Complete Genome Sequences of Six Isolates of the Oryctes rhinoceros Nudivirus.

Oryctes rhinoceros nudivirus, a double-stranded DNA virus of the family Nudiviridae, is an important biocontrol agent of the coconut rhinoceros beetle (Coleoptera: Scarabaeidae). We present the genome sequences of six isolates of Oryctes rhinoceros nudivirus collected from the Philippines, Papua New Guinea, and Tanzania between the years 1977 and 2016.

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.

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.

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.

Three-dimensional cellular aggregates formed by Beauveria pseudobassiana in liquid culture with potential for use as a biocontrol agent of the African black beetle (Heteronychus arator).

Beauveria pseudobassiana formed three-dimensional aggregates of cells (CAs) in liquid culture. CAs were formed mainly by blastospores and conidia, distinct from microsclerotia formed through adhesion of hyphae. The formation, germination and sporulation of CAs were studied, as well as the pathogenicity of conidia produced from them against adults of black beetle. After 4 days of culture, CAs were formed, becoming compact and melanised after 10 days of incubation. Electron microscopy showed three-dimensional CAs averaging 431.65 µm in length with irregular shapes and rough surfaces, where cells were trapped within an extracellular matrix. CAs germinated after 2 days of incubation on agar-plates producing hyphae and forming phialides and conidia after 4 days. Produced conidia caused 45% mortality of black beetle adults. CAs germination and sporulation on soil were directly correlated with soil moisture, reaching 80% and 100% germination on the surface of soil with 17% and 30% moisture, respectively. CAs maintained 100% germination after 2 years of storage under refrigeration. These CAs could have a similar function as microsclerotia in nature, acting as resistant structures able to protect internal cells and their ability to sporulate producing infective conidia, suggesting their potential to be used as bioinsecticides to control soil-dwelling insects.

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.

High Genetic Diversity of Microbial Cellulase and Hemicellulase Genes in the Hindgut of Holotrichia parallela Larvae.

In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95%) of glycosyl hydrolase families including GH2 (24 genes), GH8 (27 genes), GH10 (19 genes), GH11 (14 genes) and GH36 (17 genes) families was retrieved, and certain sequences of GH2 (10.61%), GH8 (3.33%), and GH11 (18.42%) families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect.

Histopathological effects of the Yen-Tc toxin complex from Yersinia entomophaga MH96 (Enterobacteriaceae) on the Costelytra zealandica (Coleoptera: Scarabaeidae) larval midgut.

Yersinia entomophaga MH96, which was originally isolated from the New Zealand grass grub, Costelytra zealandica, produces an orally active proteinaceous toxin complex (Yen-Tc), and this toxin is responsible for mortality in a range of insect species, mainly within the Coleoptera and Lepidoptera. The genes encoding Yen-Tc are members of the toxin complex (Tc) family, with orthologs identified in several other bacterial species. As the mechanism of Yen-Tc activity remains unknown, a histopathological examination of C. zealandica larvae was undertaken in conjunction with cultured cells to identify the effects of Yen-Tc and to distinguish the contributions that its individual subunit components make upon intoxication. A progressive series of events that led to the deterioration of the midgut epithelium was observed. Additionally, experiments using a cell culture assay system were carried out to determine the cellular effects of intoxication on cells after topical application and the transient expression of Yen-Tc and its individual components. While observations were broadly consistent with those previously reported for other Tc family members, some differences were noted. In particular, the distinct stepwise disintegration of the midgut shared features associated with both apoptosis and necrotic programmed cell death pathways. Second, we observed, for the first time, a contribution of toxicity from two chitinases associated with the Yen-Tc complex. Our findings were suggestive of the activities encoded within the subunit components of Yen-Tc targeting different sites along putative programmed cell death pathways. Given the observed broad host range for Yen-Tc, these targeted loci are likely to be widely shared among insects.

3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity.

Toxin complex (Tc) proteins are a class of bacterial protein toxins that form large, multisubunit complexes. Comprising TcA, B, and C components, they are of great interest because many exhibit potent insecticidal activity. Here we report the structure of a novel Tc, Yen-Tc, isolated from the bacterium Yersinia entomophaga MH96, which differs from the majority of bacterially derived Tcs in that it exhibits oral activity toward a broad range of insect pests, including the diamondback moth (Plutella xylostella). We have determined the structure of the Yen-Tc using single particle electron microscopy and studied its mechanism of toxicity by comparative analyses of two variants of the complex exhibiting different toxicity profiles. We show that the A subunits form the basis of a fivefold symmetric assembly that differs substantially in structure and subunit arrangement from its most well characterized homologue, the Xenorhabdus nematophila toxin XptA1. Histopathological and quantitative dose response analyses identify the B and C subunits, which map to a single, surface-accessible region of the structure, as the sole determinants of toxicity. Finally, we show that the assembled Yen-Tc has endochitinase activity and attribute this to putative chitinase subunits that decorate the surface of the TcA scaffold, an observation that may explain the oral toxicity associated with the complex.

Genetic and electron-microscopic characterization of Rickettsiella bacteria from the manuka beetle, Pyronota setosa (Coleoptera: Scarabaeidae).

Larvae of manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae) cause pasture damage in New Zealand by feeding on the roots of grasses. Surveys for potential biocontrol agents revealed a putative disease, expressed as whitened larvae of one of the outbreak species, Pyronota setosa. Microbial diagnosis indicated an intracoelomic, intracellular infection, and intracellular bacteria have been identified with subcellular structures characteristic of infection by Rickettsiella-like microorganisms. These bacteria were rod-shaped, often slightly bent with a mean of 628 nm in length and 220 nm in width. Numerous associated protein crystals of variable size and shape occurred within round to oval shaped "giant bodies" either singly or as clusters of smaller crystals. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the manuka beetle pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the pathotype designation 'Rickettsiella pyronotae' is proposed to refer to this organism. Moreover, genetic analysis makes it likely that--on the basis of the currently accepted organization of the genus Rickettsiella--this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening.

Esters are an important component of apple (Malus×domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, 'Royal Gala', that contain all the conserved features associated with CXE members of the α/ß hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of 'Royal Gala' (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K(m)) but decreasing turnover (k(cat)) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.

Selective sweeps at the organophosphorus insecticide resistance locus, Rop-1, have affected variation across and beyond the α-esterase gene cluster in the Australian sheep blowfly, Lucilia cuprina.

A major theoretical consequence of selection at a locus is the genetic hitchhiking of linked sites (selective sweep). The extent of hitchhiking around a gene is related to the strength of selection and the rate of recombination, with its impact diminishing with distance from the selected site. At the Rop-1 locus of the sheep blowfly, Lucilia cuprina, polymorphisms at two different sites within the LcαE7 gene encode forms of the protein that confer organophosphorus insecticide resistance. To assess the impact of selection at these two sites on variation around LcαE7, we sequenced regions within six other genes along chromosome IV across isogenic (IV) strains of L. cuprina. High levels of linkage disequilibrium, characterized by low haplotype number (K) and diversity (H), and significant R(2) values were observed for two genes, LcαE1 and LcαE10, both members of the same α-esterase gene cluster as LcαE7. A significant R(2) value was also observed for a gene predicted to be the next closest to LcαE7, AL03, but not for any of the other genes, LcRpL13a, Lcdsx, or LcAce. Skews in the site frequency spectra toward high-frequency variants were significant for LcαE1 (Fay and Wu's H = -2.91), LcαE10 (H = -1.85), and Lcdsx (H = -2.00). Since the selective sweeps, two forms of likely returning variation were observed, including variation in microsatellites in an intron of LcαE10 and a recombination event between LcαE7 and LcαE10. These data suggest that two incomplete soft sweeps have occurred at LcαE7 that have significantly affected variation across, and beyond, the α-esterase gene cluster of L. cuprina. The speed and impact of these selective sweeps on surrounding genomic variation and the ability of L. cuprina to respond to future environmental challenges are discussed.

Odorant receptors from the light brown apple moth (Epiphyas postvittana) recognize important volatile compounds produced by plants.

Moths recognize a wide range of volatile compounds, which they use to locate mates, food sources, and oviposition sites. These compounds are recognized by odorant receptors (OR) located within the dendritic membrane of sensory neurons that extend into the lymph of sensilla, covering the surface of insect antennae. We have identified 3 genes encoding ORs from the tortricid moth, Epiphyas postvittana, a pest of horticulture. Like Drosophila melanogaster ORs, they contain 7 transmembrane helices with an intracellular N-terminus, an orientation in the plasma membrane opposite to that of classical GPCRs. EpOR2 is orthologous to the coreceptor Or83b from D. melanogaster. EpOR1 and EpOR3 both recognize a range of terpenoids and benzoates produced by plants. Of the compounds tested, EpOR1 shows the best sensitivity to methyl salicylate [EC(50) = 1.8 x 10(-12) M], a common constituent of floral scents and an important signaling compound produced by plants when under attack from insects and pathogens. EpOR3 best recognizes the monoterpene citral to low concentrations [EC(50) = 1.1 x 10(-13) M]. Citral produces the largest amplitude electrophysiological responses in E. postvittana antennae and elicits repellent activity against ovipositing female moths. Orthologues of EpOR3 were found across 6 families within the Lepidoptera, suggesting that the ability to recognize citral may underpin an important behavior.

High-resolution crystal structure of plant carboxylesterase AeCXE1, from Actinidia eriantha, and its complex with a high-affinity inhibitor paraoxon.

Carboxylesterases (CXEs) are widely distributed in plants, where they have been implicated in roles that include plant defense, plant development, and secondary metabolism. We have cloned, overexpressed, purified, and crystallized a carboxylesterase from the kiwifruit species Actinidia eriantha (AeCXE1). The structure of AeCXE1 was determined by X-ray crystallography at 1.4 A resolution. The crystal structure revealed that AeCXE1 is a member of the alpha/beta-hydrolase fold superfamily, most closely related structurally to the hormone-sensitive lipase subgroup. The active site of the enzyme, located in an 11 A deep hydrophobic gorge, contains the conserved catalytic triad residues Ser169, Asp276, and His306. Kinetic analysis using artificial ester substrates showed that the enzyme can hydrolyze a range of carboxylester substrates with acyl groups ranging from C2 to C16, with a preference for butyryl moieties. This preference was supported by the discovery of a three-carbon acyl adduct bound to the active site Ser169 in the native structure. AeCXE1 was also found to be inhibited by organophosphates, with paraoxon (IC50 = 1.1 muM) a more potent inhibitor than dimethylchlorophosphate (DMCP; IC50 = 9.2 muM). The structure of AeCXE1 with paraoxon bound was determined at 2.3 A resolution and revealed that the inhibitor binds covalently to the catalytic serine residue, with virtually no change in the structure of the enzyme. The structural information for AeCXE1 provides a basis for addressing the wider functional roles of carboxylesterases in plants.

The carboxylesterase gene family from Arabidopsis thaliana.

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger alpha/beta hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.