Microbial competition for phosphorus limits the CO2 response of a mature forest.

The capacity for terrestrial ecosystems to sequester additional carbon (C) with rising CO2 concentrations depends on soil nutrient availability1,2. Previous evidence suggested that mature forests growing on phosphorus (P)-deprived soils had limited capacity to sequester extra biomass under elevated CO2 (refs. 3-6), but uncertainty about ecosystem P cycling and its CO2 response represents a crucial bottleneck for mechanistic prediction of the land C sink under climate change7. Here, by compiling the first comprehensive P budget for a P-limited mature forest exposed to elevated CO2, we show a high likelihood that P captured by soil microorganisms constrains ecosystem P recycling and availability for plant uptake. Trees used P efficiently, but microbial pre-emption of mineralized soil P seemed to limit the capacity of trees for increased P uptake and assimilation under elevated CO2 and, therefore, their capacity to sequester extra C. Plant strategies to stimulate microbial P cycling and plant P uptake, such as increasing rhizosphere C release to soil, will probably be necessary for P-limited forests to increase C capture into new biomass. Our results identify the key mechanisms by which P availability limits CO2 fertilization of tree growth and will guide the development of Earth system models to predict future long-term C storage.

The fate of carbon in a mature forest under carbon dioxide enrichment.

Atmospheric carbon dioxide enrichment (eCO2) can enhance plant carbon uptake and growth1-5, thereby providing an important negative feedback to climate change by slowing the rate of increase of the atmospheric CO2 concentration6. Although evidence gathered from young aggrading forests has generally indicated a strong CO2 fertilization effect on biomass growth3-5, it is unclear whether mature forests respond to eCO2 in a similar way. In mature trees and forest stands7-10, photosynthetic uptake has been found to increase under eCO2 without any apparent accompanying growth response, leaving the fate of additional carbon fixed under eCO2 unclear4,5,7-11. Here using data from the first ecosystem-scale Free-Air CO2 Enrichment (FACE) experiment in a mature forest, we constructed a comprehensive ecosystem carbon budget to track the fate of carbon as the forest responded to four years of eCO2 exposure. We show that, although the eCO2 treatment of +150 parts per million (+38 per cent) above ambient levels induced a 12 per cent (+247 grams of carbon per square metre per year) increase in carbon uptake through gross primary production, this additional carbon uptake did not lead to increased carbon sequestration at the ecosystem level. Instead, the majority of the extra carbon was emitted back into the atmosphere via several respiratory fluxes, with increased soil respiration alone accounting for half of the total uptake surplus. Our results call into question the predominant thinking that the capacity of forests to act as carbon sinks will be generally enhanced under eCO2, and challenge the efficacy of climate mitigation strategies that rely on ubiquitous CO2 fertilization as a driver of increased carbon sinks in global forests.

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.

Two sympatric lineages of Australian Cnestus solidus share Ambrosiella symbionts but not Wolbachia.

Sympatric lineages of inbreeding species provide an excellent opportunity to investigate species divergence patterns and processes. Many ambrosia beetle lineages (Curculionidae: Scolytinae) reproduce by predominant inbreeding through sib mating in nests excavated in woody plant parts wherein they cultivate symbiotic ambrosia fungi as their sole source of nutrition. The Xyleborini ambrosia beetle species Cnestus solidus and Cnestus pseudosolidus are sympatrically distributed across eastern Australia and have overlapping morphological variation. Using multilocus sequencing analysis of individuals collected from 19 sites spanning their sympatric distribution, we assessed their phylogenetic relationships, taxonomic status and microbial symbionts. We found no genetic differentiation between individuals morphologically identified as C. solidus and C. pseudosolidus confirming previous suggestions that C. pseudosolidus is synonymous to C. solidus. However, within C. solidus we unexpectedly discovered the sympatric coexistence of two morphologically indistinguishable but genetically distinct lineages with small nuclear yet large mitochondrial divergence. At all sites except one, individuals of both lineages carried the same primary fungal symbiont, a new Ambrosiella species, indicating that fungal symbiont differentiation may not be involved in lineage divergence. One strain of the maternally inherited bacterial endosymbiont Wolbachia was found at high prevalence in individuals of the more common lineage but not in the other, suggesting that it may influence host fitness. Our data suggest that the two Australian Cnestus lineages diverged allopatrically, and one lineage then acquired Wolbachia. Predominant inbreeding and Wolbachia infection may have reinforced reproductive barriers between these two lineages after their secondary contact contributing to their current sympatric distribution.

A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.

A Multilocus Sequence Typing Scheme for Rapid Identification of Xanthomonas citri Based on Whole-Genome Sequencing Data.

Xanthomonas citri is a plant-pathogenic bacterium associated with a diverse range of host plant species. It has undergone substantial reclassification and currently consists of 14 different subspecies or pathovars that are responsible for a wide range of plant diseases. Whole-genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of X. citri because it has a higher discriminatory power and is replicable across laboratories. WGS also allows for the improvement of multilocus sequence typing (MLST) schemes. In this study, we used genome sequences of Xanthomonas isolates from the NCBI RefSeq database to develop a seven-gene MLST scheme that yielded 19 sequence types (STs) that correlated with phylogenetic clades of X. citri subspecies or pathovars. Using this MLST scheme, we examined 2,911 Xanthomonas species assemblies from NCBI GenBank and identified 15 novel STs from 37 isolates that were misclassified in NCBI. In total, we identified 545 X. citri assemblies from GenBank with 95% average nucleotide identity to the X. citri type strain, and all were classified as one of the 34 STs. All MLST classifications correlated with a phylogenetic position inferred from alignments using 92 conserved genes. We observed several instances where strains from different pathovars formed closely related monophyletic clades and shared the same ST, indicating that further investigation of the validity of these pathovars is required. Our MLST scheme described here is a robust tool for rapid classification of X. citri pathovars using WGS and a powerful method for further comprehensive taxonomic revision of X. citri pathovars.

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.

Inhibiting essential elements of the DNA methylation pathway negatively impacts the miRNA pathway and fitness of the whitefly Bemisia tabaci.

DNA methylation is an epigenetic process that involves the chemical modification of DNA, leading to the regulation of its transcriptional activity. It is primarily known for the addition of methyl groups to cytosine in DNA. The whitefly Bemisia tabaci is a polyphagous pest insect and a vector that is responsible for transmitting numerous plant viruses, resulting in significant economic losses in agricultural crops globally. In our study, we characterized the expression of two key DNA methylation genes, the DNA methyltransferases Dnmt1 and Dnmt3, in B. tabaci. Additionally, we explored the impact of inhibiting DNMTs on the miRNA pathway and fitness of whitefly. To investigate the role of the DNA methylation pathway in B. tabaci, we found that the expression of Dnmt1 and Dnmt3 varied across different tissues and developmental stages of B. tabaci. We employed azacytidine (5-AZA) treatment of adults to inhibit DNMTs (DNMT1 and DNMT3). Administration of 5-AZA affected the survival and reproduction of this pest. Moreover, inhibition of DNMTs led to a decrease in the expression of the miRNA pathway core genes Dicer1 and Argonaute1, which subsequently resulted in reduced expression of Let-7 and miR-184 which are essential microRNAs in the physiology and biology of insects. The study suggests that DNA methyltransferases could be targeted for developing an inhibition strategy to control this pest and vector insect.

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.

Embryonic microinjection of ribonucleoprotein complex (Cas9+sgRNA) of white gene in melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae) produced white eye phenotype.

Melon fly, Zeugodacus cucurbitae (Coquillett) is a major pest of cucurbitaceous crops, and causes substantial yield losses and economic costs. CRISPR/Cas9 is a rapid and effective site-specific genome editing tool for the generation of genetic changes that are stable and heritable. The CRISPR/Cas9 tool uses synthetically designed single guide RNA (sgRNA) that is complementary to the target gene and guides the Cas9 enzyme to perform nuclease activity by making double-strand breaks in the target DNA sequences. This tool can be effectively exploited to improve traits critical for the management of insect pests by targeting specific genes encoding these traits without the need of extensive genetic information. The white gene is an important gene responsible for the transport of body pigment precursor molecules. In this study, we produced effective mutagenesis of the white gene of Z. cucurbitae using the CRISPR/Cas9 tool with double sgRNA to target multiple sites of white to increase the efficiency in the generation of frame-shift mutations resulting in the white eye phenotype in adults. This was achieved through embryonic microinjection of the ribonucleoprotein (RNP) complex in the pre-blastoderm embryo stage 1 h after embryo laying. Our success with the production of a white eye mutant fly by CRISPR/Cas9 mutagenesis is important for the research on gene function and protein-level modifications in melon fly and forms the basis for the development of new genetic control strategies such as precision guided sterile insect technique (pgSIT) for this pest of economic significance.

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.

Microbial diversity in stingless bee gut is linked to host wing size and influenced by the environment.

Stingless bees are important social corbiculate bees, fulfilling critical pollination roles in many ecosystems. However, their gut microbiota, particularly the fungal communities associated with them, remains inadequately characterised. This knowledge gap hinders our understanding of bee gut microbiomes and their impacts on the host fitness. We collected 121 samples from two species, Tetragonula carbonaria and Austroplebeia australis across 1200 km of eastern Australia. We characterised their gut microbiomes and investigated potential correlations between bee gut microbiomes and various geographical and morphological factors. We found their core microbiomes consisted of the abundant bacterial taxa Snodgrassella, Lactobacillus and Acetobacteraceae, and the fungal taxa Didymellaceae, Monocilium mucidum and Aureobasidium pullulans, but variances of their abundances among samples were large. Furthermore, gut bacterial richness of T. carbonaria was positively correlated to host forewing length, an established correlate to body size and fitness indicator in insects relating to flight capacity. This result indicates that larger body size/longer foraging distance of bees could associate with greater microbial diversity in gut. Additionally, both host species identity and management approach significantly influenced gut microbial diversity and composition, and similarity between colonies for both species decreased as the geographic distance between them increased. We also quantified the total bacterial and fungal abundance of the samples using qPCR analyses and found that bacterial abundance was higher in T. carbonaria compared to A. australis, and fungi were either lowly abundant or below the threshold of detection for both species. Overall, our study provides novel understanding of stingless bee gut microbiomes over a large geographic span and reveals that gut fungal communities likely not play an important role in host functions due to their low abundances.

Heat stress survival and thermal tolerance of Australian stingless bees.

Stingless bees (Meliponini) are important pollinators throughout the world's tropical and subtropical regions. Understanding their thermal tolerance is key to predicting their resilience to changing climates and increasingly frequent extreme heat events. We examined critical thermal maxima (CTmax), survival during 1-8 h heat periods, chill coma recovery and thermal preference for Australian meliponine species that occupy different climates across their ranges: Tetragonula carbonaria (tropical to temperate regions), T. hockingsi (tropical and subtropical regions only) and Austroplebeia australis (widely distributed including arid regions). We found interspecific differences in thermal tolerance consistent with differences in the climate variability observed in each species' range. Foragers of A. australis had a faster chill coma recovery (288 s) than foragers of T. hockingsi (1059 s) and T. carbonaria (872 s). Austroplebeia australis also had the highest CTmax of 44.5 °C, while the CTmax of the two Tetragonula species was ∼43.1 °C. After a 1-h heat exposure, T. carbonaria foragers experienced 95% mortality at 42 °C, and 100% at 45 °C. Surprisingly, larvae and pupae of both Tetragonula species were more resistant to heat exposure than foragers. Within an enclosed temperature gradient apparatus (17-38 °C), no clear preference was found for foragers; however, they were most frequently observed at ∼18 °C. Results indicate that in some regions of Australia, meliponines already experience periodic heat events exceeding their thermal maxima. Employing effective management strategies (such as nest site insulation and habitat preservation) may be crucial to colony survival under continued climate change.

Common endosymbionts affect host fitness and sex allocation via egg size provisioning.

It is hard to overemphasize the importance of endosymbionts in arthropod biology, ecology and evolution. Some endosymbionts can complement host metabolic function or provide defence against pathogens; others, such as ubiquitous Wolbachia and Cardinium, have evolved strategies to manipulate host reproduction. A common reproductive manipulation strategy is cytoplasmic incompatibility (CI) between differently infected individuals which can result in female mortality or male development of fertilized eggs in haplodiploid hosts. Recently, an additional role of endosymbionts has been recognized in the modification of sex allocation in sexually reproducing haplodiploids. This was theoretically expected due to the maternal inheritance of endosymbionts and natural selection for them to increase infected female production, yet the underlying mechanism remained unknown. Here, we tested whether and how Cardinium and Wolbachia causing different CI types interact to increase female production in a haplodiploid thrips species where sex allocation depends on both maternal condition and egg size provisioning. We found that Cardinium augmented female production by increasing maternal fitness and egg size, thereby boosting fertilization rate and offspring fitness. Wolbachia, in contrast, reduced the beneficial effects of Cardinium. Our results demonstrate different invasion strategies and antagonistic effects of endosymbiotic bacteria on host fitness and evolution of sex allocation.

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.

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.

Symbiotic microbiota may reflect host adaptation by resident to invasive ant species.

Exotic invasive species can influence the behavior and ecology of native and resident species, but these changes are often overlooked. Here we hypothesize that the ghost ant, Tapinoma melanocephalum, living in areas that have been invaded by the red imported fire ant, Solenopsis invicta, displays behavioral differences to interspecific competition that are reflected in both its trophic position and symbiotic microbiota. We demonstrate that T. melanocephalum workers from S. invicta invaded areas are less aggressive towards workers of S. invicta than those inhabiting non-invaded areas. Nitrogen isotope analyses reveal that colonies of T. melanocephalum have protein-rich diets in S. invicta invaded areas compared with the carbohydrate-rich diets of colonies living in non-invaded areas. Analysis of microbiota isolated from gut tissue shows that T. melanocephalum workers from S. invicta invaded areas also have different bacterial communities, including a higher abundance of Wolbachia that may play a role in vitamin B provisioning. In contrast, the microbiota of workers of T. melanocephalum from S. invicta-free areas are dominated by bacteria from the orders Bacillales, Lactobacillales and Enterobacteriales that may be involved in sugar metabolism. We further demonstrate experimentally that the composition and structure of the bacterial symbiont communities as well as the prevalence of vitamin B in T. melanocephalum workers from S. invicta invaded and non-invaded areas can be altered if T. melanocephalum workers are supplied with either protein-rich or carbohydrate-rich food. Our results support the hypothesis that bacterial symbiont communities can help hosts by buffering behavioral changes caused by interspecies competition as a consequence of biological invasions.

Vulnerability of island insect pollinator communities to pathogens.

Island ecosystems, which often contain undescribed insects and small populations of single island endemics, are at risk from diverse threats. The spread of pathogens is a major factor affecting not just pollinator species themselves, but also posing significant knock-on effects to often fragile island ecosystems through disruption of pollination networks. Insects are vulnerable to diverse pathogens and these can be introduced to islands in a number of ways, e.g. via the introduction of infected managed pollinator hosts (e.g. honey bees and their viruses, in particular Deformed wing virus), long-range migrants (e.g. monarch butterflies and their protozoan parasite, Ophryocystit elektroscirrha) and invasive species (e.g. social wasps are common invaders and are frequently infected with multi-host viruses such as Kashmir bee virus and Moku virus). Furthermore, these introductions can negatively affect island ecosystems through outcompeting native taxa for resources. As such, the greatest threat to island pollinator communities is not one particular pathogen, but the combination of pathogens and introduced and invasive insects that will likely carry them.

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.