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 Nema-slug®, 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.

Phasmarhabditis bohemica n. sp. is described and illustrated from the body of Deroceras reticulatum from the village of Chelčice, the Czech Republic. Morphological and molecular data showed that the new isolate is close to other species of Phasmarhabditis, particularly P. californica and P. papillosa. Females are characterised by a body length of 2079 (1777-2222) μm and a long tapering tail with prominent papilliform phasmids located laterally in the mid-tail region. Males are 1683 (1515-1818) μm long. They have a peloderan bursa, with nine pairs of rays, 1/1/1/2/1/3, and a reflexed testis 495 (434-555) μm long. Dauer juveniles are thin, 553 (474-636) μm long, with prominent lateral fields consisting of two prominent ridges and three incisures. Small subunit (18S), ITS, and D2-D3 expansion segments of the large subunit of ribosomal DNA were used to analyse the phylogenetic relationships of sequenced species in Phasmarhabditis and other closely related species. Our preliminary observations suggest that the newly described species may be a facultative mollusc-parasitic nematode that is able to survive permanently in the saprobic phase on decaying organic matter. The ecology, morphology, and phylogenetic positions of P. bohemica n. sp. are discussed.

The effect of different growing substrates on the development of the slug parasite Phasmarhabditis hermaphrodita has been studied in a series of laboratory experiments. Wild, laboratory and Nemaslug strains of P. hermaphrodita and their monoxenic counterparts with Moraxella osloensis (CCM 5605T) were reared on homogenised pig kidney, Deroceras reticulatum, Arion lusitanicus, and Galleria mellonella, the faeces of D. reticulatum and A. lusitanicus, or leaf compost. Development time, yield, lipid reserves and the body length of nematodes were assessed. All P. hermaphrodita strains were able to grow and reproduce on all tested substrates; however, yields were markedly higher on invertebrate media. Lipid content and body size varied across the substrates and strains. A uniform response of all nematode strains and the most dramatic differences among substrates were recorded in yields, which could indicate that the quality of the substrate is expressed mainly in yield. Monoxenized strains had higher yield, length, lipid content and shorter development time. In general, the strain differences between the observed parameters were most likely due to different bacterial associates. The dramatic differences in yields on invertebrate substrates, in comparison to the others, illustrate the evolutionary advantage of the association of nematodes with invertebrates.

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Ips duplicatus (Sahlberg) is an important forest pest in central Europe, but its nematode associates have seldom been studied. Therefore, nematodes associated with I. duplicatus were determined at three localities in the Czech Republic. The percentage of beetles with phoretic nematodes ranged from 18 to 65%. Micoletzkya buetschlii and other phoretic nematodes were found under elytra, on wings, and between body segments. The percentage of beetles with nematodes in the haemocoel ranged from 3 to 30%, and the nematodes included Contortylenchus diplogaster and Parasitylenchus cf. aculeatus. Juveniles of Parasitorhabditis obtusa (Fuchs 1915) were found in the intestines of 0-16% of the beetles. The most abundant species in I. duplicatus galleries were P. obtusa and M. buetschlii. Cryptaphelenchus sp., Parasitaphelenchus sp. and unidentified tylenchid juveniles also were found in the galleries. The percentage of beetles with nematodes was greater in the overwintering than in the offspring generation, and numbers of nematodes per gallery increased with gallery development.

A new nematode species belonging to the family Rhabditidae, genus Phasmarhabditis, was isolated in the city of Bari (strain BAR) and near the village Gravina (strain GRA) in Apulia, Italy, and is described herein as Phasmarhabditis apuliae n. sp. The original hosts were Milax sowerbyi and M. gagates. Females are 2623 (2262-2848) μm long with a long tapering tail. Prominent papilla-like phasmids are present. Males are 2096 (1898-2363) μm long. They have a peloderan bursa with nine pairs of rays arranged as 1 + 1 + 1 + 2 + 1 + 3. Each ray bears a single papilla. A non-paired papilla is located near the ventral appendage anterior to the cloacal aperture. Prominent papilla-like phasmids are situated close to the tail tip. Dauer juveniles have lateral fields formed from two prominent wide ridges and have three incisures. Phasmarhabditis apuliae n. sp. seems to be a facultative mollusc-parasitic nematode that is capable, in a similar manner to P. hermaphrodita, of long-term survival in the saprophytic phase on decaying organic matter. Ecology, morphology and phylogenetic relationships of Phasmarhabditis nematodes are discussed.

Phasmarhabditis bonaquaense n. sp. is described and illustrated from the body of Malacolimax tenellus, from the locality of České Švýcary near the village of Dobrá Voda, the Czech Republic. Females are characterized by a body length of 2349 (1878-2626) µm and a cupola shaped tail with a long hyaline hair-like tail tip. Extremely prominent papilla-like phasmids present. Males 1829 (1414-2121) µm long. Peloderan bursa with nine pairs of rays (papillae), 1/1/1/2/1/3. One non-paired apparent papilla-like structure located near the ventral appendage anterior to the cloaca. Prominent papilla-like phasmids located close to the tail tip. Small subunit (18S), ITS, and D2-D3 expansion segments of the large subunit of ribosomal DNA were used to analyze the phylogenetic relationships of sequenced species in the genus Phasmarhabditis and other closely related species. Phasmarhabditis bonaquaense n. sp. varied from other related nematodes both in morphological characterizations and phylogenetic analysis. The life cycle of the newly described species is not well known but it is probably a facultative, mollusc-parasitic nematode able to survive permanently in the saprobic phase on decaying organic matter.

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