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.

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.

Bacterial Communities Are Less Diverse in a Strepsipteran Endoparasitoid than in Its Fruit Fly Hosts and Dominated by Wolbachia.

Microbiomes play vital roles in insect fitness and health and can be influenced by interactions between insects and their parasites. Many studies investigate the microbiome of free-living insects, whereas microbiomes of endoparasitoids and their interactions with parasitised insects are less explored. Due to their development in the constrained environment within a host, endoparasitoids are expected to have less diverse yet distinct microbiomes. We used high-throughput 16S rRNA gene amplicon sequencing to characterise the bacterial communities of Dipterophagus daci (Strepsiptera) and seven of its tephritid fruit fly host species. Bacterial communities of D. daci were less diverse and contained fewer taxa relative to the bacterial communities of the tephritid hosts. The strepsipteran's microbiome was dominated by Pseudomonadota (formerly Proteobacteria) (> 96%), mainly attributed to the presence of Wolbachia, with few other bacterial community members, indicative of an overall less diverse microbiome in D. daci. In contrast, a dominance of Wolbachia was not found in flies parasitised by early stages of D. daci nor unparasitised flies. Yet, early stages of D. daci parasitisation resulted in structural changes in the bacterial communities of parasitised flies. Furthermore, parasitisation with early stages of D. daci with Wolbachia was associated with a change in the relative abundance of some bacterial taxa relative to parasitisation with early stages of D. daci lacking Wolbachia. Our study is a first comprehensive characterisation of bacterial communities in a Strepsiptera species together with the more diverse bacterial communities of its hosts and reveals effects of concealed stages of parasitisation on host bacterial communities.

Transmission modes and efficiency of iflavirus and cripavirus in Queensland fruit fly, Bactrocera tryoni.

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.

Endosymbionts moderate constrained sex allocation in a haplodiploid thrips species in a temperature-sensitive way.

Maternally inherited bacterial endosymbionts that affect host fitness are common in nature. Some endosymbionts colonise host populations by reproductive manipulations (such as cytoplasmic incompatibility; CI) that increase the reproductive fitness of infected over uninfected females. Theory predicts that CI-inducing endosymbionts in haplodiploid hosts may also influence sex allocation, including in compatible crosses, however, empirical evidence for this is scarce. We examined the role of two common CI-inducing endosymbionts, Cardinium and Wolbachia, in the sex allocation of Pezothrips kellyanus, a haplodiploid thrips species with a split sex ratio. In this species, irrespective of infection status, some mated females are constrained to produce extremely male-biased broods, whereas other females produce extremely female-biased broods. We analysed brood sex ratio of females mated with males of the same infection status at two temperatures. We found that at 20 °C the frequency of constrained sex allocation in coinfected pairs was reduced by 27% when compared to uninfected pairs. However, at 25 °C the constrained sex allocation frequency increased and became similar between coinfected and uninfected pairs, resulting in more male-biased population sex ratios at the higher temperature. This temperature-dependent pattern occurred without changes in endosymbiont densities and compatibility. Our findings indicate that endosymbionts affect sex ratios of haplodiploid hosts beyond the commonly recognised reproductive manipulations by causing female-biased sex allocation in a temperature-dependent fashion. This may contribute to a higher transmission efficiency of CI-inducing endosymbionts and is consistent with previous models that predict that CI by itself is less efficient in driving endosymbiont invasions in haplodiploid hosts.

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

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

Genome analyses of four Wolbachia strains and associated mitochondria of Rhagoletis cerasi expose cumulative modularity of cytoplasmic incompatibility factors and cytoplasmic hitchhiking across host populations.

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.

Host-endoparasitoid-endosymbiont relationships: concealed Strepsiptera provide new twist to Wolbachia in Australian tephritid fruit flies.

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.

Tephritid fruit flies have a large diversity of co-occurring RNA viruses.

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.

Draft Genome Sequence of a Novel "Candidatus Liberibacter" Species Detected in a Zanthoxylum Species from Bhutan.

The draft genome sequence of a novel "Candidatus Liberibacter" species detected in an unidentified species of Zanthoxylum (Rutaceae) collected in Bhutan is reported. The total length is 1,408,989 bp with 1,169 coding sequences in 96 contigs, a GC content of 37.3%, and 76 to 77% average nucleotide identity with several other "Ca Liberibacter" species.

Characterization of the bacterial communities of psyllids associated with Rutaceae in Bhutan by high throughput sequencing.

BACKGROUND: Several plant-pathogenic bacteria are transmitted by insect vector species that often also act as hosts. In this interface, these bacteria encounter plant endophytic, insect endosymbiotic and other microbes. Here, we used high throughput sequencing to examine the bacterial communities of five different psyllids associated with citrus and related plants of Rutaceae in Bhutan: Diaphorina citri, Diaphorina communis, Cornopsylla rotundiconis, Cacopsylla heterogena and an unidentified Cacopsylla sp. RESULTS: The microbiomes of the psyllids largely comprised their obligate P-endosymbiont 'Candidatus Carsonella ruddii', and one or two S-endosymbionts that are fixed and specific to each lineage. In addition, all contained Wolbachia strains; the Bhutanese accessions of D. citri were dominated by a Wolbachia strain first found in American isolates of D. citri, while D. communis accessions were dominated by the Wolbachia strain, wDi, first detected in D. citri from China. The S-endosymbionts from the five psyllids grouped with those from other psyllid taxa; all D. citri and D. communis individuals contained sequences matching 'Candidatus Profftella armatura' that has previously only been reported from other Diaphorina species, and the remaining psyllid species contained OTUs related to unclassified Enterobacteriaceae. The plant pathogenic 'Candidatus Liberibacter asiaticus' was found in D. citri but not in D. communis. Furthermore, an unidentified 'Candidatus Liberibacter sp.' occurred at low abundance in both Co. rotundiconis and the unidentified Cacopsylla sp. sampled from Zanthoxylum sp.; the status of this new liberibacter as a plant pathogen and its potential plant hosts are currently unknown. The bacterial communities of Co. rotundiconis also contained a range of OTUs with similarities to bacteria previously found in samples taken from various environmental sources. CONCLUSIONS: The bacterial microbiota detected in these Bhutanese psyllids support the trends that have been seen in previous studies: psyllids have microbiomes largely comprising their obligate P-endosymbiont and one or two S-endosymbionts. In addition, the association with plant pathogens has been demonstrated, with the detection of liberibacters in a known host, D. citri, and identification of a putative new species of liberibacter in Co. rotundiconis and Cacopsylla sp.

Parallel Sequencing of Wolbachia wCer2 from Donor and Novel Hosts Reveals Multiple Incompatibility Factors and Genome Stability after Host Transfers.

The application of Wolbachia in insect pest and vector control requires the establishment of genotypically stable host associations. The cytoplasmic incompatibility (CI) inducing Wolbachia strain wCer2 naturally occurs in the cherry fruit fly Rhagoletis cerasi as co-infection with other strains and was transferred to other fruit fly species by embryonic microinjections. We obtained wCer2 genome data from its native and three novel hosts, Drosophila simulans, Drosophila melanogaster, and Ceratitis capitata and assessed its genome stability, characteristics, and CI factor (cif) genes. De novo assembly was successful from Wolbachia cell-enriched singly infected D. simulans embryos, with minimal host and other bacterial genome traces. The low yield of Wolbachia sequence reads from total genomic extracts of one multiply infected R. cerasi pupa and one singly infected C. capitata adult limited de novo assemblies but was sufficient for comparative analyses. Across hosts wCer2 was stable in genome synteny and content. Polymorphic nucleotide sites were found in wCer2 of each host; however, only one nucleotide was different between R. cerasi and C. capitata, and none between replicated D. simulans lines. The wCer2 genome is highly similar to wAu (D. simulans), wMel (D. melanogaster), and wRec (Drosophila recens). In contrast to wMel and wRec (each with one cif gene pair) and wAu (without any cif genes), wCer2 has three pairs of Type I cif genes, and one Type V cifB gene without a cifA complement. This may explain previously reported CI patterns of wCer2, including incomplete rescue of its own CI modification in three novel host species.

Diet and irradiation effects on the bacterial community composition and structure in the gut of domesticated teneral and mature Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae).

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.

Symbionts in waiting: the dynamics of incipient endosymbiont complementation and replacement in minimal bacterial communities of psyllids.

BACKGROUND: Obligate bacterial primary (P-) endosymbionts that are maternally inherited and codiverge with hosts are widespread across insect lineages with nutritionally restricted diets. Secondary (S-) endosymbionts are mostly facultative, but in some hosts, they complement P-endosymbiont function and therefore become obligate. Phylogenetic evidence exists for host switching and replacement of S-endosymbionts. The community dynamics that precede endosymbiont replacement and complementation have been little studied across host species, yet they are fundamental to the evolution of endosymbiosis. RESULTS: We performed bacterial 16S rRNA gene amplicon sequencing of 25 psyllid species (Hemiptera, Psylloidea) across different developmental stages and ecological niches by focusing on the characterisation of the bacteria other than the universally present P-endosymbiont Carsonella (Gammaproteobacteria). Most species harboured only one dominant representative of diverse gammaproteobacterial S-endosymbionts that was consistently detected across all host individuals and populations (Arsenophonus in eight species, Sodalis or Sodalis-like bacteria in four species, unclassified Enterobacteriaceae in eight species). The identity of this dominant obligate S-endosymbiont varied across closely related host species. Unexpectedly, five psyllid species had two or three co-occurring endosymbiont species other than Carsonella within all host individuals, including a Rickettsiella-like bacterium (Gammaproteobacteria) in one psyllid species. Based on standard and quantitative PCR, all psyllids carried Carsonella, at higher titres than their dominant S-endosymbionts. Some psyllids also had Alphaproteobacteria (Lariskella, Rickettsia, Wolbachia) at varying prevalence. Incidence of other bacteria, including known plant pathogens, was low. Ecological niche of gall-forming, lerp-forming and free-living psyllid species did not impact endosymbiont communities. Two flush-feeding psyllid species had population-specific differences, and this was attributable to the higher endosymbiont diversity in native ranges and the absence of some endosymbionts in invasive ranges. CONCLUSIONS: Our data support the hypothesis of strict vertical transmission of minimal core communities of bacteria in psyllids. We also found evidence for S-endosymbiont replacement across closely related psyllid species. Multiple dominant S-endosymbionts present in some host species, including at low titre, constitute potential examples of incipient endosymbiont complementation or replacement. Our multiple comparisons of deep-sequenced minimal insect bacterial communities exposed the dynamics involved in shaping insect endosymbiosis.

Independent cytoplasmic incompatibility induced by Cardinium and Wolbachia maintains endosymbiont coinfections in haplodiploid thrips populations.

Cardinium and Wolbachia are common maternally inherited reproductive parasites that can coinfect arthropods, yet interactions between both bacterial endosymbionts are rarely studied. For the first time, we report their independent expression of complete cytoplasmic incompatibility (CI) in a coinfected host, and CI in a species of the haplodiploid insect order Thysanoptera. In Pezothrips kellyanus, Cardinium-induced CI resulted in a combination of male development (MD) and embryonic female mortality (FM) of fertilized eggs. In contrast, Wolbachia-induced CI resulted in FM together with postembryonic mortality not previously reported as a CI outcome. Both endosymbionts appeared to not influence fecundity but virgins produced more offspring than mated females. In coinfected individuals, Wolbachia density was higher than Cardinium. Wolbachia removal did not impact Cardinium density, suggesting a lack of competition within hosts. Maternal transmission was complete for Wolbachia and high for Cardinium. Our data support theoretical predictions and empirical detection of high endosymbiont prevalence in field populations of the native range of this pest thrips. However, previous findings of more frequent loss of Wolbachia than Cardinium, particularly in field populations of the host's invasive range, suggest that genetic diversity or varying environmental factors between field populations also play a role in shaping host-endosymbiont dynamics.

Codivergence of the primary bacterial endosymbiont of psyllids versus host switches and replacement of their secondary bacterial endosymbionts.

Coevolution between insects and bacterial endosymbionts contributes to the success of many insect lineages. For the first time, we tested for phylogenetic codivergence across multiple taxonomic scales, from within genera to superfamily between 36 psyllid species of seven recognised families (Hemiptera: Psylloidea), their exclusive primary endosymbiont Carsonella and more diverse secondary endosymbionts (S-endosymbionts). Within Aphalaridae, we found that Carsonella and S-endosymbionts were fixed in one Glycaspis and 12 Cardiaspina populations. The dominant S-endosymbiont was Arsenophonus, while Sodalis was detected in one Cardiaspina species. We demonstrated vertical transmission for Carsonella and Arsenophonus in three Cardiaspina species. We found strong support for strict cospeciation and validated the informative content of Carsonella as extended host genome for inference of psyllid relationships. However, S-endosymbiont and host phylogenies were incongruent, and displayed signs of host switching and endosymbiont replacement. The high incidence of Arsenophonus in psyllids and other plant sap-feeding Hemiptera may be due to repeated host switching within this group. In two psyllid lineages, Arsenophonus and Sodalis genes exhibited accelerated evolutionary rates and AT-biases characteristic of long-term host associations. Together with strict vertical transmission and 100% prevalence within host populations, our results suggest an obligate, and not facultative, symbiosis between psyllids and some S-endosymbionts.

Wolbachia pseudogenes and low prevalence infections in tropical but not temperate Australian tephritid fruit flies: manifestations of lateral gene transfer and endosymbiont spillover?

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.

Australian endemic pest tephritids: genetic, molecular and microbial tools for improved Sterile Insect Technique.

Among Australian endemic tephritid fruit flies, the sibling species Bactrocera tryoni and Bactrocera neohumeralis have been serious horticultural pests since the introduction of horticulture in the nineteenth century. More recently, Bactrocera jarvisi has also been declared a pest in northern Australia. After several decades of genetic research there is now a range of classical and molecular genetic tools that can be used to develop improved Sterile Insect Technique (SIT) strains for control of these pests. Four-way crossing strategies have the potential to overcome the problem of inbreeding in mass-reared strains of B. tryoni. The ability to produce hybrids between B. tryoni and the other two species in the laboratory has proved useful for the development of genetically marked strains. The identification of Y-chromosome markers in B. jarvisi means that male and female embryos can be distinguished in any strain that carries a B. jarvisi Y chromosome. This has enabled the study of homologues of the sex-determination genes during development of B jarvisi and B. tryoni, which is necessary for the generation of genetic-sexing strains. Germ-line transformation has been established and a draft genome sequence for B. tryoni released. Transcriptomes from various species, tissues and developmental stages, to aid in identification of manipulation targets for improving SIT, have been assembled and are in the pipeline. Broad analyses of the microbiome have revealed a metagenome that is highly variable within and across species and defined by the environment. More specific analyses detected Wolbachia at low prevalence in the tropics but absent in temperate regions, suggesting a possible role for this endosymbiont in future control strategies.

Comprehensive transcriptome analysis of early male and female Bactrocera jarvisi embryos.

BACKGROUND: Developing embryos are provided with maternal RNA transcripts and proteins, but transcription from the zygotic nuclei must be activated to control continuing embryonic development. Transcripts are generated at different stages of early development, and those involved in sex determination and cellularisation are some of the earliest to be activated. The male sex in tephritid fruit flies is determined by the presence of a Y chromosome, and it is believed that a transcript from the Y-chromosome sets in motion a cascade that determines male development, as part of the greater maternal to zygotic transition (MTZ). Here we investigate the poly(A+) transcriptome in early male and female embryos of the horticultural pest Bactrocera jarvisi (Diptera: Tephritidae). RESULTS: Bactrocera jarvisi embryos were collected over two pre-blastoderm time periods, 2-3h and 3-5h after egg laying. Embryos were individually sexed using a Y-chromosome marker, allowing the sex-specific poly(A+) transcriptome of single-sex embryo pools to be deep-sequenced and assembled de novo. Transcripts for sixteen sex-determination and two cellularisation gene homologues of Drosophila melanogaster (Diptera: Drosophilidae) were identified in early embryos of B. jarvisi, including transcripts highly upregulated prior to cellularisation. No strong candidates for transcripts derived solely from the Y chromosome were recovered from the poly(A+) fraction. CONCLUSIONS: Bactrocera jarvisi provides an excellent model for embryonic studies due to available Y-chromosome markers and the compact time frame for zygotic transcription and the sex-determined state. Our data contribute fundamental information to sex-determination research, and provide candidates for the sourcing of gene promoters for transgenic pest-management strategies of tephritid fruit flies.