Tokorhabditis n. gen. (Rhabditida, Rhabditidae), a comparative nematode model for extremophilic living.

Life in extreme environments is typically studied as a physiological problem, although the existence of extremophilic animals suggests that developmental and behavioral traits might also be adaptive in such environments. Here, we describe a new species of nematode, Tokorhabditis tufae, n. gen., n. sp., which was discovered from the alkaline, hypersaline, and arsenic-rich locale of Mono Lake, California. The new species, which offers a tractable model for studying animal-specific adaptations to extremophilic life, shows a combination of unusual reproductive and developmental traits. Like the recently described sister group Auanema, the species has a trioecious mating system comprising males, females, and self-fertilizing hermaphrodites. Our description of the new genus thus reveals that the origin of this uncommon reproductive mode is even more ancient than previously assumed, and it presents a new comparator for the study of mating-system transitions. However, unlike Auanema and almost all other known rhabditid nematodes, the new species is obligately live-bearing, with embryos that grow in utero, suggesting maternal provisioning during development. Finally, our isolation of two additional, molecularly distinct strains of the new genus-specifically from non-extreme locales-establishes a comparative system for the study of extremophilic traits in this model.

Morphological observation and first molecular characterization of Grassenema procaviae Petter, 1959 (Cosmocercoidea: Atractidae) in the stomach of Cape hyrax (Procavia capensis) raised in a zoo in Japan.

The superfamily Cosmocercoidea comprises three families: Cosmocericidae, Kathlaniidae and Atractidae. Information on the nucleotide sequences of the Cosmocercoidea is quite limited, and the molecular classification of the whole superfamily has been slow to progress. The genus Grassenema of the family Atractidae is a parasitic nematode group that occurs in the digestive tract of hyraxes and includes three species: Grassenema procaviae, G. dendrohyraci, and G. hyracis. The type species of the genus, G. procaviae, was isolated from the digestive tract of Cape hyraxes (Procavia capensis) and has the potential to cause gastric ulcers. Although G. procaviae is a common parasite of Cape hyraxes, no genetic information for the parasite is currently available. In this study, we obtained the first genomic sequences of G. procaviae and performed detailed morphological observations. Furthermore, molecular phylogenetic analysis was performed, and the taxonomic position of the parasite was evaluated using 18S and 28S rDNA sequences. Those data will be useful for molecular identification of G. procaviae and future phylogenetic analysis within the Atractidae.

Ultrastructural plasticity in the plant-parasitic nematode, Bursaphelenchus xylophilus.

Phenotypic plasticity is one of the most important strategies used by organisms with low mobility to survive in fluctuating environments. Phenotypic plasticity plays a vital role in nematodes because they have small bodies and lack wings or legs and thus, cannot move far by themselves. Bursaphelenchus xylophilus, the pathogenic nematode species that causes pine wilt disease, experiences fluctuating conditions throughout their life history; i.e., in both the phytophagous and mycetophagous phases. However, whether the functional morphology changes between the life phases of B. xylophilus remains unknown. Our study revealed differences in the ultrastructure of B. xylophilus between the two phases. Well-developed lateral alae and atrophied intestinal microvilli were observed in the phytophagous phase compared with the mycetophagous phase. The ultrastructure in the phytophagous phase was morphologically similar to that at the dauer stage, which enables the larvae to survive in harsh environments. It suggests that the living tree represents a harsh environment for B. xylophilus, and ultrastructural phenotypic plasticity is a key strategy for B. xylophilus to survive in a living tree. In addition, ultrastructural observations of obligate plant-parasitic species closely related to B. xylophilus revealed that B. xylophilus may be in the process of adapting to feed on plant cells.

Alloionema californicum n. sp. (Nematoda: Alloionematidae): a new alloionematid from USA.

A new species of the family Alloionematidae was isolated from a rotten winged gourd at White Crane Garden, San Francisco, USA, sampled by Christopher Nelson in November 2010, and a live culture is deposited in Félix Lab Strain Database (http://www.justbio.com/worms/index.php), IBENS, Paris, France. Specimens from the culture have been examined. Both morphologically and molecularly, the nematode described herein as Alloionema californicum n. sp. differs from the other alloionematid species, A. appendiculatum and Neoalloionema tricaudatum. It is characterised by having a narrow stoma, 2.5-3.5 or 4 times longer than broad in adults or dauer juveniles respectively. Lateral fields are not present in adults but occur as one prominent ridge in dauers. Males have no bursa, six pairs of genital papillae and one single papilla. Dauers have large apparent phasmids in the middle of the tail. The ecology of the newly described species is unknown but probably it is a saprobic bacteriophagous nematode preferring rotting organic material.

Myolaimus ibericus sp. n. (Nematoda, Rhabditida, Myolaimidae) from southern Iberian Peninsula, with a key to species of the genus.

A new species of the genus Myolaimus is described from agricultural areas in the southeast of the Iberian Peninsula. Myolaimus ibericus sp. n. is characterized by its 560-783 µm long body in females and 526-731 µm in males, cuticle often appearing swollen and baggy, lateral field with one longitudinal wing, lip region with six amalgamated lips having ten (6 + 4) setiform papillae, stoma consisting of a wider anterior chamber (cheilo-gymnostom) and a tubular posterior part (stegostom) separated by a well developed dorsal tooth and two small, lateral (one right and another left) teeth, glottoid-like apparatus structure appearing at metastom, pharyngeal corpus 1.7-2.6 times the isthmus length, excretory pore and deirids located at metacorpus level, deirids pore-like in females and seta-like in males, female reproductive system monodelphic-prodelphic with antidromous ovary, post-uterine sac 1.7-2.7 times the corresponding body diameter long, V = 50-56, female rectum 1.3-2.0 times the anal body diameter long, female tail conical-elongate (54-70 µm, c = 9.9-13.1, c' = 4.4-5.8) often enveloped by the baggy cuticle, male tail conoid (8-10 µm, c = 58.4-73.1, c' = 1.1-1.4) and ventrally constricted at its middle, bursal structure with seven (4+1+2) pairs of genital papillae. This is the first record of the genus from the Iberian Peninsula. Description, measurements and illustrations, including SEM photographs are provided. An illustrated compendium of the posterior ends of males in Myolaimus species is also presented.

Ultrastructural immunogold localization of major sperm protein (MSP) in spermatogenic cells of the nematode Acrobeles complexus (Nematoda, Rhabditida).

The nematode spermatozoa represent a highly modified (aberrant) type of male gametes that lack a flagellum but for which the process of spermatogenesis culminates in the production of a crawling spermatozoon on the basis of the cytoskeletal component known as "major sperm protein", or MSP. MSP is also known as an important hormone triggering oocyte maturation and ovulation in the model nematode Caenorhabditis elegans, where this protein was first identified. However, direct evidence of MSP localization and of its fate in nematode spermatogenic cells is rare. In this study, the spermatogenesis and sperm structure in the rhabditid nematode Acrobeles complexus (Rhabditida: Tylenchina: Cephalobomorpha: Cephaloboidea: Cephalobidae) has been examined with electron microscopy. Morphological observations were followed by high-pressure freezing and freeze-substitution fixation which allows post-embedding immunogold localization of MSP in all stages of sperm development using antibodies raised for MSP of C. elegans. In spermatocytes, synthetic activity results in the development of specific cellular components, fibrous bodies (FB) and membranous organelles (MO), which appear as FB-MO complexes where the filamentous matter of FB has been MSP-labeled. The spermatids subdivide into a residual body with superfluous cytoplasm, and a main cell body which contains nucleus, mitochondria and FB-MO complexes. These complexes dissociate into individual components, MO and FB, with the MSP being localized in FB. Immature spermatozoa from testes are opaque cells where a centrally located nucleus is surrounded by mitochondria, MO and FB clustered together, the MSP still being localized only in FB. Cytoplasm of mature spermatozoa from spermatheca is segregated into external pseudopods lacking organelles and a central cluster of mitochondria with intact MO surrounding the central nucleus. The FB ultimately disappear, and the MSP labeling becomes concentrated in the filamentous content of pseudopods and cytoplasm of the main cell body. Although the spermatogenesis and sperm structure of A. complexus is similar to that of many other rhabditid nematodes, their intact MO makes the morphology of the mature spermatozoa distinct from the "rhabditid pattern" and may be considered as a synapomorphy. The MSP localization in spermatogenic cells of A. complexus also follows the "rhabditid pattern" described in C. elegans and Ascaris spp. Our results and techniques of MSP labeling of A. complexus spermatogeneous cells reveal new possibilities to elucidate different research questions on MSP localization in nematodes related to C. elegans. Furthermore, the laboratory-cultured A. complexus strains can be used as a new and fascinating model to study MO and MSP functions in nematode reproduction.

The ability to survive intracellular freezing in nematodes is related to the pattern and distribution of ice formed.

A few species of nematodes can survive extensive intracellular freezing throughout all their tissues, an event that is usually thought to be fatal to cells. How are they able to survive in this remarkable way? The pattern and distribution of ice formed, after freezing at -10°C, can be observed using freeze substitution and transmission electron microscopy, which preserves the former position of ice as white spaces. We compared the pattern and distribution of ice formed in a nematode that survives intracellular freezing well (Panagrolaimus sp. DAW1), one that survives poorly (Panagrellus redivivus) and one with intermediate levels of survival (Plectus murrayi). We also examined Panagrolaimus sp. in which the survival of freezing had been compromised by starvation. Levels of survival were as expected and the use of vital dyes indicated cellular damage in those that survived poorly (starved Panagrolaimus sp. and P. murrayi). In fed Panagrolaimus sp. the intracellular ice spaces were small and uniform, whereas in P. redivivus and starved Panagrolaimus sp. there were some large spaces that may be causing cellular damage. The pattern and distribution of ice formed was different in P. murrayi, with a greater number of individuals having no ice or only small intracellular ice spaces. Control of the size of the ice formed is thus important for the survival of intracellular freezing in nematodes.

Rotylenchus sardashtensis n. sp., a monosexual species from Iran, with molecular identification and detailed morphological observations on an Iranian population of Rotylenchus cypriensis Antoniou, 1980 (Nematoda: Rhabditida: Hoplolaimidae).

Rotylenchus sardashtensis n. sp., a new monosexual species is described and illustrated based on morphological, morphometric and molecular studies. Rotylenchus sardashtensis n. sp. appears close morphologically and molecularly to eight known species of the genus, i.e. R. buxophilus Golden, 1956, R. eximius Siddiqi, 1964, R. breviglans Sher, 1965, R. cypriensis Antoniou, 1980, R. pakistanensis Maqbool & Shahina, 1986, R. vitis Cantalapiedra-Navarrete, Liébanas, Archidona-Yuste, Palomares-Rius & Castillo, 2012, R. paravitis Cantalapiedra-Navarrete, Navas-Cortés, Liébanas, Vovlas, Subbotin, Palomares-Rius & Castillo, 2013 and R. dalikhaniensis Aliramaji, Pourjam, Álvarez-Ortega, Pedram & Atighi, 2015 from which the morphological differences are discussed. The new species is characterised mainly in having a hemispherical and well set off lip region with 3-4 annuli under light microscopy and irregular cob-like appearance under scanning electron microscopy (SEM), a stylet 26-30 µm long, a vulva located at 61-77% of body length with double epiptygma, rounded tail with 2-4 annuli and ventral mucron at its tip. Morphologically, R. sardashtensis n. sp. could be distinguished from all similar species within the genus by its matrix code as follows: A3, B1, C1, D4, E1, F2, G2, H5, I2, J2, K1. Rotylenchus cypriensis collected from the rhizosphere of Prunus sp. in Gilangharb region is characterised with its morphological and molecular data. Phylogenetic analyses based on sequences of the D2-D3 expansion region of the 28S rRNA gene and the ITS1 fragment revealed the relationships of both species examined in present study and other species of the genus Rotylenchus Filipjev, 1936 and the family Hoplolaimidae Filipjev, 1934 included in analyses.

Synoecnema watinagii (Drilonematoidea: Ungellidae: Synoecneminae), a new nematode species parasitic in earthworms from the Philippines with the first molecular and SEM data for the genus.

A new species of the genus Synoecnema-S. watinagii sp. n.-is described and illustrated. The species is characterized by its small size, lack of sexual dimorphism apart from sexual characters, males with pericloacal disk, females with anterior vulva position and lacking an anus, and similar caudal organs in both sexes in the shape of long, deep slits on the surface of the posterior half of body. Sequences of D2-D3 LSU and SSU rDNA and SEM images were obtained for the first time for the genus Synoecnema. Phylogenetic analysis of these sequences supported the validity of the genus and elucidated its relationships within Synoecneminae and Drilonematoidea. The earthworm host was characterized by its CoxI mt DNA sequence.

The rectal glands of Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) hermaphrodites and their role in symbiont transmission.

Differential interference contrast, transmission electron and epifluorescence microscopy techniques were employed to examine the ultrastructure of the rectal glands in Heterorhabditis bacteriophora hermaphrodites, with special attention to the location of Photorhabdus bacteria symbionts within these structures. Three rectal glands were clearly visualized in all examined specimens, with two glands positioned sub-ventrally and another gland located dorsally. The dorsal rectal gland in all examined specimens is larger than the subventral ones. Our observations indicate that Photorhabdus bacteria do not colonize the rectal glands of H. bacteriophora hermaphrodites, but rather are present in the most posterior-intestinal cells.

Resolving phylogenetic incongruence to articulate homology and phenotypic evolution: a case study from Nematoda.

Modern morphology-based systematics, including questions of incongruence with molecular data, emphasizes analysis over similarity criteria to assess homology. Yet detailed examination of a few key characters, using new tools and processes such as computerized, three-dimensional ultrastructural reconstruction of cell complexes, can resolve apparent incongruence by re-examining primary homologies. In nematodes of Tylenchomorpha, a parasitic feeding phenotype is thus reconciled with immediate free-living outgroups. Closer inspection of morphology reveals phenotypes congruent with molecular-based phylogeny and points to a new locus of homology in mouthparts. In nematode models, the study of individually homologous cells reveals a conserved modality of evolution among dissimilar feeding apparati adapted to divergent lifestyles. Conservatism of cellular components, consistent with that of other body systems, allows meaningful comparative morphology in difficult groups of microscopic organisms. The advent of phylogenomics is synergistic with morphology in systematics, providing an honest test of homology in the evolution of phenotype.

Morphometric and molecular characteristics of Labeonema synodontisi n. comb. (Nematoda: Atractidae) from the West African fishes.

Numerous nematodes were found in the rectum of three fish species Synodontis ocellifer, S. nigrita, and S. schall (Mochokidae, Siluriformes) from the Gambia River and Mare Simenti, National Park Niokolo Koba, Senegal. A nematode species Raillietnema synodontisi Vassiliadès, 1973 (host S. ocellifer), is redescribed using morphometric (including scanning electron microscopy) and molecular characteristics and transferred into the genus Labeonema Puylaert, 1970. It is morphologically and metrically similar to Labeonema intermedium Puylaert, 1970, the other congeneric species (L. bainae Baker, 1982; L. bakeri Van Waerebeke, Chabaud, Bain et Georges, 1988; and L. africanum Moravec et Van As, 2004) differ from them either by the spicule and gubernaculum lengths, distribution and number of pre-cloacal papillae, position of the vulva, as well as hosts and geographical distribution. The partial sequences of small ribosomal subunit rDNA of L. synodontisi were analyzed and compared with other nematode sequences. Molecular analyses seem to support the position of this nematode species based on the morphological observation.

Steinernema cholashanense n. sp. (Rhabditida, Steinernematidae) a new species of entomopathogenic nematode from the province of Sichuan, Chola Shan Mountains, China.

During a random survey of entomopathogenic nematodes in the provinces of Sichuan and Gansu (eastern Tibet) in 2004, soil samples from several sites were collected and tested for the incidence of entomopathogenic nematodes. A new species was collected in this survey and it is described herein as Steinernema cholashanense n. sp. Steinernema cholashanense n. sp. is characterized by morphology and morphometry of the IJ and male. For the IJ, the new species can be recognized by the average body length 843 microm, esophagus length 125 microm, H%=39% and E%=81%. The lateral field pattern is 2, 5, 7, 4, 2. The male of the first generation is characterized by spicule shape and length and especially with prominent velum and the presence of a mucron on both generations. The average body length of the IJ of S. cholashanense n. sp. (843 microm) is shorter than that of S. oregonense (980 microm), S. kraussei (951 microm) and S. litorale (909 microm), similar to that of S. feltiae (849 microm), but longer than that of S. weiseri (740 microm), S. jollietti (711 microm) and S. hebeiense (658 microm). Esophagus length of the new species (125 microm) is closer to that of S. jollieti (123 microm) but longer than that of S. weiseri (113 microm) and shorter than that of S. oregonense (132 microm), S. kraussei (134 microm) and S. feltiae (136 microm). E% of the new species (81) is similar to that of S. kraussei (80), but smaller than that of S. jollieti (88), S. weiseri (95), S. oregonense (100) and S. feltiae (119). Spicule head length of the new species is almost the same as its width, this character is similar to that of S. kraussei but it is different from this species by its prominent velum. The new species can be recognized further by characteristics of sequences of ITS and D2D3 regions and cross hybridization with closely related species, S. feltiae, S. kraussei and S. oregonense.

Steinernema costaricense n. sp. and S. puntauvense 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.

Three-dimensional fine structural reconstruction of the nose sensory structures of Acrobeles complexus compared to Caenorhabditis elegans (Nematoda: Rhabditida).

Nematode sensory structures can be divided into two classes; cuticular sensillae, with dendrites ending outside the epidermis, and internal receptors, that typically are single dendrites terminating within the body cavity. Fine structure of the former has been described completely in more than a dozen nematode taxa, while the latter were previously only well understood in the microbial feeder Caenorhabditis elegans. The distantly related nematode Acrobeles complexus has a similar ecology and together the two span a clade representing a large proportion of nematode biodiversity. The cuticular sensillae and internal receptors of A. complexus are here shown to be remarkably similar in number, arrangement, and morphology to those of C. elegans. Several key differences are reported that likely relate to function, and suggest that this nematode has a cuticular sensillum morphology that is closer to that of the common ancestor of the two taxa. Internal sensory receptors have more elaborate termini than those of C. elegans. The existence of a novel form of mechanoreceptor in A. complexus and spatial relationships between sensillum dendrites suggest differences between two classes of sensillae in how a touch-response behavior may be mediated.

Influence of nematode age and culture conditions on morphological and physiological parameters in the bacterial vesicle of Steinernema carpocapsae (Nematoda: Steinernematidae).

Steinernema spp. third-stage infective juveniles (IJs) play a key role in the symbiotic partnership between these entomopathogenic nematodes and Xenorhabdus bacteria. Recent studies suggest that Steinernema carpocapsae IJs contribute to the nutrition and growth of their symbionts in the colonization site (vesicle) [Martens, E.C. and Goodrich-Blair, H., 2005. The S. carpocapsae intestinal vesicle contains a sub-cellular structure with which Xenorhabdus nematophila associates during colonization initiation. Cellular Microbiol. 7, 1723-1735.]. However, the morphological and physiological interactions between Xenorhabdus symbionts and Steinernema IJs are not understood in depth. This study was undertaken to assess the influence of culture conditions and IJ age on the structure, nutrition, and symbiont load (colonization level) of S. carpocapsae vesicles. Our observations indicate the vesicles of axenic IJs are shorter and wider than those of colonized IJs. Moreover, as colonized IJs age the vesicle becomes shorter and narrower and bacterial load declines. The colonization proficiency of several bacterial metabolic mutants was compared between two cultivation conditions: in vitro on lipid agar and in vivo in Galleria mellonella insects. Colonization defects were generally less severe in IJs cultivated in vivo versus those cultivated in vitro. However, IJs from both cultivation conditions exhibited similar declining bacterial load over time. These results suggest that although the vesicle forms in the absence of bacteria, the presence of symbionts within the vesicle may influence its fine structure. Moreover, these studies provide further evidence in support of the concept that the conditions under which steinernematid nematodes are cultivated and stored affect the nutritive content of the vesicle and the bacterial load, and therefore have an impact on the quality of the nematodes for their application as biological control agents.

Structure of the pharynx in the adult nematode Anguillicoloides crassus (Nematoda: Rhabditida).

The structure of the pharynx of the adult female nematode Anguillicoloides crassus (Spirurina) has been studied for the first time using light and transmission electron microscopy. The cylindrical pharynx consists of a short anterior muscular corpus and an enlarged posterior glandular and muscular postcorpus. The main cellular components of the pharynx of A. crassus include the muscle cells, the marginal cells, the nerve cells, and 1 dorsal and 2 subventral glands. New observations for nematodes include: (1) the non-contractile regions of pharyngeal musculature in the corpus have specific appearance; (2) the ventrosublateral longitudinal nerve in the pharynx has an enlarged, enucleated anterior part, with a pronounced palmate projections; and (3) abundant lysosomelike membranous bodies consisting of myelinlike figures of varied size present in marginal cells and pharyngointestinal valve. The 2 subventral glands and, apparently, the single dorsal gland, have their openings at the same level, i.e., at the border between the corpus and postcorpus. The pharyngeal-intestinal valve joins the pharynx to the intestine. Knowledge of the ultrastructure of these complex characters may be useful in understanding of functional features, and for comparative morphology as well as evolutionary considerations within the Chromadorea.

Morphological observations and the effects of artificial digestive fluids on the survival of Diploscapter coronata from a Japanese patient.

Unusual non-human parasitic nematodes and eggs were detected in the faeces of an 8-year-old Japanese female suffering from Henoch-Schönlein purpura. The worms were adult female rhabditiform nematodes measuring 325.6-441.2 micro m in length and 18.3-26.5 micro m in width. One pair of the labia oris was notched with many spiny projections, while the other pair was strongly curved outwards. The worms were identified using light and scanning electron microscopy as the free-living nematode Diploscapter coronata (Cobb) based on their characteristic morphology. The patient's faeces containing worms and eggs were cultured using a filter-paper culture technique and after 7 days of culture, male as well as female worms were recovered. Worm survival time and hatchability of the eggs were examined in vitro after treatment with an artificial gastric or intestinal fluid. Although adult worms survived for less than one minute, eggs hatched after treatment with artificial gastric fluid. This suggests that eggs accidentally ingested or produced by adult D. coronata could develop in the human gastro-intestinal tract. Some morphological features of male D. coronata are also described.

Steinernema sichuanense n. sp. (Rhabditida, Steinernematidae), a new species of entomopathogenic nematode from the province of Sichuan, east Tibetan Mts., China.

Steinernema sichuanense n. sp. is characterized by male, female and IJ. For male, the spicules are robust with prominent rostrum; gubernaculum has blunt anterior end; cuneus is arrow-shaped, pointed posteriorly. Second-generation male has a prominent mucron. For female, tail usually has one to four papillae-like projections on tail tip; post anal swelling is absent. For IJ, body length is about 710 microm; lateral field has six ridges; the formula of lateral field is 2, 5, 6, 4, 2 with two prominent submarginal ridges; tail usually has a dorsal depression. In Steinernema affine/intermedium group, the IJ of S. sichuanense n. sp. differs from S. affine by its absence of the internal tail spine; differs from Steinernema beddingi by its six ridges in lateral field compared to 4 for S. beddingi. For male mucron is absent in both generations of S. affine, S. intermedium and S. beddingi, whereas it is present in the second-generation of S. sichuanense sp. n. Morphology and morphometrics of spicules and gubernacula of the four species in S. affine/intermedium group are quite different based on SEM photographs. For female, the postanal swelling is absent in the first-generation of S. sichuanense n. sp. whereas S. affine and S. intermedium have slight swelling and S. beddingi has conspicuous swelling. The new species is further recognized by characterization of sequences of ITS and D2/D3 regions of the ribosomal DNA. The symbiotic bacterium associated to S. sichuanense belongs to the species Xenorhabdus bovienii.

Phylogenetic relationships of Steinernema Travassos, 1927 (Nematoda: Cephalobina: Steinernematidae) based on nuclear, mitochondrial and morphological data.

Entomopathogenic nematodes of the genus Steinernema are lethal parasites of insects that are used as biological control agents of several lepidopteran, dipteran and coleopteran pests. Phylogenetic relationships among 25 Steinernema species were estimated using nucleotide sequences from three genes and 22 morphological characters. Parsimony analysis of 28S (LSU) sequences yielded a well-resolved phylogenetic hypothesis with reliable bootstrap support for 13 clades. Parsimony analysis of mitochondrial DNA sequences (12S rDNA and cox 1 genes) yielded phylogenetic trees with a lower consistency index than for LSU sequences, and with fewer reliably supported clades. Combined phylogenetic analysis of the 3-gene dataset by parsimony and Bayesian methods yielded well-resolved and highly similar trees. Bayesian posterior probabilities were high for most clades; bootstrap (parsimony) support was reliable for approximately half of the internal nodes. Parsimony analysis of the morphological dataset yielded a poorly resolved tree, whereas total evidence analysis (molecular plus morphological data) yielded a phylogenetic hypothesis consistent with, but less resolved than trees inferred from combined molecular data. Parsimony mapping of morphological characters on the 3-gene trees showed that most structural features of steinernematids are highly homoplastic. The distribution of nematode foraging strategies on these trees predicts that S. hermaphroditum, S. diaprepesi and S. longicaudum (US isolate) have cruise forager behaviours.