Transcriptome Analysis Reveals a Diverse Range of Novel Viruses in Australian Sugarcane Soldier Fly (Inopus flavus) Larvae.

In Australia, Soldier flies (Inopus spp.) are economically significant pests of sugarcane that currently lack a viable management strategy. Despite various research efforts, the mechanisms underlying the damage caused by soldier fly larvae remain poorly understood. Our study aims to explore whether this damage is associated with the transmission of plant viruses during larval feeding. We also explore the larval transcriptome to identify any entomopathogenic viruses with the potential to be used as biocontrol agents in future pest management programs. Seven novel virus sequences are identified and characterised using de novo assembly of RNA-Seq data obtained from salivary glands of larvae. The novel virus sequences belong to different virus families and are tentatively named SF-associated anphevirus (SFaAV), SF-associated orthomyxo-like virus (SFaOV), SF-associated narna-like virus (SFaNV), SF-associated partiti-like virus (SFaPV), SF-associated toti-like virus (SFaTV-1 and SFaTV-2) and SF-associated densovirus (SFaDV). These newly identified viruses are more likely insect-associated viruses, as phylogenetic analyses show that they cluster with other insect-specific viruses. Small RNA analysis indicates prominent peaks at both 21 nt and 26-29 nt, suggesting the activation of host siRNA and piwiRNA pathways. Our study helps to improve understanding of the virome of soldier flies and could identify insect viruses for deployment in novel pest management strategies.

Differential gene expression and microRNA profile in corpora allata-corpora cardiaca of Aedes aegypti mosquitoes with weak juvenile hormone signalling.

The corpora allata-corpora cardiaca (CA-CC) is an endocrine gland complex that regulates mosquito development and reproduction through the synthesis of juvenile hormone (JH). Epoxidase (Epox) is a key enzyme in the production of JH. We recently utilized CRISPR/Cas9 to establish an epoxidase-deficient (epox-/-) Aedes aegypti line. The CA from epox-/- mutants do not synthesize epoxidated JH III but methyl farneosate (MF), a weak agonist of the JH receptor, and therefore have reduced JH signalling. Illumina sequencing was used to examine the differences in gene expression between the CA-CC from wild type (WT) and epox-/- adult female mosquitoes. From 18,034 identified genes, 317 were significantly differentially expressed. These genes are involved in many biological processes, including the regulation of cell proliferation and apoptosis, energy metabolism, and nutritional uptake. In addition, the same CA-CC samples were also used to examine the microRNA (miRNA) profiles of epox-/- and WT mosquitoes. A total of 197 miRNAs were detected, 24 of which were differentially regulated in epox-/- mutants. miRNA binding sites for these particular miRNAs were identified using an in silico approach; they target a total of 101 differentially expressed genes. Our results suggest that a lack of epoxidase, besides affecting JH synthesis, results in the diminishing of JH signalling that have significant effects on Ae. aegypti CA-CC transcriptome profiles, as well as its miRNA repertoire.

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.

Analysing inhibition of dengue virus in Wolbachia-infected mosquito cells following the removal of Wolbachia.

Wolbachia pipientis is known to block replication of positive sense RNA viruses. Previously, we created an Aedes aegypti Aag2 cell line (Aag2.wAlbB) transinfected with the wAlbB strain of Wolbachia and a matching tetracycline-cured Aag2.tet cell line. While dengue virus (DENV) was blocked in Aag2.wAlbB cells, we found significant inhibition of DENV in Aag2.tet cells. RNA-Seq analysis of the cells confirmed removal of Wolbachia and lack of expression of Wolbachia genes that could have been due to lateral gene transfer in Aag2.tet cells. However, we noticed a substantial increase in the abundance of phasi charoen-like virus (PCLV) in Aag2.tet cells. When RNAi was used to reduce the PCLV levels, DENV replication was significantly increased. Further, we found significant changes in the expression of antiviral and proviral genes in Aag2.tet cells. Overall, the results reveal an antagonistic interaction between DENV and PCLV and how PCLV-induced changes could contribute to DENV inhibition.

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.

Sex dependent transcriptome responses of the diamondback moth, Plutella xylostella L. to cold stress.

Temperature has fundamental influences on the performance and distribution of insects. While considerable attention has been devoted to extreme conditions, particularly extreme cold conditions, few studies have investigated effects of mild cold conditions on insects. We examined the transcriptomic changes in mid-fourth instar larvae of both sexes reared at 10 °C and 25 °C to investigate sex-dependent responses of Plutella xylostella to mild cold stress. There were 624 differentially expressed genes (DEGs) in females, the majority of which (n = 386) were down-regulated. In males 3239 genes were differentially expressed and the majority (n = 2341) were up-regulated. Only 280 DEGs were common to both sexes. In females, there were no DEGs encoding heat shock or cold shock proteins, but six of these DEGs were found in males. These differences suggest that females and males might adopt some different strategies to cope with cold stress and/or that they were affected by rearing under cold conditions to different degrees and in different ways. In addition, DEGs encoding antimicrobial peptides, cytochrome P450 monooxygenases, fatty acid-related enzymes, cuticle proteins, myofilament, and hormone-related proteins were found in both sexes under cold stress. The transcriptome study reveals unexpected sex-dependent thermal responses and provides new information of how an insect that does not diapause copes with low temperatures.

Corrigendum to ``Examination of population genetics of the Coconut Rhinoceros Beetle (Oryctes rhinoceros) and the incidence of its biocontrol agent (Oryctes rhinoceros nudivirus) in the South Pacific Islands Current'' [Research in Insect Science 1 (2021) 100015].

[This corrects the article DOI: 10.1016/j.cris.2021.100015.].

A high-quality de novo genome assembly based on nanopore sequencing of a wild-caught coconut rhinoceros beetle (Oryctes rhinoceros).

BACKGROUND: An optimal starting point for relating genome function to organismal biology is a high-quality nuclear genome assembly, and long-read sequencing is revolutionizing the production of this genomic resource in insects. Despite this, nuclear genome assemblies have been under-represented for agricultural insect pests, particularly from the order Coleoptera. Here we present a de novo genome assembly and structural annotation for the coconut rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), based on Oxford Nanopore Technologies (ONT) long-read data generated from a wild-caught female, as well as the assembly process that also led to the recovery of the complete circular genome assemblies of the beetle's mitochondrial genome and that of the biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). As an invasive pest of palm trees, O. rhinoceros is undergoing an expansion in its range across the Pacific Islands, requiring new approaches to management that may include strategies facilitated by genome assembly and annotation. RESULTS: High-quality DNA isolated from an adult female was used to create four ONT libraries that were sequenced using four MinION flow cells, producing a total of 27.2 Gb of high-quality long-read sequences. We employed an iterative assembly process and polishing with one lane of high-accuracy Illumina reads, obtaining a final size of the assembly of 377.36 Mb that had high contiguity (fragment N50 length = 12 Mb) and accuracy, as evidenced by the exceptionally high completeness of the benchmarked set of conserved single-copy orthologous genes (BUSCO completeness = 99.1%). These quality metrics place our assembly ahead of the published Coleopteran genomes, including that of an insect model, the red flour beetle (Tribolium castaneum). The structural annotation of the nuclear genome assembly contained a highly-accurate set of 16,371 protein-coding genes, with only 2.8% missing BUSCOs, and the expected number of non-coding RNAs. The number and structure of paralogous genes in a gene family like Sigma GST is lower than in another scarab beetle (Onthophagus taurus), but higher than in the red flour beetle (Tribolium castaneum), which suggests expansion of this GST class in Scarabaeidae. The quality of our gene models was also confirmed with the correct placement of O. rhinoceros among other members of the rhinoceros beetles (subfamily Dynastinae) in a phylogeny based on the sequences of 95 protein-coding genes in 373 beetle species from all major lineages of Coleoptera. Finally, we provide a list of 30 candidate dsRNA targets whose orthologs have been experimentally validated as highly effective targets for RNAi-based control of several beetles. CONCLUSIONS: The genomic resources produced in this study form a foundation for further functional genetic research and management programs that may inform the control and surveillance of O. rhinoceros populations, and we demonstrate the efficacy of de novo genome assembly using long-read ONT data from a single field-caught insect.

Discovery of a Novel Jingmenvirus in Australian Sugarcane Soldier Fly (Inopus flavus) Larvae.

In Australia, soldier flies are major pests of sugarcane, and they can cause significant yield losses in some areas, possibly due to the virus' transmission to the plants. We sequenced fly larvae salivary glands and identified a novel jingmenvirus, putatively named Inopus flavus jingmenvirus 1 (IFJV1). Phylogenetic trees confirmed that IFJV1 groups with insect-associated jingmenviruses, newly identified flavivirus-like viruses with a segmented genome. After the design and the validation of molecular detection systems for IFJV1, larval homogenates were passaged on insect and vertebrate cells, but IFJV1 could only be detected in the first two passages in insect cells and not at all in vertebrate cells. Despite this lack of consistent replication in laboratory models, this virus does replicate in its host Inopus flavus, as sequenced, small RNA from the larvae matched the IFJV1 sequences. Moreover, they were found to be predominantly 21 nucleotides long and map to the whole sequences on both strands, which is typical of an actively replicating virus. This discovery confirms the worldwide presence of jingmenviruses which, until now, had only been detected on four continents. However, the study of IFJV1 tropism and the possible pathogenicity to its host or the sugarcane it parasitizes requires the development of a stable replication model.

N6-methyladenosine modification of the Aedes aegypti transcriptome and its alteration upon dengue virus infection in Aag2 cell line.

The N6-methyladenosine (m6A) modification of RNA has been reported to affect viral infections. Studies have confirmed the role of m6A in replication of several vector-borne flaviviruses, including dengue virus (DENV), in mammalian cells. Here, we explored the role of m6A in DENV replication in the mosquito Aedes aegypti Aag2 cell line. We first determined the presence of m6A on the RNAs from mosquito cells and using methylated RNA immunoprecipitation and sequencing (MeRIP-Seq) identified m6A modification of the mosquito transcriptome and those that changed upon DENV infection. Depletion of m6A methyltransferases and the m6A binding protein YTHDF3 RNAs decreased the replication of DENV. In particular, we found that the Ae. aegypti ubiquitin carrier protein 9 (Ubc9) is m6A modified and its expression increases after DENV infection. Silencing of the gene and ectopic expression of Ubc9 led to reduced and increased DENV replication, respectively. The abundance of Ubc9 mRNA and its stability were reduced with the inhibition of m6A modification, implying that m6A modification of Ubc9 might enhance expression of the gene. We also show that the genome of DENV is m6A modified at five sites in mosquito cells. Altogether, this work reveals the involvement of m6A modification in Ae. aegypti-DENV interaction.

Transcriptomics Reveal Several Novel Viruses from Canegrubs (Coleoptera: Scarabaeidae) in Central Queensland, Australia.

Canegrubs (Coleoptera: Scarabaeidae) are major pests of sugarcane crops in Australia, but despite long-term and intensive research, no commercially viable biological control agents have been identified. We used the RNA-Seq approach to explore the viriomes of three different species of canegrubs from central Queensland, Australia to identify potential candidates for biological control. We identified six novel RNA viruses, characterized their genomes, and inferred their evolutionary relationships with other closely related viruses. These novel viruses showed similarity to other known members from picornaviruses, benyviruses, sobemoviruses, totiviruses, and reoviruses. The abundance of viral reads varied in these libraries; for example, Dermolepida albohirtum picorna-like virus (9696 nt) was built from 83,894 assembled reads while only 1350 reads mapped to Lepidiota negatoria beny-like virus (6371 nt). Future studies are essential to determine their natural incidence in different life stages of the host, biodiversity, geographical distributions, and potential as biological control agents for these important pests of sugarcane.

Transcriptional response of Wolbachia-transinfected Aedes aegypti mosquito cells to dengue virus at early stages of infection.

Mosquito-borne flaviviruses are responsible for viral infections and represent a considerable public health burden. Aedes aegypti is the principal vector of dengue virus (DENV), therefore understanding the intrinsic virus-host interactions is vital, particularly in the presence of the endosymbiont Wolbachia, which blocks virus replication in mosquitoes. Here, we examined the transcriptional response of Wolbachia-transinfected Ae. aegypti Aag2 cells to DENV infection. We identified differentially expressed immune genes that play a key role in the activation of anti-viral defence such as the Toll and immune deficiency pathways. Further, genes encoding cytosine and N6-adenosine methyltransferases and SUMOylation, involved in post-transcriptional modifications, an antioxidant enzyme, and heat-shock response were up-regulated at the early stages of DENV infection and are reported here for the first time. Additionally, several long non-coding RNAs were among the differentially regulated genes. Our results provide insight into Wolbachia-transinfected Ae. aegypti's initial virus recognition and transcriptional response to DENV infection.

Exploration of RNA-Seq data to identify a potential pathogen of the leaf-mining moth, Stomphastis thraustica (Meyrick, 1908) (Lepidoptera: Gracillariidae).

The leaf-mining moth, Stomphastis thraustica (Meyrick, 1908) was imported to Australia as a potential biological control agent of an exotic weed, bellyache bush (Jatropha gossypiifolia), from Peru. The insect colony has been maintained in the quarantine facility for over eight years but recently, significant mortality was observed in the culture. The larvae demonstrated swollen intersegments with a fragile integument. The infected larvae are cloudy muted green or yellowish whereas a healthy late instar larva is a vivid green. They slowly dehydrate and eventually die, at which point the larval body becomes rubbery and turns to black. We used next generation sequencing to identify the cause of mortality in the insects. Total RNA was extracted from 20 larvae in two cohorts, one with and one without apparent symptoms of disease, for deep sequencing on NovaSeq platform after eukaryote ribosomal RNA depletion. We identified several non-insect sequences belonging to viruses, bacteria, and fungi, but none of those showed significant abundance or enrichment in the infected dataset. The sequences related to a unicellular yeast, Saccharomyces cerevisiae, and they were among the highly expressed non-insect contigs; more than 5% of reads in both libraries mapped to the genome of this opportunistic microorganism.

Differential temperature responses between Plutella xylostella and its specialist endo-larval parasitoid Diadegma semiclausum-Implications for biological control.

Understanding the thermal dynamics of host-parasitoid interactions is crucial to predicting how biological control of pest insects by parasitoids might be affected by geographic location and climate change. We compared performance traits of Plutella xylostella (Lepidoptera: Plutellidae) and its solitary endo-larval parasitoid Diadegma semiclausum (Hymenoptera: Ichneumonidae), over a wide range of constant rearing temperatures (10-30°C). Parasitoids reared at 30°C experienced reductions in pupation rate, pupal mass, egg load, and adult life span when compared with those reared at lower temperatures. Our analyses of the fate of parasitoids and their hosts and intergenerational population growth at different rearing temperatures show that D. semiclausum and P. xylostella respond differently to temperature, leading to divergent outcomes under different temperature conditions. Some parasitoid larvae could not complete development at 30°C, the temperature at which the host biomass was least and the metabolic demands of the parasitoid could be high, suggesting that parasitoid development might be constrained by lack of host resources at higher temperatures. We discuss the potential mechanisms of parasitoid susceptibility to elevated temperatures, which likely explain the pronounced seasonal dynamics of D. semiclausum in subtropical regions and its failure to establish in lowland tropical regions, where P. xylostella is a serious pest. Similar interactions in other host-parasitoid associations would constrain the efficacy of parasitoids as biological control agents as global temperatures increase.

Diverse Host Immune Responses of Different Geographical Populations of the Coconut Rhinoceros Beetle to Oryctes Rhinoceros Nudivirus (OrNV) Infection.

Incursions of the coconut rhinoceros beetle (CRB), Oryctes rhinoceros, into different islands in the South Pacific have been detected in recent years. It has been suggested that this range expansion is related to an O. rhinoceros haplotype reported to show reduced susceptibility to the well-established classical biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). Our understanding of the genetic characteristics which distinguish the population of O. rhinoceros that has recently established in Solomon Islands from other well-established populations across the region is very limited. Here, we hypothesized that the recently established O. rhinoceros population should have greater innate immune responses when challenged by OrNV than those of well-established and native O. rhinoceros populations. We used the RNA sequencing (RNA-Seq) approach to generate gene expression profiles of midgut tissue from OrNV-infected and noninfected individuals collected in the Solomon Islands (recent incursion), Papua New Guinea and Fiji (previously established), and the Philippines (within the native range). The collections included individuals from each of the three major mitochondrial lineages (CRB-G, CRB-PNG, and CRB-S) known to the region, allowing us to explore the specific responses of each haplotype to infection. Although insects from the Philippines and Solomon Islands that were tested belong to the same mitochondrial lineage (CRB-G), their overall responses to infection were different. The number of differentially expressed genes between OrNV-infected and noninfected wild-caught individuals from the four different locations varied from 148 to 252. Persistent OrNV infection caused a high level of induced antimicrobial activity and immune responses in O. rhinoceros, but the direction and magnitude of the responses were population specific. The insects tested from the Solomon Islands displayed extremely high expression of genes which are known to be involved in immune responses (e.g. coleoptericin, cecropin, and serpin). These variations in the host immune system among insects from different geographical regions might be driven by variations in the virulence of OrNV isolates, and this requires further investigation. Overall, our current findings support the importance of immunity in insect pest incursion and an expansion of the pest's geographic range. IMPORTANCE Oryctes rhinoceros nudivirus (OrNV) is a double-stranded DNA (dsDNA) virus which has been used as a biocontrol agent to suppress coconut rhinoceros beetle (CRB) in the Pacific Islands. Recently a new wave of CRB incursions in Oceania is thought to be related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles (CRB-G). Our comparative analysis of OrNV-infected and noninfected CRBs revealed that specific sets of genes were induced by viral infection in the beetles. This induction was much stronger in beetles collected from the Solomon Islands, a newly invaded country, than in individuals collected from within the beetle's native range (the Philippines) or from longer-established populations in its exotic range (Fiji and Papua New Guinea [PNG]). Beetles from the Philippines and the Solomon Islands that were tested in this study all belonged to the CRB-G haplotype, but the country-specific responses of the beetles to OrNV infection were different.

A new dicistro-like virus from soldier fly, Inopus flavus (Diptera: Stratiomyidae), a pest of sugarcane.

Native Australian soldier flies, Inopus spp. (Diptera: Stratiomyidae), are agricultural pests of economic importance to the sugarcane industry. A screen of the salivary gland transcriptome of Inopus flavus (James) revealed the presence of viral RNA belonging to a potentially novel member of the family Dicistroviridae. The complete genome sequence consists of 9793 nucleotides with two open reading frames. The genome includes two potential internal ribosomal entry sites (IRESs): one within the 5' UTR and the other in the intergenic region (IGR). Virus particles purified from infected larvae and visualised by electron microscopy were found to be icosahedral, non-enveloped, and 30 nm in diameter.

Human blood microRNA hsa-miR-21-5p induces vitellogenin in the mosquito Aedes aegypti.

Mosquito vectors transmit various diseases through blood feeding, required for their egg development. Hence, blood feeding is a major physiological event in their life cycle, during which hundreds of genes are tightly regulated. Blood is a rich source of proteins for mosquitoes, but also contains many other molecules including microRNAs (miRNAs). Here, we found that human blood miRNAs are transported abundantly into the fat body tissue of Aedes aegypti, a key metabolic center in post-blood feeding reproductive events, where they target and regulate mosquito genes. Using an artificial diet spiked with the mimic of an abundant and stable human blood miRNA, hsa-miR-21-5p, and proteomics analysis, we found over 40 proteins showing differential expression in female Ae. aegypti mosquitoes after feeding. Of interest, we found that the miRNA positively regulates the vitellogenin gene, coding for a yolk protein produced in the mosquito fat body and then transported to the ovaries as a protein source for egg production. Inhibition of hsa-miR-21-5p followed by human blood feeding led to a statistically insignificant reduction in progeny production. The results provide another example of the involvement of small regulatory molecules in the interaction of taxonomically vastly different taxa.

The complete mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) based on long-read nanopore sequencing.

BACKGROUND: The coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is a severe and invasive pest of coconut and other palms throughout Asia and the Pacific. The biocontrol agent, Oryctes rhinoceros nudivirus (OrNV), has successfully suppressed O. rhinoceros populations for decades but new CRB invasions started appearing after 2007. A single-SNP variant within the mitochondrial cox1 gene is used to distinguish the recently-invading CRB-G lineage from other haplotypes, but the lack of mitogenome sequence for this species hinders further development of a molecular toolset for biosecurity and management programmes against CRB. Here we report the complete circular sequence and annotation for CRB mitogenome, generated to support such efforts. METHODS: Sequencing data were generated using long-read Nanopore technology from genomic DNA isolated from a CRB-G female. The mitogenome was assembled with Flye v.2.5, using the short-read Illumina sequences to remove homopolymers with Pilon, and annotated with MITOS. Independently-generated transcriptome data were used to assess the O. rhinoceros mitogenome annotation and transcription. The aligned sequences of 13 protein-coding genes (PCGs) (with degenerate third codon position) from O. rhinoceros, 13 other Scarabaeidae taxa and two outgroup taxa were used for the phylogenetic reconstruction with the Maximum likelihood (ML) approach in IQ-TREE and Bayesian (BI) approach in MrBayes. RESULTS: The complete circular mitogenome of O. rhinoceros is 20,898 bp in length, with a gene content canonical for insects (13 PCGs, two rRNA genes, and 22 tRNA genes), as well as one structural variation (rearrangement of trnQ and trnI) and a long control region (6,204 bp). Transcription was detected across all 37 genes, and interestingly, within three domains in the control region. ML and BI phylogenies had the same topology, correctly grouping O. rhinoceros with one other Dynastinae taxon, and recovering the previously reported relationship among lineages in the Scarabaeidae. In silico PCR-RFLP analysis recovered the correct fragment set that is diagnostic for the CRB-G haplogroup. These results validate the high-quality of the O. rhinoceros mitogenome sequence and annotation.

Examination of population genetics of the Coconut Rhinoceros Beetle (Oryctes rhinoceros) and the incidence of its biocontrol agent (Oryctes rhinoceros nudivirus) in the South Pacific Islands.

Recently, incursions of the Coconut rhinoceros beetle (CRB), Oryctes rhinoceros, have been detected in south Pacific countries that were previously free of the pest. It has been suggested that this range expansion is related to an O. rhinoceros haplotype that is reported to show reduced susceptibility to the well-established classical biocontrol agent, Oryctes rhinoceros nudivirus (OrNV). We investigated O. rhinoceros population genetics and the OrNV status of specimens collected in Fiji, New Caledonia, Papua New Guinea (PNG), Samoa, Solomon Islands, Tonga, Vanuatu and the Philippines. Based on the sequence of the mitochondrial CoxI gene, we found three major mitochondrial haplotype groups (CRB-G, CRB-PNG and CRB-S) across the region. Haplotype diversity varied between and within countries and a high incidence of OrNV infection was detected in all haplotypes wherever they occurred. The O. rhinoceros population in some countries was monotypic and all individuals tested belonged to a single haplotype group. However, in Samoa we detected CRB-S and CRB-PNG and in Solomon Islands we detected all three haplotype groups. Genotyping-by-Sequencing (GBS) showed genetic differentiation in the O. rhinoceros nuclear genome across populations on different islands and provided evidence for gene flow, resulting in a well-mixed population, despite the presence of different CoxI haplotypes in Solomon Islands. Evidence of admixture was also detected on both islands of Samoa. The current CoxI based method is not a reliable diagnostic marker for phenotypic traits, especially in countries such as Solomon Islands where the mitochondrial haplotypes have come back into sympatry and are mixed. To identify possible mechanisms of resistance to OrNV, further molecular analyses O. rhinoceros in response to virus infection is required. To improve biological control of O. rhinoceros, such analyses will need to be combined with an improved understanding of the population genetics of the pest and the evolutionary history of OrNV in the region.

Detection of deformed wing virus (DWV) in the Vietnamese walking stick Medauroidea extradentata (Phasmatodea).

Deformed wing virus (DWV) is a single-stranded positive sense RNA virus that mainly infects honey bees (Apis mellifera) and can have devastating impacts on the colony. Recent studies have shown the presence of this virus in several species of Apis spp. and some other Hymenoptera, but our knowledge of their host range is very limited. We screened previously sequenced RNAseq libraries from different tissues of Vietnamese Walking Stick, Medauroidea extradentata (Phasmatodea) for DWV. We only found this virus in six libraries from anterior and posterior midgut tissue. From the midgut libraries we were able to construct a complete DWV genome sequence, which consisted of 10,140 nucleotides and included one open reading frame. Pairwise genome comparison confirmed strong similarity (98.89 %) of these assembled sequences with only 113 SNPs to the original DWV genome. We hypothesize the M. extradentata acquired this virus via a foodborne transmission by consuming DWV-infected material such as pollen or leaves contaminated with virus infected bee faeces.