The European truffle beetle Leiodes cinnamomeus is the most important pest in black truffle (Tuber melanosporum) plantations. Current control methods against it are inefficient, so entomopathogenic nematodes (EPNs) could play an important role in their population regulation due to their efficacy against many soil-dwelling insect pests. A survey of EPNs and Pristionchus nematodes was conducted in truffle soils of Spain, considering environmental and physical–chemical soil factors. A total of 164 soil samples were collected from forests, productive plantations and null-low productive plantations, representing three distinct black truffle-growing habitat types. EPNs were isolated from seven soil samples (4.3%); four nematodes were identified as Steinernema feltiae and three as Heterorhabditis bacteriophora. Both species were sampled in three types of soil texture (loam, sandy loam or sandy clay loam), characterized by alkaline pH (7.5 to 8.5) and high organic matter (2.1–11.04%). The presence of these EPNs was influenced by habitat type and organic matter content. Pristionchus nematodes were isolated from truffle soil, around truffle fruit bodies and under the elytra of L. cinnamomeus, with Pristionchus maupasi being the most commonly identified species. No significant associations were found between environmental and soil factors and the occurrence of Pristionchus nematodes. These nematodes were found in alkaline soils (pH 7.75 to 8.7), across all seven sampled soil textures, with variable organic matter content (0.73%–5.92%). The ecological trends and the presence of Pristionchus may affect the occurrence of EPNs and their prospective use as biological control agents against L. cinnamomeus in black truffle plantations.

Understanding the temporal and spatial distribution of entomopathogenic nematodes is essential for determining the role of these insect parasites in soil communities and ultimately for their use in suppression of pest insect populations. We measured the vertical and horizontal distribution of endemic populations of entomopathogenic nematodes (Steinernema carpocapsae and Heterorhabditis bacteriophord) in turfgrass. Vertical distribution was determined by taking soil cores every 3 h from 05.00 to 23.00 h, over 4 days, and dividing the cores into 8, 1 cm deep sections. Steinernema carpocapsae was recovered primarily near the soil surface: 50% of positive sections were recovered in the thatch or first 1 cm of soil. S. carpocapsae recovery was lower during the middle of the day and none were recovered in the upper section. H. bacteriophora was recovered uniformly throughout the top 8 cm of soil and its vertical distribution did not change over the course of the day. Horizontal distribution was measured as the number of nematodes recovered from cores taken from 12 randomly selected 0·3 × 0·8 m sections from within four 15·3 × 15·3 m plots. Samples were collected biweekly over a 9-month period. H. bacteriophora had a patchier distribution than S. carpocapsae and both nematode species had more patchy distributions then their potential hosts. Our results support the hypothesis that these two species of nematode utilize different foraging strategies; S. carpocapsae primarily a surface adapted ambusher and H. bacteriophora as a cruise forager.

Three satellite DNAs previously isolated from the entomopathogenic nematodes Steinernema carpocapsae, Heterorhabditis bacteriophora and Heterorhabditis indicus give hybridization signals only with the S. carpocapsae, H. bacteriophora and H. indicus populations tested, indicating that these satellite sequences are species-specific. Because of their reiteration and their variabilities, we have shown that these sequences are able to discriminate at the interspecific level between the Steinernema and Heterorhabditis species, but also at the intraspecific level between S. carpocapsae strains. Furthermore, in simple squashed nematode experiments, we are able to unambiguously identify S. carpocapsae, H. bacteriophora and H. indicus populations. This last procedure is effective even on a single infective juvenile, with the main advantage that it avoids time-consuming DNA extractions.

We tested the chemotactic responses of dauer juvenile stages (DJs) of the insect parasitic nematode Heterorhabditis bacteriophora to a variety of compounds that are known to be highly attractive or highly repellent to Caenorhabditis elegans. While H. bacteriophora DJs respond to alcohols and some aromatic compounds as well as to host metabolites such as uric acid and CO2, the most notable difference in the responses of these two nematodes is that H. bacteriophora DJs are unresponsive to a large number of compounds which C. elegans finds highly attractive. The latter compounds are typical by-products of bacterial metabolism and include aldehydes, esters, ketones and short-chain alcohols. While C. elegans finds long-chain alcohols (e.g. 1-heptanol and 1-octanol) repellent and short-chain alcohols highly attractive, H. bacteriophora DJs are strongly attracted to 1-heptanol, 1-octanol and 1-nonanol and find short-chain alcohols to be only slightly attractive. Parasitic-stage H. bacteriophora nematodes show a very weak chemotactic response to volatile molecules that DJs find highly attractive. Our results suggest that, associated with the adoption of a parasitic mode of life by Heterorhabditis, there was an adaptive change in chemotactic behaviour of the infective stages, resulting in a decreased sensitivity to volatile by-products of bacterial metabolism and an increased sensitivity towards long-chain alcohols and other insect-specific volatiles and possibly also to herbivore-induced plant volatiles.

Many studies of entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) have reported that only a small proportion (typically <40%) of infective stages (dauers), even under apparently ideal conditions, actually infect a host. The ‘phased infectivity hypothesis’ is most frequently invoked to explain this pattern of low infection with entomopathogenic nematodes. It proposes that at a given point in time not all individuals are infectious i.e. infectiousness is delayed in some individuals. We tested experimentally several predictions based on this hypothesis. Specifically, if phased infectivity occurs, we should be able to expose dauers to increasing numbers of potential hosts until dauers no longer infect and still be able to recover viable dauers. These recovered dauers which did not infect should be infectious at some later point in time. However, our results do not support the phased infectivity hypothesis for 3 species of Steinernema: most dauers could be recovered in one sampling round when provided with sufficient suitable hosts. In contrast, Heterorhabditis bacteriophora frequently did not infect all available hosts, and infectious dauers were recovered in subsequent sampling rounds. This result is more consistent with the phased infectivity hypothesis, but further research is needed before we can be more confident in the hypothesis. For all species tested, the number of available hosts influenced population levels of nematode infectivity. This suggests that the infection status of hosts can influence whether a dauer infects. Our results indicate that phased infectivity is not a common phenomenon in entomopathogenic nematode dauers, despite the widespread acceptance of this hypothesis.

A specific extracellular proteinase, degrading selectively the cecropin-based defence system of insects, is secreted into the larval body during parasitism of the greater wax moth by the Heterorhabditis bacteriophora/Photorhabdus luminescens complex and by phase 1 of P. luminescens. The proteolytic digestion of insect inducible cecropin-like immune molecules was demonstrated by the disappearance of the Galleria mellonella cecropins and purified Hyalophora cecropin B peptide PAGE bands upon exposure to infected extracts, and a similar abrogation of antibacterial activity using an agar diffusion assay. Proteolytic activity of infected extracts produced by nematode/bacterial complex and phase 1 variant of P. luminescens was shown to be correlated with cecropin-inhibitory activity, suggesting that this anti-cecropin agent may be responsible for the ability of bacteria to establish infection and the insecticidal nature of H. bacteriophora. Antibacterial activity of Galleria lysozyme and that of chicken egg-white lysozyme to which P. luminescens is insensitive, was unaffected by H. bacteriophora proteinase.

There is currently no information on the genome size and complexity in the entomoparasitic nematodes Steinernema and Heterorhabditis. DNA reassociation kinetics were used to determine the genome size and complexity in 2 species: Steinernema carpocapsae and Heterorhabditis bacteriophora. C0t curves derived from renaturation kinetics of denatured DNA indicate that the genomes of these entomoparasitic rhabditids are different both in size and complexity. Genome sizes were estimated at 2·3 × 108 bp for S. carpocapsae and 3·9 × 107 bp for H. bacteriophora and repetitive DNA contents were found to represent 39% and 51% of these respective genomes.