Search

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Infections of insects with insect-specific RNA viruses are common and can affect host fitness and health. Previously, persistent RNA virus infections were detected in tephritid fruit flies, including the Queensland fruit fly (Bactrocera tryoni), Australia's most significant horticultural pest. Their transmission modes and efficiency are unclear yet may influence virus epidemiology in field and laboratory populations. Using standard RT-PCR and RT-qPCR we detected iflavirus, cripavirus and sigmavirus in five laboratory populations recently established with field-collected B.tryoni. Virus absence in some individuals suggested that virus transmission is incomplete. Random virus segregation in an isofemale experiment resulted in the establishment of isofemale lines with and without iflavirus and cripavirus. In infected lines, viral loads normalised against host gene transcripts were variable, but did not differ between pupae and adults. Iflavirus and cripavirus were transmitted horizontally, with viruses detected (including at low viral loads) in many previously uninfected individuals after four days, and in most after 12 days cohabitation with infected flies. Iflavirus, but not cripavirus, was transmitted vertically, and surface-sterilised embryos contained high loads. Furthermore, high iflavirus loads in individual females resulted in high loads in their offspring. We demonstrated that viruses are highly prevalent in laboratory populations and that it is possible to establish and maintain uninfected fly lines for the assessment of virus transmission and host effects. This is important for pest management strategies such as the sterile insect technique which requires the mass-rearing of flies, as their fitness and performance may be affected by covert virus infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

Tephritid fruit flies are amongst the most devastating pests of horticulture, and Sterile Insect Technique (SIT) programs have been developed for their control. Their interactions with viruses are still mostly unexplored, yet, viruses may negatively affect tephritid health and performance in SIT programs, and, conversely, constitute potential biological control agents. Here we analysed ten transcriptome libraries obtained from laboratory populations of nine tephritid species from Australia (six species of Bactrocera, and Zeugodacus cucumis), Asia (Bactrocera dorsalis) and Europe (Ceratitis capitata). We detected new viral diversity, including near-complete (>99%) and partially complete (>80%) genomes of 34 putative viruses belonging to eight RNA virus families. On average, transcriptome libraries included 3.7 viruses, ranging from 0 (Z. cucumis) to 9 (B. dorsalis). Most viruses belonged to the Picornavirales, represented by fourteen Dicistroviridae (DV), nine Iflaviridae (IV) and two picorna-like viruses. Others were a virus from Rhabdoviridae (RV), one from Xinmoviridae (both Mononegavirales), several unclassified Negev- and toti-like viruses, and one from Metaviridae (Ortervirales). Using diagnostic PCR primers for four viruses found in the transcriptome of the Bactrocera tryoni strain bent wings (BtDV1, BtDV2, BtIV1, and BtRV1), we tested nine Australian laboratory populations of five species (B. tryoni, Bactrocera neohumeralis, Bactrocera jarvisi, Bactrocera cacuminata, C. capitata), and one field population each of B. tryoni, B. cacuminata and Dirioxa pornia. Viruses were present in most laboratory and field populations yet their incidence differed for each virus. Prevalence and co-occurrence of viruses in B. tryoni and B. cacuminata were higher in laboratory than field populations. This raises concerns about the potential accumulation of viruses and their potential health effects in laboratory and mass-rearing environments which might affect flies used in research and control programs such as SIT., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Wolbachia are widespread endosymbionts that affect arthropod reproduction and fitness. Mostly maternally inherited, Wolbachia are occasionally transferred horizontally. Previously, two Wolbachia strains were reported at low prevalence and titres across seven Australian tephritid species, possibly indicative of frequent horizontal transfer. Here, we performed whole-genome sequencing of field-caught Wolbachia-positive flies. Unexpectedly, we found complete mitogenomes of an endoparasitic strepsipteran, Dipterophagus daci, suggesting that Wolbachia in the flies are linked to concealed parasitization. We performed the first genetic characterization of D. daci and detected D. daci in Wolbachia-positive flies not visibly parasitized, and most but not all Wolbachia-negative flies were D. daci-negative, presumably reflecting polymorphism for the Wolbachia infections in D. daci. We dissected D. daci from stylopized flies and confirmed that Wolbachia infects D. daci, but also found Wolbachia in stylopized fly tissues, likely somatic, horizontally transferred, non-heritable infections. Furthermore, no Wolbachia cif and wmk genes were detected and very low mitogenomic variation in D. daci across its distribution. Therefore, Wolbachia may influence host fitness without reproductive manipulation. Our study of 13 tephritid species highlights that concealed early stages of strepsipteran parasitization led to the previous incorrect assignment of Wolbachia co-infections to tephritid species, obscuring ecological studies of this common endosymbiont and its horizontal transmission by parasitoids., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: The endosymbiont Wolbachia can manipulate arthropod reproduction and invade host populations by inducing cytoplasmic incompatibility (CI). Some host species are coinfected with multiple Wolbachia strains which may have sequentially invaded host populations by expressing different types of modular CI factor (cif) genes. The tephritid fruit fly Rhagoletis cerasi is a model for CI and Wolbachia population dynamics. It is associated with at least four Wolbachia strains in various combinations, with demonstrated (wCer2, wCer4), predicted (wCer1) or unknown (wCer5) CI phenotypes., Results: We sequenced and assembled the draft genomes of the Wolbachia strains wCer1, wCer4 and wCer5, and compared these with the previously sequenced genome of wCer2 which currently invades R. cerasi populations. We found complete cif gene pairs in all strains: four pairs in wCer2 (three Type I; one Type V), two pairs in wCer1 (both Type I) and wCer4 (one Type I; one Type V), and one pair in wCer5 (Type IV). Wolbachia genome variant analyses across geographically and genetically distant host populations revealed the largest diversity of single nucleotide polymorphisms (SNPs) in wCer5, followed by wCer1 and then wCer2, indicative of their different lengths of host associations. Furthermore, mitogenome analyses of the Wolbachia genome-sequenced individuals in combination with SNP data from six European countries revealed polymorphic mitogenome sites that displayed reduced diversity in individuals infected with wCer2 compared to those without., Conclusions: Coinfections with Wolbachia are common in arthropods and affect options for Wolbachia-based management strategies of pest and vector species already infected by Wolbachia. Our analyses of Wolbachia genomes of a host naturally coinfected by several strains unravelled signatures of the evolutionary dynamics in both Wolbachia and host mitochondrial genomes as a consequence of repeated invasions. Invasion of already infected populations by new Wolbachia strains requires new sets of functionally different cif genes and thereby may select for a cumulative modularity of cif gene diversity in invading strains. Furthermore, we demonstrated at the mitogenomic scale that repeated CI-driven Wolbachia invasions of hosts result in reduced mitochondrial diversity and hitchhiking effects. Already resident Wolbachia strains may experience similar cytoplasmic hitchhiking effects caused by the invading Wolbachia strain., (© 2021. The Author(s).)

Background: Mass-rearing, domestication and gamma irradiation of tephritid fruit flies used in sterile insect technique (SIT) programmes can negatively impact fly quality and performance. Symbiotic bacteria supplied as probiotics to mass-reared fruit flies may help to overcome some of these issues. However, the effects of tephritid ontogeny, sex, diet and irradiation on their microbiota are not well known., Results: We have used next-generation sequencing to characterise the bacterial community composition and structure within Queensland fruit fly, Bactrocera tryoni (Froggatt), by generating 16S rRNA gene amplicon libraries derived from the guts of 58 individual teneral and mature, female and male, sterile and fertile adult flies reared on artificial larval diets in a laboratory or mass-rearing environment, and fed either a full adult diet (i.e. sugar and yeast hydrolysate) or a sugar only adult diet. Overall, the amplicon sequence read volume in tenerals was low and smaller than in mature adult flies. Operational taxonomic units (OTUs), belonging to the families Enterobacteriaceae (8 OTUs) and Acetobacteraceae (1 OTU) were most prevalent. Enterobacteriaceae dominated laboratory-reared tenerals from a colony fed a carrot-based larval diet, while Acetobacteraceae dominated mass-reared tenerals from a production facility colony fed a lucerne chaff based larval diet. As adult flies matured, Enterobacteriaceae became dominant irrespective of larval origin. The inclusion of yeast in the adult diet strengthened this shift away from Acetobacteraceae towards Enterobacteriaceae. Interestingly, irradiation increased 16S rRNA gene sequence read volume., Conclusions: Our findings suggest that bacterial populations in fruit flies experience significant bottlenecks during metamorphosis. Gut bacteria in teneral flies were less abundant and less diverse, and impacted by colony origin. In contrast, mature adult flies had selectively increased abundances for some gut bacteria, or acquired these bacteria from the adult diet and environment. Furthermore, irradiation augmented bacterial abundance in mature flies. This implies that either some gut bacteria were compensating for damage caused by irradiation or irradiated flies had lost their ability to regulate bacterial load. Our findings suggest that the adult stage prior to sexual maturity may be ideal to target for probiotic manipulation of fly microbiota to increase fly performance in SIT programmes.

Background: Maternally inherited Wolbachia bacteria infect many insect species. They can also be transferred horizontally into uninfected host lineages. A Wolbachia spillover from an infected source population must occur prior to the establishment of heritable infections, but this spillover may be transient. In a previous study of tephritid fruit fly species of tropical Australia we detected a high incidence of identical Wolbachia strains in several species as well as Wolbachia pseudogenes. Here, we have investigated this further by analysing field specimens of 24 species collected along a 3,000 km climate gradient of eastern Australia., Results: Wolbachia sequences were detected in individuals of nine of the 24 (37 %) species. Seven (29 %) species displayed four distinct Wolbachia strains based on characterisation of full multi locus sequencing (MLST) profiles; the strains occurred as single and double infections in a small number of individuals (2-17 %). For the two remaining species all individuals had incomplete MLST profiles and Wolbachia pseudogenes that may be indicative of lateral gene transfer into host genomes. The detection of Wolbachia was restricted to northern Australia, including in five species that only occur in the tropics. Within the more widely distributed Bactrocera tryoni and Bactrocera neohumeralis, Wolbachia also only occurred in the north, and was not linked to any particular mitochondrial haplotypes., Conclusions: The presence of Wolbachia pseudogenes at high prevalence in two species in absence of complete MLST profiles may represent footprints of historic infections that have been lost. The detection of identical low prevalence strains in a small number of individuals of seven species may question their role as reproductive manipulator and their vertical inheritance. Instead, the findings may be indicative of transient infections that result from spillover events from a yet unknown source. These spillover events appear to be restricted to northern Australia, without proliferation in host lineages further south. Our study highlights that tropical fruit fly communities contain Wolbachia pseudogenes and may be exposed to frequent horizontal Wolbachia transfer. It also emphasises that global estimates of Wolbachia frequencies may need to consider lateral gene transfer and Wolbachia spillover that may be regionally restricted, transient and not inherited.

Tephritid fruit fly species display a diversity of host plant specialisation on a scale from monophagy to polyphagy. Furthermore, while some species prefer ripening fruit, a few are restricted to damaged or rotting fruit. Such a diversity of host plant use may be reflected in the microbial symbiont diversity of tephritids and their grade of dependency on their microbiomes. Here, we investigated the microbiome of six tephritid species from three genera, including species that are polyphagous pests (Bactrocera tryoni, Bactrocera neohumeralis, Bactrocera jarvisi, Ceratitis capitata) and a monophagous specialist (Bactrocera cacuminata). These were compared with the microbiome of a non-pestiferous but polyphagous tephritid species that is restricted to damaged or rotting fruit (Dirioxa pornia). The bacterial community associated with whole fruit flies was analysed by 16S ribosomal DNA (rDNA) amplicon pyrosequencing to detect potential drivers of taxonomic composition. Overall, the dominant bacterial families were Enterobacteriaceae and Acetobacteraceae (both Proteobacteria), and Streptococcaceae and Enterococcaceae (both Firmicutes). Comparisons across species and genera found different microbial composition in the three tephritid genera, but limited consistent differentiation between Bactrocera species. Within Bactrocera species, differentiation of microbial composition seemed to be influenced by the environment, possibly including their diets; beyond this, tephritid species identity or ecology also had an effect. The microbiome of D. pornia was most distinct from the other five species, which may be due to its ecologically different niche of rotting or damaged fruit, as opposed to ripening fruit favoured by the other species. Our study is the first amplicon pyrosequencing study to compare the microbiomes of tephritid species and thus delivers important information about the turnover of microbial diversity within and between fruit fly species and their potential application in pest management strategies.

Wolbachia are endosymbiotic bacteria that infect 40-65% of arthropod species. They are primarily maternally inherited with occasional horizontal transmission for which limited direct ecological evidence exists. We detected Wolbachia in 8 out of 24 Australian tephritid species. Here, we have used multilocus sequence typing (MLST) to further characterize these Wolbachia strains, plus a novel quantitative polymerase chain reaction method for allele assignment in multiple infections. Based on five MLST loci and the Wolbachia surface protein gene (wsp), five Bactrocera and one Dacus species harboured two identical strains as double infections; furthermore, Bactrocera neohumeralis harboured both of these as single or double infections, and sibling species B. tryoni harboured one. Two Bactrocera species contained Wolbachia pseudogenes, potentially within the fruit fly genomes. A fruit fly parasitoid, Fopius arisanus shared identical alleles with two Wolbachia strains detected in one B. frauenfeldi individual. We report an unprecedented high incidence of four shared Wolbachia strains in eight host species from two trophic levels. This suggests frequent exposure to Wolbachia in this tropical tephritid community that shares host plant and parasitoid species, and also includes species that hybridize. Such insect communities may act as horizontal transmission platforms that contribute to the ubiquity of the otherwise maternally inherited Wolbachia., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

In tephritids, the sex-determination pathway follows the sex-specific splicing of transformer (tra) mRNA, and the cooperation of tra and transformer-2 (tra-2) to effect the sex-specific splicing of doublesex (dsx), the genetic double-switch responsible for male or female somatic development. The Dominant Male Determiner (M) is the primary signal that controls this pathway. M, as yet uncharacterized, is Y-chromosome linked, expressed in the zygote and directly or indirectly diminishes active TRA protein in male embryos. Here we first demonstrated the high conservation of tra, tra-2 and dsx in two Australian tephritids, Bactrocera tryoni and Bactrocera jarvisi. We then used quantitative reverse transcription PCR on single, sexed embryos to examine expression of the key sex-determination genes during early embryogenesis. Individual embryos were sexed using molecular markers located on the B. jarvisi Y-chromosome that was also introgressed into a B. tryoni line. In B. jarvisi, sex-specific expression of tra transcripts occurred between 3 to 6 h after egg laying, and the dsx isoform was established by 7 h. These milestones were delayed in B. tryoni lines. The results provide a time frame for transcriptomic analyses to identify M and its direct targets, plus information on genes that may be targeted for the development of male-only lines for pest management., (© 2014 The Royal Entomological Society.)

We report the heritable germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP. A transformation frequency of 5-10% was obtained. Inheritance of the transgenes has remained stable over more than 15 generations despite the presence of endogenous piggyBac sequences in the B. tryoni genome. The sequence of insertion sites shows the usual canonical pattern of piggyBac integraton into TTAA target sites. An investigation of endogenous piggyBac elements in the B. tryoni genome reveals the presence of sequences almost identical to those reported recently for the B. dorsalis complex of fruit flies and two noctuid moths, suggesting a common origin of piggyBac sequences in these species. The availability of transformation protocols for B. tryoni has the potential to deliver improvements in the performance of the Sterile Insect Technique for this pest species.

Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) or "Qfly," is the most serious horticultural pest in Australia, with a bioclimatic range that extends from the tropical north to the temperate south. Various Australian horticultural exports depend on certification that they originated from B. tryoni-free areas. To eliminate, rather than suppress, B. tryoni in production areas, a sterile insect technique (SIT) campaign directed at B. tryoni has been in operation in southeastern Australia since 1997. Like many other SIT programs around the world, the B. tryoni SIT program relies on fluorescent dust to mark the sterile insects. However, fluorescent dust marking does not provide 100% accuracy in the identification of sterile insects, as required where the aim is to declare regions completely free of fruit fly. Here, we show that novel mitochondrial markers can be introduced into a strain of B. tryoni by interspecies hybridization between B. tryoni and a related but well-differentiated species, Bactrocera jarvisi (Tryon), followed by backcrossing of the hybrid strain with the parental B. tryoni strain. These novel markers do not affect the viability of the strain as measured by pupation and eclosion rates. A simple polymerase chain reaction-based test is described that distinguishes the marked B. tryoni from wild B. tryoni. As required in practice, the test was shown to work reliably on DNA extracted from dead flies that had remained in field traps for up to two weeks.