Aedes aegypti gut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes.

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or the field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, many more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities is associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique for investigating host-microbe interactions in mosquitoes.

Aedes aegypti gut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes.

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, far more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities are associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique.

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.

Vertical and Horizontal Transmission of Cell Fusing Agent Virus in Aedes aegypti.

Cell fusing agent virus (CFAV) is an insect-specific flavivirus (ISF) found in Aedes aegypti mosquitoes. ISFs have demonstrated the ability to modulate the infection or transmission of arboviruses such as dengue, West Nile, and Zika viruses. It is thought that vertical transmission is the main route for ISF maintenance in nature. This has been observed with CFAV, but there is evidence of horizontal and venereal transmission in other ISFs. Understanding the route of transmission can inform strategies to spread ISFs to vector populations as a method of controlling pathogenic arboviruses. We crossed individually reared male and female mosquitoes from both a naturally occurring CFAV-positive Ae. aegypti colony and its negative counterpart to provide information on maternal, paternal, and horizontal transmission. RT-PCR was used to detect CFAV in individual female pupal exuviae and was 89% sensitive, but only 42% in male pupal exuviae. This is a possible way to screen individuals for infection without destroying the adults. Female-to-male horizontal transmission was not observed during this study. However, there was a 31% transmission rate from mating pairs of CFAV-positive males to negative female mosquitoes. Maternal vertical transmission was observed with a filial infection rate of 93%. The rate of paternal transmission was 85% when the female remained negative, 61% when the female acquired CFAV horizontally, and 76% overall. Maternal and paternal transmission of CFAV could allow the introduction of this virus into wild Ae. aegypti populations through male or female mosquito releases, and thus provides a potential strategy for ISF-derived arbovirus control. IMPORTANCE Insect-specific flaviviruses (ISFs), are a group of nonpathogenic flaviviruses that only infect insects. ISFs can have a high prevalence in mosquito populations, but their transmission routes are not well understood. The results of this study confirm maternal transmission of cell fusing agent virus (CFAV) and demonstrate that paternal transmission is also highly efficient. Horizontal transmission of CFAV was also observed, aided by evaluation of the pupal infection status before mating with an infected individual. This technique of detecting infection in discarded pupae exuviae has not been evaluated previously and will be a useful tool for others in the field of studying viral transmission in mosquitoes. Identifying these routes of transmission provides information about how CFAV could be maintained in wild populations of mosquitoes and can aid future studies focusing on interactions of CFAV with their hosts and other viruses that infect mosquitoes.

Cold case: The disappearance of Egypt bee virus, a fourth distinct master strain of deformed wing virus linked to honeybee mortality in 1970's Egypt.

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission by Varroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

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.

Temporal changes in the microbiome of stingless bee foragers following colony relocation.

Maintaining beneficial interactions with microbial symbionts is vital for animal health. Yet, for social insects, the stability of microbial associations within and between cohorts is largely unknown. We investigated temporal changes in the microbiomes of nine stingless bee (Tetragonula carbonaria) colonies at seven timepoints across a 10-month period when moved between two climatically and florally different sites. Bacterial 16S rRNA gene and fungal ITS amplicon sequencing confirmed that microbiomes varied considerably between colonies initially at site one. However, following relocation, considerable changes occurred in bacterial community composition within each colony, and the microbiome composition became more similar across colonies. Notably, Snodgrassella disappeared and Zymobacter appeared as relatively abundant taxa. Remarkably, bacterial communities within colonies continued to shift over time but remained similar across colonies, becoming dominated by Acinetobacter six months after returning to the original site. Our results indicate that the stingless bee microbiome can undergo major changes in response to the environment, and that these changes can be long-lasting. Such legacy effects have not been reported for corbiculate bees. Further understanding the microbial ecology of stingless bees will aid future management of colonies used in agricultural production.

RNAseq of Deformed Wing Virus and Other Honey Bee-Associated Viruses in Eight Insect Taxa with or without Varroa Infestation.

The global spread of a parasitic mite (Varroa destructor) has resulted in Deformed wing virus (DWV), a previously rare pathogen, now dominating the viromes in honey bees and contributing to large-scale honey bee colony losses. DWV can be found in diverse insect taxa and has been implicated in spilling over from honey bees into associated ("apiary") and other ("non-apiary") insects. Here we generated next generation sequence data from 127 insect samples belonging to diverse taxa collected from Hawaiian islands with and without Varroa to identify whether the mite has indirectly affected the viral landscapes of key insect taxa across bees, wasps, flies and ants. Our data showed that, while Varroa was associated with a dramatic increase in abundance of (predominantly recombinant) DWV in honey bees (and no other honey bee-associated RNA virus), this change was not seen in any other taxa sampled. Honey bees share their environment with other insect populations and exist as a homogenous group, frequently sharing common viruses, albeit at low levels. Our data suggest that the threat of Varroa to increase viral load in an apiary does not automatically translate to an increase in virus load in other insects living in the wider community.

Linking the Phyllosphere Microbiome to Plant Health.

The phyllosphere harbors diverse microbial communities that influence ecosystem functioning. Emerging evidence suggests that plants impaired in genetic networks harbor an altered microbiome and develop dysbiosis in the phyllosphere, which pinpoints plant genetics as a key driver of the phyllosphere microbiome assembly and links the phyllosphere microbiome to plant health.

Microbiome-Mediated Stress Resistance in Plants.

Plants are subjected to diverse biotic and abiotic stresses in life. These can induce changes in transcriptomics and metabolomics, resulting in changes to root and leaf exudates and, in turn, altering the plant-associated microbial community. Emerging evidence demonstrates that changes, especially the increased abundance of commensal microbes following stresses, can be beneficial for plant survival and act as a legacy, enhancing offspring fitness. However, outstanding questions remain regarding the microbial role in plant defense, many of which may now be answered utilizing a novel synthetic community approach. In this article, building on our current understanding on stress-induced changes in plant microbiomes, we propose a 'DefenseBiome' concept that informs the design and construction of beneficial microbial synthetic communities for improving fundamental understanding of plant-microbial interactions and the development of plant probiotics.

Occurrence of honey bee-associated pathogens in Varroa-free pollinator communities.

Australia remains the last significant land mass free of Varroa, a parasitic mite which has caused dramatic honey bee (Apis mellifera) colony losses across the globe, due to its association with the pathogenic deformed wing virus (DWV). As such, Australia continues to maintain relatively healthy honey bee populations, despite recent work showing apiaries harbor a surprisingly high prevalence of microbial pathogens. We sought to determine the prevalence of these microbial pathogens in honey bees and native pollinators actively co-foraging on mass flowering crops and to understand the extent to which they may be shared between taxa. We found high prevalences of black queen cell virus (BQCV) and sacbrood virus (SBV) in the honey bees (88% and 41% respectively), and correspondingly, these were the most common honey bee pathogens detected in native pollinator taxa, albeit at much lower prevalence; the maximum prevalence for any pathogen in a native pollinator group was 24% (BQCV in Halictidae spp.). The viral pathogens Israeli acute paralysis virus and Lake Sinai viruses 1 and 2, and the fungal parasites Nosema apis and Nosema ceranae, were only rarely detected. Phylogenetic analyses of the most common pathogens revealed similar genotypes circulating between species. Our data suggest that, in Australian orchards, pathogen prevalence in honey bees is a good predictor of pathogen prevalence in native pollinators, which raises concerns about how the viral landscape may change in native taxa if, or when, Varroa arrives.

Deformed Wing Virus in Honeybees and Other Insects.

Deformed wing virus (DWV) has become the most well-known, widespread, and intensively studied insect pathogen in the world. Although DWV was previously present in honeybee populations, the arrival and global spread of a new vector, the ectoparasitic mite Varroa destructor, has dramatically altered DWV epidemiology. DWV is now the most prevalent virus in honeybees, with a minimum average of 55% of colonies/apiaries infected across 32 countries. Additionally, DWV has been detected in 65 arthropod species spanning eight insect orders and three orders of Arachnida. Here, we describe the significant progress that has been made in elucidating the capsid structure of the virus, understanding its ever-expanding host range, and tracking the constantly evolving DWV genome and formation of recombinants. The construction of molecular clones, working with DWV in cell lines, and the development of immunohistochemistry methods will all help the community to move forward. Identifying the tissues in which DWV variants are replicating and understanding the impact of DWV in non-honeybee hosts are major new goals.

RNAseq Analysis Reveals Virus Diversity within Hawaiian Apiary Insect Communities.

Deformed wing virus (DWV) is the most abundant viral pathogen of honey bees and has been associated with large-scale colony losses. DWV and other bee-associated RNA viruses are generalists capable of infecting diverse hosts. Here, we used RNAseq analysis to test the hypothesis that due to the frequency of interactions, a range of apiary pest species would become infected with DWV and/or other honey bee-associated viruses. We confirmed that DWV-A was the most prevalent virus in the apiary, with genetically similar sequences circulating in the apiary pests, suggesting frequent inter-species transmission. In addition, different proportions of the three DWV master variants as indicated by BLAST analysis and genome coverage plots revealed interesting DWV-species groupings. We also observed that new genomic recombinants were formed by the DWV master variants, which are likely adapted to replicate in different host species. Species groupings also applied when considering other viruses, many of which were widespread in the apiaries. In social wasps, samples were grouped further by site, which potentially also influenced viral load. Thus, the apiary invertebrate community has the potential to act as reservoirs of honey bee-associated viruses, highlighting the importance of considering the wider community in the apiary when considering honey bee health.

Plant Defense by VOC-Induced Microbial Priming.

The plant holobiont extends the plant's capacity for nutrient acquisition and stress protection. Recent studies show that under biotic stress plants can promote the acquisition of certain beneficial bacteria to their rhizosphere. Active emission of volatile organic compounds (VOCs) is a newly identified mechanism utilized by plants for this process.

Evidence of Varroa-mediated deformed wing virus spillover in Hawaii.

Varroa destructor, a parasitic mite of honey bees, is also a vector for viral diseases. The mite displays high host specificity and requires access to colonies of Apis spp. to complete its lifecycle. In contrast, the Deformed Wing Virus (DWV), one of the many viruses transmitted by V. destructor, appears to have a much broader host range. Previous studies have detected DWV in a variety of insect groups that are not directly parasitized by the mite. In this study, we take advantage of the discrete distribution of the Varroa mite in the Hawaiian archipelago to compare DWV prevalence on non-Apis flower visitors, and test whether Varroa presence is linked to a "viral spillover". We selected two islands with different viral landscapes: Oahu, where V. destructor has been present since 2007, and Maui, where the mite is absent. We sampled individuals of Apis mellifera, Ceratina smaragdula, Polistes aurifer, and Polistes exclamens, to assess and compare the DWV prevalence in the Hymenoptera community of the two islands. The results indicated that, as expected, honey bee colonies on Oahu have much higher incidence of DWV compared to Maui. Correspondingly, DWV was detected on the Non-Apis Hymenoptera collected from Oahu, but was absent in the species examined on Maui. The study sites selected shared a similar geography, climate, and insect fauna, but differed in the presence of the Varroa mite, suggesting an indirect, but significant, increase on DWV prevalence in the Hymenoptera community on mite-infected islands.

Novel RNA Virus Genome Discovered in Ghost Ants (Tapinoma melanocephalum) from Hawaii.

Here, we report the full-genome sequence of Milolii virus, a novel single-stranded (positive-sense) RNA virus discovered from Tapinoma melanocephalum ants in Hawaii. The genome is 10,475 nucleotides long, encoding a polyprotein of 3,304 amino acids.

A Comparison of Deformed Wing Virus in Deformed and Asymptomatic Honey Bees.

Deformed wing virus (DWV) in association with Varroa destructor is currently attributed to being responsible for colony collapse in the western honey bee (Apis mellifera). The appearance of deformed individuals within an infested colony has long been associated with colony losses. However, it is unknown why only a fraction of DWV positive bees develop deformed wings. This study concerns two small studies comparing deformed and non-deformed bees. In Brazil, asymptomatic bees (no wing deformity) that had been parasitised by Varroa as pupae had higher DWV loads than non-parasitised bees. However, we found no greater bilateral asymmetry in wing morphology due to DWV titres or parasitisation. As expected, using RT-qPCR, deformed bees were found to contain the highest viral loads. In a separate study, next generation sequencing (NGS) was applied to compare the entire DWV genomes from paired symptomatic and asymptomatic bees from three colonies on two different Hawaiian islands. This revealed no consistent differences between DWV genomes from deformed or asymptomatic bees, with the greatest variation seen between locations, not phenotypes. All samples, except one, were dominated by DWV type A. This small-scale study suggests that there is no unique genetic variant associated with wing deformity; but that many DWV variants have the potential to cause deformity.