterase (CarE) family. Quantitative PCR was used to verify eight, six, and two genes of GSTs, P450s, and CarEs, respectively, in different P. utilis tissues and at different developmental stages. The detoxification enzyme genes were mainly expressed in the foregut and midgut. Moreover, the unigenes were higher expressed in the larvae, pupae, and 3-day adults, while they were expressed at lower levels in eggs. These transcriptomic data provide a valuable molecular resource for better understanding the function of the P. utilis alimentary canal. These identified genes could be pinpoints to address the molecular mechanisms of P. utilis interacting with toxic plant host.

Two Gut-Associated Yeasts in a Tephritid Fruit Fly have Contrasting Effects on Adult Attraction and Larval Survival.

Yeast-insect interactions have been well characterized in drosophilid flies, but not in tephritid fruit flies, which include many highly polyphagous pest species that attack ripening fruits. Using the Queensland fruit fly (Bactrocera tryoni) as our model tephritid species, we identified yeast species present in the gut of wild-collected larvae and found two genera, Hanseniaspora and Pichia, were the dominant isolates. In behavioural trials using adult female B. tryoni, a fruit-agar substrate inoculated with Pichia kluyveri resulted in odour emissions that increased the attraction of flies, whereas inoculation with Hanseniaspora uvarum, produced odours that strongly deterred flies, and both yeasts led to decreased oviposition. Larval development trials showed that the fruit-agar substrate inoculated with the 'deterrent odour' yeast species, H. uvarum, resulted in significantly faster larval development and a greater number of adult flies, compared to a substrate inoculated with the 'attractive odour' yeast species, P. kluyveri, and a yeast free control substrate. GC-MS analysis of volatiles emitted by H. uvarum and P. kluyveri inoculated substrates revealed significant quantitative differences in ethyl-, isoamyl-, isobutyl-, and phenethyl- acetates, which may be responsible for the yeast-specific olfactory responses of adult flies. We discuss how our seemingly counterintuitive finding that female B. tryoni flies avoid a beneficial yeast fits well with our understanding of female choice of oviposition sites, and how the contrasting behavioural effects of H. uvarum and P. kluyveri raises interesting questions regarding the role of yeast-specific volatiles as cues to insect vectors. A better understanding of yeast-tephritid interactions could assist in the future management of tephritid fruit fly pests through the formulation of new "attract and kill" lures, and the development of probiotics for mass rearing of insects in sterile insect control programs.

A new species of Gastrozona Bezzi (Diptera: Tephritidae: Dacinae: Gastrozonini) with an updated key to species from India.

Gastrozona nigrifemur sp. nov., is described from India. Postabdominal structures of G. fasciventris (both male and female), G. soror (female) and G. montana (male) are described and illustrated. An updated key to Indian species of Gastrozona is provided.

Redescription, lectotype designation and new records of Anastrepha luederwaldti Lima (Diptera, Tephritidae).

The previously poorly known species Anastrepha luederwaldti Lima, 1934 is redescribed based on a reexamination of the syntypes from São Paulo and additional specimens from Santa Catarina and Rio Grande do Sul, Brazil. A lectotype is designated.

Do Fruit Ripening Volatiles Enable Resource Specialism in Polyphagous Fruit Flies?

Frugivorous tephritid fruit flies have lineages with high levels of host generalism. These insects use olfaction to locate fruits, but how they are able to recognize the odors of so many different host species is poorly understood. We used a series of behavioral experiments to investigate the role of fruit ripening volatiles as host cues in the Queensland fruit fly, Bactrocera tryoni (Froggatt), a polyphagous pest in Australia. Odors of mature guava (Psidium guajava) attracted female and male flies more strongly than three other ripening stages and guava pulp. We analyzed volatiles from guava odor and selected eleven compounds, all of which elicited an electrophysiological response in the antenna of female flies. Three of these, ethyl acetate, ethyl butyrate, and ethyl propionate, were released at the highest rates from the most attractive ripening stage. In behavioral trials, these three esters were not attractive individually, whereas a combination was necessary and sufficient in attracting female flies. The three-component blend was as attractive as the entire 11-component blend, which without these key volatiles was not attractive. Moreover, injecting low ranking hosts (squash and cucumber) with the three volatiles increased attraction in ovipositing female flies. These fruit flies are classed as generalists, but like many polyphagous insects they could be regarded as resource specialists, preferring specific plant reproductive stages with predictable odor cues. Exploring olfaction from this perspective could improve our understanding of host choice in polyphagous insects, and the selection of volatiles to be used as attractants in insect pest management.

A new genus and species of Xyphosiini (Diptera: Tephritidae) from Iran.

Valera, a monotypic new genus (Tephritidae, Tephritinae, Xyphosiini) and Valera ariana n. sp., the type and only known species, are described from Iran. Systematic relationships with other Xyphosiini are discussed and a key to the genera of the tribe Xyphosiini is presented.

New species of Rhagoletotrypeta (Diptera: Tephritidae) from the Dominican Republic and southern Brazil and Paraguay.

Rhagoletotrypeta chapecensis Norrbom & Savaris, new species, and R. gelabertae Norrbom & Savaris, new species, are described and illustrated from specimens from southern Brazil (Paraná, Santa Catarina) and Paraguay, and from the Dominican Republic, respectively. The larvae of R. chapecensis develop in fruits of Celtis iguanaea (Jacq.) Sarg. (Ulmaceae). A key to the species of the xanthogastra species group is provided.

Three new species of Acidiostigma Hendel (Diptera: Tephritidae: Trypetinae) and an updated key to species from the eastern Palaearctic and Oriental Regions.

Three new species of Acidiostigma Hendel from China are described and illustrated. The new species are A. nigrofasciola Chen and Zhang, sp. nov., A. subpostsignatum Chen and Zhao, sp. nov. and A. tongmaiense Chen, sp. nov. An updated key to the known species of the genus is provided. The male of A. bimaculatum Wang and female of A. postsignatum (Chen) are newly recorded, described and illustrated. The biology and behaviour of A. bimaculatum Wang and A. postsignatum (Chen) are observed in the field and recorded for the first time.

Two new species and a new record of Bactrocera Macquart (Diptera: Tephritidae: Dacinae: Dacini) from India.

Two new species of Bactrocera Macquart, namely Bactrocera (Calodacus) harrietensis Ramani & David, sp. nov. and Bactrocera (Calodacus) chettalli David & Ranganath, sp. nov., are described from the Andaman and Nicobar Islands and Karnataka, India, respectively. Bactrocera (Zeugodacus) semongokensis Drew & Romig is recorded for the first time from India.

A new species and new records of Cryptodacus (Diptera: Tephritidae) from Colombia, Bolivia and Peru.

Cryptodacus bernardoi Rodriguez & Rodriguez, new species, is described from Colombia. It was reared from fruits of Phoradendron sp. near piperoides (Kunth) Trel. New distribution records are reported for Cryptodacus ornatus Norrbom from Colombia and Peru, for Cryptodacus trinotatus Norrbom & Korytkowski from Colombia, and for Cryptodacus obliquus Hendel from Bolivia and Peru. The female abdomen and terminalia of C. obliquus is described for the first time. The Norrbom & Korytkowski (2008)`s key to species was modified to include C. bernardoi n. sp.

New taxa of Carpomyini, with special emphasis on Goniglossum (Diptera: Tephritidae: Trypetinae).

Goniglossum Rondani, hitherto a monobasic genus, is reinstated from synonymy under Carpomya Costa. A second species, Goniglossum liat n. sp., is described nearly two centuries after the description of the first species and 160 years after the description of Goniglossum. G. liat is described from Israel (also known from Turkey) and compared with G. wiedemanni (Meigen), an essentially European species, which is the type-species of Goniglossum and the only other known congener. The two species differ in details of the mesonotal and pleural color pattern (e.g., tip of scutellum with three small black spots in G. wiedemanni, whereas with one large spot in G. liat), structure of the male and female terminalia (e.g., the aculeus of G. wiedemanni is distinctly narrower apically than that of G. liat, and its tip has significantly fewer teeth), and host plant associations (Bryonia alba L. and B. dioica Jacq. are hosts of G. wiedemanni, whereas Bryonia cretica L. and B. syriaca Boiss. are hosts of G. liat). Myiopardalis Bezzi is also reinstated from synonymy under Carpomya. C. tica Norrbom, originally described in Carpomya, is transferred to Norrbomella, new genus, as its type and only included species.

Toward an Automated Identification of Anastrepha Fruit Flies in the fraterculus group (Diptera, Tephritidae).

In this study, we assess image analysis techniques as automatic identifiers of three Anastrepha species of quarantine importance, Anastrepha fraterculus (Wiedemann), Anastrepha obliqua (Macquart), and Anastrepha sororcula Zucchi, based on wing and aculeus images. The right wing and aculeus of 100 individuals of each species were mounted on microscope slides, and images were captured with a stereomicroscope and light microscope. For wing image analysis, we used the color descriptor Local Color Histogram; for aculei, we used the contour descriptor Edge Orientation Autocorrelogram. A Support Vector Machine classifier was used in the final stage of wing and aculeus classification. Very accurate species identifications were obtained based on wing and aculeus images, with average accuracies of 94 and 95%, respectively. These results are comparable to previous identification results based on morphometric techniques and to the results achieved by experienced entomologists. Wing and aculeus images produced equally accurate classifications, greatly facilitating the identification of these species. The proposed technique is therefore a promising option for separating these three closely related species in the fraterculus group.