Zealand Grass Grub Costelytra Zealandica Research Articles

Dose reduction and alternatives to the phenol pheromone in monitoring and management of the grass grub Costelytra zealandica.

Endemic New Zealand grass grub Costelytra zealandica is a pest of introduced pasture that uses phenol as a sex pheromone. The pheromone could be used to monitor and manage grass grub populations, but the irritating properties and toxicity of phenol for human handlers, as well as the possible ecotoxicological effects, pose obstacles to the deployment of the pheromone. This study aimed to limit the use of phenol by dose-response studies and investigation into alternative attractants and synergists to phenol. No difference in trap catch was seen across the range of 1-100 mg of phenol, while rates below this (0.001-0.1 mg) caused a large drop in catches. Our results indicated that 1 mg loading in lures was enough to indicate beetle presence over 1 week. 4-Hydroxybenzaldehyde and p-cresol proved unattractive in this study, both as single attractants and as synergists with phenol. Phenyl acetate, phenyl benzoate and diphenyl carbonate all formed phenol under hydrolytic conditions to act as successful propheromones, while phenyl acetate was found to be as attractive as phenol on its own. This study described several ways to reduce or avoid the use of phenol in the field while maintaining lure effectiveness. © 2017 Society of Chemical Industry.

Yersinia entomophaga sp. nov., isolated from the New Zealand grass grub Costelytra zealandica

A Gram-negative, rod-shaped, non-spore-forming bacterium (MH96(T)) was isolated from diseased larvae of the New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae). On the basis of 16S rRNA gene sequence similarity, strain MH96(T) is a member of the genus Yersinia, which is a member of the class Gammaproteobacteria. The most similar 16S rRNA gene sequence to that of MH96(T) is that of the type strain of Yersinia mollaretii (98.5 % similarity) followed by those of the type strains of Yersinia aldovae, Y. frederiksenii and Y. rohdei (all 98.4 % similarity). Multilocus sequence typing of five housekeeping genes (dnaJ, glnA, gyrB, groEL and recA) identified Yersinia ruckeri (81-92 % similarity) as the closest relative. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MH96(T) from the four most closely related Yersinia species with validly published names, including a Y. ruckeri isolate. Strain MH96(T) therefore represents a novel species, for which the name Yersinia entomophaga sp. nov. is proposed, with the type strain MH96(T) ( = DSM 22339(T) = ATCC BAA-1678(T)).

Nucleotide sequence of the Serratia entomophila plasmid pADAP and the Serratia proteamaculans pU143 plasmid virulence associated region

Some strains of Serratia entomophila and S. proteamaculans cause amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The disease determinants of S. entomophila, are encoded on a 153,404-bp plasmid, termed pADAP for amber disease associated plasmid. The S. proteamaculans strain 143 ( Sp143) exhibits an unusual pathotype, where only 60–70% of C. zealandica larvae infected with the bacterium succumb to disease. DNA sequence analysis of the Sp143 pU143 virulence associated region identified high DNA similarity to the pADAP sep virulence associated region, with DNA sequence variation in the sepA gene and the variable region of the sepC component. No pADAP anti-feeding prophage orthologue was detected in the Sp143 genome. The region of pADAP replication was cloned and found to replicate in S. entomophila but not in Escherichia coli. DNA sequence analysis of the plasmid pSG348 repA gene from the French isolate of Serratia grimesii, identified 93% DNA identity to the pADAP repA gene. A comparison of the pU143 virulence associated region with the completed pADAP nucleotide sequence is given.

Structural Study of the Serratia entomophila Antifeeding Prophage: Three-Dimensional Structure of the Helical Sheath

The sheath of the Serratia entomophila antifeeding prophage, which is pathogenic to the New Zealand grass grub Costelytra zealandica, is a 3-fold helix formed by a 4-fold symmetric repeating motif disposed around a helical inner tube. This structure, determined by electron microscopy and image processing, is distinct from that of the other known morphologically similar bacteriophage sheaths.

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 α-amylase were still detected in the midgut of diseased larvae.

Amber Disease Reduces Trypsin Activity in Midgut of Costeltyra zealandica (Coleoptera; Scarabaeidae) Larvae

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 α-amylase were still detected in the midgut of diseased larvae.

Pathology of Amber Disease in the New Zealand Grass Grub Costelytra zealandica (Coleoptera: Scarabaeidae)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is a chronic infection of the larval gut caused by strains of the bacteria Serratia entomophila or S. proteamaculans. Following ingestion of pathogenic bacterial cells, larvae cease feeding after 2-5 days and become amber colored due to gut clearance. However, death does not occur until 1-3 months from initial infection. Light and electron microscopy demonstrated that bacteria colonize the gut, most specifically the foregut in the region of the cardiac valve. Bacteria adhere to the foregut intima and grow across the crop membrane, forming large clusters in folds of the crop. Adhesion appears to be mediated by fimbriae of the bacterial cells. As the disease progresses the larvae become shrunken due to a general degradation of the fat cells. The bacteria are not directly invasive, penetration of the hemocoel not occurring until the late stages of the disease when general septicemia is accompanied by death of the insect. Culturing of the hemolymph confirmed that invasion of the hemocoel was restricted to the final stages of disease. Enumeration experiments confirmed that the majority of the bacteria in the foregut were Serratia spp.

Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.

Serratia entomophila sp. nov. Associated with Amber Disease in the New Zealand Grass Grub Costelytra zealandica

Serratia entomophila sp. nov. is a homogeneous deoxyribonucleic acid relatedness group most closely related to Serratia ficaria and Serratia marcescens. All 19 strains studied resembled S. marcescens (the phenotypically closest species) by their inability to ferment or utilize l-rhamnose, l-rhamnose, d-melibiose, dulcitol, and d-raffinose but differed from S. marcescens by their inability to ferment or utilize sorbitol and their lack of lysine and ornithine decarboxylases. All strains utilized itaconate, a unique characteristic among Serratia species. Two biotypes could be distinguished. Strains of biotype 1 utilized d-arabitol but not l-arabitol or d-xylose. Strains of biotype 2 utilized l-arabitol and d-xylose but not d-arabitol. S. entomophila has been isolated from insect larvae and water. Most strains were isolated in New Zealand from the grass grub Costelytra zealandica infected with amber disease. None of the strains were isolated from infected humans, animals (other than insects), or plants. The type strain is strain A1T (ATCC 43705T).