Diplogasteroides asiaticus n. sp. is Associated with Monochamus alternatus in Japan

Diplogasteroides asiaticus n. sp. is described and illustrated, and its molecular profile and phylogenetic status within the family Diplogastridae are inferred. Morphologically, the new species is characterized by its stomatal structure, a tube-like stoma with three small, rod-like dorsal teeth and two subventral ridges; a spicule clearly ventrally bent at 1/3 from the anterior end; a gubernaculum with a rounded anterior end and sharply pointed distal end in lateral view; nine pairs of genital papillae with an arrangement of <v1, (v2, v3d), C, v4, ad, ph, (v5, v6, v7), pd>; a short tail spike in males; and a well-developed receptaculum seminis, i.e., the antiparallel blind sacs of the uteri beyond the vulva region and elongated conical tail in females. This new species is morphologically similar to D. haslacheri, but it can be distinguished by the morphology of the somewhat shorter tail in females. D. asiaticus n. sp. shares high sequence conservation with D. andrassyi as there is only one base pair difference in the nearly full-length 18S rDNA and seven base pair differences in the D2-D3 expansion segments of the 28S rDNA. Despite this sequence conservation, the species status of D. asiaticus n. sp. was confirmed using the biological species concept, as D. asiaticus n. sp. and D. andrassyi failed to generate viable F₂ progeny in hybridization tests.     

Steinernema akhursti sp. n. (Nematoda: Steinernematidae) from Yunnan, China

A new species of entomopathogenic nematode, herein described as Steinernema akhursti sp. n., was recovered from soil samples collected from Yunnan Province, the People's Republic of China. Both morphological and molecular data show congruently that S. akhursti sp. n. belongs to the Steinernema feltiae group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For the first generation male, the new species can be recognized by spicule length 90 +/- 4.6 microm, spicule tip blunt with an aperture on the ventral side, gubernaculum with a long and needle-shaped cuneus, and tail conoid with a prominent mucron on the tip and a concave on ventral side. For the infective juvenile, the combination of the following characters: body length 812 +/- 19 microm, distance from anterior end to excretory pore 59 +/- 1.5 microm, tail length 73 +/- 2.9 microm, E% 77 +/- 4.5, lateral field with six evenly distributed and identical ridges at the middle body portion, and tail with long and slightly constrict hyaline portion can be used to separate the new species from other nematodes. For the female, the new species is characterized by: tail conoid with a short mucron and slightly swelling anal portion and a symmetrical, slightly protruding vulva with conspicuous double-flapped epiptygma. The nematode can be separated from other described species of Steinernema by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA and from the closely related species S. feltiae and Steinernema oregonense by cross-breeding tests.     

Steinernema biddulphi n. sp., a New Entomopathogenic Nematode (Nematoda: Steinernematidae) from South Africa

A new species of entomopathogenic nematode (EPN), Steinernema biddulphi n. sp., was isolated from a maize field in Senekal, Free State Province of South Africa. Morphological and molecular studies indicated the distinctness of S. biddulphi n. sp. from other Steinernema species. Steinernema biddulphi n. sp. is characterized IJs with average body length of 663 μm (606–778 μm), lateral fields with six ridges in mid-body region forming the formula 2,6,2. Excretory pore located anterior to mid-pharynx (D% = 46). Hyaline layer occupies approximately half of tail length. Male spicules slightly to moderately curved, with a sharp tip and golden brown in color. The first generation of males lacking a mucron on the tail tip while the second generation males with a short filamentous mucron. Genital papillae with 11 pairs and one unpaired preanal papilla. The new species is further characterized by sequences of the internal transcribed spacer (ITS) and partial 28S regions (D2-D3) of the ribosomal DNA (rDNA). Phylogenetic data show that S. biddulphi n. sp. belongs to the “bicornutum” clade within the Steinernematidae family.     

Cardicola opisthorchis n. sp. (Trematoda: Aporocotylidae) from the Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel, 1844), cultured in Japan

A new aporocotylid blood fluke is described, based on specimens from the ventricle of the Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel), cultured in Wakayama and Nagasaki Prefectures, Japan. The new species is morphologically similar to the members of the genus Cardicola Short, 1953, but shows distinct differences in the body form, location of the testis and the orientation of the ootype. The body of the new species is long and slender, whereas other Cardicola species are small and generally lanceolate. The testis is mostly located posterior to the caeca and anterior to the ovary, occupying 31–45% of body length, in contrast to the known Cardicola species, whose testis is typically intercaecal. The ootype is oriented anteriorly, while in most congeners, it is directed posteriorly or horizontally. Phylogenetic analyses of this aporocotylid, together with Cardicola orientalis Ogawa, Tanaka, Sugihara et Takami, 2010 from the same host, were conducted based on DNA sequences of the ITS2 rDNA and the 28S region of ribosomal RNA. The analyses revealed that the new blood fluke belongs to the genus Cardicola despite the marked morphological differences. Thus, this aporocotylid is named Cardicola opisthorchis n. sp. and the generic diagnosis is emended in this paper. In addition, 100% identity among the ITS2 sequences from the present species, Cardicola sp. from T. orientalis in Mexico and Cardicola sp. from the northern bluefin tuna, Thunnus thynnus (Linnaeus) in Spain suggests that C. opisthorchis n. sp. has a broad geographical distribution and that it infects both the Pacific and northern bluefin tuna.     

Description and Bionomics of Mesomermis camdenensis n. sp. (Mermithidae), a Parasite of Black Flies (Simuliidae)

Mesomermis camdenensis n. sp. is described from larvae of Simulium tuberosum (lundstroem) collected in Camden Valley Creek, Washington County, New York. This species possesses a barrel-shaped vagina, vulval flap. two short separate spicules, terminal mouth, six longitudinal chords, six cephalic papillae, large sexually dimorpbic anaphids, an esophagns of uniform width which extends for less than one-third of the body length, and a cone-shaped tail directed ventrally without appendage. Juveniles also are described and illustrated.A detailed morphological comparison with the mermithid M. flumenalis Welch is presented. The most pronounced morphological differences between these species are in the shape of the vulva, juvenile tail, and infective stage. Cross-mating trials support the integrity of the new species.The life cycle of M. camdenensis is closely synchronized with that of its primary host, S. tuberosum larvae. Infected S. tuberosum larvae were first collected in May. Emergence of postparasites from late instars took place from mid-June through mid-October. Sampling data indicate a lower susceptibility to infection among S. venuslum Say larvae.     

Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms

The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.     

The life cycle and host specificity of Psychodiella sergenti n. sp. and Ps. tobbi n. sp. (Protozoa: Apicomplexa) in sand flies Phlebotomus sergenti and Ph. tobbi (Diptera: Psychodidae)

Two new gregarines in the recently erected genus Psychodiella (formerly Ascogregarina), Psychodiella sergenti n. sp. and Psychodiella tobbi n. sp., are described based on morphology and life cycle observations conducted on larvae and adults of their natural hosts, the sand flies Phlebotomus sergenti and Phlebotomus tobbi, respectively. The phylogenetic analyses inferred from small subunit ribosomal DNA (SSU rDNA) sequences indicate the monophyly of newly described species with Psychodiella chagasi. Ps. sergenti n. sp. and Ps. tobbi n. sp. significantly differ from each other in the life cycle and in the size of life stages. The sexual development of Ps. sergenti n. sp. (syzygy, formation of gametocysts and oocysts) takes place exclusively in blood-fed Ph. sergenti females, while the sexual development of Ps. tobbi n. sp. takes place also in males and unfed females of Ph. tobbi. The susceptibility of Phlebotomus perniciosus, Phlebotomus papatasi, Ph. sergenti, Ph. tobbi, and Phlebotomus arabicus to both gregarines was examined by exposing 1st instar larvae to parasite oocysts. High host specificity was observed, as both gregarines were able to fully develop and complete regularly the life cycle only in their natural hosts. Both gregarines are considered as serious pathogens in laboratory-reared colonies of Old World sand flies.     

Molecular Phylogeny and Surface Morphology of Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp. (Gregarinasina,Apicomplexa) Isolated from a Deep Sea Oradarea sp. (Amphipoda) in the West Pacific

In an effort to broaden our understanding of the biodiversity and distribution of gregarines infecting crustaceans, this study describes two new species of gregarines, Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp., parasitizing a deep sea amphipod (Oradarea sp.). Amphipods were collected using the ROV Hyper‐Dolphin at a depth of 855 m while on a cruise in Sagami Bay, Japan. Gregarine trophozoites and gamonts were isolated from the gut of the amphipod and studied with light and scanning electron microscopy, and phylogenetic analysis of 18S rDNA. Thiriotia hyperdolphinae n. sp. was distinguished from existing species based on morphology, phylogenetic position, as well as host niche and geographic locality. Cephaloidophora oradareae n. sp. distinguished itself from existing Cephaloidophora, based on a difference in host (Oradarea sp.), geographic location, and to a certain extent morphology. We established this latter new species with the understanding that a more comprehensive examination of diversity at the molecular level is necessary within Cephaloidophora. Results from the 18S rDNA molecular phylogeny showed that T. hyperdolphinae n. sp. was positioned within a clade consisting of Thiriotia spp., while C. oradareae n. sp. grouped within the Cephaloidophoridae. Still, supplemental genetic information from gregarines infecting crustaceans will be needed to better understand relationships within this group of apicomplexans.     

The symbiont Capsaspora owczarzaki,nov. gen. nov. sp., isolated from three strains of the pulmonate snail Biomphalaria glabrata is related to members of the Mesomycetozoea

While investigating the resistance of some strains of Biomphalaria glabrata to infection with Schistosoma mansoni, a unicellular eukaryotic symbiont was noted in the snail haemolymph. It was similar in appearance to Nuclearia sp. reported from B. glabrata. Sequences comprising the 18S, ITS1, 5.8S, ITS2 and the beginning of the 28S rDNA gene regions were obtained from symbionts isolated from three strains of B. glabrata, and compared with the same sequences obtained from a culture of Nuclearia sp. 18S rDNA sequences were identical for all four isolates. 18S rDNA sequences were used in a phylogenetic analysis to produce minimum evolution, maximum parsimony, maximum likelihood and Bayesian trees. All four analyses indicated that the B. glabrata symbiont is not closely related to Nuclearia but instead to the Mesomycetozoea, a recently recognised clade of symbiotic eukaryotes. Based on phylogenetic analysis, life history and morphological differences, the symbiont is described as a new genus and species, Capsaspora owczarzaki. Distinguishing characters are the presence of life cycle stage(s) that occur within snail haemolymph; ability to kill and ingest digenetic trematode larvae; ability to undergo asexual fission to produce daughter cells; absence of flagella, a mucous sheath and membranes containing chitin, elastin, or collagen; and presence of long unbranching pseudopodia and a penetration process. Using both polymerase chain reaction (PCR) and culturing techniques, the S. mansoni-resistant Salvador and 13-16-R1 strains were found to be significantly more likely to harbour the symbiont than the susceptible M line strain. Small but consistent sequence differences were noted among symbiont isolates from different snail strains, raising the possibility that the symbiont has diverged in different snail lineages. This suggests further that the symbiont is not restricted to albino lab-reared snails. A role, if any, of the symbiont in resistance awaits further study.     

Steinernema scapterisci n. sp. (Rhabditida: Steinernematidae)

Steinernema scapterisci n. sp., isolated in Uruguay from the mole cricket Scapteriscus vicinus, can be distinguished from other members in the genus by the presence of prominent cheilorhabdions, an elliptically shaped structure associated with the excretory duct, and a double-flapped epitygma in the first-generation female. The spicules of the male are pointed, tapering smoothly to a small terminus, and the shaft (calomus) is long, bearing a sheath. The gubernaculum has a long, upward-bent anterior part. The ratio of head to excretory pore divided by tail length of the third-stage juvenile is greater for S. scapterisci n. sp. than for S. carpocapsae. Steinernema scapterisci n. sp. did not hybridize with S. carpocapsae strain Breton. In laboratory tests, S. scapterisci n. sp. killed 10% or less of non-orthopteran insects, including the wax moth larva, a universal host for other species of Steinernema.     

Ergasilus thatcheri n. sp. (Copepoda, Poecilostomatoida, Ergasilidae) from the gills of Rhamdia quelen (Teleostei, Siluriformes, Pimelodidae) from southern Brazil

The female of Ergasilus thatcheri n. sp. (Copepoda, Poecilostomatoida, Ergasilidae) is described from the gills of the "jundiá," Rhamdia quelen (Teleostei, Siluriformes, Pimelodidae), from fish ponds in the State of Rio Grande do Sul, Brazil. The new species has a falciform semipinnate terminal seta on the first exopod, which suggests phylogenetic affinity to 10 other congeners, all from fishes of the Amazon Basin. Based on general morphology, the new species shows great similarity to E. callophysus Thatcher and Boeger, 1984, a parasite of another pimelodid fish Callophysus macropterus. Ergasilus thatcheri n. sp., however, can be easily differentiated from E. callophysus in its possession of a more distal sensillum not located on a cuticular elevation, and a row of spinules on the inner margin of the second antennal segment (first endopodal segment).     

Aphelenchoides microstylus n. sp. and Seinura onondagensis n. sp. (Nemata: Aphelenchina) from New York

Aphelenchoides microstylus n. sp. and Seinura onondagensis n. sp., a nematode predator, are described from dead Scots pine (Pinus sylvestris L.) in Onondaga County, New York. Females of A. microstylus are 370 to 485 µm long. The body is slender and tapers posteriorly to an amucronate, pointed terminus. The head is continuous with the body, and lips bear a stylet guide. Diagnostic characters of females are three incisures in the lateral field, a short stylet (6-7.5 µm) with small basal knobs, a single row of oocytes, and a long postuterine sac (25-50 µm). Males are characterized by small spicules (10-11µm); two pairs of post-anal, subventral papillae; and a single row of spermatocytes. A bursa and gubernaculum are absent. Seinura onondagensis females are characterized by a body of moderate length (475-595 µm), finely annulated cuticle, and a slightly set-off head. Diagnostic characters are four incisures in the lateral field, long stylet without basal knobs (17-22 µm), single row of oocytes, and presence of a postuterine sac (14-38 µm). Males are unknown. The monospecific genus Indaphelenchus is proposed as a synonym of Seinura, and S. siddiqii n. comb. is proposed for the only species, I. siddiqii.     

Oxnerella micra sp. n. (Oxnerellidae fam. n.), a tiny naked centrohelid, and the diversity and evolution of heliozoa

We describe a new tiny naked centrohelid heliozoan, Oxnerella micra, and sequenced its 18S and 28S rRNA genes. Its extremely slender axopodia have prominent extrusomes and are normally stretched across the substratum like those of many tiny granofilosean Cercozoa. Phylogenetic analysis of 18S rDNA shows that Oxnerella does not branch within any of the six known centrohelid families but very deeply in the order Pterocystida, between Choanocystidae and Pterocystidae; therefore we place it in a new family, Oxnerellidae. Oxnerella arose from ancestors with siliceous scales by losing them; as independently did Heterophryidae and Marophryidae, which replaced them by organic spicules, and Chlamydaster that is not truly naked but retains a mucilage coat and nests extremely shallowly within Pterocystidae. 28S rDNA has a group I intron. Concatenated Bayesian 18S/28S rRNA phylogeny shows centrohelids weakly as sisters to the naked non-centrohelid heliozoan Microheliella maris (Microhelida: Heliozoa). The centrohelid Marophrys marina possesses an elongation factor α-like (EFL) protein related to that of Polyplacocystis; Microheliella also has EFL. We also analysed Hsp90 and 18S rDNA sequences from 'Pinaciophora sp.' ATCC50355; they must be from a centrohelid, probably misidentified as Pinaciophora, the rDNA sequence branching deeply within Pterocystida. We reclassify two Polyplacocystis, Luffisphaera, Phaeodaria and Rotosphaerida.     

Development, ultrastructure, natural occurrence, and molecular characterization of Liebermania patagonica n. g., n. sp., a microsporidian parasite ot the grasshopper Tristira magellanica (Orthoptera: Tristiridae)

A new microsporidium, Liebermannia patagonica n. gen., n. sp., is described from midgut and gastric caecum epithelial cells of Tristira magellanica, an apterous grasshopper species of southern Patagonia, Argentina. L.patagonica is diplokaryotic, apansporoblastic, homosporous, and polysporoblastic. Transitional (from merogony to sporogony) stages and sporonts of L. patagonica were surrounded by host rough endoplasmic reticulum. The ovocylindrical spores measured 2.9 +/- 0.09 x 1.2 +/- 0.04 microm (fresh, n = 50), and they had an isofilar polar filament of only three coils and a cluster of tubules instead of a classical posterior vacuole. Prevalence was high (up to 80.6%) at the type locality for the four years sampled . Maximum likelihood , neighbor joining, maximum parismony analyses of the small submit rDNA all placed L.patagonica(Accession No. DQ 239917) in one with Orthosomella operophterae.     

Steinernema aciari sp. n. (Nematoda: Steinernematidae), a new entomopathogenic nematode from Guangdong, China

A new species of entomopathogenic nematode, Steinernema aciari sp. n. was described. It was recovered from a soil sample collected from Haimen town, Shantou district in the eastern coast of Guangdong province, the People's Republic of China during a survey for entomopathogenic nematodes. S. aciari sp. n. belongs to the Steinernema glaseri group. It can be separated from all described Steinernema species by the combined morphological and morphometrical characters of various stages of the nematodes. For male, the new species can be recognized by spicule length (86+/-6.3 microm); spicule tip blunt with a hook-like structure; gubernaculum with a short and Y-shaped cuneus and corpus well-separated posteriorly. For infective juvenile, the combination of the following characters: body length (1113+/-68 microm), distance from anterior end to excretory pore (95+/-3.7 microm), tail length (78+/-5.2 microm), and E % (123+/-7) can be used to differentiate the new species from other nematodes. For female, the tail (conoid with a long mamillate terminus and a distinct postanal swelling) and vulva (slightly protruding from body surface with conspicuous double flapped epiptygma) shapes can be used as diagnostic characters for the new species. The new species can also be distinguished from other Steinernema species by DNA sequences of either a partial 28S rDNA or the internal transcribed spacer regions of rDNA, and from the close related species S. glaseri, Steinernema longicaudum CWL05, and Steinernema guangdongense by cross-breeding test.     

Bursaphelenchus anatolius n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Bees in the Genus Halictus

Bursaphelenchus anatolius n. sp., a phoretic associate of Halictus bees from Ankara, Turkey, is described and illustrated. Bursaphelenchus anatolius n. sp. is closest to B. kevini, which is phoretically associated with Halictus bees from the Pacific Northwest. Bursaphelenchus anatolius n. sp. and B. kevini appear to be sister taxa based upon several shared morphological features, similar life histories involving phoresy with soil-dwelling Halictus bees, and molecular analysis of the near-full-length small subunit rDNA, D2D3 expansion segments of the large subunit rDNA, and partial mitochondrial DNA COI. Bursaphelenchus anatolius n. sp. can be differentiated from all other species of Bursaphelenchus based upon spicule morphology. The paired spicules are uniquely shaped and ventrally recurved, and both B. anatolius n. sp. and B. kevini possess extending flaps that open when the spicules are protracted beyond the cloaca. Population growth of B. anatolius n. sp. was measured at 23 °C in the laboratory on cultures of the fungus Monilinia fructicola grown on lactic acid-treated, 5% glycerol-supplemented potato dextrose agar. Nematode population densities rapidly increased from 110 to about 110,000/9-cm-diam. dish within 21 days.     

Heterorhabditidoides rugaoensis n. sp. (Rhabditida: Rhabditidae), a Novel Highly Pathogenic Entomopathogenic Nematode Member of Rhabditidae

A novel entomopathogenic nematode species, Heterorhabditidoides rugaoensis n. sp. RG081015, collected from Rugao, China, is described. The new species is morphologically very similar to H. chongmingensis but can be distinguished from it on the basis of some morphological characteristics, combined with molecular data and a cross-hybridization test. Males of the new species can be recognized on the basis of body length averaging 1396.2 μm; lateral field with one ridge; metastome isoglottoid with one hemispherical swellings comprised of two to three well-developed warts; asymmetric spicules; peloderan bursa. In IJs, EP = 134.5 μm; ES = 149.3 μm; tail length = 82.5 μm; and a = 20.5. Hermaphroditic females have four to five lateral ridges. The 18S rDNA and ITS sequences of the two nematodes share 99% and 98% identity, respectively. Phylogenetic trees of 18S rDNA and ITS indicate that the new species is most closely related to H. chongmingensis; thus, the two nematodes belong to the same genus. Failure of cross-hybridization between them indicates that nematode strain RG081015 is a novel species and is described herein as H. rugaoensis n. sp. The LC₅₀ of the novel species against Galleria mellonella were 24.35 IJs / ml within 48 hours of infection. Morphological characteristics, genetic similarity analyses, and phylogenetic relationships provide strong evidence that some species of Oscheius/Insectivora-group should be reassigned to the genus Heterorhabditidoides.     

Descriptions of Eumonhystera borealis n. sp., Sphaerolaimus occidentalis n. sp., and a Redescription of S. gracilis de Man 1876 (Nemata) from Bothnian Bay,Baltic Sea

The free-living nematodes Eumonhystera borealis n. sp., Sphaerolaimus occidentalis n. sp., and S. gracilis de Man 1876 from Bothnian Bay in the northern Baltic Sea are described and illustrated. Eumonhystera borealis n. sp. differs from other species by its small body size (314-393 μm), narrow body (a = 37-49), and large anterior amphids. In Sphaerolaimus occidentalis n. sp. the amphids are posterior to the buccal cavity, and it differs from other similar species by having two sclerotized rings in the posterior part of the buccal cavity. An intersex is reported for S. gracilis. Sphaerolaimus gracilis is cannibalistic or a predator of other species, with a preference for E. borealis n. sp. Sphaerolaimus occidentalis n. sp. coexists with S. gracilis at depths of 80 m but not at 12 m.     

<Emphasis Type="Italic">Steinernema costaricense</Emphasis> n. sp. and <Emphasis Type="Italic">S. puntauvense</Emphasis> n. sp. (Rhabditida: Steinernematidae), two new entomopathogenic nematodes from Costa Rica

Steinernema costaricense n. sp. and S. puntauvense n. sp. were recovered during a survey for native entomopathogenic nematodes in Costa Rica. Morphological data, molecular (28S rDNA sequence data) studies and cross-hybridisation tests were used for diagnostic and identification purposes. Additionally, 28S rDNA sequence data were used to assess the evolutionary relationships of the new species with other Steinernema spp. Morphological diagnostic features for S. costaricense n. sp. include: the body size of the infective juvenile (av. 1,696); the presence of protruding 'horn-like' cephalic papillae; the position of the excretory pore in the infective juvenile (av. 77 microm) and the first generation male (av. 117 microm); the D% value of the infective juvenile (av. 53) and the first generation male (av. 56); the E% value of the infective juvenile (av. 85); and the morphology of the spicules and gubernaculum of the first generation male. Diagnostic traits for S. puntauvense n. sp. are: the position of the excretory pore in the infective juveniles (av. 25 microm); the shape and size of the spicules and gubernaculum of the first generation male; and the shape of the tail of the first generation female. In addition to these traits, 28S rDNA sequences analysis and hybridisation tests showed that both new Steinernema species are distinct and unique entities.