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.

A new species of Rhabdias (Nematoda: Rhabdiasidae), a lung parasite of Pseudopaludicola pocoto (Anura: Leptodactylidae) from north-eastern Brazil: description and phylogenetic analyses.

Rhabdias pocoto n. sp. is herein described from the lungs of the swamp frog Pseudopaludicola pocoto Magalhães, Loebmann, Nogueira, Kokubum, Baptista, Haddad & Garda, 2014, from the Caatinga biome in the state of Ceará, in north-eastern Brazil. The new species is characterized by a body that dilates posteriorly, six small lips (protuberances) and two rounded lateral expansions of cuticular inflation on the anterior end, each containing an amorphous gland-like structure inside and a short and conical tail. Additionally, molecular analysis and comparison of the partial mitochondrial cytochrome c oxidase I sequence of R. pocoto n. sp. revealed genetic divergence between the new species and the sequences of Rhabdias spp. previously deposited in GenBank. Phylogenetic analysis grouped the new taxon into the R. pseudosphaerocephala species complex + R. glaurungi clade. The new discovery represents the 19th species of Rhabdias spp. described in the Neotropical region, the ninth in Brazil and the first species of Rhabdias found parasitizing South American frogs of the genus Pseudopaludicola, as well as the first Caatinga biome species of Rhabdias.

Molecular characterization of human isolates of Strongyloides stercoralis and Rhabditis spp. based on mitochondrial cytochrome c oxidase subunit 1 (cox1).

BACKGROUND: Due to the similarity of Strongyloides stercoralis with free-living nematodes of Rhabditis species they might be miss-diagnosed with each other in microscopical examination of stool samples. The aim of this study was molecular characterization and differentiation of human derived isolates of S. stercoralis and Rhabditis species based on the mitochondrial gene of cytochrome c oxidase subunit 1 (cox1) amplification. METHODS: Using parasitological methods, ten isolates of S. stercoralis and three isolates of Rhabditis spp. were obtained from fresh stool samples of patients and the genomic DNA of the samples were extracted. PCR amplification of cox1 gene was carried out for all the isolates and the products were sequenced. RESULTS: The phylogenetic analysis illustrated that S. stercoralis and Rhabditis spp. isolates were placed in two distinguishable separate clades. Inter-species genetic variation between isolates of S. stercoralis and Rhabditis spp. were ranged from 13.5 to 14.5%. CONCLUSIONS: Cox1 gene was a suitable marker for discrimination of S. stercoralis from Rhabditis spp. retrieved from human in the current study. The availability of gene sequence information will be helpful in the future development and validation of discriminatory PCR-based assays of these nematodes.

Molecular detection and quantification of slug parasitic nematodes from the soil and their hosts.

Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques based on qPCR methods for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (>32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Alloionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras larvae compared to the resistant Arion vulgaris. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes.

Survey of slug-parasitic nematodes in East and West Flanders, Belgium and description of Angiostoma gandavensis n. sp. (Nematoda: Angiostomidae) from arionid slugs.

A survey for slug-associated nematodes in five locations of East and West Flanders in Belgium revealed the presence of one new and six known slug-parasitic nematodes, Agfa flexilis (Dujardin, 1845), Alloionema appendiculatum (Schneider, 1859), Angiostoma dentiferum (Mengert, 1953), Angiostoma limacis (Dujardin, 1845), Angiostoma norvegicum (Ross et al., 2017) and Phasmarhabditis hermaphrodita (Schneider, 1859). Angiostoma norvegicum and P. hermaphrodita are recorded for the first time in Belgium. The six known species are documented by light microscopy (LM) microphotographs and informative DNA sequences. Angiostoma gandavensis n. sp. (Angiostomatidae), discovered from arionid slugs, is described based on light microscopy, scanning electron microscopy (SEM) and molecular data. Based on analyses of D2D3 expansion segment of 28S and 18S rDNA sequences, this new species is found to be related to A. limacis, A. norvegicum, A. margaretae (Ross et al., 2011) and A. milacis (Ivanova and Wilson, 2009). The new species can be distinguished from these others based on morphological characters such as the distinctive mucronate structures at the tail tip of both sexes, presence of lateral ala, reflexed female ovaries and the number and arrangement pattern of male genital papillae.

Development of Phasmarhabditis hermaphrodita (and members of the Phasmarhabditis genus) as new genetic model nematodes to study the genetic basis of parasitism.

The genetic mechanisms of how free-living nematodes evolved into parasites are unknown. Current genetic model nematodes (e.g. Caenorhabditis elegans) are not well suited to provide the answer, and mammalian parasites are expensive and logistically difficult to maintain. Here we propose the terrestrial gastropod parasite Phasmarhabditis hermaphrodita as a new alternative to study the evolution of parasitism, and outline the methodology of how to keep P. hermaphrodita in the lab for genetic experiments. We show that P. hermaphrodita (and several other Phasmarhabditis species) are easy to isolate and identify from slugs and snails from around the UK. We outline how to make isogenic lines using 'semi-natural' conditions to reduce in-lab evolution, and how to optimize growth using nematode growth media (NGM) agar and naturally isolated bacteria. We show that P. hermaphrodita is amenable to forward genetics and that unc and sma mutants can be generated using formaldehyde mutagenesis. We also detail the procedures needed to carry out genetic crosses. Furthermore, we show natural variation within our Phasmarhabditis collection, with isolates displaying differences in survival when exposed to high temperatures and pH, which facilitates micro and macro evolutionary studies. In summary, we believe that this genetically amenable parasite that shares many attributes with C. elegans as well as being in Clade 5, which contains many animal, plant and arthropod parasites, could be an excellent model to understand the genetic basis of parasitism in the Nematoda.

Gastropod parasitic nematodes (Phasmarhabditis sp.) are attracted to hyaluronic acid in snail mucus by cGMP signalling.

Phasmarhabditis hermaphrodita is a parasitic nematode of terrestrial gastropods that has been formulated into a biological control agent for farmers and gardeners to kill slugs and snails. In order to locate slugs it is attracted to mucus, faeces and volatile cues; however, there is no information about whether these nematodes are attracted to snail cues. It is also unknown how wild isolates of P. hermaphrodita or different Phasmarhabditis species behave when exposed to gastropod cues. Therefore, we investigated whether P. hermaphrodita (commercial and wild isolated strains), P. neopapillosa and P. californica were attracted to mucus from several common snail species (Cepaea nemoralis, Cepaea hortensis, Arianta arbustorum and Cornu aspersum). We also examined whether snails (C. aspersum) collected from different locations around the UK differed in their attractiveness to wild isolates of P. hermaphrodita. Furthermore, we also investigated what properties of snail mucus the nematodes were attracted to, including hyaluronic acid and metal salts (FeSO4, ZnSO4, CuSO4 and MgSO4). We found that the commercial strain of P. hermaphrodita responded poorly to snail mucus compared to wild isolated strains, and C. aspersum collected from different parts of the UK differed in their attractiveness to the nematodes. We found that Phasmarhabditis nematodes were weakly attracted to all metals tested but were strongly attracted to hyaluronic acid. In a final experiment we also showed that pharmacological manipulation of cyclic guanosine monophosphate (cGMP) increased chemoattraction to snail mucus, suggesting that the protein kinase EGL-4 may be responsible for Phasmarhabditis sp. chemoattraction.

Nematodes that associate with terrestrial molluscs as definitive hosts, including Phasmarhabditis hermaphrodita (Rhabditida: Rhabditidae) and its development as a biological molluscicide.

Terrestrial molluscs (Mollusca: Gastropoda) are important economic pests worldwide, causing extensive damage to a variety of crop types, and posing a health risk to both humans and wildlife. Current knowledge indicates that there are eight nematode families that associate with molluscs as definitive hosts, including Agfidae, Alaninematidae, Alloionematidae, Angiostomatidae, Cosmocercidae, Diplogastridae, Mermithidae and Rhabditidae. To date, Phasmarhabditis hermaphrodita (Schneider, 1859) Andrássy, 1983 (Rhabditida: Rhabditidae) is the only nematode that has been developed as a biological molluscicide. The nematode, which was commercially released in 1994 by MicroBio Ltd, Littlehampton, UK (formally Becker Underwood, now BASF) under the tradename Nemaslug®, is now sold in 15 different European countries. This paper reviews nematodes isolated from molluscs, with specially detailed information on the life cycle, host range, commercialization, natural distribution, mass production and field application of P. hermaphrodita.

Heterorhabditis pakistanense n. sp. (Nematoda: Heterorhabditidae) a new entomopathogenic nematode from Pakistan.

A new entomopathogenic nematode species of Heterorhabditis, described as H. pakistanense n. sp., was isolated from soil samples around the roots of grass at Malir, Karachi, Sindh, Pakistan. The new species is characterized morphologically by features of males: body size 819 µm (720-1013 µm), D% ((distance from anterior end to excretory pore divided by pharynx length) × 100) 119 (110-126), SW% ((spicule length divided by anal body diameter) × 100) 156 (144-191), GS% ((gubernaculum length divided by spicule length) × 100) 58 (48-65) and variations in the number of bursal papillae of the terminal group: 8th and 9th papillae sometimes absent on both sides, sometimes eight papillae present on the right side whereas six papillae present on the left side. On the right side the arrangement of papillae is 1 + 2 + 3 + 2 whereas on the left side it is 1 + 2 + 3. The hermaphrodite has a prominent post-anal swelling and a conoid tail 82 µm (64-95 µm) long with a pointed terminus. Hermaphrodites of H. pakistanense n. sp. can be distinguished from all species of Heterorhabditis except H. downesi by having a mucronate tail. Infective juveniles have a medium-sized body (581 µm (558-624 µm)), long pharynx (117 µm (113-125 µm)), ensheathed tail (99 µm (95-110 µm)) and E% ((distance from anterior end to excretory pore divided by tail length) × 100) 100 (95-107). The new species can be distinguished from all species of Heterorhabditis by the absence of the 7th, 8th and 9th bursal papillae. Heterorhabditis pakistanense is further characterized by the internal transcribed spacer (ITS) and the D2D3 region of the 28S rDNA gene. The closest species H. indica, H. gerrardi, H. amazonensis and H. noenieputensis being separated by 9, 7, 66 and 15 bp, respectively, in the ITS region. Molecular phylogenetic trees based on sequences of ITS rDNA, D2D3 regions and the mitochondrial 12S rRNA gene support the description of H. pakistanense as a new species.

Morphometric and phylogenetic analyses of Serpentirhabdias viperidicus n. sp. (Nematoda: Rhabdiasidae) from the lancehead snake Bothrops moojeni Hoge, 1966 (Reptilia: Serpentes: Viperidae) in Brazil.

Serpentirhabdias viperidicus n. sp. (Nematoda: Rhabdiasidae) is described from the lungs of the 'Brazilian lancehead' Bothrops moojeni (Hoge, 1966) from the savannah in São Paulo State, Brazil. The new species is the eighth species of Serpentirhabdias described in the Neotropical region, and differs from other species mainly by a combination of characters: lips slightly notable, presence of fine striations at posterior ends, presence of two parallel lines with intercalated pores, a pore-shaped phasmid situated at the level of the anal aperture and another two in the posterior half of the tail. It is the first species of Serpentirhabdias reported in this snake host and the second species of this genus found parasitizing South American viperidian snakes. Molecular phylogenetic analysis using ribosomal (ITS and 28S partial) genes confirms Serpentirhabdias viperidicus n. sp. as a new species that clustered in the Serpentirhabdias clade, sister taxon to Serpentirhabdias fuscovenosa and Serpentirhabdias elaphe. This is the first description of Serpentirhabdias species from Brazil using molecular approaches and morphological characters to confirm the monophyly of this recent genus.

Ecological factors responsible for the geographic distribution of Rhabdias joaquinensis: where do lungworms infect anurans in nature?

The lungworm Rhabdias joaquinensis (Nematoda: Rhabdiasidae) is a common parasite of anurans in eastern Nebraska, yet absent from the same hosts in western Nebraska. This study investigated the ecology of the lungworm's free-living stages that reside in host feces and surrounding soils to establish the worm's free-living niche in eastern Nebraska. Using a comparative method, this study also investigated the absence of lungworms in western Nebraska's Sandhills. Soil composition, moisture, and temperature were experimentally varied in the laboratory to assess their effects on juvenile development and survival. Field mesocosm and host defecation experiments were used to determine where in nature lungworms survive and eventually infect frogs and toads and to discover if vegetation facilitates lungworm transmission to hosts. The results found loam soils were amenable to lungworm development, whereas soils with high clay or sand content produced few infective lungworms. Soil moisture <50 % did not support lungworm development. Infective juveniles successfully developed between 5 and 35 °C, albeit at different rates, whereas only a limited number of non-infective worms developed at 40 °C. Field studies found that shoreline environments supported lungworm development, and the majority of lungworm infections appear to occur within a zone of infection adjacent to shorelines in eastern Nebraska. The prevalence in vegetation mesocosms was 100 %, and a significantly higher mean abundance was found in toads from containers with vegetation than without. Overall, these experiments suggest that the spatial distribution of R. joaquinensis in Nebraska is constrained by the worm's ability to survive and reproduce in a soil matrix.

Outer ear canal infection with Rhabditis sp. nematodes in a human.

Here we report the first human case of an outer ear canal infection with a free-living nematode of the genus Rhabditis. Otomicroscopy revealed viable worms in the outer ear canal of a patient suffering from chronic otorrhea and hearing loss. The nematode was identified by microscopy and internal transcribed spacer (ITS)-PCR.

Heterorhabditis noenieputensis n. sp. (Rhabditida: Heterorhabditidae), a new entomopathogenic nematode from South Africa.

A new entomopathogenic nematode in the genus Heterorhabditis is described from South Africa, from two singular isolates found 1000 km from each other, from beneath a fig tree and in a citrus orchard, respectively. Morphological and molecular studies indicate both isolates to be the same and a new undescribed Heterorhabditis species. Comparison of sequences of the internal transcribed spacer (ITS) rDNA and the D2D3 region of the 28S rDNA gene with available sequences of other described species within the genus, indicate the two isolates as a new species. Phylogenetic analysis of the sequence data concerned placed the new species, H. noenieputensis n. sp., closest to H. indica and H. gerrardi in the indica-group. The new species, H. noenieputensis n. sp., is distinguished from other species in the genus by a combination of several morphological traits of the males and the infective juveniles (IJs). The new species differs from all other species previously described, as regards the body length of the IJs, except for H. indica and H. taysearae, in which the IJ is smaller. The IJ also differs from that of H. indica in the length of the oesophagus, the body diameter, the length of the tail and the E%. In addition, males of H. noenieputensis n. sp. differ from their closest relative, H. indica, in the position of the excretory pore, SW% and D%; and from H. gerrardi in the length of the oesophagus and SW%. The seventh pair of genital papillae of H. noenieputensis n. sp. are normally developed, while for H. indica they are often branched or swollen at the base, while 8 and 9 are usually absent in both species.

Description of Rhabdias breviensis n. sp. (Rhabditoidea: Rhabdiasidae) in two Neotropical frog species.

Nematodes of the genus Rhabdias Stiles & Hassall, 1905 (Rhabditoidea: Rhabdiasidae) have a dioecious free-living stage and a hermaphroditic stage that parasitises the lungs of amphibians and reptiles. Approximately 94 species of Rhabdias have been described. Because the similar morphological characteristics such as the labial structures, the location of the vulva and the shape of the tail of Rhabdias spp. hinder their identification, molecular biology techniques and scanning electron microscopy have been employed to diagnose species of this genus. This study describes Rhabdias breviensis n. sp., parasitic in the lungs of two Neotropical frog species Leptodactylus petersii Steindachner and Leptodactylus macrosternum Miranda-Ribeiro. The description of this species integrates classical taxonomy, scanning electron microscopy and a molecular analysis of the mitochondrial COI gene. The new species differs from all other Rhabdias species parasitic in Neotropical hosts in certain morphometric parameters, the position of the vulva, the host group and the cephalic characters.

Rhabdias esculentarum n. sp. (Nematoda: Rhabdiasidae) from green frogs of the Rana esculenta species complex in Italy: molecular evidence, morphological description and genetic differentiation from its congeners in frogs and toads.

A new taxon, Rhabdias esculentarum n. sp., is described based on DNA sequence analysis at multiple loci (i.e. mtDNA cox-1, 12S rRNA, ITS-1 and partial ITS-2 regions of the nuclear rDNA) and morphometric analysis carried out on specimens collected from the green frogs of the Rana esculenta species complex in Italy (i.e. R. lessonae Camerano and R. esculenta Linnaeus, identified genetically by diagnostic allozyme loci). Rhabdias esculentarum n. sp. was differentiated genetically, at both mitochondrial and nuclear levels, from Rh. bufonis (Schrank, 1788) (sensu Hartwich, 1972) and Rh. sphaerocephala Goodey, 1924 recovered from the toad Bufo bufo Linnaeus collected sympatrically with the specimens of Rana lessonae and R. esculenta examined in the present study. Moreover, the new taxon proved to be different from the other species of Rhabdias from anurans, which had previously been sequenced using the same genes and deposited in GeneBank. Phylogenetic analyses (MP and ML) inferred from mitochondrial (mtDNA cox-1 and 12S ribosomal RNA) and nuclear (ITS-1 and ITS-2 of the rDNA regions) sequences datasets were congruent in depicting Rh. esculentarum n. sp. as forming a highly supported clade distinct from the sympatric species Rh. bufonis, as well as from Rh. sphaerocephala, characterised on the basis of the same loci. Morphometric analysis and the differential diagnosis of genetically characterised specimens of the new species have revealed differences in several features in comparison with the type-species, Rh. bufonis. Material of the latter species included voucher specimens from Germany deposited by Hartwich (1972) and other specimens collected from B. bufo in Italy. Among the diagnostic characters, the particular cup-shaped buccal capsule characterising Rh. esculentarum is clearly different from the tear-shaped buccal capsule observed in material of R. bufonis obtained from Berlin Museum and collected in the same geographical area as the green frogs under study. Rh. esculentarum was also found to differ in some measurements and allometric characters from Rh. bufonis (sensu Moravec et al., 1997). The data so far collected appear to indicate a host-preference of Rh. esculentarum for Rana lessonae and R. esculenta, which belong to the R. esculenta hybridogenetic species complex in Italy.

Prevalence of parasitic contamination of raw vegetables in villages of Qazvin Province, Iran.

We conducted a study to determine the extent of parasitic contamination of raw vegetables distributed by wholesalers in villages of Qazvin Province, Iran. Vegetables collected for the study were leek, parsley, lettuce, coriander, radish, spring onion, tarragon, basil, spearmint, and cress. Samples of each vegetable were collected at monthly intervals for 6 months from May to October 2008, yielding a total of 654 samples. Samples of each vegetable collected at each monthly interval were selected randomly and allocated to be washed or to remain unwashed before being analyzed for parasitic contamination. Of the 654 samples collected over the study period, 218 samples were analyzed for parasitic contamination without washing and 436 were washed before being analyzed, with the wash procedure consisting of submersion in a solution of 200 ppm of calcium hypochlorite and rinsing in an automated vegetable washer for 10 minutes. The samples were analyzed according to the method used by the U.S. Food and Drug Administration for identifying parasitic contamination of raw fruits and vegetables, with specimens weighing 200 g each being prepared from the vegetable samples, followed by sonication of each specimen for 10 minutes in 1.5 L of detergent wash solution, collection and centrifugation of a 50-mL aliquot of the wash solution for 15 minutes at 1500 g, and examination of the resulting sediment for parasites by light microscopy. No parasitic contamination was found on any of the 436 washed samples of vegetables. Of the 218 unwashed samples of vegetables, 82 (37.6%) were found to be contaminated with parasites, of which 69 (31.6%) were contaminated with metazoa (helminth eggs in 9.6% and rhabditoid larvae in 22.0%), and 13 (6.0%) were contaminated with protozoa (Entamoeba coli cysts in 2.8%, Entamoeba histolytica/E. dispar cysts in 1.4%, and Giardia lamblia cysts in 1.8%). The difference in parasitic contamination of the washed and unwashed groups of vegetables was significant at p < 0.05. Helminth eggs detected in unwashed samples included those of nematodes (Ascaris lumbricoides [2.3%], Trichuris trichiura [0.9%], and Trichostrongylus spp. [2.8%]), trematodes (Dicrocoelium dendriticum [1.4%]), and cestodes (Taenia spp. [1.8%] and Hymenolepis nana [0.5%]). The eggs recovered in greatest number were those of nematodes (n = 13), followed by those of cestodes (n = 5) and trematodes (n = 3). No helminth eggs were found on unwashed samples of spearmint, tarragon, coriander, or radish. The percent parasitic contamination of unwashed samples was highest for leek (66.7%) and lowest for radish (20.7%). The highest percent parasitic contamination was with rhabditoid larvae (22.0%) and the lowest with H. nana eggs (0.5%). These findings emphasize the importance of properly washing and disinfecting raw vegetables before they are consumed.

Rhabdias filicaudalis n. sp. (Nematoda: Rhabdiasidae) from the snake Spilotes pullatus (Serpentes: Colubridae) in Brazil.

Rhabdias filicaudalis n. sp. (Nematoda: Rhabdiasidae) from the lung of Spilotes pullatus (Serpentes: Colubridae) is described. The host snake was captured in the municipality of Avaré, São Paulo State, Brazil. Rhabdias filicaudalis n. sp. differs from all other species by the combination of the following characters: straight body, truncated anterior end, six weakly developed lips arranged in two opposite groups of three, pre-equatorial vulva, oesophagus length/body length ratio (%) 4.9-7.5 (5.8 +/- 0.6), nerve ring distance from anterior end/oesophagus length ratio (%) 36.9-61.1 (49.8 +/- 6.4), tail length/body length ratio (%) 3.0-5.5 (4.0 +/- 0.5), vulva distance from anterior end/body length ratio (%) 39.9-51.7 (45.9 +/- 3.5), and a cuticular filiform tail tip.

Angiostoma glandicola sp. n. (Nematoda: Angiostomatidae): a parasite in the land snail Megaustenia sp. from the Cat Tien Forest, Vietnam.

The nematode Angiostoma glandicola sp. n. found parasitizing Megaustenia sp. snails (Pulmonata: Helicarionidae) collected in Dong Nai Province, Vietnam, is described and illustrated. The new species is characterized by its unusual host location (the digestive gland) and in having: two circles of cephalic probolae, six lips each bearing two prominent papillae, amphids situated at posterior of lips, the presence of lateral alae, an enlarged stoma, a club-shaped pharynx with cuticularized valves in the bulb, the nerve ring situated on the junction of the corpus and isthmus and the excretory pore opposite the bulb. Oviparous females have a mid-body vulva position, long reflexed ovaries and short divergent uteri, and a conical tail with a slightly triangular tip. Males have long, curved, not distinctly cephalate spicules and a small gubernaculum, nine pairs of pedunculate genital papillae (GP) incorporated into the bursa (three precloacal, six postcloacal in two groups: GP 4-7 closely behind the cloaca and GP 8-9 distanced), of which GP 5 and 8 terminate on dorsal edge of the bursa and GP 8 and 9 are fused at the base, the bursa nearly reaches the tail tip, unpaired precloacal GP and a pair of ventral circumcloacal GP not incorporated into bursa. It clearly differs from other species of the genus by having cephalic probolae vs. no cephalic probolae and the specific number and disposition of male GP. A partial sequence of D2D3 large subunit (LSU) rDNA was obtained and subjected to phylogenetic analyses. Relationships within the Angiostoma genus are discussed.

Rhabdias lacertae n. sp. (Nematoda: Rhabdiasidae), the first rhabdiasid species parasitising lizards in Europe.

A new nematode species, Rhabdias lacertae n. sp. (Rhabdiasidae), is described from the body-cavity of the common lizard Lacerta vivipara Jacquin (Lacertidae) from the Ridge of Malá Fatra (Sokolie Hill), north-western Slovakia. The new species differs from its congeners mainly in possessing 3 min cuticular spikes at the tail tip and some other features. This is the first species of Rhabdias Stiles & Hassall, 1905 described from lizards in Europe and the first species of this genus parasitising hosts belonging to the Lacertidae.

Isolation and characterization of the native entomopathogenic nematode, Heterorhabditis brevicaudis, and its symbiotic bacteria from Taiwan.

The occurrence of Heterorhabditis brevicaudis (Rhabditida: Heterorhabditidae) and its symbiotic bacteria, Photorhabdus luminescens subsp. akhurstii in Taiwan were recorded for the first time. H. brevicaudis was described by Liu in 1994, but it was unavailable and no molecular data has ever been published for it ever since. The native entomopathogenic nematode (EPN), H. brevicaudis TG01 was recovered from sandy coastal soils in moist bamboo forest, as observed in this article. The bacterial symbiont was isolated from H. brevicaudis for the first time. On the basis of biochemical tests and 16S rDNA it was identified as P. luminescens subsp. akhurstii. This is also the first report of novel nucleotide sequences of the internal transcribed spacer (ITS) from H. brevicaudis. The phylogenetic relationships of ITS sequences were established using Neighbor-Joining, Maximum Parsimony, and Maximum Likelihood methods. The inferred trees strongly support that H. brevicaudis TG01 is specifically related to H. indica and H. hawaiiensis. But the tail length of the infective juveniles (IJ) of H. brevicaudis TG01 in our study, which was less than 80 microm, shorter than that of other species indeed, fall within the original description for H. brevicaudis. Moreover, comparing with morphometrics of IJ and male of H. brevicaudis and H. indica, we recognize that the H. brevicaudis TG01 does not represent variation among populations of H. indica and H. hawaiiensis. This article will answer questions regarding the status of H. brevicaudis and would also provide this species for further investigation.