The biocontrol nematode Phasmarhabditis hermaphrodita infects and increases mortality of Monadenia fidelis, a non-target terrestrial gastropod species endemic to the Pacific Northwest of North America, in laboratory conditions.

Inundative biological control (biocontrol) efforts in pest management lead to the mass distribution of commercialized biocontrol agents. Many 'biocontrol gone awry' incidents have resulted in disastrous biodiversity impacts, leading to increased scrutiny of biocontrol efforts. The nematode Phasmarhabditis hermaphrodita is sold as a biocontrol agent on three continents and targets pest gastropods such as Deroceras reticulatum, the Grey Field Slug; P. hermaphrodita is not presently approved for use in the United States. Investigations into the potential for P. hermaphrodita to infect non-target gastropod species of conservation relevance, however, are limited. We examined the effects of three strains of P. hermaphrodita on mortality in Monadenia fidelis, the Pacific Sideband, a snail species endemic to the Pacific Northwest of North America, in laboratory conditions. Across a 71-day laboratory infectivity assay, snails exposed to each of the three nematode strains, each analyzed at two doses, experienced a mean 50% mortality by days 20-42. All nematode-treated snails were dead by the end of the study. By contrast, 30/30 water-control snails experienced no mortality. Nematodes killed smaller, juvenile-stage snails significantly faster than those in larger and more developmentally advanced hosts. Our results provide direct evidence that the biocontrol nematode P. hermaphrodita infects and kills M. fidelis, a non-target gastropod species endemic to the Pacific Northwest, in laboratory conditions. This study suggests that introduction of P. hermaphrodita to new ecosystems might negatively impact endemic gastropod biodiversity and advocates for further investigation of non-target effects, including in conditions closer to the natural environments of non-target species.

Molecular phylogeny of the family Rhabdiasidae (Nematoda: Rhabditida), with morphology, genetic characterization and mitochondrial genomes of Rhabdias kafunata and R. bufonis.

BACKGROUND: The family Rhabdiasidae (Nematoda: Rhabditida) is a globally distributed group of nematode parasites, with over 110 species parasitic mainly in amphibians and reptiles. However, the systematic position of the family Rhabdiasidae in the order Rhabditida remains unsolved, and the evolutionary relationships among its genera are still unclear. Moreover, the present knowledge of the mitochondrial genomes of rhabdiasids remains limited. METHODS: Two rhabdiasid species: Rhabdias kafunata Sata, Takeuchi & Nakano, 2020 and R. bufonis (Schrank, 1788) collected from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) in China, were identified based on morphology (light and scanning electron microscopy) and molecular characterization (sequencing of the nuclear 28S and ITS regions and mitochondrial cox1 and 12S genes). The complete mitochondrial genomes of R. kafunata and R. bufonis were also sequenced and annotated for the first time. Moreover, phylogenetic analyses based on the amino acid sequences of 12 protein-coding genes (PCGs) of the mitochondrial genomes were performed to clarify the systematic position of the family Rhabdiasidae in the order Rhabditida using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic analyses based on the 28S + ITS sequences, were also inferred to assess the evolutionary relationships among the genera within Rhabdiasidae. RESULTS: The detailed morphology of the cephalic structures, vulva and eggs in R. kafunata and R. bufonis was revealed using scanning electron microscopy (SEM) for the first time. The characterization of 28S and ITS regions of R. kafunata was reported for the first time. The mitogenomes of R. kafunata and R. bufonis are 15,437 bp and 15,128 bp long, respectively, and both contain 36 genes, including 12 PCGs (missing atp8). Comparative mitogenomics revealed that the gene arrangement of R. kafunata and R. bufonis is different from all of the currently available mitogenomes of nematodes. Phylogenetic analyses based on the ITS + 28S data showed Neoentomelas and Kurilonema as sister lineages, and supported the monophyly of Entomelas, Pneumonema, Serpentirhabdias and Rhabdias. Mitochondrial phylogenomic results supported Rhabdiasidae as a member of the superfamily Rhabditoidea in the suborder Rhabditina, and its occurrance as sister to the family Rhabditidae. CONCLUSIONS: The complete mitochondrial genome of R. kafunata and R. bufonis were reported for the first time, and two new gene arrangements of mitogenomes in Nematoda were revealed. Mitogenomic phylogenetic results indicated that the family Rhabdiasidae is a member of Rhabditoidea in Rhabditina, and is closely related to Rhabditidae. Molecular phylogenies based on the ITS + 28S sequence data supported the validity of Kurilonema, and showed that Kurilonema is sister to Neoentomelas. The present phylogenetic results also indicated that the ancestors of rhabdiasids seem to have initially infected reptiles, then spreading to amphibians.

Nematodes associated with terrestrial gastropod molluscs in Belgium and additional characterisation of Pellioditis californica and P. hermaphrodita.

A survey for slug- and snail-associated nematodes was conducted in forests, parks, botanical gardens, and nature reserves at 13 localities in Belgium to uncover more diversity of gastropod mollusc-associated nematodes and to characterise Pellioditis populations found in the country. A total of 319 slugs and snails belonging to nine species were examined. Arion vulgaris was the most commonly found mollusc species in this study (eight locations), and 19.4% of the examined mollusc specimens were found infected by nematodes. The highest prevalence of nematodes was observed in Cornu aspersum (60%) followed by A. vulgaris (34.8%), Limax maximus (28.6%), and Cepaea sp. (20%). Eleven nematode species belonging to eight families were isolated and identified from the mollusc hosts including Alloionema appendiculatum, Angiostoma dentiferum, A. gandavense, Angiostrongylus vasorum, Cosmocerca longicauda, Panagrolaimus cf. subelongatus, Pellioditis californica, P. hermaphrodita, Rhabditis sp., Tetrameres cf. fissispina, and Troglostrongylus cf. brevior.Pellioditis was the most commonly found nematode genus (at nine localities) and C. longicauda and P. californica were reported in Belgium for the first time. Co-infections of more than one nematode species were observed in eight (2.5%) molluscs specimens. Most co-infections consisted of two nematode species. In one A. vulgaris specimen, a co-infection of three nematode species (A. vasorum, P. hermaphrodita, and Tetrameres cf. fissispina) was observed. Four ex vivo cultures of P. californica and six ex vivo cultures of P. hermaphrodita were established from single hermaphrodites, and both species were described based on light microscopy, scanning electron microscopy, and morphometric, morphological, and molecular data.

To live free or being a parasite: The optimal foraging behavior may favor the evolution of entomopathogenic nematodes.

Facultative parasites can alternate between a free-living and a parasitic existence to complete their life cycle. Yet, it remains uncertain which lifestyle they prefer. The optimal foraging theory suggests that food preferences align with fitness benefits. To test this hypothesis, we investigated the facultative parasite nematode Rhabditis regina, assessing its host preference and the associated benefits. Two experiments were conducted using wild nematode populations collected from Phyllophaga polyphylla, their natural host. In the first experiment, we used a behavioral arena to assess host preference between the natural host and two experimental hosts: Spodoptera frugiperda which is an alternative host and dead Tenebrio molitor, which simulates a saprophytic environment. In the second experiment, we subjected wild nematodes to "experimental evolution" lasting 50 generations in S. frugiperda and 53 generations in T. molitor carcass. We then compared life history traits (the size, survival, number of larvae, and glycogen and triglycerides as energy reserves) of dauer larvae with those nematodes from P. polyphylla (control group). We found a significant preference for P. polyphylla, which correlated with higher values in the nematode's life history traits. In contrast, the preference for S. frugiperda and the saprophytic environment was lower, resulting in less efficient life history traits. These findings align with the optimal foraging theory, as the nematode's parasitic preferences are in line with maximizing fitness. This also indicates that R. regina exhibits specificity to P. polyphylla and is better adapted to a parasitic lifestyle than a free-living one, suggesting an evolutionary pathway towards parasitism.

A biological invasion modifies the dynamics of a host-parasite arms race.

By imposing novel selection pressures on both participants, biological invasions can modify evolutionary 'arms races' between hosts and parasites. A spatially replicated cross-infection experiment reveals strong spatial divergence in the ability of lungworms (Rhabdias pseudosphaerocephala) to infect invasive cane toads (Rhinella marina) in Australia. In areas colonized for longer than 20 years, toads are more resistant to infection by local strains of parasites than by allopatric strains. The situation reverses at the invasion front, where super-infective parasites have evolved. Invasion-induced shifts in genetic diversity and selective pressures may explain why hosts gain advantage over parasites in long-colonized areas, whereas parasites gain advantage at the invasion front.

Metarhabditis amsactae: A potential biopesticide isolated from Punjab (India) with potent insecticidal activity and immunomodulatory effects against Galleria mellonella (Lepidoptera: Pyralidae).

A survey was undertaken to isolate entomopathogenic nematodes from Amritsar district of Punjab, India. Out of 20 soil samples collected, two were found positive for the presence of nematodes. 18S and ITS rDNA gene sequencing revealed their identity as Metarhabditis amsactae. To assess its biocontrol potential, Galleria mellonella larvae were treated with concentrations of 20, 40, 80 and 160 IJs/L (infective juveniles/larva) and mortality was recorded from 24 h up to 96 h of nematode exposure. Distilled water without nematodes was used as an untreated control. M. amsactae showed potent larvicidal activity against G. mellonella that was found to be concentration and time dependent. Nematode infection caused 93.33 % larval mortality at 80 IJs/L after 72 h of treatment. 100 % mortality was observed after 96 h. No mortality was observed in control. To evaluate the immunomodulatory effects of M. amsactae, G. mellonella larvae were infected with 100 IJs/L and activities of antioxidant and detoxifying enzymes viz., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), phenol oxidase (PO), glutathione-S-transferase (GST) and acetylcholine esterase (AChE) were appraised after 12, 24, 36 and 48 h of nematode exposure. Malondialdehyde content was also determined. The results obtained demonstrated a significant elevation in all the enzyme activities at all time intervals in treated larvae when compared with untreated control. MDA levels were also enhanced in response to nematode infection. Thus, the present study revealed high insecticidal potential and immunomodulatory effects of M. amsactae on G. mellonella that should be further explored on other insect pests as well.

Synopsis of gastropod-associated nematodes of Ciscaucasia (Russian Federation) with the description of a new species of Pellioditis Dougherty, 1953 (syn. Phasmarhabditis Andrássy, 1976).

Caucasus is known as one of the few biodiversity hotspots in Europe and is characterised by rich gastropod fauna while the nematode fauna in association with gastropods has remained largely understudied. Surveys conducted in 2019 and 2021 in the North Caucasus of the Russian Federation (Stavropol Upland and western and central parts of Krasnodar Krai) has revealed the presence of three new species of Pellioditis, a facultative parasite of land gastropods, and two species of obligate parasites, the intestinal parasite Angiostoma kimmeriense and a new, still undescribed species of a larval ectoparasite Alloionema sp. The new associations of Cruznema sp. and Rhabditophanes sp. with land gastropods were recorded for the first time in the Russian Federation. The new species of Pellioditis Dougherty, 1953 described here is based on the analysis of morphology and molecular studies of two distant and morphologically distinct strains, thermalis and sindicae. Pellioditis thermalis n. sp. was characterised by females possessing a tail of about 95-100 µm long, broadly conical in shape in the thermalis and sindicae strains, with a rounded anterior part and a subulate terminal part as long as the former; prominent phasmids located at the mid-tail, equatorial vulva position, a lateral field of three ridges (four incisions), males with spicules featuring a hole at the distal tip, ensheathed infective juveniles with average length 717 µm in the thermals strain and 771 µm in the sindicae strain, and exsheathed ones 644 µm and 682 µm, respectively. ITS-based phylogenetic analyses revealed that all Pellioditis species found in Ciscaucasia and Transcaucasia probably belong to two separate clades, with independent evolutionary histories of colonisation of this area. The entire Caucasus range area appears to serve as a biodiversity hotspot for the genus Pellioditis, presumably due to its complicated geological history and repeated isolation events for its terrestrial mollusc hosts.

Enhancing mass production of Heterorhabditis bacteriophora: influence of different bacterial symbionts (Photorhabdus spp.) and inoculum age on dauer juvenile recovery.

The entomopathogenic nematode Heterorhabditis bacteriophora (Nematoda: Rhabditidae) is used in biological insect control. Their dauer juveniles (DJs) are free-living and developmentally arrested, invading host insects. They carry cells of their bacterial symbiont Photorhabdus spp. in the intestine. Once inside the insect´s hemolymph the DJs perceive a food signal, triggering them to exit the DJ stage and regurgitate the Photorhabdus cells into the insect's haemocoel, which kill the host and later provide essential nutrients for nematode reproduction. The exit from the DJ stage is called "recovery". For commercial pest control, nematodes are industrially produced in monoxenic liquid cultures. Artificial media are incubated with Photorhabdus before DJs are added. In absence of the insect's food signal, DJs depend on unknown bacterial food signals to trigger exit of the DJ stage. A synchronized and high DJ recovery determines the success of the industrial in vitro production and can significantly vary between nematode strains, inbred lines and mutants. In this study, fourteen bacterial strains from H. bacteriophora were isolated and identified as P. laumondii, P. kayaii and P. thracensis. Although the influence of bacterial supernatants on the DJ recovery of three inbred lines and two mutants differed significantly, the bacterial impact on recovery has a subordinate role whereas nematode factors have a superior influence. Recovery of inbred lines decreased with age of the DJs. One mutant (M31) had very high recovery in bacterial supernatant and spontaneous recovery in Ringer solution. Another mutant (M88) was recovery defective.

Taxonomic and molecular characterization of a new entomopathogenic nematode species, Heterorhabditis casmirica n. sp., and whole genome sequencing of its associated bacterial symbiont.

BACKGROUND: Nematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural insect pests. This study describes a new species of Heterorhabditis. METHODS: Six Heterorhabditis nematode populations were recovered from agricultural soils in Jammu and Kashmir, India. An initial examination using mitochondrial and nuclear genes showed that they belong to a new species. To describe this new species, a variety of analyses were conducted, including reconstructing phylogenetic relationships based on multiple genes, characterizing the nematodes at the morphological and morphometric levels, performing self-crossing and cross-hybridization experiments, and isolating and characterizing their symbiotic bacteria. RESULTS: The newly discovered species, Heterorhabditis casmirica n. sp., shares 94% mitochondrial cytochrome C oxidase subunit I gene (COI) sequence identity with Heterorhabditis bacteriophora and Heterorhabditis ruandica, and 93% with Heterorhabditis zacatecana. Morphologically, it differs from H. bacteriophora in its infective juvenile phasmids (present vs. inconspicuous) and bacterial pouch visibility in the ventricular portion of the intestine (invisible vs. visible); genital papilla 1 (GP1) position (at manubrium level vs. more anterior), and in its b ratio (body length/neck length), c ratio (tail length/bulb width), and D% [(excretory pore/neck length) × 100]. Other morphological differences include anterior end to the nerve ring distance (77-100 vs. 121-130 µm), V% [(anterior end of vulva/body length) × 100] (46-57 vs. 41-47) in hermaphroditic females; rectum size (slightly longer than the anal body diameter vs. about three times longer), phasmids (smaller vs. inconspicuous), body length (0.13-2.0 vs. 0.32-0.39 mm), body diameter (73-150 vs. 160-220 µm), anterior end to the excretory pore distance (135-157 vs. 174-214 µm), and demanian ratios in amphimictic females. Morphological differences with H. ruandica and H. zacatecana were also observed. Furthermore, H. casmirica n. sp. did not mate or produce fertile progeny with other Heterorhabditis nematodes reported from India. It was also discovered that H. casmirica n. sp. is associated with Photorhabdus luminescence subsp. clarkei symbiotic bacteria. CONCLUSIONS: The discovery of H. casmirica n. sp. provides novel insights into the diversity and evolution of Heterorhabditis nematodes and their symbiotic bacteria. This new species adds to the catalog of entomopathogenic nematodes in India.

Pellioditis pelhamensis n. sp. (Nematoda: Rhabditidae) and Pellioditis pellio (Schneider, 1866), earthworm associates from different subclades within Pellioditis (syn. Phasmarhabditis Andrássy, 1976).

Recently, much attention has been focused on a group of rhabditid nematodes called Phasmarhabditis, a junior synonym of Pellioditis, as a promising source of biocontrol agents for invasive slugs. Pellioditis pelhamensis n. sp. was first isolated from earthworms near Pelham Bay Park in Bronx, New York, USA, in 1990 and has been found to be pathogenic to slugs as well as some earthworms. It has also been used in several comparative developmental studies. Here, we provide a description of this species, as well as a redescription of a similar earthworm-associated nematode, Pellioditis pellio Schneider, 1866, re-isolated from the type locality. Although P. pelhamensis n. sp. and P. pellio are morphologically similar, they are reproductively isolated. Molecular phylogenetic analysis places both species in a clade that includes all species previously described as Phasmarhabditis which are associated with gastropods. Phasmarhabditis Andrássy, 1976 is therefore a junior synonym of Pellioditis Dougherty, 1953. Also, Pellioditis bohemica Nermut', Puza, Mekete & Mrácek, 2017, described to be a facultative parasite of slugs, is found to be a junior synonym of Pellioditis pellio (Schneider, 1866), adding to evidence that P. pellio is associated with both slugs and earthworms. The earthworm-associated species P. pelhamensis n. sp. and P. pellio represent different subclades within Pellioditis, suggesting that Pellioditis species in general have a broader host range than just slugs. Because of this, caution is warranted in using these species as biological control agents until more is understood about their ecology.

Programmed DNA elimination in Mesorhabditis nematodes.

Some species undergo programmed DNA elimination (PDE), whereby portions of the genome are systematically destroyed in somatic cells. PDE has emerged independently in several phyla, but its function is unknown. Although the mechanisms are partially solved in ciliates, PDE remains mysterious in metazoans because the study species were not yet amenable to functional approaches. We fortuitously discovered massive PDE in the free-living nematode genus Mesorhabditis, from the same family as C. elegans. As such, these species offer many experimental advantages to start elucidating the PDE mechanisms in an animal. Here, we used cytology to describe the dynamics of chromosome fragmentation and destruction in early embryos. Elimination occurs once in development, at the third embryonic cell division in the somatic blastomeres. Chromosomes are first fragmented during S phase. Next, some of the fragments fail to align on the mitotic spindle and remain outside the re-assembled nuclei after mitosis. These fragments are gradually lost after a few cell cycles. The retained fragments form new mini chromosomes, which are properly segregated in the subsequent cell divisions. With genomic approaches, we found that Mesorhabditis mainly eliminate repeated regions and also about a hundred genes. Importantly, none of the eliminated protein-coding genes are shared between closely related Mesorhabditis species. Our results strongly suggest PDE has not been selected for regulating genes with important biological functions in Mesorhabditis but rather mainly to irreversibly remove repeated sequences in the soma. We propose that PDE may target genes, provided their elimination in the soma is invisible to selection.

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.

Phasmarhabditis eagyptiaca n. sp. (Nematoda: Rhabditidae) Isolated from Egyptian Terrestrial Snails and Its Role as Control Bio-agent to Gastropods.

INTRODUCTION: To date nineteen nematode species in the genus Phasmarhabditis Andrássy, 1976 recorded and described. This paper describes Phasmarhabditis eagyptiaca n. sp. (Nematoda: Rhabditidae), morphologically and phylogenatically and investigate its role as bio control agent for gastropods. METHODS: Snails infected with nematode collected from Great Cairo Egypt. Sequences of the 18S ribosomal (18S rRNA) gene performed and used for phylogenetic studies. Morphological parameter measured and the nematode photographed and illustrated. Bioassay conducted on some snails and slugs species. RESULTS: Phasmarhabditis eagyptiaca n. sp. is the second new species of the genus Phasmarhabditis recorded and described in Egypt after Phasmarhabditis tawfiki Azzam2003, the fourth species from Africa and the 20th from the world. Morphological and molecular parameter showed that the new isolate is close to other species of Phasmarhabditis, especially Phasmarhabditis neopapillosa Andrássy, 1983, Phasmarhabditis hermaphrodita (Schneider, 1859) and P. tawfiki Azzam with high bootstrap supported values (99.25%, 98.85%, and 98.64%, respectively). This nematode could infect and killed all snails and slugs exposed to infection in laboratory. Phasmarhabditis eagyptiaca n. sp. differs from all previously recorded species by shorter tail of female spikey tail of male and the arrangement of genital papillae formula which different from all previous species. CONCLUSION: It could be confirmed that this nematode is a new species of Phasmarhabditis. This nematode could be considering a biological control agent for snails and slugs.

Pestiferous slugs and their associated nematodes in agricultural fields, greenhouses, and nurseries in Alberta, Canada.

Some slug species are considered a nuisance in agriculture and horticulture worldwide, causing economic losses to growers. Phasmarhabditis is a genus of bacteria-feeding nematodes that can parasitize slugs and snails and thus potentially serve as a biological control agent. Canada had no record of Phasmarhabditis until a survey conducted in 2019 reported a Canadian strain of Phasmarhabditis californica from a single Arion rufus slug. To build on this discovery, we surveyed three major agricultural sites, ten greenhouses, and nurseries in Alberta from June to September 2021 to collect pest slug species and investigate their associated nematodes, specifically P. californica. Slugs were collected from the field and returned to the laboratory to check for emerging nematodes on White traps. We collected 1331 slugs belonging to nine species, with Deroceras reticulatum being the most common. Only 45 (3.38%) slug samples were positive for nematodes, and the majority were identified to species level: Alloionema appendiculatum, Caenorhabditis briggsae, Caenorhabditis elegans, Panagrolaimus subelongatus, and Mesorhabditis spiculigera. We did not isolate P. californica from any of the slugs collected from these survey sites, which included the original site where P. californica was discovered. However, four D. reticulatum slugs retrieved from a residential garden sample were infected with P. californica. These findings suggest the possibility of a fragmented distribution of P. californica across Alberta. Future research should focus on extensively surveying agriculture and horticulture sites and residential gardens in different provinces across Canada.

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.

A proposal to synonymize Loffienema with Haematozoon, Quercorhabditis with Diploscapteroides, and Stegorhabditis with Stomachorhabditis (Nematoda, Rhabditida, Rhabditomorpha), including keys to species identification.

The morphology of three genera of the family Rhabditidae, Loffienema, Quercorhabditis and Stegorhabditis, described recently from Jammu and Kashmir (India), is analyzed in detail. After the revision of their morphology, these genera are proposed to be synonymized with other three known genera: Loffienema with Haematozoon, Quercorhabditis with Diploscapteroides, and Stegorhabditis with Stomachorhabditis. In addition, the taxonomic status of the genus Distorhabditis is discussed. Dichotomous keys for the identification of species of the genera Haematozoon, Diploscapteroides and Stomachorhabditis are included.

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.

A new isolate of Mesorhabditis monhystera (Bütschli, 1873) Dougherty, 1955 (Rhabditida: Rhabditidae): re-evaluated with molecular data and scanning electron microscopic observations.

A new isolate of Mesorhabditis monhystera (Bütschli, ) Dougherty, is described and illustrated with morphological and molecular data. The phylogenetic analysis based on the D2/D3 segment of 28S rDNA using the Bayesian inference method, revealed monophyly of the genus Mesorhabditis as the subordinate taxa clustered in one clade. The clade further divided into two subclades representing the Monhystera-group and Spiculigera-group with 100% posterior probability values. However, GenBank sequences of several species constituting the Monhystera-group, showed high similarity and very little genetic divergence (98-99%) of up to 4-5 bases. In order to ascertain the status of those isolates, detailed morphological comparison is provided along with a pictorial key. A sequence-based phylogeography of haplogroups of Mesorhabditis using the median-joining network method, was also inferred. The results suggested the need for morphological validation of a species before its sequences are deposited in GenBank.

Nematodes and trematodes associated with terrestrial gastropods in Nottingham, England.

A parasitological survey of terrestrial slugs and snails was conducted at popular dog walking locations across the city of Nottingham, with the intensions of finding gastropods infected with parasites of medical (or veterinary) importance such as lungworm (metastrongyloid nematodes) and trematodes. A total of 800 gastropods were collected from 16 sites over a 225 km2 area. The extracted nematodes and trematodes were identified by molecular barcoding. Of the 800 gastropods collected, 227 were infected (172 had nematode infections, 37 had trematode infections and 18 had both nematode and trematode infections). Of the nematode infected gastropods genotyped, seven species were identified, Agfa flexilis, Angiostoma gandavense, Angiostoma margaretae, Cosmocerca longicauda, Phasmarhabditis hermaphrodita, Phasmarhabditis neopapillosa and an unknown Cosmocercidae species. Of the trematode infected gastropods genotyped, four species were identified, Brachylaima arcuate, Brachylaima fuscata, Brachylaima mesostoma and an unknown Plagiorchioidea species. No lungworm species were found within the city of Nottingham. To our knowledge, this study represents the first survey of gastropod-associated nematodes and trematodes in the East midlands of the United Kingdom.

RNA-Sequencing of Heterorhabditis nematodes to identify factors involved in symbiosis with Photorhabdus bacteria.

BACKGROUND: Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria. RESULTS: A de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified. CONCLUSIONS: The symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.