S.D.G. Marshall

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.

The scarab gut: A potential bioreactor for bio-fuel production

Abstract  Cellulose and hemicelluloses are the most prevalent sources of carbon in nature. Currently many approaches employ micro‐organisms and their enzyme products to degrade plant feedstocks for production of bioenergy. Scarab larvae are one such model. They consume celluloses from a variety of sources including plant roots, soil organic matter and decaying wood, and are able to extract nutrients and energy from these sources. In this paper, we review the physicochemical properties of the scarab larval gut, the diversity and digestive role that microflora play in the scarab gut and discuss the potential for applying these digestive processes in bioreactors for improving bio‐fuel production. Scarab larvae are characterised by their highly alkaline midgut which is dominated by serine proteinase enzymes, and a modified hindgut which harbors the majority of the intestinal microbiota under anaerobic conditions. Evidence suggests that digestion of recalcitrant organic matter in scarab larvae likely results from a combination of endogenous gut proteinases and cellulolytic enzymes produced by symbiotic micro‐organisms. Most of the easily digestible proteins are mobilized and absorbed in the midgut by endogenous proteinases. The hindgut contents of scarab larvae are characterized by high concentrations of volatile fatty acids, the presence of fermenting bacteria, and typical anaerobic activities, such as methanogenesis. The hindgut typically contains a wide diversity of micro‐organisms, some of which appear to be obligate symbionts with cellulolytic potential. As a result, the scarab larval gut can be regarded as a small bioreactor resembling the rumen of sheep or cattle, where solid food particles composed of cellulose, hemicellulose, pectin and polysaccharides are degraded through enzymatic and fermentation processes. Together these observations suggest scarab larvae have potential to assist the bio‐fuel industry by providing new sources of (hemi)cellulolytic bacteria and bacterial (hemi)cellulolytic enzymes.

Histopathological Effects of the Yen-Tc Toxin Complex from Yersinia entomophaga MH96 (Enterobacteriaceae) on the Costelytra zealandica (Coleoptera: Scarabaeidae) Larval Midgut

ABSTRACT 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.

Multilocus sequence analysis (MLSA) of ‘Rickettsiella costelytrae' and ‘Rickettsiella pyronotae’, intracellular bacterial entomopathogens from New Zealand

Larvae of scarab beetles live in the soil and are frequently hosts for microbial pathogens. In New Zealand, larvae of the grass grub, Costelytrae zealandica (Coleoptera: Scarabaeidae), and manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae), have been collected from field populations showing loss of vigour and a whitened appearance. Diagnosis indicated an intracellular infection of fat body tissues by Rickettsiella‐like micro‐organisms. Rickettsiella bacteria are under evaluation as a possible new source of insect bio‐control agents for important agricultural pests as, e.g. scarabaeid and elaterid larvae. The present study aimed at the unequivocal molecular taxonomic identification and comparison of the bacteria associated with Costelytra and Pyronota.

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and alpha-amylase were still detected in the midgut of diseased larvae.

Formation of microsclerotia in three species of Beauveria and storage stability of a prototype granular formulation

ABSTRACT Beauveria bassiana is the only species from the Beauveria genus that has been previously described as forming microsclerotia [Wang, H.-H., Wang, J.-L., Li, Y.-P., Liu, X., Wen, J.-Z., & Lei, Z.-R. (2011). Liquid culturing of microsclerotia of Beauveria bassiana, an entomopathogenic fungus to control western flower thrip, Frankliniella occidentalis in Journal of Applied Entomology, 48(3), 588–595]. Microsclerotia (MS) are compact, melanised aggregates that have been recognised in plant pathogenic fungi as overwintering structures. MS were successfully produced in shake flask cultures from three different Beauveria species (B. bassiana, B. brogniartii and B. pseudobassiana) using liquid media containing a low carbon to nitrogen ratio and replacing glucose with a complex carbon source. Maximum MS yield after 10 days fermentation was 8.46 × 103 MS/mL obtained with B. pseudobassiana AgR-F704, a New Zealand fungus isolated from the African black beetle (Heteronychus arator F.). Using this isolate, MS were produced using two C:N compositions, formulated with diatomaceous earth and air dried and monitored for germination and storage stability. Germination of MS granules varied with C:N ratio and storage temperature. MS produced using a higher carbon concentration maintained 100% germination after six months of storage at 4°C, but when stored at 20°C, the germination rate was reduced to 68%. In contrast, MS produced using a lower carbon concentration showed reduced germination (0 to 18%) after storage for more than two months at either temperature and had lost viability completely after six months. Conidia production from surviving MS was estimated at 109 conidia/g of granules. Results demonstrated that Beauveria species are able to form MS in submerged culture and those produced in the relatively higher carbon medium were capable of surviving under low moisture, ambient temperature storage and are able to produce conidia after germination; all of these attributes suggest that MS can be incorporated into granules as a new sustained release strategy to control soil-dwelling insects.