Christine T. Griffin

Biology and Behaviour

Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae are lethal pathogens of insects. These pathogens contribute to the regulation of natural populations of insects, but the main interest in them is as an inundatively applied biocontrol agent. Their success in this role can be attributed to the unique partnership between a host-seeking nematode and a lethal insect-pathogenic bacterium. Because of their biocontrol potential, considerable attention has been directed over the past few decades to Heterorhabditis and Steinernema and their respective bacterial partners, Photorhabdus and Xenorhabdus.

Occurrence of Insect-Parasitic Nematodes (Steinernematidae, Heterorhabditidae) in the Republic of Ireland

Insect parasitic nematodes were surveyed in the Republic of Ireland between October 1986 and October 1987. A total of 551 soil samples was tested for the presence of nematodes by baiting with Galleria mellonella larvae. Steinernema feltiae (= S. bibionis) and S. affinis were recovered from 7.1 and 3.3% of samples, respectively. Heterorhabditis sp. was found in one sample. There was a significant association between locality (county) and frequency of nematode recovery. Nematodes were more frequently recovered from sandy and peaty soils than from clays and clay loams, but differences between soil types were not significant. Nematodes were present in tilled fields (41/365 samples), grassland (13/143) and woodland (4/43). There was a significant association between sampling time and frequency of recovery. Nematodes were less likely to be recovered in May-June than at other times of the year.

The identification of biological species in the genus Heterorhabditis (Nematoda: Heterorhabditidae) by cross-breeding second-generation amphimictic adults

Entomopathogenic nematodes of the genus Heterorhabditis are morphologically conservative, consequently the majority of isolates remain unassigned at the species level. When a Heterorhabditis infective juvenile infects an insect host, it develops into a protandrous hermaphrodite female. These first-generation females give rise to a second generation which contains both males and females. Because of this complex life-cycle and also because of uncertainty as to whether second-generation females are amphimictic, cross-breeding studies to facilitate species determination have not been carried out previously. We demonstrate here that second-generation Heterorhabditis females are amphimictic. Because of this finding, we have been able to develop a successful cross-breeding technique for the purposes of species determination in Heterorhabditis. Interstrain crosses using second-generation males and females from the appropriate strains have been successfully set up in Xenorhabdus luminescens-treated G. mellonella cadavers and also on agar plates. Using the techniques described here we confirm that H . bacteriophora (Brecon strain), H . megidis and H . zealandica are distinct biological species, we note that the H. bacteriophora group contains at least 2 species and we provide evidence for the existence of a new Irish species of Heterorhabditis.

Dispersal Behaviour and Transmission Strategies of the Entomopathogenic Nematodes Heterorhabditis and Steinernema

Entomopathogenic nematodes of the Heterorhabditidae and Steinernematidae appear to be capable of long-distance dispersal and local migration. Their transmission strategies include both highly active seek-and-destroy behaviours and ambusher strategies, and they may be sensitive to sex-related factors in their own populations. Their host-finding abilities are poorly understood, despite the fact that these abilities are fundamental to their success as biocontrol agents in soil. Like the vast numbers of exotic hymenopterans and other natural enemies that have been released for biological control over the past century, they may be used in their ecologically competent wild-type form. On the other hand, because they are applied inundatively, they may be tailored, by breeding or transformation, to their intended purpose and to ecological incompetence, improving both their efficacy and their ecological safety.

A self-fertile species of Steinernema from Indonesia: further evidence of convergent evolution amongst entomopathogenic nematodes?

More than 20 species of the entomopathogenic nematode Steinernema have been described; to date, all reproduce exclusively by cross-fertilization of male and female individuals. Steinernema sp. strain T87 from Indonesia was found to consist largely of self-fertile hermaphrodites. Progeny were produced by morphological females both in insects (Galleria mellonella) and in hanging drops of insect haemolymph inoculated with a single infective juvenile. Sperm were present in the oviduct of unmated morphological females. Approximately 1% of infective juveniles developed into males, and males were also present in the second generation where they constituted 1-6% of the population. Under the same conditions the related species Steinernema longicaudum strain CB2B displayed typical steinernematid reproduction: cross-fertilization and a 1:1 sex ratio. It is argued that the development of hermaphroditism in Steinernema sp. T87 represents convergent evolution with Heterorhabditis, the other major genus of entomopathogenic nematode.

ESTABLISHMENT, PERSISTENCE, AND INTROGRESSION OF ENTOMOPATHOGENIC NEMATODES IN A FOREST ECOSYSTEM

Entomopathogenic nematodes (EPN) are currently marketed worldwide for use in inundative biological control, where the applied natural enemy population (rather than its offspring) is expected to reduce insect numbers. Unlike classical biological control, in inundative control natural enemy establishment is not crucial in order to achieve pest suppression. Field trials in Irish forestry provided the opportunity to test predictions regarding the establishment of two exotic (Steinernema carpocapsae and Heterorhabditis megidis) and two indigenous (Steinernema feltiae and Heterorhabditis downesi) species. Nematodes were inundatively applied to pine stumps to control populations of pine weevil, Hylobius abietis, on three clearcut sites, and their persistence and spread monitored for up to five years. All species were recovered three years after application but only S. feltiae was recovered in years 4 and 5. Limited horizontal dispersal to 20 cm (but not 100 cm) was observed, but the majority of nematodes were recovered close to the area of application. Steinernema feltiae was also recovered from nearby stumps to which it had not been applied, indicating possible phoretic dispersal by weevils or other stump-associated fauna. EPN were not recovered from stumps outside the treated area, suggesting that such dispersal is quite localized. Two strains of S. feltiae (Irish and exotic) were applied. Amplified fragment length polymorphism (AFLP) analysis on 11 populations isolated from soil four years later showed that all had a much closer affinity to the applied Irish strain, suggesting persistence of this genotype and extinction of the exotic one. Some strains were clustered close together, and this is interpreted in the light of possible population genetic scenarios. The findings from the field study confirm predictions based on background knowledge of the species and demonstrate the importance of medium-term studies, as a 3-year study would have overestimated the risk of establishment of exotic species. Short-term persistence and spread of S. carpocapsae, S. feltiae, and H. downesi was also studied in pine forest mesocosms. Similar trends to field results, such as limited horizontal dispersal, even vertical distribution, and more abundant recovery of S. feltiae than of other species, point to the utility of mesocosm studies as a predictive tool.

Morphological and molecular characterisation of Steinernema hermaphroditum n. sp. (Nematoda: Steinernematidae), an entomopathogenic nematode from Indonesia, and its phylogenetic relationships with other members of the genus

Steinernema hermaphroditum n. sp., a new entomopathogenic nematode isolated from soil samples in the Moluccan islands, Indonesia, is described. Morphological observations as well as biological evidence (cross-hybridisation studies) indicate the distinctness of S. hermaphroditum n. sp. from other Steinernema spp. This new species is characterised by the presence of hermaphrodites in the first adult generation. Key morphological diagnostic characters include: a digitate tail with a mucro and a glandular spermatheca filled with sperm in the first generation hermaphrodite; the value of D%; the morphology of the male spicules and gubernaculum and the number and arrangement of the genital papillae; the values of D%, E% and the pattern of the lateral field of the third-stage infective juvenile. Additionally, molecular evidence obtained from ITS rDNA RFLP profiles, 28S rDNA sequence analyses, and phylogenetic reconstruction provide further evidence to establish this nematode as a new species.

Occurrence and distribution of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis indica in Indonesia

Soil samples from 79 sites on five islands of Indonesia were baited with insects for the recovery of entomopathogenic nematodes. Heterorhabditis and Steinernema were equally prevalent, and were recovered from 11.7% of samples representing 20.3% of sites sampled. Both genera were recovered from coastal sites only. Entomopathogenic nematodes were more prevalent on the Moluccan islands of Ambon and Seram than on Java or Bali. They were not detected on Sulawesi, where non-coastal sites only were sampled. RFLP analysis was used in the identification of nematode isolates. Heterorhabditis indica was the only heterorhabditid identified. Two RFLP types of Steinernema were identified.

Low Temperature Activity in Heterorhabditis Sp. (Nematoda: Heterorhabditidae)

Four isolates of Heterorhabditis sp. (H181, Hf85 and Hnhl 86 from the Netherlands and K122 from Ireland) were compared in laboratory bioassays. Galleria mellonella larvae were exposed to infective juveniles in sand for 2-5 days. There were significant differences between isolates in the number of infective juveniles that found and entered the larvae at 9° and 12°C. At 9°C, . Hf85 was significantly better than all the other isolates. Success of Hf85 was considerably reduced at 7°C, and there was no parasitisation at 5°C. The infectivity for late instar Tenebrio .. molitor larvae was tested on filter paper at 5°, 7°, 9°, 12°, and 20°C. There was no mortality at 5°C. At 7°C, the LD50 of Hf85 was 23 IJs/insect; this was significantly lower than for the other three isolates. The LD50s of isolates differed significantly at 9° and 12° but not at 20°C. Differences between LT50s were significant at 20°C. The rank order of efficacy of the three Dutch isolates in the bioassays was the same as that established by other authors in pot trials at low temperature.

Simulated roots and host feeding enhance infection of subterranean insects by the entomopathogenic nematode Steinernema carpocapsae

Steinernema carpocapsae can be effective against root-feeding insects despite its reputation as a sedentary ambusher. In pot experiments, using twigs as surrogate roots and pine weevil larvae as targets, we tested the hypothesis that roots serve as physical routeways and conduits of feeding-associated stimuli, thus enhancing the success of S. carpocapsae applied at the surface against subterranean hosts. Insect mortality was lowest (25%) in the absence of plant material, increased to 48% when twigs linked nematodes and insects, and further increased to 69% when the insects were allowed feed on the twigs. This is the first experimental support for the root-routeway hypothesis.