Female semiochemicals stimulate male courtship but dampen female sexual receptivity.

Chemical communication plays a vital role in mate attraction and discrimination among many insect species. Here, we document a unique example of semiochemical parsimony, where four chemicals act as both aphrodisiacs and anti-aphrodisiacs in different contexts in Bactrocera dorsalis. Specifically, we identified four female-specific semiochemicals, ethyl laurate, ethyl myristate, ethyl cis-9-hexadecenoate, and ethyl palmitate, which serve as aphrodisiacs to attract male flies and arouse male courtship. Interestingly, these semiochemicals, when sexually transferred to males during mating, can function as anti-aphrodisiacs, inhibiting the receptivity of subsequent female mates. We further showed that the expression of elongase11, a key enzyme involved in the biosynthesis of these semiochemicals, is under the control of doublesex, facilitating the exclusive biosynthesis of these four semiochemicals in females and guaranteeing effective chemical communication. The dual roles of these semiochemicals not only ensure the attractiveness of mature females but also provide a simple yet reliable mechanism for female mate discrimination. These findings provide insights into chemical communication in B. dorsalis and add elements for the design of pest control programs.

A viral mutualist employs posthatch transmission for vertical and horizontal spread among parasitoid wasps.

Heritable symbionts display a wide variety of transmission strategies to travel from one insect generation to the next. Parasitoid wasps, one of the most diverse insect groups, maintain several heritable associations with viruses that are beneficial for wasp survival during their development as parasites of other insects. Most of these beneficial viral entities are strictly transmitted through the wasp germline as endogenous viral elements within wasp genomes. However, a beneficial poxvirus inherited by Diachasmimorpha longicaudata wasps, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), is not integrated into the wasp genome and therefore may employ different tactics to infect future wasp generations. Here, we demonstrated that transmission of DlEPV is primarily dependent on parasitoid wasps, since viral transmission within fruit fly hosts of the wasps was limited to injection of the virus directly into the larval fly body cavity. Additionally, we uncovered a previously undocumented form of posthatch transmission for a mutualistic virus that entails external acquisition and localization of the virus within the adult wasp venom gland. We showed that this route is extremely effective for vertical and horizontal transmission of the virus within D. longicaudata wasps. Furthermore, the beneficial phenotype provided by DlEPV during parasitism was also transmitted with perfect efficiency, indicating an effective mode of symbiont spread to the advantage of infected wasps. These results provide insight into the transmission of beneficial viruses among insects and indicate that viruses can share features with cellular microbes during their evolutionary transitions into symbionts.

miR-275/305 cluster is essential for maintaining energy metabolic homeostasis by the insulin signaling pathway in Bactrocera dorsalis.

Increasing evidence indicates that miRNAs play crucial regulatory roles in various physiological processes of insects, including systemic metabolism. However, the molecular mechanisms of how specific miRNAs regulate energy metabolic homeostasis remain largely unknown. In the present study, we found that an evolutionarily conserved miR-275/305 cluster was essential for maintaining energy metabolic homeostasis in response to dietary yeast stimulation in Bactrocera dorsalis. Depletion of miR-275 and miR-305 by the CRISPR/Cas9 system significantly reduced triglyceride and glycogen contents, elevated total sugar levels, and impaired flight capacity. Combined in vivo and in vitro experiments, we demonstrated that miR-275 and miR-305 can bind to the 3'UTR regions of SLC2A1 and GLIS2 to repress their expression, respectively. RNAi-mediated knockdown of these two genes partially rescued metabolic phenotypes caused by inhibiting miR-275 and miR-305. Furthermore, we further illustrated that the miR-275/305 cluster acting as a regulator of the metabolic axis was controlled by the insulin signaling pathway. In conclusion, our work combined genetic and physiological approaches to clarify the molecular mechanism of metabolic homeostasis in response to different dietary stimulations and provided a reference for deciphering the potential targets of physiologically important miRNAs in a non-model organism.

miR-309a is a regulator of ovarian development in the oriental fruit fly Bactrocera dorsalis.

Fecundity is arguably one of the most important life history traits, as it is closely tied to fitness. Most arthropods are recognized for their extreme reproductive capacity. For example, a single female of the oriental fruit fly Bactrocera dorsalis, a highly invasive species that is one of the most destructive agricultural pests worldwide, can lay more than 3000 eggs during its life span. The ovary is crucial for insect reproduction and its development requires further investigation at the molecular level. We report here that miR-309a is a regulator of ovarian development in B. dorsalis. Our bioinformatics and molecular studies have revealed that miR-309a binds the transcription factor pannier (GATA-binding factor A/pnr), and this activates yolk vitellogenin 2 (Vg 2) and vitellogenin receptor (VgR) advancing ovarian development. We further show that miR-309a is under the control of juvenile hormone (JH) and independent from 20-hydroxyecdysone. Thus, we identified a JH-controlled miR-309a/pnr axis that regulates Vg2 and VgR to control the ovarian development. This study has further enhanced our understanding of molecular mechanisms governing ovarian development and insect reproduction. It provides a background for identifying targets for controlling important Dipteran pests.

The Global Epidemic of Bactrocera Pests: Mixed-Species Invasions and Risk Assessment.

Throughout the past century, the global spread of Bactrocera pests has continued to pose a significant threat to the commercial fruit and vegetable industry, resulting in substantial costs associated with both control measures and quarantine restrictions. The increasing volume of transcontinental trade has contributed to an escalating rate of Bactrocera pest introductions to new regions. To address the worldwide threat posed by this group of pests, we first provide an overview of Bactrocera. We then describe the global epidemic, including border interceptions, species diagnosis, population genetics, geographical expansion, and invasion tracing of Bactrocera pests. We further consider the literature concerning the invasion co-occurrences, life-history flexibility, risk assessment, bridgehead effects, and ongoing implications of invasion recurrences, as well as a case study of Bactrocera invasions of California. Finally, we call for global collaboration to effectively monitor, prevent, and control the ongoing spread of Bactrocera pests and to share experience and knowledge to combat it.

Urban socioeconomic variation influences the ecology and evolution of trophic interactions.

As urbanization expands, it is becoming increasingly important to understand how anthropogenic activity is affecting ecological and evolutionary processes. Few studies have examined how human social patterns within cities can modify eco-evolutionary dynamics. We tested how socioeconomic variation corresponds with changes in trophic interactions and natural selection on prey phenotypes using the classic interaction between goldenrod gall flies (Eurosta solidaginis) and their natural enemies: birds, beetles, and parasitoid wasps. We sampled galls from 84 sites across neighbourhoods with varying socioeconomic levels, and quantified the frequency of predation/parasitism on flies and natural selection by each enemy. We found that bird predation was higher in the highest income neighbourhoods, increasing the strength of selection for smaller galls. Wasp and beetle attack, but not their strength of selection, increased in lower income neighbourhoods. We show that socioeconomic variation in cities can have strong unintended consequences for the ecology and evolution of trophic interactions.

Variation in Thermal Sensitivity of Diapause Development among Individuals and over Time Predicts Life History Timing in a Univoltine Insect.

AbstractPhysiological time is important for understanding the development and seasonal timing of ectothermic animals but has largely been applied to developmental processes that occur during spring and summer, such as morphogenesis. There is a substantial knowledge gap in the relationship between temperature and development during winter, a season that is increasingly impacted by climate change. Most temperate insects overwinter in diapause, a developmental process with little obvious morphological change. We used principles from the physiological time literature to measure and model the thermal sensitivity of diapause development rate in the apple maggot fly Rhagoletis pomonella, a univoltine fly whose diapause duration varies substantially within and among populations. We show that diapause duration can be predicted by modeling a relationship between temperature and development rate that is shifted toward lower temperatures compared with typical models of morphogenic, nondiapause development. However, incorporating interindividual variation and ontogenetic variation in the temperature-to-development rate relationship was critical for accurately predicting fly emergence, as diapause development proceeded more quickly at high temperatures later in diapause. We conclude that the conceptual framework may be flexibly applied to other insects and discuss possible mechanisms of diapause timers and implications for phenology with warming winters.

Transmission mode predicts coinfection patterns of insect-specific viruses in field populations of the Queensland fruit fly.

Insect-specific viruses (ISVs) can affect insect health and fitness, but can also interact with other insect-associated microorganisms. Despite this, ISVs are often studied in isolation from each other, in laboratory populations. Consequently, their diversity, prevalence and associations with other viruses in field populations are less known, yet these parameters are important to understanding virus epidemiology. To help address this knowledge gap, we assessed the diversity, prevalence and coinfections of three ISVs (horizontally transmitted cripavirus, biparentally transmitted sigmavirus and maternally transmitted iflavirus) in 29 field populations of Queensland fruit fly, Australia's most significant horticultural pest, in the context of their different transmission modes. We detected new virus variant diversity. In contrast to the very high virus prevalence in laboratory populations, 46.8% of 293 field flies carried one virus and 4.8% had two viruses. Cripavirus and sigmavirus occurred in all regions, while iflavirus was restricted to subtropical and tropical regions. Cripavirus was most prevalent (37.5%), followed by sigmavirus (13.7%) and iflavirus (4.4%). Cripavirus coinfected some flies with either one of the two vertically transmitted viruses. However, sigmavirus did not coinfect individuals with iflavirus. Three different modelling approaches detected negative association patterns between sigmavirus and iflavirus, consistent with the absence of such coinfections in laboratory populations. This may be linked with their maternal transmission and the ineffective paternal transmission of sigmavirus. Furthermore, we found that, unlike sigmavirus and iflavirus, cripavirus load was higher in laboratory than field flies. Laboratory and mass-rearing conditions may increase ISV prevalence and load due to increased transmission opportunities. We conclude that a combination of field and laboratory studies is needed to uncover ISV interactions and further our understanding of ISV epidemiology.

A classic screening marker does not affect antennal electrophysiology but strongly regulates reproductive behaviours in Bactrocera dorsalis.

The key phenotype white eye (white) has been used for decades to selectively remove females before release in sterile insect technique programs and as an effective screening marker in genetic engineering. Bactrocera dorsalis is a representative tephritid pest causing damage to more than 150 fruit crops. Yet, the function of white in important biological processes remains unclear in B. dorsalis. In this study, the impacts of the white gene on electrophysiology and reproductive behaviour in B. dorsalis were tested. The results indicated that knocking out Bdwhite disrupted eye pigmentation in adults, consistent with previous reports. Bdwhite did not affect the antennal electrophysiology response to 63 chemical components with various structures. However, reproductive behaviours in both males and females were significantly reduced in Bdwhite-/- . Both pre-copulatory and copulation behaviours were significantly reduced in Bdwhite-/- , and the effect was male-specific. Mutant females significantly delayed their oviposition towards γ-octalactone, and the peak of oviposition behaviour towards orange juice was lost. These results show that Bdwhite might not be an ideal screening marker in functional gene research aiming to identify molecular targets for behaviour-modifying chemicals. Instead, owing to its strong effect on B. dorsalis sexual behaviours, the downstream genes regulated by Bdwhite or the genes from white-linked areas could be alternate molecular targets that promote the development of better behavioural modifying chemical-based pest management techniques.

Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain.

Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.

The hAT family hopper transposon exists as highly similar yet discontinuous elements in the Bactrocera tephritid fly genus.

The hAT family transposable element, hopper, was originally discovered as a defective 3120-bp full-length element in a wild-type strain of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), and subsequently a functional 3131-bp element, hopperBdwe, was isolated from a white eye mutant strain. The latter study showed that closely related elements exist in melonfly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), a closely related subgenus, suggesting that hopper could have a widespread presence in the Bactrocera genus. To further understand the distribution of hopper within and beyond the B. dorsalis species complex, primer pairs from hopperBdwe and its adjacent genomic insertion site were used to survey the presence and relatedness of hopper in five species within the complex and four species beyond the complex. Based on sequence identity of a 1.94 kb internal nucleotide sequence, the closest relationships were with mutated elements from B. dorsalis s.s. and species synonymized with B. dorsalis including B. papayae, B. philippinensis and B. invadens, ranging in identity between 88.4% and 99.5%. Notably, Bactrocera carambolae (Drew & Hancock) (Diptera: Tephritidae), which is most closely related to B. dorsalis beyond the synonymized species, shared hopper identities of 97.3%-99.5%. Beyond the B. dorsalis complex, Z. cucurbitae, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) and Bactrocera zonata (Saunders) (Diptera: Tephritidae) shared identities of 83.1%-97.1%, while hopper was absent from the Bactrocera oleae (Gmelin) (Diptera: Tephritidae) strain tested. While the functional autonomous hopperBdwe element was not detected in these species, another closely related hopper element isolated from a B. dorsalis genetic sexing strain has an uninterrupted transposase open reading frame. The discontinuous presence of hopper in the Bactrocera genus has implications for its use for genomic manipulation and understanding the phylogenetic relationship of these species.

Suppressing the expression of glutathione S-transferase gene GSTd10 increases the sensitivity of Zeugodacus cucurbitae against ß-cypermethrin.

Zeugodacus cucurbitae Coquillett (Diptera: Tephritidae) is an agriculturally and economically important pest worldwide that has developed resistance to ß-cypermethrin. Glutathione S-transferases (GSTs) have been reported to be involved in the detoxification of insecticides in insects. We have found that both ZcGSTd6 and ZcGSTd10 were up-regulated by ß-cypermethrin induction in our previous study, so we aimed to explore their potential relationship with ß-cypermethrin tolerance in this study. The heterologous expression of ZcGSTd6 and ZcGSTd10 in Escherichia coli showed significantly high activities against 1-chloro-2,4-dinitrobenzene (CDNB). The kinetic parameters of ZcGSTd6 and ZcGSTd10 were determined by Lineweaver-Burk. The Vmax and Km of ZcGSTd6 were 0.50 µmol/min·mg and 0.3 mM, respectively. The Vmax and Km of ZcGSTd10 were 1.82 µmol/min·mg and 0.53 mM. The 3D modelling and molecular docking results revealed that ß-cypermethrin exhibited a stronger bounding to the active site SER-9 of ZcGSTd10. The sensitivity to ß-cypermethrin was significantly increased by 18.73% and 27.21%, respectively, after the knockdown of ZcGSTd6 and ZcGSTd10 by using RNA interference. In addition, the inhibition of CDNB at 50% (IC50) and the inhibition constants (Ki) of ß-cypermethrin against ZcGSTd10 were determined as 0.41 and 0.33 mM, respectively. The Ki and IC50 of ß-cypermethrin against ZcSGTd6 were not analysed. These results suggested that ZcGSTd10 could be an essential regulator involved in the tolerance of Z. cucurbitae to ß-cypermethrin.

Genomic signals of local adaptation across climatically heterogenous habitats in an invasive tropical fruit fly (Bactrocera tryoni).

Local adaptation plays a key role in the successful establishment of pest populations in new environments by enabling them to tolerate novel biotic and abiotic conditions experienced outside their native range. However, the genomic underpinnings of such adaptive responses remain unclear, especially for agriculturally important pests. We investigated population genomic signatures in the tropical/subtropical Queensland fruit fly, Bactrocera tryoni, which has an expanded range encompassing temperate and arid zones in Australia, and tropical zones in the Pacific Islands. Using reduced representation sequencing data from 28 populations, we detected allele frequency shifts associated with the native/invasive status of populations and identified environmental factors that have likely driven population differentiation. We also determined that precipitation, temperature, and geographic variables explain allelic shifts across the distribution range of B. tryoni. We found spatial heterogeneity in signatures of local adaptation across various climatic conditions in invaded areas. Specifically, disjunct invasive populations in the tropical Pacific Islands and arid zones of Australia were characterised by multiple significantly differentiated single nucleotide polymorphisms (SNPs), some of which were associated with genes with well-understood function in environmental stress (e.g., heat and desiccation) response. However, invasive populations in southeast Australian temperate zones showed higher gene flow with the native range and lacked a strong local adaptive signal. These results suggest that population connectivity with the native range has differentially affected local adaptive patterns in different invasive populations. Overall, our findings provide insights into the evolutionary underpinnings of invasion success of an important horticultural pest in climatically distinct environments.

Impact of Larval Food Source on the Stability of the Bactrocera dorsalis Microbiome.

Bacterial symbionts are crucial to the biology of Bactrocera dorsalis. With larval diet (fruit host) being a key factor that determines microbiome composition and with B. dorsalis using more than 400 fruits as hosts, it is unclear if certain bacterial symbionts are preserved and are passed on to B. dorsalis progenies despite changes in larval diet. Here, we conducted a fly rearing experiment to characterize diet-induced changes in the microbiome of female B. dorsalis. In order to explicitly investigate the impacts of larval diet on the microbiome, including potential stable bacterial constituents of B. dorsalis, we performed 16S rRNA sequencing on the gut tissues of teneral female flies reared from four different host fruits (guava, mango, papaya, and rose apple) infested using a single cohort of wild B. dorsalis that emerged from tropical almond (mother flies). Although B. dorsalis-associated microbiota were predominantly shaped by the larval diet, some major bacterial species from the mother flies were retained in progenies raised on different larval diets. With some variation, Klebsiella (ASV 1 and 2), Morganella (ASV 3), and Providencia (ASV 6) were the major bacterial symbionts that were stable and made up 0.1-80% of the gut and ovipositor microbiome of female teneral flies reared on different host fruits. Our results suggest that certain groups of bacteria are stably associated with female B. dorsalis across larval diets. These findings provide a basis for unexplored research on symbiotic bacterial function in B. dorsalis and may aid in the development of novel management techniques against this devastating pest of horticultural importance.

Shaping the Microbial Landscape: Parasitoid-Driven Modifications of Bactrocera dorsalis Microbiota.

Koinobiont endoparasitoids regulate the physiology of their hosts through altering host immuno-metabolic responses, processes which function in tandem to shape the composition of the microbiota of these hosts. Here, we employed 16S rRNA and ITS amplicon sequencing to investigate whether parasitization by the parasitoid wasps, Diachasmimorpha longicaudata (Ashmaed) (Hymenoptera: Braconidae) and Psyttalia cosyrae (Wilkinson) (Hymenoptera: Braconidae), induces gut dysbiosis and differentially alter the gut microbial (bacteria and fungi) communities of an important horticultural pest, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). We further investigated the composition of bacterial communities of adult D. longicaudata and P. cosyrae to ascertain whether the adult parasitoids and parasitized host larvae share microbial taxa through transmission. We demonstrated that parasitism by D. longicaudata induced significant gut perturbations, resulting in the colonization and increased relative abundance of pathogenic gut bacteria. Some pathogenic bacteria like Stenotrophomonas and Morganella were detected in both the guts of D. longicaudata-parasitized B. dorsalis larvae and adult D. longicaudata wasps, suggesting a horizontal transfer of microbes from the parasitoid to the host. The bacterial community of P. cosyrae adult wasps was dominated by Arsenophonus nasoniae, whereas that of D. longicaudata adults was dominated by Paucibater spp. and Pseudomonas spp. Parasitization by either parasitoid wasp was associated with an overall reduction in fungal diversity and evenness. These findings indicate that unlike P. cosyrae which is avirulent to B. dorsalis, parasitization by D. longicaudata induces shifts in the gut bacteriome of B. dorsalis larvae to a pathobiont-dominated community. This mechanism possibly enhances its virulence against the pest, further supporting its candidacy as an effective biocontrol agent of this frugivorous tephritid fruit fly pest.

Antennal transcriptome analysis of Psyttalia incisi (silvestri) (Hymenoptera: Braconidae): identification and tissue expression profiling of candidate odorant-binding protein genes.

BACKGROUND: Olfaction plays an important role in host-seeking by parasitoids, as they can sense chemical signals using sensitive chemosensory systems. Psyttalia incisi (Silvestri) (Hymenoptera: Braconidae) is the dominant parasitoid of Bactrocera dorsalis (Hendel) in fruit-producing regions of southern China. The olfactory behavior of P. incisi has been extensively studied; however, the chemosensory mechanisms of this species are not fully understood. RESULTS: Bioinformatics analysis of 64,515 unigenes from the antennal transcriptome of both male and female adults P. incisi identified 87 candidate chemosensory genes. These included 13 odorant-binding proteins (OBPs), seven gustatory receptors (GRs), 55 odorant receptors (ORs), 10 ionotropic receptors (IRs), and two sensory neuron membrane proteins (SNMPs). Phylogenetic trees were constructed to predict evolutionary relationships between these chemosensory genes in hymenopterans. Moreover, the tissue expression profiles of 13 OBPs were analyzed by quantitative real-time PCR, revealing high expression of seven OBPs (1, 3, 6, 7, 8, 12, and 13) in the antennae. CONCLUSION: This study represents the first identification of chemosensory genes and the determination of their expression patterns in different tissues of P. incisi. These results contribute to a better understanding of the function of the chemosensory system of this parasitoid species.

Insights on reproduction-related genes in the striped fruit fly, Zeugodacus scutellata (Hendel) (Diptera: Tephritidae).

The striped fruit fly, Zeugodacus scutellata is a significant pest in East and Southeast Asia by damaging Cucurbitaceae blossoms and fruits. To control this pest, a novel strategy to suppress the gene(s) associated with sexually dimorphic phenotypes has been devised and implemented in a laboratory scale. However, comprehensive transcriptomic analysis related to this sex differentiation of Z. scutellata was necessary to determine effective target genes for the genetic control. We performed de novo assembly of the transcript obtained by paired-end sequencing using an Illumina HiSeq platform and let to 217,967 unigenes (i.e., unique genes) with a minimum length of 200 bp. The female produced 31, 604, 442 reads with 97.93% of Q20, 94.76% of Q30, and the male produced 130, 592, 828 reads with 97.93% of Q20 and 94.76 of Q30%. The differentially expressed genes were used to predict genetic factors associated with sex differentiation, which included Rho1, extra-macrochaetae (emc), hopscotch (hop), doublesex (dsx), sex-lethal (sxl), transformer-2 (tra-2), testis-specific serine/threonine-protein kinase (tssk1), tektin1 (tkt1) and 2 (tkt2), odorant binding proteins (OBPs), fruitless (fru), vitellogenin receptor, and hormone receptors in Z. scutellata. In addition, this transcriptome analysis provides the additional gene associated with sex determination and mating behaviors, which would be applied to develop a novel sterile insect technique against Z. scutellata.

RNA virus diversity and prevalence in field and laboratory populations of melon fly throughout its distribution.

Insects have a rich diversity of RNA viruses that can either cause acute infections or persist in host populations without visible symptoms. The melon fly, Zeugodacus cucurbitae (Tephritidae) causes substantial economic losses through infestation of diverse cucurbit and other crops. Of Indomalayan origin, it is now established in many tropical regions of the world. The virome diversity of Z. cucurbitae is largely unknown across large parts of its distribution, including the Indian subcontinent. We have analysed three transcriptomes each of one field-collected and one laboratory-reared Z. cucurbitae population from Bangalore (India) and discovered genomes of ten putative RNA viruses: two sigmaviruses, one chimbavirus, one cripavirus, one noda-like virus, one nora virus, one orbivirus, one partiti-like virus, one sobemovirus and one toti-like virus. Analysis of the only available host genome of a Hawaiian Z. cucurbitae population did not detect host genome integration of the detected viruses. While all ten viruses were found in the Bangalore field population only seven were detected in the laboratory population, indicating that these seven may cause persistent covert infections. Using virus-specific RNA-dependent RNA polymerase gene primers, we detected nine of the RNA viruses with an overall low variant diversity in some but not all individual flies from four out of five Indian regions. We then screened 39 transcriptomes of Z. cucurbitae laboratory populations from eastern Asia (Guangdong, Hainan, Taiwan) and the Pacific region (Hawaii), and detected seven of the ten virus genomes. We found additional genomes of a picorna-like virus and a negev-like virus. Hawaii as the only tested population from the fly's invasive range only had one virus. Our study provides evidence of new and high RNA virus diversity in Indian populations within the original range of Z. cucurbitae, as well as the presence of persistent covert infections in laboratory populations. It builds the basis for future research of tephritid-associated RNA viruses, including their host effects, epidemiology and application potential in biological control.

Rearing and 60Co radiation do not affect attractiveness but alter the volatile profiles released by Anastrepha obliqua calling males.

Calling males of Anastrepha obliqua release volatile compounds to attract conspecific males to form leks and females to mate. Male volatiles from Mexican and Brazilian populations of A. obliqua have been previously identified. However, there are differences in the number and identity of volatile compounds between the populations. These differences in volatile profiles may be due to male origin (e.g. wild or mass-reared flies) or methodological issues (e.g. sampling techniques). In this study, we evaluated the attractiveness of wild, laboratory non-irradiated, and laboratory-irradiated flies under semi-field conditions. Male volatiles were collected using dynamic headspace sampling (DHS) and solid-phase microextraction (SPME) techniques, and identified using gas chromatography-coupled mass spectrometry. The results showed no difference in the attractiveness of wild, laboratory non-irradiated, and irradiated males to females. However, the number of captured females differed according to the origin; wild and non-irradiated females were captured more frequently than the irradiated flies. A total of 21 compounds were found using SPME, whereas only 12 were collected using DHS, although the relative amounts of these compounds were higher than those obtained using the former sampling technique. In addition, only laboratory non-irradiated males released α-pinene and menthol, which have not been previously reported in this fruit fly species. Additionally, we identified novel compounds in A. obliqua; however, certain compounds previously reported were not detected. This study suggests that despite the qualitative and quantitative variations in the volatile profiles of A. obliqua males, their attractiveness was unaffected.