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.

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.

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.

Susceptibility and immune response of Deroceras reticulatum, Milax gagates and Limax pseudoflavus exposed to the slug parasitic nematode Phasmarhabditis hermaphrodita.

We exposed three slug species (Deroceras reticulatum (Müller), Milax gagates (Draparnaud) and Limax pseudoflavus L.) to the parasitic nematode Phasmarhabditis hermaphrodita Schneider. P. hermaphrodita was able to cause mortality and feeding inhibition to both D. reticulatum and M. gagates but did not negatively affect L. pseudoflavus. On dissection of surviving L. pseudoflavus large numbers of P. hermaphrodita were found encapsulated in the shell of the slug. We found that by increasing shell size, the slug was able to trap invading nematodes, which could be an immune response to P. hermaphrodita invasion. This is the first report of a slug defense mechanism to inhibit P. hermaphrodita.