Ganpati B. Jagdale

BOTH HEAT-SHOCK AND COLD-SHOCK INFLUENCE TREHALOSE METABOLISM IN AN ENTOMOPATHOGENIC NEMATODE

Heat-shock response is highly conserved in animals and microorganisms, and it results in the synthesis of heat-shock proteins. In yeast, heat-shock response has also been reported to induce trehalose accumulation. We explored the relationship between heat- (35 C) or cold-shock (1 and 10 C) and trehalose metabolism in the entomopathogenic nematode, Heterorhabditis bacteriophora. Because both heat- and cold-shocks may precede desiccation stress in natural soil environments, we hypothesized that nematodes may accumulate a general desiccation protectant, trehalose, under both situations. Indeed, both heat- and cold-shocks influenced trehalose accumulation and activities of enzymes of trehalose metabolism in H. bacteriophora. Trehalose increased by 5- and 6-fold in heat- and cold-shocked infective juveniles, respectively, within 3 hr of exposure, compared with the nematodes maintained at 25 C (culture temperature). The activity of trehalose-6-phosphate synthase (T6PS), an enzyme involved in the synthesis of trehalose, also significantly increased in both heat- and cold-shocked nematodes during the first 3 hr of exposure. Generally, the trehalose levels and activities of T6PS declined to their original levels within 3 hr when nematodes were transferred back to 25 C. In both heat- and cold-shocked nematodes, trehalase activity decreased significantly within the first 3 hr and generally returned to the original levels within 3 hr when these nematodes were transferred back to 25 C. The results demonstrate that the trehalose concentrations in H. bacteriophora are influenced by both heat- and cold-shocks and are regulated by the action of 2 trehalose-metabolizing enzymes, T6PS and trehalase. The accumulated trehalose may enhance survival of nematodes under both cold and warm conditions, but it may also provide simultaneous protection against desiccation that may result from subsequent evaporation or freezing. This is the first report of the relationship between trehalose metabolism and heat-shock for the Nematoda.

Suppression of plant-parasitic nematodes by application of live and dead infective juveniles of an entomopathogenic nematode, Steinernema carpocapsae, on boxwood (Buxus spp.)

Effects of live and dead (heat-killed) infective juveniles (IJs) of the entomopathogenic nematode Steinernema carpocapsae on nematodes associated with boxwood Buxus spp. were evaluated in field experiments during 1999 and 2000. Both living and dead IJs of S. carpocapsae were equally effective, causing more than 50% reduction in total populations of plant-parasitic nematodes relative to the control 15 and 30 days after treatment in both years. No significant differences were observed between reductions in populations of plant-parasitic nematodes following entomopathogenic nematode and chemical nematicide (ethoprop) treatments. In 2000, populations of Criconemella, Hoplolaimus, Longidorus, and Rotylenchus were significantly reduced in all treatments relative to the control 30 days after treatment. However, in 1999 these genera were unaffected by treatments even though total plant-parasitic nematode population was decreased. The population of Tylenchorynchus was significantly reduced in all treatments 30 days after treatment in 1999. Further, the population of Tylenchus was significantly reduced in ethoprop and dead S. carpocapsae treatments 15 days after treatment relative to the control whereas the population of Aphelenchoides was unaffected by these treatments in 1999. Live S. carpocapsae showed no significant effect on the population of both these genera either 15 or 30 days after treatment in 1999. No viable symbiotic bacteria were observed in the dead nematodes used in this study indicating that dead nematodes alone or dead nematodes in combination with dead symbiotic bacteria produced the suppressive effect. In contrast to the plant-parasitic nematodes, populations of non-stylet-bearing nematodes were not affected by the application of entomopathogenic nematodes. Although ethoprop reduced population of non-stylet-bearing nematodes relative to the untreated control 15 days after treatment in 2000, no significant differences were observed 30 days after treatment. These findings suggest that allelochemicals produced by dead or live nematodes and/or by their symbiotic bacteria may be selectively acting against plant-parasitic nematodes. Our results demonstrate that both live and dead infective juveniles of S. carpocapsae may provide a possible control strategy for plant-parasitic nematodes on boxwood.

Indicative Value of Soil Nematode Food Web Indices and Trophic Group Abundance in Differentiating Habitats with a Gradient of Anthropogenic Impact

Indicative value of nematode food web indices and trophic group abundance was assessed for differentiating habitats in two soil types. A total of 604 soil samples were collected from croplands, grassy borders, turfgrass lawns, shrublands and forests in Huron County, Ohio characterized by muck soils. Also, a total of 242 soil samples were collected from two habitats, turfgrass lawns and croplands, in Wayne County, Ohio characterized by mineral soils. All nematodes extracted from soil samples were identified to genus level, categorized to trophic groups and various nematode community indices were calculated. Multivariate analysis of variance using composite of nematode measures revealed that croplands were significantly different from forests, shrublands, turfgrass lawns and grassy borders in the muck soils. Turfgrass lawns in muck soils were not different from forests but were different from shrublands. No differences were observed between forests and shrublands. Turfgrass lawns in mineral soils differed s...

Genetic variation and relationships between isolates and species of the entomopathogenic nematode genus Heterorhabditis deciphered through isozyme profiles.

We studied variation in isozyme patterns of 8 metabolic enzymes in 5 species of Heterorhabditis (H. bacteriophora, H. indica, H. marelata, H. megidis, and H. zealandica) comprising 18 isolates. Isozyme banding patterns of all the 8 enzymes were species specific; however, 3 enzymes, i.e., arginine kinase, fumarate hydratase, and malate dehydrogenase, displayed distinct patterns among all the 18 isolates. Phylogenetic analysis of the isozyme patterns produced dendrograms depicting a high degree of genetic variation among Heterorhabditis species, with the average pairwise distance of 0.2000. Trees constructed using different phylogenetic methods showed a relatively close genetic relationship between H. megidis and H. zealandica and between H. bacteriophora and H. indica. Also, H. bacteriophora HP88 was the most distant species from H. megidis (UK isolate), H. marelatus (Oregon isolate), and H. zealandica (X1 isolate) with pairwise distance of 0.1957, 0.2228, and 0.2120, respectively. Phylogenetic analysis also revealed genetic variation among H. bacteriophora isolates with the average pairwise distance of 0.1507. GPS2 and GPS3 were the most closely related isolates with the average distance of only 0.0870, followed by GPS1 and GPS2 with average distance of 0.1087. In contrast, KMD19 and HP88, OH25, and HP88, and OH25 and Acows isolates were the most divergent populations with a pairwise distance of 0.2011 and 37 character differences. Pairwise distance analysis also revealed that genetic divergence among populations of H. bacteriophora is relatively independent of geographic distance. Overall, these results demonstrate strong subspecies structuring in H. bacteriophora.

Differences in Immune Defense Evasion of Selected Inbred Lines of Heterorhabditis Bacteriophora in Two White Grub Species

We determined virulence of seven Heterorhabditis bacteriophora strain GPS11 inbred lines possessing superior infective juvenile longevity, and heat and ultra violet radiation tolerance against white grubs Popillia japonica and Cyclocephala borealis. At 1 and 2 weeks after treatment, inbred line A2 was significantly more virulent towards P. japonica compared to the parent strain GPS11 and inbred lines A7, A8, A12 and A21; and line A2 caused significantly higher C. borealis mortality than lines A6 and A12. Penetration, encapsulation and survival of two inbred lines, A2 and A12, that showed the highest and lowest virulence against both grub species were then assessed. There were no differences between the two lines for the total number of nematodes penetrated in either P. japonica or C. borealis within the first 24 h, but a significantly higher percentage of penetrated nematodes were alive in line A2 compared to the line A12 in both grub species. P. japonica immune response over time to hemocoel-injected nematodes of A2, A12 and the parent strain was further investigated. While all injected nematodes were encapsulated at 6 h post injection, non-encapsulated living nematodes were detected at 12 and 24 h post injection, showing the breakage out of encapsulation. A higher percentage of non-encapsulated living nematodes and a lower percentage of dead nematodes were found in line A2 as compared to the line A12 after 12 h post injection. These data suggest that virulence differences in the studied H. bacteriophora inbred lines are not due to differences in nematode penetration or recognition by the grub immune system, but are related to the ability of the infective juveniles to break out of encapsulation.