The Imd Pathway Is Involved in Antiviral Immune Responses in Drosophila

Cricket Paralysis virus (CrPV) is a member of the Dicistroviridae family of RNA viruses, which infect a broad range of insect hosts, including the fruit fly Drosophila melanogaster. Drosophila has emerged as an effective system for studying innate immunity because of its powerful genetic techniques and the high degree of gene and pathway conservation. Intra-abdominal injection of CrPV into adult flies causes a lethal infection that provides a robust assay for the identification of mutants with altered sensitivity to viral infection. To gain insight into the interactions between viruses and the innate immune system, we injected wild type flies with CrPV and observed that antimicrobial peptides (AMPs) were not induced and hemocytes were depleted in the course of infection. To investigate the contribution of conserved immune signaling pathways to antiviral innate immune responses, CrPV was injected into isogenic mutants of the Immune Deficiency (Imd) pathway, which resembles the mammalian Tumor Necrosis Factor Receptor (TNFR) pathway. Loss-of-function mutations in several Imd pathway genes displayed increased sensitivity to CrPV infection and higher CrPV loads. Our data show that antiviral innate immune responses in flies infected with CrPV depend upon hemocytes and signaling through the Imd pathway.     

An insect picornavirus may have genome organization similar to that of caliciviruses.

Computer-assisted analysis of the amino acid sequence of the product encoded by the sequenced 3' portion of the cricket paralysis virus (CrPV), an insect picornavirus, genome showed that this protein is homologous not to the RNA-directed RNA polymerases, as originally suggested, but to the capsid proteins of mammalian picornaviruses. Alignment of the CrPV protein sequence with those of picornavirus and calicivirus capsid proteins demonstrated that the sequenced portion of the insect picornavirus genome encodes the C-terminal part of VP3 and the entire VP1. Thus CrPV seems to have a genome organization distinct from that of other picornaviruses but closely resembling that of caliciviruses, with the capsid proteins encoded in the 3' part of the genome. On the other hand, the tentative phylogenetic trees generated from the VP3 alignment revealed grouping of CrPV with hepatitis A virus, a true picornavirus, not with caliciviruses. Thus CrPV may be a picornavirus with a calicivirus-like genome organization. Different options for CrPV genome expression are discussed.     

Aromatized to Find Mates: α-Pinene Aroma Boosts the Mating Success of Adult Olive Fruit Flies

Background

Contrary to other Tephritidae, female but also male olive flies, Bactrocera oleae release pheromones during their sexual communication. Alpha-pinene, a common plant volatile found in high amounts in unripe olive fruit and leaves has been detected as one of the major components of the female pheromone. However, possible effects of α-pinene and that of other host volatiles on the mating behavior of the olive fly have not been investigated.

Methodology

Using wild olive flies, reared on olive fruit for 3 generations in the laboratory, we explored whether exposure of male and female olive flies to α-pinene affects their sexual performance.

Results

Exposure of sexually mature adult olive flies to the aroma of α-pinene significantly increases the mating performance over non-exposed individuals. Interestingly, exposure to α-pinene boosts the mating success of both males and female olive flies.

Conclusions

This is the first report of such an effect on the olive fly, and the first time that a single plant volatile has been reported to induce such a phenomenon on both sexes of a single species. We discuss the possible associated mechanism and provide some practical implications.     

Cricket paralysis virus and drosophila C virus: serological analysis and comparison of capsid polypeptides and host range.

Five strains of Drosophila C virus (DCV) were found to be serologically indistinguishable. By using antisera against DVC strains and cricket paralysis virus (CrPV), a relationship was shown to exist between the two viruses. All DCV strains exhibited the same polypeptide profile when analyzed on SDS-polyacrylamide gels and, while basically similar, analysis of CrPV polypeptides revealed that they were slightly larger than those of DCV. Three strains of DCV could be distinguished from each other by their pathogenicity in Drosophila melanogaster or Galleria mellonella, while only CrPV was able to multiply in Gryllus bimaculatus. Also, CrPV but not DCV, could multiply in an established cell line of Lymantria dispar.     

昆虫小RNA病毒研究进展

昆虫小RNA病毒依据基因组结构特点分为家蚕软化病毒组、蟋蟀痹病毒组和豌豆蚜病毒组。昆虫小RNA病毒蛋白质翻译具有独特性：不依赖于帽子结构,不需要启始因子eIF1、eIF1A和帽子结合蛋白eIF4E;而且蟋蟀痹病毒组和豌豆蚜病毒组结构蛋白翻译从内部核糖体进入位点独立启始,启始密码子是CUU。该文还简介了昆虫小RNA病毒复制机理及与其它动、植物小RNA病毒或类小RNA病毒的亲缘关系等的研究进展。     

Mediterranean Fruit Fly as a Potential Vector of Bacterial Pathogens

The Mediterranean fruit fly (Ceratitis capitata) is a cosmopolitan pest of hundreds of species of commercial and wild fruits. It is considered a major economic pest of commercial fruits in the world. Adult Mediterranean fruit flies feed on all sorts of protein sources, including animal excreta, in order to develop eggs. After reaching sexual maturity and copulating, female flies lay eggs in fruit by puncturing the skin with their ovipositors and injecting batches of eggs into the wounds. In view of the increase in food-borne illnesses associated with consumption of fresh produce and unpasteurized fruit juices, we investigated the potential of Mediterranean fruit fly to serve as a vector for transmission of human pathogens to fruits. Addition of green fluorescent protein (GFP)-tagged Escherichia coli to a Mediterranean fruit fly feeding solution resulted in a dose-dependent increase in the fly's bacterial load. Flies exposed to fecal material enriched with GFP-tagged E. coli were similarly contaminated and were capable of transmitting E. coli to intact apples in a cage model system. Washing contaminated apples with tap water did not eliminate the E. coli. Flies inoculated with E. coli harbored the bacteria for up to 7 days following contamination. Fluorescence microscopy demonstrated that the majority of fluorescent bacteria were confined along the pseudotrachea in the labelum edge of the fly proboscis. Wild flies captured at various geographic locations were found to carry coliforms, and in some cases presumptive identification of E. coli was made. These findings support the hypothesis that the common Mediterranean fruit fly is a potential vector of human pathogens to fruits.     

Crop domestication relaxes both top-down and bottom-up effects on a specialist herbivore

Domestication of crop plants selects for numerous traits that often distinguish them dramatically from their wild progenitors. In some cases, these modifications lead to increased herbivory, by enhancing their attractiveness to herbivorous insects or reducing the efficiency of natural enemies, or both. This study investigated the effects of fruit enlargement on the olive (Olea europaea L.), the specialist olive fruit fly, Bactrocera oleae (Rossi), and its specialized larval parasitoids. Wild olive fruit are small (<2 mm pulp thickness) and the larval parasitoids associated with B. oleae have short ovipositors (<3 mm), while cultivated fruit are larger (4–8 mm pulp thickness). Female flies allocate more offspring to large than to small fruit within or across different-sized commercial cultivars, without reducing the fitness of their offspring. Fly larvae move deeper into the olive pulp with their increasing age and fruit size. In contrast, the specialist larval parasitoid, Psyttalia lounsburyi (Silvestri), more effectively parasitizes hosts in smaller than larger fruit. The inverse relationship between the performance of the fly and its co-evolved parasitoids on fruit of increasing sizes indicates that olive cultivation favors the success of the fly by providing a better food resource and more enemy-free space. These findings offer some explanation for the failure of the decades-old classical biological efforts to manage B. oleae using specialized larval parasitoids in the Mediterranean Basin and provide further evidence that crop domestication can alter host–parasitoid interactions.     

Biosystematics of the Psyttalia concolor Species Complex (Hymenoptera: Braconidae: Opiinae): The Identity of Populations Attacking Ceratitis capitata (Diptera: Tephritidae) in Coffee in Kenya

The identity of a species of Psyttalia, a parasitoid of tephritid fruit flies in Kenya, was investigated. Individuals reared from coffee infested with Ceratitis capitata (Wiedemann) (Medfly) and two other tephritid species in Kenya were compared with individuals of Psyttalia concolor (Szépligeti) from a laboratory culture in Italy used in augmentative biological control of olive fly, Bactrocera oleae (Gmelin). Reciprocal crosses showed full compatibility, with production of viable female offspring. A preliminary morphometric analysis demonstrated that most individuals originating from Italy could be separated from most of those from Kenya based largely on differences in overall size.     

Trapping tephritid fruit flies (Diptera: Tephritidae) in citrus groves of Fujian Province of China

Certain tephritid fruit flies, such as the oriental fruit fly, Bactrocera dorsalis, the Chinese citrus fly, B. minax and the Japanese orange fly, B. tsuneonis (Diptera: Tephritidae: Dacinae), are destructive citrus pests in China. A two-year trapping study was conducted in pomelo, Citrus maxima, groves in Fujian Province of China. The objectives of this study were to investigate the species, the abundance of tephritid fruit flies in the orchards, as well as the efficacy of the selected lure traps to these flies. Four lure traps or devices, i.e. methyl eugenol + Steiner trap (ST), cuelure + ST, ammonium acetate + putrescine + ST, and sticky spheres, were deployed from June to November 2017 and April to October 2018. Six economically significant Dacini pests were trapped during the period. These flies are B. dorsalis, the melon fruit fly, Zeugodacus cucurbitae, the pumpkin fruit fly, Z. tau, the Malaysian fruit fly, B. latifrons, and other two species - B. rubigina and Z. scutellatus. B. dorsalis was the most abundant, accounting for more than 50% of the capture, followed by Z. cucurbitae. The remaining four species accounted for less than 2% of the total capture. B. minax and B. tsuneonis, two destructive citrus-damaging tephritid fruit flies in China, were not found during the trapping period. Methyl eugenol trapped the highest number of fruit flies, followed by cuelure.     

Differential Adsorption of Occluded and Nonoccluded Insect-Pathogenic Viruses to Soil-Forming Minerals

Soil represents the principal environmental reservoir of many insect-pathogenic viruses. We compared the adsorption and infectivity of one occluded and two nonoccluded viruses, Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) (Baculoviridae), Cricket paralysis virus (CrPV) (Dicistroviridae), and Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae), respectively, in mixtures with a selection of soil-forming minerals. The relative infective titers of HaSNPV and CrPV were unchanged or slightly reduced in the presence of different minerals compared to their titers in the absence of the mineral. In contrast, the infective titer of IIV-6 varied according to the mineral being tested. In adsorption studies, over 98% of HaSNPV occlusion bodies were adsorbed by all the minerals, and a particularly high affinity was observed with ferric oxide, attapulgite, and kaolinite. In contrast, the adsorption of CrPV and IIV-6 differed markedly with mineral type, with low affinity to bentonites and high affinity to ferric oxide and kaolinite. We conclude that interactions between soil-forming minerals and insect viruses appear to be most important in nucleopolyhedroviruses, followed by invertebrate iridescent viruses, and least important in CrPV, which may reflect the ecology of these pathogens. Moreover, soils with a high content of iron oxides or kaolinite would likely represent highly effective reservoirs for insect-pathogenic viruses.     

The Drosophila Nora virus is an enteric virus, transmitted via feces

The biology of the Drosophila viruses has not been intensely investigated. Here we have investigated the biology of the Nora virus, a persistent Drosophila virus. We find that injected Nora virus is able to replicate in the files, reaching a high titer that is maintained in the next generation. There is a remarkable variation in the viral loads of individual flies in persistently infected stocks; the titers can differ by three orders of magnitude. The Nora virus is mainly found in the intestine of infected flies, and the histology of these infected intestines show increased vacuolization. The virus is excreted in the feces and is horizontally transmitted. The Nora virus infection has a very mild effect on the longevity of the flies, and no significant effect on the number of eggs laid and the percent of eggs that develop to adults.     

Microsatellite analysis revealed a different approach of control of olive fly population (Bactrocera oleae) in Slovenia

The olive fruit fly (Bactrocera oleae Gmelin) is the most important olive pest in the north‐eastern Adriatic coast region. Despite the importance of olive production in the region, and the significance of the olive fruit fly, no information with respect to genetic diversity, population structure or dispersion patterns of this pest is available. The aims of this study were to investigate the genetic structure of the olive fly population in the Slovenian Istria region using microsatellite markers to determine olive fruit fly migration between locations and to establish an appropriate and effective strategy for controlling the pest population. Analysis was performed on a sample of 117 flies, collected from attacked olive fruits at three different locations. Olive fruit flies were genotyped using eight microsatellite loci. Sixty‐six alleles were identified over all microsatellite loci with an average of 8.25 alleles per locus. The population structure was determined with methods based on Bayesian principles using the BAPS 6.0 and STRUCTURE 2.3 programs. Genetic analysis confirmed unlimited migration and random mating between individuals of different microlocations, which suggests time‐coordinated first treatment in the region would be the best solution.     

Determination of volatile substances in olives and their effect on reproduction of the olive fruit fly

Females of the olive fruit fly Bactrocera oleae lay their eggs in olives mainly using fruit volatile stimuli. Using GC-MS analysis, we determined the chemical composition of the volatile blend emitted from field-collected olive fruit of cv. Megaritiki, at different stages of maturity. GC-MS analysis demonstrated qualitative and quantitative differences in the headspace blend emitted by the olive fruit. Certain chemicals such as toluene, n-octane, α-pinene, limonene, ethyl hexanol, nonanal n-dodecane, decanal and n-tetradecane were detected in greater amounts, irrespective of the growth stage of the fruit. The flies’ exposure to a number of these chemicals, such as n-octane and α-pinene, as well as to a mixture consisting of n-octane, α-pinene, limonene, ethyl hexanol, nonanal, n-dodecane, decanal and n–tetradecane favoured successful mating and egg production. The results may contribute to the improvement of the mass rearing of the fly, through these findings, which illustrate the positive effect that certain fruit volatile chemicals have on the fly's reproduction and offer a better understanding of the relation between the fly and the host fruit.     

Oxidative Stress Correlates with Wolbachia-Mediated Antiviral Protection in Wolbachia-Drosophila Associations

Wolbachia mediates antiviral protection in insect hosts and is being developed as a potential biocontrol agent to reduce the spread of insect-vectored viruses. Definition of the molecular mechanism that generates protection is important for understanding the tripartite interaction between host insect, Wolbachia, and virus. Elevated oxidative stress was previously reported for a mosquito line experimentally infected with Wolbachia, suggesting that oxidative stress is important for Wolbachia-mediated antiviral protection. However, Wolbachia experimentally introduced into mosquitoes impacts a range of host fitness traits, some of which are unrelated to antiviral protection. To explore whether elevated oxidative stress is associated with antiviral protection in Wolbachia-infected insects, we analyzed oxidative stress of five Wolbachia-infected Drosophila lines. In flies infected with protective Wolbachia strains, hydrogen peroxide concentrations were 1.25- to 2-fold higher than those in paired fly lines cured of Wolbachia infection. In contrast, there was no difference in the hydrogen peroxide concentrations in flies infected with nonprotective Wolbachia strains compared to flies cured of Wolbachia infection. Using a Drosophila mutant that produces increased levels of hydrogen peroxide, we investigated whether flies with high levels of endogenous reactive oxygen species had altered responses to virus infection and found that flies with high levels of endogenous hydrogen peroxide were less susceptible to virus-induced mortality. Taken together, these results suggest that elevated oxidative stress correlates with Wolbachia-mediated antiviral protection in natural Drosophila hosts.     

The Drosophila alcohol dehydrogenase gene may have evolved independently of the functionally homologous medfly, olive fly, and flesh fly genes

cDNAs for alcohol dehydrogenase (ADH) isozymes were cloned and sequenced from two tephritid fruit flies, the medfly Ceratitis capitata and the olive fly Bactrocera oleae. Because of the high sequence divergence compared with the Drosophila sequences, the medfly cDNAs were cloned using sequence information from the purified proteins, and the olive fly cDNAs were cloned by functional complementation in yeast. The medfly peptide sequences are about 83% identical to each other, and the corresponding mRNAs have the tissue distribution shown by the corresponding isozymes, ADH-1 and ADH-2. The olive fly peptide sequence is more closely related to medfly ADH-2. The tephritid ADHs share less than 40% sequence identity with Drosophila ADH and ADH-related genes but are >57% identical to the ADH of the flesh fly Sarcophaga peregrina, a more distantly related species. To explain this unexpected finding, it is proposed that the ADH: genes of the family Drosophilidae may not be orthologous to the ADH: genes of the other two families, Tephritidae and Sarcophagidae.     

Host genetics and pathogen species modulate infection-induced changes in social aggregation behaviour

Identifying how infection modifies host behaviours that determine social contact networks is important for understanding heterogeneity in infectious disease dynamics. Here, we investigate whether group social behaviour is modified during bacterial infection in fruit flies (Drosophila melanogaster) according to pathogen species, infectious dose, host genetic background and sex. In one experiment, we find that systemic infection with four different bacterial species results in a reduction in the mean pairwise distance within infected female flies, and that the extent of this change depends on pathogen species. However, susceptible flies did not show any evidence of avoidance in the presence of infected flies. In a separate experiment, we observed genetic- and sex-based variation in social aggregation within infected, same-sex groups, with infected female flies aggregating more closely than infected males. In general, our results confirm that bacterial infection induces changes in fruit fly behaviour across a range of pathogen species, but also highlight that these effects vary between fly genetic backgrounds and can be sex-specific. We discuss possible explanations for sex differences in social aggregation and their consequences for individual variation in pathogen transmission.     

The Cricket Paralysis Virus Suppressor Inhibits microRNA Silencing Mediated by the Drosophila Argonaute-2 Protein

Small RNAs are potent regulators of gene expression. They also act in defense pathways against invading nucleic acids such as transposable elements or viruses. To counteract these defenses, viruses have evolved viral suppressors of RNA silencing (VSRs). Plant viruses encoded VSRs interfere with siRNAs or miRNAs by targeting common mediators of these two pathways. In contrast, VSRs identified in insect viruses to date only interfere with the siRNA pathway whose effector Argonaute protein is Argonaute-2 (Ago-2). Although a majority of Drosophila miRNAs exerts their silencing activity through their loading into the Argonaute-1 protein, recent studies highlighted that a fraction of miRNAs can be loaded into Ago-2, thus acting as siRNAs. In light of these recent findings, we re-examined the role of insect VSRs on Ago-2-mediated miRNA silencing in Drosophila melanogaster. Using specific reporter systems in cultured Schneider-2 cells and transgenic flies, we showed here that the Cricket Paralysis virus VSR CrPV1-A but not the Flock House virus B2 VSR abolishes silencing by miRNAs loaded into the Ago-2 protein. Thus, our results provide the first evidence that insect VSR have the potential to directly interfere with the miRNA silencing pathway.     

Prevalence of Candidatus Erwinia dacicola in wild and laboratory olive fruit fly populations and across developmental stages

The microbiome of the olive fruit fly, Bactrocera oleae (Gmelin), a worldwide pest of olives (Olea europaea L.), has been examined for >100 yr as part of efforts to identify bacteria that are plant pathogens vectored by the fly or are beneficial endosymbionts essential for the fly's survival and thus targets for possible biological control. Because tephritid fruit flies feed on free-living bacteria in their environment, distinguishing between the transient, acquired bacteria of their diet and persistent, resident bacteria that are vertically transmitted endosymbionts is difficult. Several culture-dependent and -independent studies have identified a diversity of species in the olive fruit fly microbiome, but they have not distinguished the roles of the microbes. Candidatus Erwinia dacicola, has been proposed to be a coevolved endosymbiont of the olive fruit fly; however, this was based on limited samples from two Italian populations. Our study shows that C. Erwinia dacicola was present in all New and Old World populations and in the majority of individuals of all life stages sampled in 2 yr. Olive fruit flies reared on olives in the laboratory had frequencies of C. Erwinia dacicola similar to that of wild populations; however, flies reared on artificial diets containing antibiotics in the laboratory rarely had the endosymbiont. The relative abundance of C. Erwinia dacicola varied across development stages, being most abundant in ovipositing females and larvae. This uniform presence of C. Erwini dacicola suggests that it is a persistent, resident endosymbiont of the olive fruit fly.     

Kinetics of Colonization of Adult Queensland Fruit Flies (Bactrocera tryoni) by Dinitrogen-Fixing Alimentary Tract Bacteria

The average total population of bacteria remained constant in the alimentary tracts of adult laboratory-raised Queensland fruit flies (Bactrocera tryoni) although the insects had ingested large numbers of live bacteria as part of their diet. The mean number of bacteria (about 13 million) present in the gut of the insects from 12 to 55 days after emergence was not significantly modified when, at 5 days after emergence, the flies were fed antibiotic-resistant bacteria belonging to two species commonly isolated from the gut of field-collected B. tryoni. Flies were fed one marked dinitrogen-fixing strain each of either Klebsiella oxytoca or Enterobacter cloacae, and the gastrointestinal tracts of fed flies were shown to be colonized within 7 days by antibiotic-resistant isolates of K. oxytoca but not E. cloacae. The composition of the microbial population also appeared to be stable in that the distribution and frequency of bacterial taxa among individual flies exhibited similar patterns whether or not the flies had been bacteria fed. Isolates of either E. cloacae or K. oxytoca, constituting 70% of the total numbers, were usually dominant, with oxidative species including pseudomonads forming the balance of the population. Antibiotic-resistant bacteria could be spread from one cage of flies to the adjacent surfaces of a second cage within a few days and had reached a control group several meters distant by 3 weeks. Restriction of marked bacteria to the population of one in five flies sampled from the control group over the next 30 days suggested that the bacterial population in the gut of the insect was susceptible to alteration in the first week after emergence but that thereafter it entered a steady state and was less likely to be perturbed by the introduction of newly encountered strains. All populations sampled, including controls, included at least one isolate of the dinitrogen-fixing family Enterobacteriaceae; many were distinct from the marked strains fed to the flies. Nitrogenase activity detected by the acetylene reduction assay was associated with flies fed dinitrogen-fixing bacteria as well as with control groups given either no supplement or free access to a yeast hydrolysate preparation. Nitrogen fixed from the atmosphere may supplement the nutrition of the alimentary tract microbial population of B. tryoni. Transmission electron microscopy showed that the principal site of bacterial colonization in the abdominal alimentary tract was the lumen of the midgut inside the peritrophic membrane. No intracellular symbionts were seen in the gut tissues nor were bacteria found attached to the cuticular folds of the hindgut. The ultrastructure of the gut resembled that of other fly genera except that the intercellular spaces between rectal epithelial cells were more extensive, suggesting a role for unspecialized epithelium in water and solute uptake in B. tryoni.     

Physiological state influences the antennal response of Anastrepha obliqua to male and host volatiles

The sexual and host‐related behaviours of the fruit fly Anastrepha obliqua Macquart (Diptera: Tephritidae) are mediated by volatile compounds. However, whether the physiological state of this species affects its antennal and behavioural responses to semiochemicals is unknown. The effects of age, mating status, diet and the topical application of methoprene, a Juvenile hormone analogue (JHA), on the antennal sensitivity of this tephritid fruit fly species to selected male [(Z)‐3‐nonenol] and host fruit volatiles (ethyl benzoate, ethyl hexanoate, ethyl butyrate and trans‐β‐ocimene) are investigated using electroantennography (EAG). Overall, (Z)‐3‐nonenol and ethyl benzoate elicit the highest EAG responses in both sexes. Flies of both sexes aged 1, 5 and 10 days old show higher EAG responses to the tested compounds compared with flies aged 20 days old. Virgin females and males show higher EAG responses to volatile compounds than mated flies. Females and males fed with sugar plus protein show higher antennal responses to volatiles compared with flies fed sugar or protein alone. Flies of both sexes treated with methoprene show higher antennal responses than flies treated with acetone (control). These results suggest that the peripheral olfactory system in A. obliqua is modulated by the physiological state of the flies.