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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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Through transposon mutagenesis and DNA sequence analysis, the main disease determinant of the entomopathogenic bacterium Yersinia entomophaga MH96 was localized to an ~32-kb pathogenicity island (PAI) designated PAI(Ye₉₆). Residing within PAI(Ye₉₆) are seven open reading frames that encode an insecticidal toxin complex (TC), comprising not only the readily recognized toxin complex A (TCA), TCB, and TCC components but also two chitinase proteins that form a composite TC molecule. The central TC gene-associated region (~19 kb) of PAI(Ye₉₆) was deleted from the Y. entomophaga MH96 genome, and a subsequent bioassay of the ΔTC derivative toward Costelytra zealandica larvae showed it to be innocuous. Virulence of the ΔTC mutant strain could be restored by the introduction of a clone containing the entire PAI(Ye₉₆) TC gene region. As much as 0.5 mg of the TC is released per 100 ml of Luria-Bertani broth at 25°C, while at 30 or 37°C, no TC could be detected in the culture supernatant. Filter-sterilized culture supernatants derived from Y. entomophaga MH96, but not from the ΔTC strain grown at temperatures of 25°C or less, were able to cause mortality. The 50% lethal doses (LD₅₀s) of the TC toward diamondback moth Plutella xylostella and C. zealandica larvae were defined as 30 ng and 50 ng, respectively, at 5 days after ingestion. Histological analysis of the effect of the TC toward P. xylostella larva showed that within 48 h after ingestion of the TC, there was a general dissolution of the larval midgut.

Serratia entomophila and Serratia proteamaculans cause amber disease of the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae). Three genes required for virulence, sepABC, are located on a large plasmid, pADAP. The translated products of the sep genes are members of the toxin complex (Tc) family of insecticidal toxins that reside in the genomes of some Enterobacteriaceae. Each of the sep genes was placed either singly or as various combinations under the control of an inducible arabinose promoter, allowing their inductive expression. Western Immunoblot confirmed that each of the Sep proteins migrated at their predicted size on sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. Bioassays of sonicated filtrates derived from the various arabinose-induced para-SEP constructs showed that only when sepA, sepB and sepC were coexpressed were amber disease symptoms observed in grass grub larvae. Fourteen days after ingestion of the Sep protein filtrate, approximately 64% of the larvae reverted from a diseased to a healthy phenotype. Redosing the revertents with a fresh Sep protein filtrate reinitiated the amber pathotype, indicating that the Sep proteins are needed to be continuously present to exert an effect.

The Serratia entomophila antifeeding prophage (Afp) is thought to form a virus-like structure that has activity towards the New Zealand grass grub, Costelytra zealandica. Through the trans based expression of AnfA1, an RfaH - like transcriptional antiterminator, the Afp, was able to be induced. The expressed Afp was purified and visualized by electron microscopy. The Afp resembled a phage tail-like bacteriocin, exhibiting two distinct morphologies: an extended and a contracted form. The purified Afp conferred rapid activity towards C. zealandica larvae, causing cessation of feeding and a change to an amber colouration within 48 h postinoculation, with increased dose rates causing larval mortality.

Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.

The introduction of Oryctes virus into outbreak areas of the rhinoceros beetle, Oryctes rhinoceros (Coleoptera: Scarabaeidae), has been a major success for "classical" biocontrol with a virus and has led to a dramatic reduction in palm damage in many areas of the Asia/Pacific region. In recent years, however, there have been new reports of high levels of rhinoceros beetle damage to palms. Damage has been especially intense in SE Asia following the introduction of no-burn polices for land clearance and replanting, but outbreaks have also been reported from some Pacific Islands where control seems to have diminished over time. SE Asian studies show that there is considerable genetic variation among endemic Oryctes virus isolates and studies in new island release areas have shown rapid evolution of the virus. The consequences of such genetic variation are in need of further study. Furthermore, the taxonomic position of the virus is unclear, with its removal from the Baculoviridae to an "unassigned' virus, reflecting its novel characteristics. Genomic sequencing could help resolve the taxonomy of the virus and provide a basis for studying strain variation. Oryctes virus has achieved wide success in the past without the benefit of molecular analysis and identification techniques. In order to fully take advantage of this unique pathogen for protection of palms, a renewed, coordinated effort centered on genetic selection and distribution of effective strains is required.

Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae) cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 155-kb plasmid, pADAP, carries the genes sepA, sepB, and sepC, which are essential for production of amber disease symptoms. Transposon insertions in any of the sep genes in pADAP abolish gut clearance but not cessation of feeding, indicating the presence of an antifeeding gene(s) elsewhere on pADAP. Based on deletion analysis of pADAP and subsequent sequence data, a 47-kb clone was constructed, which when placed in either an Escherichia coli or a Serratia background exerted strong antifeeding activity and often led to rapid death of the infected grass grub larvae. Sequence data show that the antifeeding component is part of a large gene cluster that may form a defective prophage and that six potential members of this prophage are present in Photorhabdus luminescens subsp. laumondii TTO1, a species which also has sep gene homologues.

Amber disease in the grass grub (Costelytra zealandica White) (Coleoptera: Scarabaeidae), caused by strains of the bacteria Serratia entomophila or S. proteamaculans, is characterised by cessation of feeding and clearance of the midgut. Analysis of the midgut enzyme activity in diseased grass grub larvae showed that proteolytic activity was reduced to low levels. The endopeptidases, trypsin, elastase, and chymotrypsin, were all markedly reduced in activity whereas the exopeptidases (leucine-aminopeptidase and carboxypeptidase A and B) were much less affected. There was no effect on the non-proteolytic enzymes, esterase and alpha-amylase. Sequential analysis of enzyme levels in the gut during onset of disease showed that proteolytic activity dropped after cessation of feeding and preceded gut clearance. In starved, uninfected larvae enzyme activity levels remained high, indicating that decline in enzyme activity is not associated with absence of food and cessation of feeding, but with the onset of disease.

A new nematode, Elaeolenchus parthenonema n. g., n. sp., is described from the palm-pollinating weevil Elaeidobius kamerunicus Faust. The new genus is placed in the Anandranematidae n. fam., which, together with the genus Anandranema Poinar et al., 1993, is characterised by nematodes having only a single autotokous generation in the insect host. This is the first report of a member of this superfamily reproducing only parthenogenetically. The development of E. parthenonema and its effect on the weevil host is discussed, along with a phylogenetic synopsis of the families of the Sphaerularioidea Lubbock 1861. The Beddingiidae n. fam. is proposed for Beddingia Blinova & Korenchenko, 1986, comprising the original Deladenus parasites of Hymenoptera that possess both free-living and parasitic amphimictic generations in their life-cycles. This family is considered to have the most primitive type of development in the superfamily.

A series of constitutive green fluorescent protein (pGFPuv) derivatives of the bacterium Serratia entomophila (Enterobacteriaceae) were constructed, allowing the fate of cells causing amber disease ingested by the New Zealand grass grub (Costelytra zealandica, Coleoptera: Scarabaeidae) to be monitored. Examination of tissue and contents of the alimentary tract over time from ingestion, under fluorescence microscopy, revealed that the major site of S. entomophila colonisation in the grass grub is intestinal particulate matter. Visual examinations showed that wild type pathogenic strain persisted in high numbers in the grass grub intestinal tract, notably in the area of the hindgut, but the S. entomophila pADAP-free strain 5.6RC and the pADK mutant derivatives (pADK-4, -10, -13) that gave a non-feeding without gut clearance phenotype, were unable to colonise the gut. The indiscriminate colonisation of the intestinal tract particulate matter by pathogenic bacteria, rather than the colonisation of a specific site of activity, suggests that the bacterial toxins are induced and released from the bacteria while they live freely in the grass grub intestinal tract.