Sequences Encoding a Novel Toursvirus Identified from Southern and Northern Corn Rootworms (Coleoptera: Chrysomelidae).

Sequences derived from a novel toursvirus were identified from pooled genomic short read data from U.S. populations of southern corn rootworm (SCR, Diabrotica undecimpunctata howardi Barber) and northern corn rootworm (NCR, Diabrotica barberi Smith & Lawrence). Most viral sequences were identified from the SCR genomic dataset. As proteins encoded by toursvirus sequences from SCR and NCR were almost identical, the contig sets from SCR and NCR were combined to generate 26 contigs. A total of 108,176 bp were assembled from these contigs, with 120 putative toursviral ORFs identified indicating that most of the viral genome had been recovered. These ORFs included all 40 genes that are common to members of the Ascoviridae. Two genes typically present in Ascoviridae (ATP binding cassette transport system permeases and Baculovirus repeated open reading frame), were not detected. There was evidence for transposon insertion in viral sequences at different sites in the two host species. Phylogenetic analyses based on a concatenated set of 45 translated protein sequences clustered toursviruses into a distinct clade. Based on the combined evidence, we propose taxonomic separation of toursviruses from Ascoviridae.

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.

Confirmation of Oryctes rhinoceros nudivirus infections in G-haplotype coconut rhinoceros beetles (Oryctes rhinoceros) from Palauan PCR-positive populations.

Coconut rhinoceros beetle (CRB), Oryctes rhinoceros, is a pest of palm trees in the Pacific. Recently, a remarkable degree of palm damage reported in Guam, Hawaii, Papua New Guinea and Solomon Islands has been associated with a particular haplotype (clade I), known as "CRB-G". In the Palau Archipelago, both CRB-G and another haplotype (clade IV) belonging to the CRB-S cluster coexist in the field. In this study, more than 75% of pheromone trap-captured adults of both haplotypes were Oryctes rhinoceros nudivirus (OrNV)-positive by PCR. No significant difference in OrNV prevalence between the haplotypes was detected. In PCR-positive CRB-G tissue specimens from Palau, viral particles were observed by electron microscopy. Hemocoel injection of CRB larvae with crude virus homogenates from these tissues resulted in viral infection and mortality. OrNV isolated from Palauan-sourced CRB was designated as OrNV-Palau1. Both OrNV-Palau1 and OrNV-X2B, a CRB biological control isolate released in the Pacific, were propagated using the FRI-AnCu-35 cell line for production of inoculum. However, the OrNV-Palau1 isolate exhibited lower viral production levels and longer larval survival times compared to OrNV-X2B in O. rhinoceros larvae. Full genome sequences of the OrNV-Palau1 and -X2B isolates were determined and found to be closely related to each other. Altogether these results suggest CRB adults in Palau are infected with a less virulent virus, which may affect the nature and extent of OrNV-induced pathology in Palauan populations of CRB.

Nudivirus Sequences Identified from the Southern and Western Corn Rootworms (Coleoptera: Chrysomelidae).

Analysis of pooled genomic short read sequence data revealed the presence of nudivirus-derived sequences from U.S. populations of both southern corn rootworm (SCR, Diabrotica undecimpunctata howardi Barber) and western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte). A near complete nudivirus genome sequence was assembled from sequence data for an SCR population with relatively high viral titers. A total of 147,179 bp was assembled from five contigs that collectively encode 109 putative open reading frames (ORFs) including 20 nudivirus core genes. In contrast, genome sequence recovery was incomplete for a second nudivirus from WCR, although sequences derived from this virus were present in three geographically dispersed populations. Only 48,989 bp were assembled with 48 putative ORFs including 13 core genes, representing about 20% of a typical nudivirus genome. Phylogenetic analysis indicated that both corn rootworm nudiviruses grouped with the third known nudivirus of beetles, Oryctes rhinoceros nudivirus in the genus Alphanudivirus. On the basis of phylogenetic and additional analyses, we propose further taxonomic separation of nudiviruses within Alphanudivirus and Betanudivirus into two subfamilies and five genera. Identification of nudivirus-derived sequences from two species of corn rootworm highlights the diversity of viruses associated with these agricultural insect pests.

Complete genome analysis of a novel iflavirus from a leaf beetle, Aulacophora lewisii.

The leaf beetle Aulacophora lewisii (family Chrysomelidae, order Coleoptera) is a common insect pest of cucurbitaceous vegetables. In this study, the complete genome sequence of a novel virus from a single leaf beetle was determined using metagenomic sequencing and rapid amplification of cDNA ends. A homology search and phylogenetic analysis suggested that the new virus belongs to the genus Iflavirus, family Iflaviridae, and it was tentatively named "Aulacophora lewisii iflavirus 1" (ALIV1). ALIV1 has a single positive-stranded RNA genome of 9655 nucleotides in length (excluding the polyA tail) that is predicted to encode typical conserved domains of iflaviruses, including two picornavirus-like capsid protein domains, a helicase domain, and an RNA-dependent RNA polymerase (RdRp) domain. Sequence comparisons showed that the full genome sequence of ALIV1 is most similar to that of Brevicoryne brassicae picorna-like virus, with 42.4% sequence identity, and it shares 60% sequence identity in the coat protein region with its closest homolog, Watson virus. The average coverage of the ALIV1 sequence was approximately 5000X, suggesting that it might actively replicate in the host. Phylogenetic analysis based on deduced amino acid sequences suggested that ALIV1 is closely related to Dinocampus coccinellae paralysis virus. To the best of our knowledge, ALIV1 is the first virus discovered in A. lewisii and is also the first iflavirus identified in a member of the genus Aulacophora.

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.

Identification of a Novel Picorna-like Virus in Coconut Rhinoceros Beetles (Oryctes rhinoceros).

A novel Picorna-like virus, tentatively named Oryctes rhinoceros Picorna-like virus 1 (OrPV1), was identified in coconut rhinoceros beetle (Oryctes rhinoceros) larvae in Taiwan. The complete genome sequence consisted of 9,665 nucleotides with a polyA tail and included one open reading frame. Conserved structural domains such as Picornavirus capsid protein, RNA helicase, Peptidase and RNA-dependent RNA polymerase (RdRp) were identified through Pfam domain searches. The genome shares approximately 27-28% identity with other unclassified Picornavirales that infect honey bees (Darwin bee virus 2, Bundaberg bee virus 5, and Sacbrood virus) and a recently reported virus from Asian lady beetle (Harmonia axyridis virus 1). We did not detect this virus in any other geographical populations of O. rhinoceros collected from the South Pacific Islands and the Philippines. Analysis of the deduced RdRp amino acid sequences showed that the virus clustered with other Picorna-like viruses and separated from other members of family Dicistroviridae and Iflaviridae.

Sequencing and phylogenetic characterization of a novel RNA virus genome from Harmonia axyridis.

As a natural predator of many insect pests on its native Asian range, Harmonia axyridis remains amongst the insects whose pathogenic or beneficial microorganisms are yet to be studied. The genome nucleotide (nt) and amino acid sequences of open reading frames (ORFs) of the novel RNA virus were identified. Neighbor-joining (NJ) were constructed using MEGA7 software packages with nt sequences and conserved amino acid sequences of predicted RNA-dependent RNA polymerase (RdRp).The complete genome of a novel virus named Harmonia axyridis virus 1 was determined by RNA-seq and rapid amplification of cDNA ends from H. axyridis, which had a single-stranded RNA genome of 8868 nts in length and contains two putative ORFs. ORF1 encodes a polypeptide of 2182 amino acids, which contained conserved domains for 2 picornavirus-like capsid proteins and one RNA helicase. ORF2 encodes a polypeptide of 655 amino acids, which contained 1 RdRp domain. Phylogenetic analysis of whole genome nt sequences and RdRp deduced amino acid sequences suggested that the virus clustered with several unclassified Hubei picorna-like virus. To our knowledge, this is the first full annotated genome of a novel member of the unclassified group of RNA viruses, infecting H. axyridis in natural field conditions.

Deformed wings of small hive beetle independent of virus infections and mites.

Insect wing deformities can be caused by viruses, mites and other environmental stressors during development. Here we conducted differential diagnostics of deformed wings in small hive beetles, Aethina tumida (SHB). Adult SHB with and without deformed wings from individual and mass reared scenarios were evaluated for the mite Tyrophagus putrescentiae and for deformed wing virus. Viral load and mite number were similar for SHB with deformed wings and unaffected beetles. Because deformed wings were only observed in individually pupating SHB, a humidity challenge most likely explains the observed clinical symptoms. Our observations support the importance of differential diagnostics.

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.

The Mexican bean beetle (Epilachna varivestis) regurgitome and insights into beetle-borne virus specificity.

For nearly 400 million years, insects and plants have been embattled in an evolutionary arms race. Insects have developed diverse feeding strategies and behaviors in an effort to circumvent and overcome an extensive collection of plant defense tactics. Sap-sucking insects often inject saliva into hosts plants, which contains a suite of effector proteins and even microbial communities that can alter the plant's defenses. Lacking salivary glands, leaf-feeding beetles represent an interesting group of phytophagous insects. Feeding beetles regurgitate onto leaf surfaces and it is thought that these oral secretions influence insect-plant interactions and even play a role in virus-vector specificity. Since the molecular and biological makeup of the regurgitant is virtually unknown, we carried out RNA sequencing and 16S rDNA analysis on a major soybean pest, Epilachna varivestis, to generate the first ever beetle "regurgitome" and characterize its microbiome. Interestingly, the regurgitant is comprised of a rich molecular assortment of genes encoding putative extracellular proteins involved in digestion, molting, immune defense, and detoxification. By carrying out plant inoculation assays, we reinforced the fundamental role of the regurgitant in beetle-borne virus specificity. Ultimately, these studies begin to characterize the importance of regurgitant in virus transmission and beetle-plant interactions.

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.

A new cell line derived from embryonic tissues of Holotrichia parallela (Coleoptera:Scarabaeidae).

Holotrichia parallela is an important agricultural underground insect pest and also an edible and medicinal insect. Establishing a new cell line of H. parallela will provide a rapid and convenient tool for the studies on its physiology, pathology, and gene functions. In this study, by using the embryonic tissue of H. parallela as the material, we established a new cell line named Hp-E-1. The microscopic observation of its morphological characteristics revealed that its cellular morphology was mainly in the spherical morphology with a mean cellular diameter of 17.71 ± 2.34 µm, accounting for 67% of the total cells. The spindle-shaped cells accounted for 33% of the total cells with a mean size of 23.51 ± 4.37 × 13.98 ± 2.05 µm. The chromosomal number varied from 7 to 40, with about 50% of the cells having a diploid chromosome number of 2n = 20. Random amplified polymorphic DNA (RAPD) analysis indicated that the profiles of PCR-amplified fragments of this cell line were basically similar to those of the embryonic tissues of H. parallela but were obviously different from those of cell line BTI-Tn5B1-4 of Trichoplusia ni and cell line Sf-9 of Spodoptera frugiperda. The DNA fragment encoding mitochondrial cytochrome C oxidase subunit I (COI) gene of this cell line shared 99.7% homology with that of the embryonic tissue of H. parallela, confirming that this cell line is indeed derived from H. parallela. The results of growth curve measurement indicated that the population doubling time of this cell line was 136.7 h. Cell line Hp-E-1 could not be infected by three viruses Autographa californica multiple nucleopolyhedrovirus (AcMNPV), Bombyx mori nucleopolyhedrovirus (BmNPV), and Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV).

Leaf crinkle disease in urdbean (Vigna mungo L. Hepper): An overview on causal agent, vector and host.

Urdbean leaf crinkle disease (ULCD) is an economically significant widespread and devastating disease resulting in extreme crinkling, puckering and rugosity of leaves inflicting heavy yield losses annually in major urdbean-producing countries of the world. This disease is caused by urdbean leaf crinkle virus (ULCV). Urdbean (Vigna mungo L. Hepper) is relatively more susceptible than other pulses to leaf crinkle disease. Urdbean is an important and useful crop cultivated in various parts of South-East Asia and well adapted for cultivation under semi-arid and subtropical conditions. Aphids, insects and whiteflies have been reported as vectors of the disease. The virus is also transmitted through sap inoculation, grafting and seed. The loss in seed yield in ULCD-affected urdbean crop ranges from 35 to 81%, which is dependent upon type of genotype location and infection time. The diseased material and favourable climatic conditions contribute for the widespread viral disease. Anatomical and biochemical changes take place in the affected diseased plants. Genetic variations have been reported in the germplasm screening which suggest continuous screening of available varieties and new germplasm to search for new traits (new genes) and identify new sources of disease resistance. There are very few reports on breeding programmes for the development and release of varieties tolerant to ULCD. Mostly random amplified polymorphic DNA (RAPD) as well as inter-simple sequence repeat (ISSR) molecular markers have been utilized for fingerprinting of blackgram, and a few reports are there on sequence-tagged micro-satellite site (STMS) markers. There are so many RNA viruses which have also developed strategies to counteract silencing process by encoding suppressor proteins that create hindrances in the process. But, in the case of ULCV, there is no report available indicating which defence pathway is operating for its resistance in the plants and whether same silencing suppression strategy is also followed by this virus causing leaf crinkle disease in urdbean. The antiviral principles (AVP) present in leaf extracts of several plants are known to inhibit infection by many viruses. Many chemicals have been reported as inhibitors of virus replication in plants. Raising the barrier crops also offers an effective solution to control the spread of virus.

First report of Oryctes rhinoceros nudivirus (Coleoptera: Scarabaeidae) causing severe disease in Allomyrina dichotoma in Korea.

Oryctes rhinoceros nudivirus (OrNV) has been known to cause severe disease in coconut palm rhinoceros beetle, Oryctes rhinoceros, in Southeastern Asia and is used as a biological control to reduce the pest population. Here, we report for the first time that the OrNV may have landed on Korea and may be the major pathogen for diseased larvae of Korean horn beetle, Allomyrina dichotoma. After peroral inoculation, over 60% of infected larvae perished in 6 wk. This viral disease spreads very fast in several locations throughout Korea. This threat not only makes economic loss of local farms rearing A. dichotoma larvae but also may disturb the ecosystem by transmitting to wild A. dichotoma.

Afp14 is involved in regulating the length of Anti-feeding prophage (Afp).

The anti-feeding prophage (Afp), a phage-tail-like particle that causes cessation of feeding in the New Zealand grass grub, Costelytra zealandica, is encoded by 18 open reading frames (afp1-18). C-terminal truncations of afp14 resulted in shortened Afp particles, suggesting that Afp14 is involved in Afp length determination. We constructed an Afp assembly system (afp1-18), wherein Afp14 was truncated after the N-terminal 88 residues. This construct, when expressed in trans in Escherichia coli expressing a N-terminal 98-amino acid Afp14 construct, yielded fully assembled Afp but no assembled Afp was detected in the case of a N-terminal 96-amino acid Afp14 construct. These results suggested that the 98 N-terminal, amino acid residues of Afp14 is crucial for the initiation of Afp assembly via baseplate formation. Trans-based expression of wild-type afp14 resulted in Afp particles of varying lengths, all of which were shorter than the wild-type Afp particle. On the other hand, similar expression of Afp14 harboring a C-terminal extension (KLLEH(6)) resulted in elongated Afp particles. This information, combined with bioinformatics data, allowed us to propose a model delineating the mechanism and role of Afp14 in the maturation of the Afp particle.

Who is the puppet master? Replication of a parasitic wasp-associated virus correlates with host behaviour manipulation.

Many parasites modify their host behaviour to improve their own transmission and survival, but the proximate mechanisms remain poorly understood. An original model consists of the parasitoid Dinocampus coccinellae and its coccinellid host, Coleomegilla maculata; during the behaviour manipulation, the parasitoid is not in contact with its host anymore. We report herein the discovery and characterization of a new RNA virus of the parasitoid (D. coccinellae paralysis virus, DcPV). Using a combination of RT-qPCR and transmission electron microscopy, we demonstrate that DcPV is stored in the oviduct of parasitoid females, replicates in parasitoid larvae and is transmitted to the host during larval development. Next, DcPV replication in the host's nervous tissue induces a severe neuropathy and antiviral immune response that correlate with the paralytic symptoms characterizing the behaviour manipulation. Remarkably, virus clearance correlates with recovery of normal coccinellid behaviour. These results provide evidence that changes in ladybeetle behaviour most likely result from DcPV replication in the cerebral ganglia rather than by manipulation by the parasitoid. This offers stimulating prospects for research on parasitic manipulation by suggesting for the first time that behaviour manipulation could be symbiont-mediated.

Biology and management of palm dynastid beetles: recent advances.

Coconut, oil, and date palms are important crops in the tropics and are attacked by dynastids that cause loss of production or death of hosts. Knowledge of their breeding sites has been extended since a previous review in 1980. The fungus Metarhizium anisopliae has potential as a biopesticide against immature stages in friable breeding sites. The molecular biology and ultrastructure of Oryctes rhinoceros Nudivirus (OrNV), disseminated by adults, have been studied, and this pathogen can reduce O. rhinoceros populations and damage when introduced into new locations, especially where damage had been high. New PCR techniques may enable reliable quantification of dosages ingested and hence virulence of different isolates. Male-produced aggregation pheromones have been identified in several species, for which they may have management potential, having been used commercially for trapping O. rhinoceros in oil palm plantations in Southeast Asia, and tested against O. monoceros in Africa.