North American crayfish harbour diverse members of the Nudiviridae.

Three novel crayfish-infecting nudiviruses from crayfish in North America represent the first genomic confirmation of nudiviruses in crayfish: Faxonius propinquus nudivirus (FpNV), Faxonius rusticus nudivirus (FrNV), and Faxonius virilis nudivirus (FvNV). Histopathology and electron microscopy revealed nuclear infections, including nuclear hypertrophy in hepatopancreatic epithelial cells and the presence of membrane-bound bacilliform virions. Metagenomic sequencing resulted in complete circular genome assembly, and phylogenetic analyses (based on nudivirus core genes) placed these viruses within the unofficial Epsilonnudivirus genus. One of the nudiviruses was detected in the antennal gland of its host, and another is correlated with invasive crayfish decline in one infected lake ecosystem - suggesting a potential route for viral transmission through water, and possible population level impact. This study highlights the importance of genomic and ecological data in elucidating the diversity and evolutionary relationships of the Nudiviridae, while expanding their known diversity and range of host species.

Viral Prevalence and Genomic Xenology in the Coevolution of HzNV-2 (Nudiviridae) with Host Helicoverpa zea (Lepidoptera: Noctuidae).

Insect viruses have been described from numerous lineages, yet patterns of genetic exchange and viral prevalence, which are essential to understanding host-virus coevolution, are rarely studied. In Helicoverpa zea, the virus HzNV-2 can cause deformity of male and female genitalia, resulting in sterility. Using ddPCR, we found that male H. zea with malformed genitalia (agonadal) contained high levels of HzNV-2 DNA, confirming previous work. HzNV-2 was found to be prevalent throughout the United States, at more than twice the rate of the baculovirus HaSNPV, and that it contained several host-acquired DNA sequences. HzNV-2 possesses four recently endogenized lepidopteran genes and several more distantly related genes, including one gene with a bacteria-like sequence found in both host and virus. Among the recently acquired genes is cytosolic serine hydroxymethyltransferase (cSHMT). In nearly all tested H. zea, cSHMT contained a 200 bp transposable element (TE) that was not found in cSHMT of the sister species H. armigera. No other virus has been found with host cSHMT, and the study of this shared copy, including possible interactions, may yield new insights into the function of this gene with possible applications to insect biological control, and gene editing.

Complete genome sequence of an oryctes rhinoceros nudivirus isolated from Korean rhinoceros beetles (Trypoxylus dichotomus) in Korea.

Nudivirus-infected Korean rhinoceros beetles (Trypoxylus dichotomus) were first identified in 2015, and while a complete genome sequence of the virus has long been uploaded to the NCBI database, it has not been examined in detail. Here, we describe the genomic characteristics of Trypoxylus dichotomus nudivirus (TdNV), which represents a new Oryctes rhinoceros nudivirus (OrNV) strain, isolated from infected T. dichotomus in the Republic of Korea. We examined factors derived by the cross-species infection of OrNV from nucleotide levels to the whole genome level. Our genomic analysis study suggests that TdNV-Korea is highly conserved with other OrNVs in terms of genomic structures and genome size. Our investigation of the genomic structure revealed that TdNV-Korea has the least number of open reading frames (ORFs) of all available OrNV genomes; three hypothetical genes were notably absent only in TdNV-Korea. In addition, the genomic alteration of the nudivirus core genes discloses that various amino acid mutations caused by single-nucleotide polymorphism and short indels (insertion/deletion) were found in most of the nudivirus core genes of TdNV-Korea. Our findings provide a valuable resource for those seeking a greater understanding of cross-species nudivirus transmission and will certainly provide valuable insight for reconstruction and reinterpretation of future and previously identified OrNV strains.

A chromosome scale assembly of the parasitoid wasp Venturia canescens provides insight into the process of virus domestication.

The parasitoid wasp Venturia canescens is an important biological control agent of stored products moth pests and serves as a model to study the function and evolution of domesticated endogenous viruses (DEVs). The DEVs discovered in V. canescens are known as virus-like particles (VcVLPs), which are produced using nudivirus-derived components and incorporate wasp-derived virulence proteins instead of packaged nucleic acids. Previous studies of virus-derived components in the V. canescens genome identified 53 nudivirus-like genes organized in six gene clusters and several viral pseudogenes, but how VcVLP genes are organized among wasp chromosomes following their integration in the ancestral wasp genome is largely unknown. Here, we present a chromosomal scale genome of V. canescens consisting of 11 chromosomes and 56 unplaced small scaffolds. The genome size is 290.8 Mbp with a N50 scaffold size of 24.99 Mbp. A high-quality gene set including 11,831 protein-coding genes were produced using RNA-Seq data as well as publicly available peptide sequences from related Hymenoptera. A manual annotation of genes of viral origin produced 61 intact and 19 pseudogenized nudivirus-derived genes. The genome assembly revealed that two previously identified clusters were joined into a single cluster and a total of 5 gene clusters comprising of 60 intact nudivirus-derived genes were located in three chromosomes. In contrast, pseudogenes are dispersed among 8 chromosomes with only 4 pseudogenes associated with nudivirus gene clusters. The architecture of genes encoding VcVLP components suggests it originates from a recent virus acquisition and there is a link between the processes of dispersal and pseudogenization. This high-quality genome assembly and annotation represents the first chromosome-scale assembly for parasitoid wasps associated with VLPs, and is publicly available in the National Center for Biotechnology Information Genome and RefSeq databases, providing a valuable resource for future studies of DEVs in parasitoid wasps.

Conserved Viral Transcription Plays a Key Role in Virus-Like Particle Production of the Parasitoid Wasp Venturia canescens.

Nudiviruses are large double-stranded DNA viruses related to baculoviruses known to be endogenized in the genomes of certain parasitic wasp species. These wasp-virus associations allow the production of viral particles or virus-like particles that ensure wasp parasitism success within lepidopteran hosts. Venturia canescens is an ichneumonid wasp belonging to the Campopleginae subfamily that has endogenized nudivirus genes belonging to the Alphanudivirus genus to produce "virus-like particles" (Venturia canescens virus-like particles [VcVLPs]), which package proteic virulence factors. The main aim of this study was to determine whether alphanudivirus gene functions have been conserved following endogenization. The expression dynamics of alphanudivirus genes was monitored by a high throughput transcriptional approach, and the functional role of lef-4 and lef-8 genes predicted to encode viral RNA polymerase components was investigated by RNA interference. As described for baculovirus infections and for endogenized nudivirus genes in braconid wasp species producing bracoviruses, a transcriptional cascade involving early and late expressed alphanudivirus genes could be observed. The expression of lef-4 and lef-8 was also shown to be required for the expression of alphanudivirus late genes allowing correct particle formation. Together with previous literature, the results show that endogenization of nudiviruses in parasitoid wasps has repeatedly led to the conservation of the viral RNA polymerase function, allowing the production of viruses or viral-like particles that differ in composition but enable wasp parasitic success. IMPORTANCE This study shows that endogenization of a nudivirus genome in a Campopleginae parasitoid wasp has led to the conservation, as for endogenized nudiviruses in braconid parasitoid wasps, of the viral RNA polymerase function, required for the transcription of genes encoding viral particles involved in wasp parasitism success. We also showed for the first time that RNA interference (RNAi) can be successfully used to downregulate gene expression in this species, a model in behavioral ecology. This opens the opportunity to investigate the function of genes involved in other traits important for parasitism success, such as reproductive strategies and host choice. Fundamental data acquired on gene function in Venturia canescens are likely to be transferable to other parasitoid wasp species used in biological control programs. This study also renders possible the investigation of other nudivirus gene functions, for which little data are available.

Coconut Rhinoceros Beetle in Samoa: Review of a Century-Old Invasion and Prospects for Control in a Changing Future.

It is now more than 100 years since the coconut rhinoceros beetle (CRB: Oryctes rhinoceros L.) was first detected in the Pacific Island state of Samoa. The exotic pest from Asia became the principal pest of coconut palms in Samoa and, from this first point of invasion, spread to several surrounding countries in the South-West Pacific Ocean. An intensive control operation was initiated, but the beetle could not be eliminated. Various pest management strategies were attempted but had limited success until the introduction of a biological control agent (BCA), Oryctes rhinoceros nudivirus (OrNV), during the late 1960s and early 1970s. The biocontrol release was very successful and became the prime example of "classical biological control" of an insect pest by a virus. Changing economic and social conditions in Samoa and other islands of the Pacific require a re-evaluation of the threat of CRB to coconut production to suggest how the IPM system may be modified to meet future needs. Therefore, it is timely to review the history of CRB in Samoa and summarize experiences in development of an integrated pest management (IPM) system limiting the impact of the pest. We also present results from a recent study conducted in 2020 on the island of Upolu to define the current status of the CRB population and its BCA, OrNV. The lessons from Samoa, with its long history of containment and management of CRB, are applicable to more recent invasion sites. Recommendations are provided to modify the IPM programme to enhance the sustainable control of CRB and support the ongoing coconut replantation program promoted by the Samoan government.

Monodon baculovirus (MBV) infects wild mud crab, Scylla serrata.

During a survey of farmed and wild crustaceans from India for viruses, spherical baculovirosis otherwise known as Penaeus monodon-type baculovirus (MBV) was detected in field-collected juvenile/sub-adult mud crab, Scylla serrata using a nested polymerase chain reaction (PCR)-based amplification of the hepatopancreatic DNA. Eight out of 115 mud crab (7.0%) examined during the study were found to be positive in the nested PCR resulting in a 361 nt amplicon. Mud crab, S. olivacea and other crustaceans such as marine crab, Portunus sanguinolentus and farmed penaeid shrimp, Penaeus vannamei and P. monodon were tested negative for the virus. Further, degenerate primers reported to amplify polyhedrin protein gene of MBV also showed PCR amplification in one of the MBV-positive crab samples resulting in a 250 nt amplicon. Sequencing of the two target amplicons (MBV- 361 nt and MBV polyhedrin - 216 nt) revealed more than 97.5 % and 92.8% sequence identity, respectively with the Penaeus monodon nudivirus and Penaeus monodon nucleopolyhedrovirus (MBV) reported from shrimp. Further, histological analysis of mud crab revealed nuclear hypertrophy, chromatin margination and intranuclear eosinophilic/basophilic inclusions in tubule epithelium of hepatopancreas. The hepatopancreatic tissue also showed unusually large, eosinophilic/basophilic inclusion-like structures. These inclusions resembled the viral inclusions reported from S. serrata from Australia. This is the first record of monodon-type baculovirus from a crab host and the second from a non-penaeid crustacean. Interestingly, some of the crab samples also showed deeply basophilic intranuclear inclusion-like bodies resembling hepatopancreatic parvovirus group of viruses (HPV). However, none of the crab samples subjected to PCR amplification using HPV-specific primers showed any amplification. The histological observations made in the present study indicate the possibility of the presence of two hepatopancreas-infecting viruses in S. serrata from India.

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.

Identification and Full Characterisation of Two Novel Crustacean Infecting Members of the Family Nudiviridae Provides Support for Two Subfamilies.

Multiple enveloped viruses with rod-shaped nucleocapsids have been described, infecting the epithelial cell nuclei within the hepatopancreas tubules of crustaceans. These bacilliform viruses share the ultrastructural characteristics of nudiviruses, a specific clade of viruses infecting arthropods. Using histology, electron microscopy and high throughput sequencing, we characterise two further bacilliform viruses from aquatic hosts, the brown shrimp (Crangon crangon) and the European shore crab (Carcinus maenas). We assembled the full double stranded, circular DNA genome sequences of these viruses (~113 and 132 kbp, respectively). Comparative genomics and phylogenetic analyses confirm that both belong within the family Nudiviridae but in separate clades representing nudiviruses found in freshwater and marine environments. We show that the three thymidine kinase (tk) genes present in all sequenced nudivirus genomes, thus far, were absent in the Crangon crangon nudivirus, suggesting there are twenty-eight core genes shared by all nudiviruses. Furthermore, the phylogenetic data no longer support the subdivision of the family Nudiviridae into four genera (Alphanudivirus to Deltanudivirus), as recently adopted by the International Committee on Taxonomy of Viruses (ICTV), but rather shows two main branches of the family that are further subdivided. Our data support a recent proposal to create two subfamilies within the family Nudiviridae, each subdivided into several genera.

The discovery, distribution, and diversity of DNA viruses associated with Drosophila melanogaster in Europe.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here, we report fourteen DNA viruses detected in a metagenomic analysis of 6668 pool-sequenced Drosophila, sampled from forty-seven European locations between 2014 and 2016. These include three new nudiviruses, a new and divergent entomopoxvirus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of galbut virus, a double-stranded RNA partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a bidnavirus, may be composed of up to twelve segments and thus represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2 per cent or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D.melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

Can Biological Control Overcome the Threat From Newly Invasive Coconut Rhinoceros Beetle Populations (Coleoptera: Scarabaeidae)? A Review.

The coconut rhinoceros beetle (CRB: Oryctes rhinoceros Linnaeus) is one of the most damaging pests to coconut and oil palms in Asia and the Pacific Islands. Adults bore into the crown and damage developing fronds, which affects tree development and yield. The insect is native to South and Southeast Asia and was inadvertently introduced into the Pacific in 1909. It has since spread to several Pacific island nations and territories, causing significant economic impact on these important coconut and palm-growing regions. In the 1950s and 1960s, an international biological control effort was initiated to search for and release natural enemy species. Release of the Oryctes rhinoceros nudivirus Huger (OrNV) and the species complex of Metarhizium Sorokin (Hypocreales: Clavicipitaceae) was successful in controlling CRB in its invaded range. Recently a new biotype of the beetle, known as CRB-G, has spread into the Pacific Islands causing unprecedented levels of damage due to the failure of previously successful biological control agents (BCAs) to suppress this biotype. The re-emergence of CRB as a serious pest warrants a rigorous re-evaluation of potential BCAs and a new search for effective natural enemies if necessary. In this article, we review literature on CRB to 1) analyze past introductions of BCAs and their effectiveness; 2) identify potentially important natural enemies and their geographical origins; and 3) assess possible approaches for utilization of BCAs against the new wave of CRB invasion. Research gaps and directions deserving future attention are highlighted and a strategy for renovation of biological controls for CRB suggested.

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.

Transcription Profile and Genomic Variations of Oryctes Rhinoceros Nudivirus in Coconut Rhinoceros Beetles.

Oryctes rhinoceros nudivirus (OrNV) is a double-stranded DNA (dsDNA) virus which has been used as a biocontrol agent to suppress the coconut rhinoceros beetle (Oryctes rhinoceros) in Southeast Asia and the Pacific Islands. A new wave of O. rhinoceros incursions in Oceania is thought to be related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles. In this study, chronically infected beetles were collected from Philippines, Fiji, Papua New Guinea (PNG), and the Solomon Islands (SI). RNA sequencing (RNA-seq) was performed to investigate the global viral gene expression profiles and for comparative genomic analysis of structural variations. Maximum likelihood phylogenic analysis indicated that OrNV strains from the SI and Philippines are closely related, while OrNV strains from PNG and Fiji formed a distinct adjacent clade. We detected several polymorphic sites with a frequency higher than 35% in 892 positions of the viral genome. Nonsynonymous mutations were detected in several hypothetical proteins and 15 nudivirus core genes, such as gp034, lef-8, lef-4, and vp91 We found limited evidence of variation in viral gene expression among geographic populations. Only a few genes, such as gp01, gp022, and gp107, were differentially expressed among different strains. Additionally, small RNA sequencing from the SI population suggested that OrNV is targeted by the host RNA interference (RNAi) response with abundant 21-nucleotide small RNAs. Some of these genomic changes are specific to the geographic population and could be related to particular phenotypic characteristics of the strain, such as viral pathogenicity or transmissibility, and this requires further investigation.IMPORTANCE Oryctes rhinoceros nudivirus has been an effective biocontrol agent against the coconut rhinoceros beetle in Southeast Asia and the Pacific Islands for decades. The recent outbreak of these beetles in many South Pacific islands has had a significant impact on livelihoods in the region. It has been suggested that the resurgence and spread of the pest are related to the presence of low-virulence isolates of OrNV or virus-tolerant haplotypes of beetles. We examined viral genomic and transcriptional variations in chronically infected beetles from different geographical populations. A high number of polymorphic sites among several geographical strains of OrNV were identified, but potentially only a few of these variations in the genome are involved in functional changes and can potentially alter the typical function. These findings provide valuable resources for future studies to improve our understanding of the OrNV genetic variations in different geographic regions and their potential link to virus pathogenicity.

Endogenous viral elements integrated into the genome of the soybean aphid, Aphis glycines.

Sequence analysis of the genomic DNA isolated from four biotypes of the soybean aphid, Aphis glycines (AG), revealed that in addition to the commonly observed retrovirus-related retrotransposons, viral sequences derived from multiple RNA and DNA viruses have integrated into the genome. Notably, sequences of more than 60 nudiviral genes were identified from de novo assembled DNA contigs, and mapped to assembled genomic scaffolds of AG, indicating that an ancient nudivirus, named Aphis glycines endogenous nudivirus (AgENV), had integrated into the AG genome. Furthermore, sequences derived from a similar endogenous nudivirus, Melanaphis sacchari endogenous nudivirus (MsENV), were identified from the genomic scaffolds of the sugarcane aphid, Melanaphis sacchari. Analysis of transcriptome and small RNA sequence data derived from AG did not provide evidence for transcription of the integrated AgENV genes. Hence, the genes of AgENV may be present as pseudogenes. Phylogenetic analysis based on nudivirus core genes indicated that these aphid ENVs belong to the genus Alphanudivirus.

Chalcid wasp paleoviruses bridge the evolutionary gap between bracoviruses and nudiviruses.

Polydnaviruses are obligate mutualists of parasitoid wasps and are divided into two genera, Bracovirus and Ichnovirus. Bracoviruses are thought to originate from a single integration of an ancestral nudivirus into the ancestor of microgastroid complex ~100 million years ago. However, all the known nudiviruses are only distantly related to bracoviruses, and much remains obscure about the origin of bracoviruses. Here we employ a paleovirological method to screen endogenous nudivirus-like elements across arthropods. Interestingly, we identify many endogenous nudivirus-like elements within the genome of Eurytoma brunniventris, a species of the Chalcidoidea superfamily. Among them, we find 14 core gene sequences are likely to be derived from a betanudivirus (designated EbrENV-ß), suggesting that betanudivirus has been circulating in parasitoid wasps. Phylogenomic analysis suggests that EbrENV-ß is the known closest relative of bracoviruses. Synteny analyses show the order of core genes is not well conserved between EbrENV-ß and nudiviruses, revealing the dynamic nature of the evolution of nudivirus genome structures. Our findings narrow down the evolutionary gap between bracoviruses and nudiviruses and provide novel insights into the origin and evolution of polydnaviruses.

Complete genome sequence of Oryctes rhinoceros nudivirus isolated from the coconut rhinoceros beetle in Solomon Islands.

Oryctes rhinoceros nudivirus (OrNV) has been an effective biocontrol agent against the insect pest Oryctes rhinoceros (Coleoptera: Scarabaeidae) for decades, but there is evidence that resistance could be evolving in some host populations. We detected OrNV infection in O. rhinoceros from Solomon Islands and used Oxford Nanopore Technologies (ONT) long-read sequencing to determine the full length of the virus genomic sequence isolated from an individual belonging to a mitochondrial lineage (CRB-G) that was previously reported as resistant to OrNV. The complete circular genome of the virus consisted of 125,917 nucleotides, 1.698 bp shorter than the originally-described full genome sequence of Ma07 strain from Malaysia. We found 130 out of 139 previously annotated ORFs (seven contained interrupted/non-coding sequences, two were identified as duplicated versions of the existing genes), as well as a putatively inverted regions containing four genes. These results demonstrate the usefulness of a long-read sequencing technology for resolving potential structural variations when describing new virus isolates. While the Solomon Islands isolate exhibited 99.41 % nucleotide sequence identity with the originally described strain, we found several genes, including a core gene (vlf-1), that contained multiple amino acid insertions and/or deletions as putative polymorphisms of large effect. Our complete annotated genome sequence of a newly found isolate in Solomon Islands provides a valuable resource to help elucidate the mechanisms that compromise the efficacy of OrNV as a biocontrol agent against the coconut rhinoceros beetle.

Transpacific coalescent pathways of coconut rhinoceros beetle biotypes: Resistance to biological control catalyses resurgence of an old pest.

Biological control agents have several advantages over chemical control for pest management, including the capability to restore ecosystem balance with minimal non-target effects and a lower propensity for targets to develop resistance. These factors are particularly important for invasive species control. The coconut rhinoceros beetle (Oryctes rhinoceros Linnaeus) is a major palm pest that invaded many Pacific islands in the early 20th century through human-mediated dispersal. Application of the Oryctes nudivirus in the 1960s successfully halted the beetle's first invasion wave and made it a textbook example of successful biological control. However, a recently discovered O. rhinoceros biotype that is resistant to the nudivirus appears to be correlated with a new invasion wave. We performed a population genomics analysis of 172 O. rhinoceros from seven regions, including native and invasive populations, to reconstruct invasion pathways and explore correlation between recent invasions and biotypes. With ddRAD sequencing, we generated data sets ranging from 4,000 to 209,000 loci using stacks and ipyrad software pipelines and compared genetic signal in downstream clustering and phylogenetic analyses. Analysis suggests that the O. rhinoceros resurgence is mediated by the nudivirus-resistant biotype. Genomic data have been proven essential to understanding the new O. rhinoceros biotype's invasion patterns and interactions with the original biotype. Such information is crucial to optimization of strategies for quarantine and control of resurgent pests. Our results demonstrate that while invasions are relatively rare events, new introductions can have significant ecological consequences, and quarantine vigilance is required even in previously invaded areas.

Nutritional demands and metabolic characteristics of the DSIR-HA-1179 insect cell line during growth and infection with the Oryctes nudivirus.

The DSIR-HA-1179 coleopteran cell line has been identified as a susceptible and permissive host for the in vitro replication of the Oryctes nudivirus, which can be used as a biopesticide against the coconut rhinoceros beetle, pest of palms. The major challenge to in vitro large-scale Oryctes nudivirus production is ensuring process economy. This rests, among other requisites, on the use of low-cost culture media tailored to the nutritional and metabolic needs of the cell line, both in uninfected and infected cultures. The aim of the present study was to characterize the nutritional demands and the metabolic characteristics of the DSIR-HA-1179 cell line during growth and subsequent infection with Oryctes nudivirus in the TC-100 culture medium. Serum-supplementation of the culture medium was found to be critical for cell growth, and addition of 10% fetal bovine serum v/v led to a maximum viable cell density (16.8 × 105 cells ml-1) with a population doubling time of 4.2 d. Nutritional and metabolic characterization of the cell line revealed a trend of glucose and glutamine consumption but minimal uptake of other amino acids, negligible production of lactate and ammonia, and the accumulation of alanine, both before and after infection. The monitoring of virus production kinetics showed that the TC-100 culture medium was nutritionally sufficient to give a peak yield of 7.38 × 107 TCID50 ml-1 of OrNV at the 6th day post-infection in attached cultures of DSIR-HA-1179 cells in 25 cm2 T-flasks. Knowledge of the cell line's nutritional demands and virus production kinetics will aid in the formulation of a low-cost culture medium and better process design for large-scale OrNV production in future.

A new haplotype of the coconut rhinoceros beetle, Oryctes rhinoceros, has escaped biological control by Oryctes rhinoceros nudivirus and is invading Pacific Islands.

The coconut rhinoceros beetle (CRB; Oryctes rhinoceros) is a major pest of coconut and oil palm, but the discovery and release of Oryctes rhinoceros nudivirus (OrNV) in the 1960s and 70s suppressed the pest such that no new invasions of uninfested islands by CRB were reported for over 30years after implementation of the biocontrol programme. Surprisingly, a highly damaging outbreak was reported from Guam (2007), which could not be controlled by OrNV. Subsequently, new invasions have been reported from Port Moresby, Papua New Guinea (2009); O'ahu, Hawai'i (2013); and Honiara, Solomon Islands (2015). We have found that all of these outbreaks have been caused by a previously unrecognized haplotype, CRB-G, which appears to be tolerant to OrNV. PCR analysis shows that OrNV is generally present at high incidence in established populations of CRB, but is generally absent from the invasive CRB-G populations. CRB-G from Guam was not susceptible to OrNV infection by oral delivery, but injection of the virus did cause mortality. Further genetic analysis shows that CRB populations can be divided into a number of clades that coincide with the endemic and invasive history of the beetle. Analysis suggests that CRB-G originated in Asia, though the precise location remains to be discovered.

Dr. Alois Huger - Laying the foundations for an integrated insect pathology.

Dr. Rer. Nat. Alois M. Huger had a long and distinguished career at the Institut für biologische Schädlingsbekämpfung (Institute for Biological Pest Control) Darmstadt, Germany, where he became one of the world's leading insect pathologists. He applied his experience and understanding of insect tissues and specialist skills in microscopy to diagnosis and elucidation of the pathology of insect diseases. During his career he discovered the Oryctes nudivirus and Bacillus thuringiensis subsp. tenebrionis, and was a member of teams unravelling amber disease in scarab beetles and the role of bacteria in parthenogenesis of parasitoids. He researched and described the life cycles of microsporidian and rickettsial pathogens of insects and was the first to describe the ultrastructure of a microsporidian spore. Dr. Huger carried out diagnosis of diseased insects over a period of 55years and has left us with many publications and a database containing thousands of records for ongoing investigation. Working with multiple pathogens in different systems, Dr. Huger obtained an overview second to none in the complexities of insect/pathogen relationships and has been at the forefront of making these discoveries benefit humanity. He is a worthy recipient of the Founders' Lecture Honoree Award, the highest honour of the Society for Invertebrate Pathology.