Costelytra Zealandica Research Articles

Ultrastructure of the attachment of the bacteria Serratia entomophila to foregut cuticle of Costelytra zealandica (Coleoptera : Scarabidae) and a review of nomenclature for insect epicuticular layers

The adherence of a pathogenic bacterium Serratia entomophila to foregut cuticle or Costelytra zealandica (Coleoptera : Scarabidae) is described ultrastructurally. The bacteria adhere by fimbriae to the epicuticular superficial layer which often strips off with bacteria remaining attached. The superficial layer stains with ruthenium red, but it is not known if fimbriae attach to specific carbohydrate receptors. Previous terminology used for insect epicuticle layers is reviewed and a standard nomenclature proposed.

Preliminary characterization of bacteriophages of Serratia entomophila

C.R. WILSON, T.A. JACKSON AND H.K. MAHANTY. 1993. Bacteriophage was found for the first time associated with the bacterium Serratia entomophila, a pathogen of the New Zealand grass grub (Costelytra zealandica). Phage was isolated from the homogenized guts of field‐collected grass grubs from sites throughout New Zealand. The main phage type, φCW1, produced opaque plaques in sensitive bacterial lawn and had a lamboid structure consisting of an icosahedral head (55 nm) and a long non‐contractile tail (175 times 17 nm) with a bar across the base of the tail. Nucleic acid from φCW1 was digested to nucleotides by DNAse, suggesting double stranded DNA. On further examination of the homogenates, five phage types, φCW1‐5, could be distinguished on the basis of plaque morphology. Bacterial host range was determined by testing against a selection of Serratia spp. and other bacteria. All five phage types lysed Ser. entomophila only. Differences in susceptibility to the phage types were found within this species. Lysogeny was demonstrated in φCW1 by immunity to superinfection and induction of free bacteriophage from suspected lysogens. A restriction map for φCW1 was determined with BamHI, EcoRI and HindIII and a postulated origin of replication (ori) and cohesive site (cos) was suggested. The possible implications of bacteriophage on the use of Ser. entomophila as a biological control agent are discussed.

Beauveria vermiconia is an entomopathogenic fungus

Beauveria vermiconia, previously described from volcanic ash in Chile, is reported as a pathogen of insects for the first time. The species was found causing an epizootic among larvae of Hylamorpha elegans (Coleoptera: Scarabaeidae) in Osorno, Chile. A strain recovered from larvae showed some activity against larvae of Costelytra zealandica, an endemic scarab of New Zealand.

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.

Evaluation of a technique for the early detection of pasture damage by grass grub, <i>Costelytra zealandica</i>, and porina, <i>Wiseana</i> sp

Evaluation of a technique for the early detection of pasture damage by grass grub, <i>Costelytra zealandica</i>, and porina, <i>Wiseana</i> sp

Sequential sampling of grass grub,Costelytra zealandica

Abstract Sequential sampling schemes for testing a paddock for infestation by grass grub (Costelytra zealandica (White) (Coleoptera: Scarabaeidae)) larvae are developed. Instead of taking a fixed number of samples per paddock, decisions are made during sampling as to whether sampling needs to be continued. The hypothesis that the larvae have a negative binomial distribution is tested and accepted. The performance of the schemes is assessed theoretically for larvae populations with varying degrees of clumping. Their performance in practice is compared with that of schemes which use a fixed number of samples, and it is found that the sequential schemes significantly reduce the number of samples required with little loss of accuracy.

Behavioural and physiological responses of grass grub larvae (Costelytra zealandica) feeding on protease inhibitors

Abstract Growth of first instar Costelytra zealandica larvae was significantly reduced after 6 weeks when reared on an artificial diet containing 0.3 and 1% soybean trypsin inhibitor (SBTI), 0.1% and 0.3% potato inhibitor II, and 0.3% potato protease inhibitor I and cowpea trypsin inhibitor. Limabean trypsin inhibitor at 1% significantly stimulated growth compared with that on diet with corresponding levels of added casein. A direct relationship between increased free-trypsin activity and decreased larval growth was observed. Sequential measurement of enzyme activity in third instar larvae feeding on SBTI was compared with that of larvae feeding on casein. The increase in enzyme activity was observed after 14 days in larvae feeding on SBTI. Larvae preferred to feed on SBTI-free diet when given a choice between diet containing this inhibitor at 0.3% and added casein at 0.3%.

Further electron microscopic evidence of inapparent viral infections in the midgut of the New Zealand grass grub, Costelytra zealandica white (Coleoptera: Scarabaeidae)

Further electron microscopic evidence of inapparent viral infections in the midgut of the New Zealand grass grub, Costelytra zealandica white (Coleoptera: Scarabaeidae)

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.

Adhesion of bacteria (Serratiaspp.) to the foregut of grass grub (Costelytra zealandica(white)) larvae and its relationship to the development of amber disease

Adhesion of pathogenic strains of Serratia spp. to the foregut tissue of the New Zealand grass grub (Costelytra zealandica) was shown to be associated with the development of amber disease. Bacteria were always found adhering to the crop in the region of the cardiac valve in larvae showing disease symptoms after in vivo treatment with pathogenic bacteria while no significant colonization was observed in larvae treated with wild‐type, non‐pathogenic strains. The in vitro inoculation of excised crops with pathogenic and non‐pathogenic strains resulted in a similar pattern of adhesion. It is suggested that adhesion is an early step in pathogenesis and that farther bacterial mediated factors could be required for fall expression of amber disease.

Selection, development and testing of phage‐resistant strains ofSerratia entomophilafor grass grub control

Use of the bacterium Serratia entomophila as an inundative biological control agent for the New Zealand grass grub (Costelytra zealandica) depends on the consistent production of high yields of the bacterium in liquid fermentation. Following the phage related failure of several S. entomophila fermentations, a programme was initiated to isolate phage‐resistant strains. No naturally occurring strains were found to be resistant to eight stocks of phage isolated from either grass grub larvae or the fermenter. Therefore, ethylmethane sulphonate was used to generate phage‐resistant mutants. Strains which showed cross resistance to all eight phage test stocks were tested for pathogenicity towards grass grub. Several strains showing unimpaired pathogenicity were selected for further tests. Four of these phage‐resistant strains produced high cell yields, even when grown in the presence of high numbers of fermenter‐derived phage. Phage‐resistant strains have subsequently been produced in bulk in industrial fermente...

Hirsutella stylophoraMains, a pathogen ofCostelytra zealandica(Coleoptera: Scarabaeidae) in New Zealand

The entomopathogenic fungus, Hirsutella stylophora, is described as a pathogen of larvae of the New Zealand grass grub, Costelytra zealandica, for the first time. This species is the anamorph of Cordyceps stylophora. Infected grass grub larvae were dull brown in colour, usually with a distinctive stromatal growth extending from the head capsule. Despite the frequent production of stromata on diseased larvae, no ascospores (sexual stage) were found. Collections of diseased C. zealandica were made from several sites in the southern half of the South Island.

Serological identification of Serratia entomophila, a bacterial pathogen of the New Zealand grass grub ( Costelytra zealandica)

A competitive inhibition enzyme-linked immunosorbent assay was used to examine the antigenic relationships between strains of the bacterium Serratia entomophila and other members of the genus. S. entomophila causes amber disease of the New Zealand grass grub ( Costelytra zealandica) and is currently being developed as a biological agent to control this pasture pest. The assay distinguished S. entomophila from other Serratia species and indicated variability in the antigenic structure of the species, suggesting the existence of several serotypes. The method is potentially useful as an identification method as an alternative to biochemical testing.

Pathogenicity of<i> Beauvaria, metarhizium, Paecilomyces</i> and <i>Fusarium</i> isolates against early instar grass grub (<i>Costelytra zealandica</i>) larvae

Pathogenicity of<i> Beauvaria, metarhizium, Paecilomyces</i> and <i>Fusarium</i> isolates against early instar grass grub (<i>Costelytra zealandica</i>) larvae

Electron microscope evidence of a new virus in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae)

Electron microscope evidence of a new virus in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae)

Responses of a New Zealand North Island hill perennial ryegrass collection to nitrogen, moisture stress, and grass grub (Costelytra zealandica) infestation

Abstract A hill country perennial ryegrass collection from 60 sites throughout the North Island hill country in New Zealand was screened for tolerance of three important stresses in hill country: low nitrogen (N), moisture stress, and grass grub infestation. The screening was carried out in a simulated hill soil profile using 1800 hill plants (30 plants from 60 sites), four standard cultivars, and two local ecotypes each represented by 30 individuals. Plants were subjected to: (1) 0 versus 7 kg N/ha per week; (2) no water for 12 weeks versus regular watering; and (3) the addition of 600 grass grub larvae/m2. When grown with added N, plants removed from steep slopes i.e., conditions of severe N deficiency, produced less dry matter than plants removed from less stressed environments. Plants removed from northern steep slopes i.e., subjected to seasonal severe moisture stress, had a low yield compared with plants removed from wetter areas when grown with a regular water supply. Plant N concentration was simi...

Ultrastructure and tissue specificity ofNosema costelytraeandNosema takapauensis, microsporidian pathogens of grass grubs,Costelytra zealandica, (Coleoptera: Scarabaeidae) in New Zealand

Abstract Nosema costelytrae and Nosema takapauensis, microsporidian pathogens of grass grub beetle larvae of the scarabaeid pasture pest species, Costelytra zealandica, are redescribed. N. costelytrae has spherical meronts (6.34 µm) and elongate-ovoid or fusiform sporonts (6.81 × 3.27 µm). Stages have one, two, or four nuclei, usually in diplokaryotic arrangement. Spores are ovoid (4.37 × 2.50 µm, fresh), with a distinct point at one end. Spores develop in the fat body, tracheole cells, nerve ganglia, and muscle. There is no evidence of dimorph-ism when Planotortrix excessana (Lepidoptera) are experimentally infected with N. costelytrae at 15 or 22°C. N. takapauensis has spherical meronts (7.74 µm), with one, two, or four nuclei and spherical sporonts (3.70 µm), with one or two nuclei. Spores are ovoid to cylindrical (6.37 × 2.82 µm, fresh), with some macrospores. Fat body, tracheole cells, and ocnocytes support growth of this parasite. Both species have a fine structure that is usual for Nosema species.

Biological control of grass grub in Canterbury

The grass grub (Costelytra zealandica) is a major pest of Canterbury pastures. Grass grub numbers are low in young pastures and then commonly rise to a peak 4-6 years from sowing, before declining. Grass grub numbers in older pastures fluctuate but rarely reach the same levels as the early peak. Biological control agents such as bird and invertebrate predators, parasites and diseases cause mortality in grass grub populations; the effect of predators and parasites is limited. Pathogens are common in grass grub populations. Amber disease, caused by the bacteria Serratia spp., was the disease most frequently found in population surveys in Canterbury, while milky disease (Bacillus popilliae) and protozoan diseases were less common. The level of amber disease was reduced in pastures where insecticide had been applied. Inundative applications of entomophagous nematodes and the bacterium Serratia entomophila have been tested against grass grub. A bacterial productbased on S. entomophila is now being marketed for grass grub control. Keywords grass grub, biological control, pathogens

Control of grass grub (Costelytra zealandica) adults with soil insecticides

Abstract Four soil insecticides (lindane, isazophos, fensulfothion, and carbofuran) were tested primarily for use against grass grub adults and compared with foliar applications of a synthetic pyrethroid (cypermethrin). Separate treatments of the four soil chemicals were applied at 2 times just before the adult emergence and mating period, again just after this period, and in the autumn when mature larvae were present. Lindane was also applied 2 and 4 months before adult emergence. The progress of all soil treatments was monitored by larval and adult bioassays and chemical residue analysis on soil collected from the treated field plots. The lindane treatments were shown to be ineffective against mature larvae, but all treatments applied before adult emergence gave a significant reduction (85–95%) in the subsequent generation. Isazophos and fensulfothion were not sufficiently active against adults to be of practical use, but they affected both immature and mature larvae. Carbofuran was not active. The cype...

Integrated management of grass grub,Costelytra zealandica(White), in pasture using fensulfothion insecticide and nitrogen fertiliser alone and combined

Abstract Combinations of four rates of nitrogen (N) fertiliser and four rates of fensulfothion insecticide were applied to pasture in early autumn at three sites in northern Southland containing high (980/m2), medium (101–590/m2), and low (300/m2) densities of grass grub (Costelytra zealandica). The treatment producing the greatest additional dry matter (DM) of pasture per dollar invested for grass grub densities above 500/m2 was either 0.5 kg/ha of fensulfothion alone which gave a 77% mortality of grass grub resulting in an increase of 9 kg DM/ha per day pasture in the autumn, or alternatively, 0.5 kg/ha fensulfothion combined with 25 kgN/ha producing an additional 19 kg DM/ha per day compared with control plots. At the sites containing less than 500 larvae/m2, 50 kg N/ha alone gave the best DM return per dollar invested. Pasture responses from N at sites containing high and medium densities of grass grub continued through to spring. There were no significant interactions between N and insecticide on pas...