Raymond J. Akhurst

Morphological and Functional Dimorphism in Xenorhabdus spp., Bacteria Symbiotically Associated with the Insect Pathogenic Nematodes Neoaplectana and Heterorhabditis

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with the nematodes Neoaplectana and Heterorhabditis, occur in two forms. In general, only one form, designated the primary form, is transmitted into new hosts by the infective stage of the nematode. The significance of the relationship between the two forms has been examined with X. nematophilus, the symbiont of N. feltiae. The forms of X. nematophilus can be differentiated by their colony characteristics but by only two biochemical tests. The two forms of X. nematophilus are equally pathogenic when injected into the haemocoel of Galleria larvae. However, the primary form when injected into Galleria larvae with axenic nematodes provides better conditions for reproduction of the nematodes than the secondary form, for which a role has not been determined. Although the primary form readily converts to the secondary form in vitro and occasionally in vivo, the secondary form is usually stable. Possible causes of the instability have been investigated.

Antibiotic Activity of Xenorhabdus spp., Bacteria Symbiotically Associated with Insect Pathogenic Nematodes of the Families Heterorhabditidae and Steinernematidae

A wide range of micro-organisms, including yeasts, was found to be inhibited by the primary form of Xenorhabdus spp., but not by the secondary form. Only one Xenorhabdus strain, the symbiont of Neoaplectana glaseri, did not inhibit any of the micro-organisms tested; it is suggested that this strain may not have been isolated in the primary form. Gram-positive bacteria were sensitive to all active isolates of Xenorhabdus; each of the yeasts and almost all of the Gram-negative bacteria were sensitive to some but not all Xenorhabdus isolates. Each Xenorhabdus isolate was sensitive to some other Xenorhabdus isolates. The antibiotic activity of X. nematophilus was unaffected by autoclaving but was lost after dialysis. Anaerobically incubated Xenorhabdus spp. did not exhibit antibiotic activity.

DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov.

The levels of DNA relatedness for a broad sample of Xenorhabdus strains isolated from different species of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) and from different geographical sources were estimated by the hydroxyapatite method. The level of DNA-DNA relatedness for the two phases of each isolate tested was not significantly different from 100%, demonstrating unequivocally that the phase variation demonstrated by all Xenorhabdus spp. is not due to contamination. The isolates of the described Xenorhabdus species coalesced into different DNA relatedness groups, confirming that Xenorhabdus nematophilus, Xenorhabdus bovienii, Xenorhabdus poinarii, and Xenorhabdus beddingii, defined on the basis of phenotypic differences, are valid species. The symbiont of Steinernema intermedia also coalesced with the X. bovienii isolates. This was the only symbiont of seven recently described and unamed Steinernema spp. (including Steinernema ritteri, Steinernema rara, and Steinernema anomali) that formed a group with any of the previously described Xenorhabdus species; new species descriptions are required to accommodate the other taxa, but too few isolates were available to allow satisfactory descriptions of them. The DNA relatedness data also showed that the bacteria currently classified as Xenorhabdus luminescens are significantly different from all other Xenorhabdus strains. These data strongly support indications from previous studies of phenotypic characteristics, cellular fatty acids, and DNA relatedness that X. luminescens should be classified as a separate genus. A new genus, Photorhabdus, with an amended description of the type species, Photorhabdus luminescens, is proposed.

Biochemical and Physiological Characterization of Colony Form Variants in Xenorhabdus spp. (Enterobacteriaceae)

SUMMARY: Primary and secondary form variants of Xenorhabdus isolated from 21 strains (13 species) of Steinernematidae and Heterorhabditidae were tested for 240 biochemical and physiological characters. Primary form variants, isolated from the infective stage nematodes, could always be distinguished from the secondary by adsorption of neutral red from MacConkey agar. Lecithinase, antibiotic activity and/or adsorption of bromothymol blue were useful for distinguishing the variants of most strains. The variants of all strains also differed for other characteristics but the distinguishing characteristics varied from strain to strain. The importance of including both variants of each strain and of using appropriate methods in the study of Xenorhabdus taxonomy was demonstrated.

The nature of the intestinal vesicle in nematodes of the family steinernematidae

Abstract The intestinal vesicle is a modification of the ventricular portion of the intestine of infective larvae of nematodes of the family Steinernematidae. This structure, which normally houses the bacterium Xenorhabdus nematophilus in Steinernema species, was examined in a number of species either in the living state using differential interference contrast optics or in sections under the electron microscope. In Steinernema bibionis and S. kraussei the vesicle was found to be a thick walled, tapered structure packed with bacteria. In S. glaseri the wall of the vesicle was more irregular in thickness and the bacteria were not so tightly packed, whilst in S. feltiae the wall was thinner still. Development of the vesicle is not dependent on the presence of bacteria; a similar but empty structure was produced in axenically grown infective larvae of S. bibionis . The vesicle of an undescribed steinernematid designated Q1 was found to differ substantially from those mentioned above in that it had distinct microvilli. The lumen of this structure was filled with an amophorus matrix in which bacteria were embedded some distance apart from each other. There appears to be considerable variation in the morphology of the intestinal vesicle in the infective larvae of different steinernematids.

Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton

The susceptibilities of the major pests of cotton in Australia, Helicoverpa armigera and Helicoverpa punctigera, to some insecticidal proteins from Bacillus thuringiensis were tested by bioassay. A commercial formulation, DiPel, and individual purified insecticidal proteins were tested. H. armigera was consistently more tolerant to B. thuringiensis insecticidal proteins than was H. punctigera, although both were susceptible to only a limited range of these proteins. Only Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Vip3A killed H. armigera at dosages that could be considered acceptable. There was no significant difference in the toxicities of Cry1Fa and Cry1Ac for H. punctigera but Cry1Fa had little toxicity for H. armigera. The five instars of H. armigera did not differ significantly in their susceptibility to DiPel on the basis of LC(50). However, there were significant differences in the susceptibility to Cry1Ac and Cry2Aa of three strains of H. armigera. Bioassays conducted with Cry1Ac and Cry2Aa showed that there was a small but significant negative interaction between these delta-endotoxins.

Heterorhabditis spp., Neoaplectana spp., and Steinernema kraussei: Interspecific and intraspecific differences in infectivity for insects

The effectiveness of various dosages of different species/strains of nematodes was compared for Galleria mellonella and various pest insects that live in or pupate in soil. Neoaplectana feltiae (= carpocapsae), the only nematode species tested by most other workers, was never the most infective for any of the insect species tested and was least infective for two. All species/strains of nematode were able to kill insects of each species. The degree of infectivity of each of the nematode species/strains for different hosts varied considerably, and no one species/strain of nematode was the most infective for all insect species. This indicates the importance of testing a number of nematode species against any particular insect before commencing field evaluations for biological control.

Neoaplectana species: Specificity of association with bacteria of the genus Xenorhabdus

Each of five Neoaplectana (Nematoda: Steinernematidae) species was cultured monoxenically with various Xenorhabdus (Eubacteriales: Enterobacteriaceae) isolates. The nematodes were usually able to reproduce when cultured with the bacterial symbiont of any one of the five Neoaplectana spp. but never with Xenorhabdus luminescens, symbiotic with Heterorhabditis spp., or with the Xenorhabdus sp. isolated from an undescribed steinernematid species. Only Neoaplectana bibionis could be cultured with the Xenorhabdus symbiont of Steinernema kraussei. A high proportion of infectives were able to carry within their intestine X. nematophilus isolated from other strains of the same nematode species; a small proportion of infectives were able to carry X. nematophilus isolated from other nematode species.

Taxonomic Study of Xenorhabdus, a Genus of Bacteria Symbiotically Associated with Insect Pathogenic Nematodes

The taxonomy of the bacteria symbiotically associated with the insect-pathogenic nematodes Neoaplectana and Heterorhabditis was examined. The bacteria studied were isolated from 33 populations obtained from Australasia, Europe, and the United States. The symbionts of all species of Neoaplectana and Heterorhabditis examined were members of the genus Xenorhabdus, but they differed in several respects from the description of the genus Xenorhabdus, including the guanine-plus-cytosine content of the deoxyriboneucleic acid and the production of acid from carbohydrates. All bacteria isolated from Heterorhabditis spp. were identified as members of Xenorhabdus luminescens. The bacteria isolated from one Neoaplectana species were similar and were distinguishable from the bacteria isolated from other Neoaplectana species. The following three subspecies of Xenorhabdus nematophilus are proposed: Xenorhabdus nematophilus subsp. nematophilus (bacteria symbiotic with Neoaplectana feltiae [= Neoaplectana carpocapsae]; type strain ATCC 19061), Xenorhabdus nematophilus subsp. bovienii (bacteria symbiotic with Neoaplectana bibionis; type strain, UQM 2210T), and Xenorhabdus nematophilus subsp. poinarii (bacteria symbiotic with Neoaplectana glaseri; type strain, UQM 2216). These subspecies vary in host nematode, pigmentation, maximum temperature for growth, responses to tests for phenylalanine deaminase by secondary-form isolates and for lipase (Tween 80 test) and lecithinase by primary-form isolates, and coloration of primary-form isolates on MacConkey agar and media containing bromthymol blue.

Relative fitness of Cry1a-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on conventional and transgenic cotton

Abstract Glasshouse and laboratory experiments were conducted to evaluate the relative fitness of Cry1A-susceptible and laboratory-selected resistant strains of Helicoverpa armigera (Hübner). Life history parameters of H. armigera larvae feeding on young cotton plants showed a significant developmental delay of up to 7 d for the resistant strain compared with the susceptible strain on non-Bacillus thuringiensis (Bt) cotton. This fitness cost was not evident on artificial diet. There was no developmental delay in the F1 hybrid progeny from the reciprocal backcross of the resistant and susceptible strains, indicating that the fitness cost is recessive. In two cohorts tested, survival to pupation of resistant larvae on Bt cotton expressing Cry1Ac was 54 and 51% lower than on non-Bt cotton, whereas all susceptible and F1 larvae tested on Cry1Ac cotton were killed. Mortality of susceptible larvae occurred in the first or second instar, whereas the F1 larvae were able to develop to later instars before dying, demonstrating that resistance is incompletely recessive. The intrinsic rate of increase was reduced by >50% in the resistant strain on Cry1Ac cotton compared with the susceptible strain on non-Bt cotton. There was a significant reduction in the survival of postdiapausal adults from the resistant strain and the F1 strains, indicating that there is a nonrecessive overwintering cost associated with Cry1A resistance in H. armigera.