Thirty years of slug control using the parasitic nematode Phasmarhabditis hermaphrodita and beyond.

Several slug species are highly pestiferous and threaten global sustainable agriculture. Current control methods rely heavily on metaldehyde pellets, which are often ineffective, harm nontarget organisms and have been banned in some countries. A viable alternative is the parasitic nematode Phasmarhabditis hermaphrodita (and recently P. californica), which has been formulated into a biological control agent (Nemaslug®) to control slugs across northern Europe. Nematodes are mixed with water and applied to soil where they seek out slugs, penetrate behind the mantle and kill them in 4-21 days. Phasmarhabditis hermaphrodita has been on the market since 1994 and since then there has been ample research on its use. Here we review the research carried out on P. hermaphrodita over the last 30 years since its development and release as a commercial product. We provide information on life cycle, worldwide distribution, history of commercialisation, gastropod immunity, host range, ecological and environmental factors that affect its success in the field, bacterial relationships, and summarise results of field trials. Finally, we suggest future directions for P. hermaphrodita research (and other Phasmarhabditis species) to enhance its use as a biological control agent to control slugs for the next 30 years. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A parasitic nematode induces dysbiosis in susceptible but not resistant gastropod hosts.

Animals' gut microbiomes affect a wide array of biological processes including immunity and protection from pathogens. However, how the microbiome changes due to infection by parasites is still largely unknown, as is how the microbiome changes in hosts that differ in their susceptibility to parasites. To investigate this, we exposed two slug species of differing susceptibility to the parasitic nematode Phasmarhabditis hermaphrodita (Deroceras reticulatum is highly susceptible and Ambigolimax valentianus resistant to the nematode) and profiled the gut microbiota after 7 and 14 days. Before infection, both slug species' microbiota was dominated by similar bacterial genera: Pseudomonas (by far the most abundant), Sphingobacterium, Pedobacter, Chryseobacterium, and Flavobacterium. In the resistant host A. valentianus, there was no significant change in the bacterial genera after infection, but in D. reticulatum, the bacterial profile changed, with a decrease in the abundance of Pseudomonadaceae and an increase in the abundance of Flavobacteriaceae and Sphingobacteriaceae after 7 days postinfection. This suggests nematode infection causes dysbiosis in hosts that are susceptible to infection, but the microbiome of resistant species remains unaltered. In summary, the regulation of the immune system is tightly linked with host survival, and nematode infection can alter the microbiome structure.

Avoidance and attraction behaviour of slugs exposed to parasitic nematodes.

Avoidance of pathogens and parasites is the first line of defense to survive. Several slug species avoid the parasitic nematode Phasmarhabditis hermaphrodita to reduce infection however, there is nothing known about whether slugs avoid other members of the Phasmarhabditis genus. I exposed two slug species (Deroceras invadens and Limax maculatus) to Phasmarhabditis californica and P. neopapillosa. D. invadens avoided P. californica but was strangely attracted to P. neopapillosa. L. maculatus did not avoid P. californica, but on day 1 and 3 significantly more slugs were found with P. neopapillosa. Reasons for host attraction to P. neopapillosa are discussed.

Microbiome Analysis of Malacopathogenic Nematodes Suggests No Evidence of a Single Bacterial Symbiont Responsible for Gastropod Mortality.

Nematodes and bacteria are prevalent in soil ecosystems, and some have evolved symbiotic relationships. In some cases, symbionts carry out highly specialized functions: a prime example being entomopathogenic nematodes (EPNs), which vector bacteria (Xenorhabdus or Photorhabdus) into insect hosts, killing them to provide a food source for the nematodes. It is thought that the commercially available malacopathogenic (kills slugs and snails) biocontrol nematode Phasmarhabditis hermaphrodita vectors a bacterium (Moraxella osloensis) into slugs to kill them. To investigate this further we used a metagenomic approach to profile the bacteria present in the commercial strain of P. hermaphrodita, a wild strain of P. hermaphrodita and two other Phasmarhabditis species (P. californica and P. neopapillosa), after they had killed their slug host (Deroceras invadens). We show that these nematodes do not exclusively associate with one bacterium but a range of species, with members of the phyla Pseudomonadota, Bacillota, Actinobacteriota and Bacteroidota the most prevalent. The commercial strain of P. hermaphrodita had the least diverse bacterial community. Furthermore, we found that the bacterium P. hermaphrodita has been cultured on for 25 years is not the expected species M. osloensis but is Psychrobacter spp. and the only strain of the Phasmarhabditis species to associate with Psychrobacter spp. was the commercial strain of P. hermaphrodita. In summary, we found no evidence to show that P. hermaphrodita rely exclusively on one bacterium to cause host mortality but found variable and diverse bacterial communities associated with these nematodes in their slug hosts.

Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand.

Biological control (biocontrol) as a component of pest management strategies reduces reliance on synthetic chemicals, and seemingly offers a natural approach that minimizes environmental impact. However, introducing a new organism to new environments as a classical biocontrol agent can have broad and unanticipated biodiversity effects and conservation consequences. Nematodes are currently used in a variety of commercial biocontrol applications, including the use of Phasmarhabditis hermaphrodita as an agent targeting pest slug and snail species. This species was originally discovered in Germany, and is generally thought to have European origins. P. hermaphrodita is sold under the trade name Nemaslug®, and is available only in European markets. However, this nematode species was discovered in New Zealand and the western United States, though its specific origins remained unclear. In this study, we analyzed 45 nematode strains representing eight different Phasmarhabditis species, collected from nine countries around the world. A segment of nematode mitochondrial DNA (mtDNA) was sequenced and subjected to phylogenetic analyses. Our mtDNA phylogenies were overall consistent with previous analyses based on nuclear ribosomal RNA (rRNA) loci. The recently discovered P. hermaphrodita strains in New Zealand and the United States had mtDNA haplotypes nearly identical to that of Nemaslug®, and these were placed together in an intraspecific monophyletic clade with high support in maximum likelihood and Bayesian analyses. We also examined bacteria that co-cultured with the nematode strains isolated in Oregon, USA, by analyzing 16S rRNA sequences. Eight different bacterial genera were found to associate with these nematodes, though Moraxella osloensis, the bacteria species used in the Nemaslug® formulation, was not detected. This study provided evidence that nematodes deriving from the Nemaslug® biocontrol product have invaded countries where its use is prohibited by regulatory agencies and not commercially available.

Pathogenicity of wild and commercial Phasmarhabditis hermaphrodita exposed to the pestiferous slug Deroceras invadens.

Many terrestrial gastropods are pestiferous and pose a significant threat to agriculture, horticulture and floriculture. They are usually controlled by metaldehyde based pellets but an alternative control method is the slug parasitic nematode Phasmarhabditis hermaphrodita, which has been formulated into a biological control agent (Nemaslug®) for use by farmers and gardeners to kill certain pestiferous slug species in 4-21 days. The current strain of P. hermaphrodita (called DMG0001) has been used in commercial production since 1994, but there is little information about the pathogenicity of wild strains of P. hermaphrodita towards slugs. Here, we exposed the pestiferous slug Deroceras invadens to nine wild isolated strains of P. hermaphrodita (DMG0002, DMG0003, DMG0005, DMG0006, DMG0007, DMG0008, DMG0009, DMG0010 and DMG0011) and the commercial strain (DMG0001) to three doses (0, 500 and 1000 nematodes per ml). Survival and feeding were recorded over 14 days. All wild P. hermaphrodita strains (other than DMG0010) and P. hermaphrodita (DMG0001), applied at 500 nematodes per ml, caused significant mortality to D. invadens compared to an uninfected control. Similarly, all P. hermaphrodita strains applied at 1000 nematodes per ml, caused significant mortality to D. invadens compared to an uninfected control. Overall, all wild P. hermaphrodita strains (other than DMG0011) caused significantly more mortality than P. hermaphrodita DMG0001 at one or both nematode concentrations. In summary, we have found some wild P. hermaphrodita strains were more virulent than P. hermaphrodita (DMG0001). Ultimately, these strains could potentially be developed as alternative, efficient biological control agents for use against slugs.

A nematode that can manipulate the behaviour of slugs.

The ability of parasites to manipulate the behaviour of their hosts has evolved multiple times, and has a clear fitness benefit to the parasite in terms of facilitating growth, reproduction and transfer to suitable hosts. The mechanisms by which these behavioural changes are induced are poorly understood, but in many cases parasite manipulation of serotonergic signalling in the host brain is implicated. Here we report that Phasmarhabditis hermaphrodita, a parasite of terrestrial gastropod molluscs, can alter the behaviour of slugs. Uninfected slugs (Deroceras panormitanum, Arion subfuscus and Arion hortensis) avoid areas where P. hermaphrodita is present, but slugs infected with P. hermaphrodita are more likely to be found where the nematodes are present. This ability is specific to P. hermaphrodita and other nematodes (Steinernema carpocapsae and Heterorhabditis bacteriophora) do not induce this behavioural change. To investigate how P. hermaphrodita changes slug behaviour we exposed slugs to fluoxetine (a selective serotonin reuptake inhibitor) and cyproheptadine (a serotonin receptor antagonist). Uninfected slugs fed fluoxetine no longer avoided areas where P. hermaphrodita was present; and conversely, infected slugs fed cyproheptadine showed no increased attraction to areas with nematodes. These findings suggest that a possible mechanism by which P. hermaphrodita is able to manipulate parasite avoidance behaviour in host slugs is by manipulating serotonergic signalling in the brain, and that increased serotonin levels are potentially associated with a reduction in parasite avoidance.

Genome-Wide RNAi Screens in C. elegans to Identify Genes Influencing Lifespan and Innate Immunity.

RNA interference is a rapid, inexpensive, and highly effective tool used to inhibit gene function. In C. elegans, whole genome screens have been used to identify genes involved with numerous traits including aging and innate immunity. RNAi in C. elegans can be carried out via feeding, soaking, or injection. Here we outline protocols used to maintain, grow, and carry out RNAi via feeding in C. elegans and determine whether the inhibited genes are essential for lifespan or innate immunity.

A functional genomic screen for evolutionarily conserved genes required for lifespan and immunity in germline-deficient C. elegans.

The reproductive system regulates lifespan in insects, nematodes and vertebrates. In Caenorhabditis elegans removal of germline increases lifespan by 60% which is dependent upon insulin signaling, nuclear hormone signaling, autophagy and fat metabolism and their microRNA-regulators. Germline-deficient C. elegans are also more resistant to various bacterial pathogens but the underlying molecular mechanisms are largely unknown. Firstly, we demonstrate that previously identified genes that regulate the extended lifespan of germline-deficient C. elegans (daf-2, daf-16, daf-12, tcer-1, mir-7.1 and nhr-80) are also essential for resistance to the pathogenic bacterium Xenorhabdus nematophila. We then use a novel unbiased approach combining laser cell ablation, whole genome microarrays, RNAi screening and exposure to X. nematophila to generate a comprehensive genome-wide catalog of genes potentially required for increased lifespan and innate immunity in germline-deficient C. elegans. We find 3,440 genes to be upregulated in C. elegans germline-deficient animals in a gonad dependent manner, which are significantly enriched for genes involved in insulin signaling, fatty acid desaturation, translation elongation and proteasome complex function. Using RNAi against a subset of 150 candidate genes selected from the microarray results, we show that the upregulated genes such as transcription factor DAF-16/FOXO, the PTEN homolog lipid phosphatase DAF-18 and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6) are essential for both lifespan and immunity of germline deficient animals. We also identify a novel role for genes including par-5 and T12G3.6 in both lifespan-extension and increased survival on X. nematophila. From an evolutionary perspective, most of the genes differentially expressed in germline deficient C. elegans also show a conserved expression pattern in germline deficient Pristionchus pacificus, a nematode species that diverged from C. elegans 250-400 MYA.

System wide analysis of the evolution of innate immunity in the nematode model species Caenorhabditis elegans and Pristionchus pacificus.

The evolution of genetic mechanisms used to combat bacterial infections is critical for the survival of animals and plants, yet how these genes evolved to produce a robust defense system is poorly understood. Studies of the nematode Caenorhabditis elegans have uncovered a plethora of genetic regulators and effectors responsible for surviving pathogens. However, comparative studies utilizing other free-living nematodes and therefore providing an insight into the evolution of innate immunity have been lacking. Here, we take a systems biology approach and use whole genome microarrays to profile the transcriptional response of C. elegans and the necromenic nematode Pristionchus pacificus after exposure to the four different pathogens Serratia marcescens, Xenorhabdus nematophila, Staphylococcus aureus and Bacillus thuringiensis DB27. C. elegans is susceptible to all four pathogens whilst P. pacificus is only susceptible to S. marcescens and X. nematophila. We show an unexpected level of specificity in host responses to distinct pathogens within and across species, revealing an enormous complexity of effectors of innate immunity. Functional domains enriched in the transcriptomes on different pathogens are similar within a nematode species but different across them, suggesting differences in pathogen sensing and response networks. We find translation inhibition to be a potentially conserved response to gram-negative pathogens in both the nematodes. Further computational analysis indicates that both nematodes when fed on pathogens up-regulate genes known to be involved in other stress responses like heat shock, oxidative and osmotic stress, and genes regulated by DAF-16/FOXO and TGF-beta pathways. This study presents a platform for comparative systems analysis of two nematode model species, and a catalog of genes involved in the evolution of nematode immunity and identifies both pathogen specific and pan-pathogen responses. We discuss the potential effects of ecology on evolution of downstream effectors and upstream regulators on evolution of nematode innate immunity.

Genome-wide analysis of germline signaling genes regulating longevity and innate immunity in the nematode Pristionchus pacificus.

Removal of the reproductive system of many animals including fish, flies, nematodes, mice and humans can increase lifespan through mechanisms largely unknown. The abrogation of the germline in Caenorhabditis elegans increases longevity by 60% due to a signal emitted from the somatic gonad. Apart from increased longevity, germline-less C. elegans is also resistant to other environmental stressors such as feeding on bacterial pathogens. However, the evolutionary conservation of this pathogen resistance, its genetic basis and an understanding of genes involved in producing this extraordinary survival phenotype are currently unknown. To study these evolutionary aspects we used the necromenic nematode Pristionchus pacificus, which is a genetic model system used in comparison to C. elegans. By ablation of germline precursor cells and subsequent feeding on the pathogen Serratia marcescens we discovered that P. pacificus shows remarkable resistance to bacterial pathogens and that this response is evolutionarily conserved across the Genus Pristionchus. To gain a mechanistic understanding of the increased resistance to bacterial pathogens and longevity in germline-ablated P. pacificus we used whole genome microarrays to profile the transcriptional response comparing germline ablated versus un-ablated animals when fed S. marcescens. We show that lipid metabolism, maintenance of the proteasome, insulin signaling and nuclear pore complexes are essential for germline deficient phenotypes with more than 3,300 genes being differentially expressed. In contrast, gene expression of germline-less P. pacificus on E. coli (longevity) and S. marcescens (immunity) is very similar with only 244 genes differentially expressed indicating that longevity is due to abundant gene expression also involved in immunity. By testing existing mutants of Ppa-DAF-16/FOXO and the nuclear hormone receptor Ppa-DAF-12 we show a conserved function of both genes in resistance to bacterial pathogens and longevity. This is the first study to show that the influence of the reproductive system on extending lifespan and innate immunity is conserved in evolution.

The importance of being regular: Caenorhabditis elegans and Pristionchus pacificus defecation mutants are hypersusceptible to bacterial pathogens.

Bacterial pathogens have shaped the evolution and survival of organisms throughout history, but little is known about the evolution of virulence mechanisms and the counteracting defence strategies of host species. The nematode model organisms, Caenorhabditis elegans and Pristionchus pacificus, feed on a wealth of bacteria in their natural soil environment, some of which can cause mortality. Previously, we have shown that these nematodes differ in their susceptibility to a range of human and insect pathogenic bacteria, with P. pacificus showing extreme resistance compared with C. elegans. Here, we isolated 400 strains of Bacillus from soil samples and fed their spores to both nematodes. Spores of six Bacillus strains were found to kill C. elegans but not P. pacificus. While the majority of Bacillus strains are benign to nematodes, observed pathogenicity is restricted to either the spore or the vegetative stage. We used the rapid C. elegans killer strain (Bacillus sp. 142) to conduct a screen for hypersusceptible P. pacificus mutants. Two P. pacificus mutants with severe muscle defects and an extended defecation cycle that die rapidly on Bacillus spores were isolated. These genes were identified to be homologous to C. elegans, unc-22 and unc-13. To test whether a similar relationship between defecation and bacterial pathogenesis exists in C. elegans, we used five known defecation mutants. Quantification of the defecation cycle in mutants also revealed a severe effect on survival in C. elegans. Thus, intestinal peristalsis is critical to nematode health and contributes significantly to survival when fed Gram-positive bacteria.

A subset of naturally isolated Bacillus strains show extreme virulence to the free-living nematodes Caenorhabditis elegans and Pristionchus pacificus.

The main food source of free-living nematodes in the soil environment is bacteria, which can affect nematode development, fecundity and survival. In order to occupy a reliable source of bacterial food, some nematodes have formed specific relationships with an array of invertebrate hosts (where bacteria proliferate once the hosts dies), thus forming a tritrophic system of nematode, bacteria and insect or other invertebrates. We isolated 768 Bacillus strains from soil (from Germany and the UK), horse dung and dung beetles and fed them to the genetically tractable free-living nematodes Caenorhabditis elegans and Pristionchus pacificus to isolate nematocidal strains. While C. elegans is a bacteriovorous soil nematode, P. pacificus is an omnivorous worm that is often found in association with scarab beetles. We found 20 Bacillus strains (consisting of B. cereus, B. weihenstephanensis, B. mycoides and Bacillus sp.) that were pathogenic to C. elegans and P. pacificus causing 70% to 100% mortality over 5 days and significantly affect development and brood size. The most pathogenic strains are three B. cereus-like strains isolated from dung beetles, which exhibit extreme virulence to C. elegans in less than 24 h, but P. pacificus remains resistant. C. elegans Bre mutants were also highly susceptible to the B. cereus-like strains indicating that their toxins use a different virulence mechanism than B. thuringiensis Cry 5B toxin. Also, mutations in the daf-2/daf-16 insulin signaling pathway do not rescue survival. We profiled the toxin genes (bcet, nhe complex, hbl complex, pcpl, sph, cytK, piplc, hly2, hly3, entFM and entS) of these three B. cereus-like strains and showed presence of most toxin genes but absence of the hbl complex. Taken together, this study shows that the majority of naturally isolated Bacillus from soil, horse dung and Geotrupes beetles are benign to both C. elegans and P. pacificus. Among 20 pathogenic strains with distinct virulence patterns against the two nematodes, we selected three B. cereus-like strains to investigate resistance and susceptibility immune responses in nematodes.

Quantitative assessment of the nematode fauna present on Geotrupes dung beetles reveals species-rich communities with a heterogeneous distribution.

Pristionchus spp. nematodes exhibit several traits that might serve as pre-adaptations to parasitism. Under harsh environmental conditions, these nematodes can arrest development and form dauer larvae. In addition, they have been shown to live in necromenic association with a range of beetles, including dung beetles ( Geotrupes stercorosus ) on which, for example, Pristionchus entomophagus is commonly found. It has been argued that the formation of dauer larvae and the association with invertebrates represent intermediate steps towards parasitism. To better understand necromenic associations, and to gain information on Pristionchus spp. abundance and the general species composition on dung beetles, we extracted all the nematode fauna present on 114 individuals of G. stercorosus. By direct sequencing using the 18S SSU, we provide a barcode for all nematodes isolated from the beetle samples. In total, 5,002 dauer-stage nematodes were sequenced, which included Pristionchus spp., Koerneria spp. (Diplogastridae), Pelodera spp. (Rhabditidae), and Strongyloidea as well as Spirurida. Intensities of infection varied from over 1,000 nematodes isolated from a single G. stercorosus to none, with Pelodera spp. being the most abundant group isolated. This study presents the first quantitative data on the Pristionchus spp. infection of beetles.

The slug parasitic nematode Phasmarhabditis hermaphrodita associates with complex and variable bacterial assemblages that do not affect its virulence.

Phasmarhabditis hermaphrodita is a nematode parasite of slugs that is commercially reared in monoxenic culture with the bacterium Moraxella osloensis and sold as a biological molluscicide. However, its bacterial associations when reared in vivo in slugs are unknown. We show that when reared in vivo in slugs, P. hermaphrodita does not retain M. osloensis and associates with complex and variable bacterial assemblages that do not influence its virulence. This is in marked contrast to the entomopathogenic nematodes that form highly specific mutualistic associations with Enterobacteriaceae that are specifically retained during in vivo growth.

Chemoattraction and host preference of the gastropod parasitic nematode Phasmarhabditis hermaphrodita.

Phasmarhabditis hermaphrodita is a parasitic nematode that has been formulated into a biological control agent for slugs. The nematode responds to slug-associated cues such as mucus and feces in order to locate potential hosts. We assessed the olfactory response of P. hermaphrodita to mucus from 9 species of slugs, 2 snails, and 2 earthworms (non-hosts). We then examined the susceptibility of each invertebrate test species to high doses of P. hermaphrodita to determine whether susceptible species are more attractive than non-susceptible species to the nematode. We also studied the numbers of infective juveniles produced in each test species, as well as infectivity. Phasmarhabditis hermaphrodita showed strong attraction to mucus from the non-susceptible slug Arion subfuscus, the snail Helix aspersa, and the highly susceptible slug Deroceras reticulatum. In reproduction experiments, P. hermaphrodita produced the highest number of infective juveniles in D. reticulatum and Deroceras panormitanum; however, there was no significant relationship with attraction. Phasmarhabditis hermaphrodita caused significant mortality in 5-11 gastropod species tested (and showed no chemotactic preference for susceptible or non-susceptible species). There was a significant positive relationship between numbers of P. hermaphrodita penetrating into non-susceptible species and chemotaxis response. These necromenic species represent ideal hosts for P. hermaphrodita in terms of providing protection against abiotic and biotic factors as well as transport to many diverse areas.

Isolation of naturally associated bacteria of necromenic Pristionchus nematodes and fitness consequences.

Nematodes and bacteria are major components of the soil ecosystem. Many nematodes use bacteria for food, whereas others evolved specialized bacterial interactions ranging from mutualism to parasitism. Little is known about the biological mechanisms by which nematode-bacterial interactions are achieved, largely because in the laboratory nematodes are often cultured under artificial conditions. We investigated the bacterial interactions of nematodes from the genus Pristionchus that have a strong association with scarab beetles. Pristionchus has a different feeding strategy than Caenorhabditis and meta-genomic 16S sequence analysis of Pristionchus individuals showed a diversity of living bacteria within the nematode gut and on the nematode cuticle. Twenty-three different bacterial strains were isolated from three Pristionchus-beetle associations and were used to study nematode-bacterial interactions under controlled laboratory conditions. We show a continuum of bacterial interactions from dissemination, to reduction in brood size and nematode mortality caused by bacteria derived from insect hosts. Olfactory discrimination experiments show distinct chemoattraction and fitness profiles of Pristionchus nematodes when exposed to different bacteria. For example, Pristionchus pacificus avoids Serratia marcescens possibly because of pathogenicity. Also, P. pacificus avoids Bacillus thuringiensis and insect pathogenic bacteria but is resistant to the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa, unlike Caenorhabditis elegans. Pristionchus specifically recognize and respond to bacteria that cause ill health. Bringing the nematode-bacterial interaction into the laboratory allows detailed functional studies, including the genetic manipulation of the interaction in both nematodes and bacteria.

Pristionchus pacificus: A Genetic Model System for the Study of Evolutionary Developmental Biology and the Evolution of Complex Life-History Traits.

INTRODUCTIONPristionchus pacificus is a nematode that has been established as a model system for evolutionary developmental biology. Initially, P. pacificus was used as a convenient nematode with which to compare the processes of vulva and gonad development as well as sex determination to Caenorhabditis elegans, one of the best-studied animal models. P. pacificus shares many features with C. elegans, including a short generation time, its ability to be easily cultured in the laboratory, and self-fertilization as a mode of reproduction. These features allowed forward and reverse genetic tools to be developed for this species. The application of these tools for genetic and molecular analysis of vulva formation revealed substantial differences between P. pacificus and C. elegans. The genome of P. pacificus has recently been sequenced and showed an expansion of protein-coding genes compared with C. elegans. Interestingly, the P. pacificus genome encodes some genes, such as cellulases, that are known to be present only in plant-parasitic nematodes. Many of the putative functions of the predicted genes in the genome are related to the ecology of P. pacificus and other Pristionchus species. Pristionchus nematodes can be isolated from beetles and soil, indicating that the ecology of P. pacificus is strikingly different from that of C. elegans. Generally, Pristionchus species show an unexpected level of species specificity in their beetle associations, providing a unique opportunity to study the genetic and molecular mechanisms underlying the interactions of organisms in the environment. Thus, P. pacificus is not only an established model system for evolutionary developmental biology, but also an emerging model system for the evolution of complex life-history traits.

Isolation of pristionchus nematodes from beetles.

INTRODUCTIONIn this procedure, nematodes disembark from a beetle carcass and feed on Escherichia coli OP50. The nematodes are then monitored for a few days and identified using simple morphological characteristics. This method is rapid, easy, and biased for Pristionchus species.