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The Japanese threadfin bream N. japonicus (Bloch) (Perciformes: Nemipteridae) is an important marine food fish in Asia. However, our present knowledge of the occurrence of its nematode parasites is still limited. In the present study, the species composition and infection rate of ascaridoid nematodes in N. japonicus from the South China Sea, were studied for the first time. Five ascaridoid species, namely Anisakis typica (L3), Hysterothylacium amoyense (L3), Hysterothylacium sp. IV-A (L3), adult of H. thalassini and Raphidascaris lophii (L3), were identified using integrative taxonomy. Hysterothylacium amoyense was the most prevalent species (prevalence 47.2%, mean intensity 14.9 ± 17.1). Hysterothylacium thalassini and R. lophii were reported in the Japanese threadfin bream for the first time. Two different genotypes of A. typica (overall prevalence of 3.4%; mean intensity 1.7 ± 0.9) were found in the South China Sea for the first time. The unique restriction polymorphism patterns of three species of Hysterothylacium are provided for rapid diagnosis. Our present results indicate that RFLP analysis of ITS region, using the restriction enzymes HhaI and RsaI, represents a simple and practical method for large-scale surveys of Hysterothylacium for seafood industry.

Background The whitespotted conger Conger myriaster (Brevoort) (Anguilliformes: Congridae) is an extremely marketable food fish, commonly consumed as sashimi or sushi in some Asian countries (i.e. Japan, Korea and China). Conger myriaster is also suspected as being an extremely important source of human anisakidosis. However, there is currently very little information on the levels of infection with ascaridoid nematode parasites in this economically important marine fish. The aims of the present study are to determine the species composition, prevalence and mean intensity of ascaridoid parasites of C. myriaster caught in the Zhoushan Fishery. Results A total of 1142 third-stage ascaridoid larvae were isolated from 204 C. myriaster. The overall prevalence of infection was 100% (mean intensity 5.6). Nine species of such larvae were accurately identified using integrative taxonomic techniques involving both morphological and genetic data; these included Anisakis pegreffii, A. typica and A. simplex (sensu stricto) × A. pegreffii, Hysterothylacium fabri, H. aduncum, H. sinense, H. amoyense, H. zhoushanense and Raphidascaris lophii. Although high levels of infection and species richness were revealed in C. myriaster, most of the ascaridoid parasites (1135 individuals) were collected from the body cavity and visceral organs of the fish and only seven individuals of A. pegreffii were found in the musculature. Conclusions This study represents the first report C. myriaster from the Zhoushan Fishery being heavily infected with third-stage ascaridoid larvae. Among the ascaridoid larvae parasitic in this fish, an important etiological agent of human anisakidosis, A. pegreffii (L3), represents the predominant species. The genus Hysterothylacium has the highest species richness, with H. fabri (L3) being the most prevalent species. This high level of infection of A. pegreffii (L3) in C. myriaster suggests a high risk of anisakidosis or associated allergies for people consuming raw or poorly cooked fish originating from this marine area. These findings provide important basic information on the occurrence and infection parameters of ascaridoid nematodes in this economically important marine fish. They also have significant implications for the prevention and control of human anisakidosis when conger eels from the Zhoushan Fishery are consumed.

Seuratascaris numidica (Seurat, 1917) is a specialized nematode species parasitizing amphibians only. In the present study, the detailed morphology of this poorly known species was studied using light and scanning electron microscopy based on the newly material collected from Hoplobatrachus chinensis (Osbeck) (Amphibia: Anura) in China. We found that the relative length of intestinal caecum in our male specimens (representing 68.4–71.1% of oesophageal length) is slighter longer than the previously reported data (not over 60.0% of oesophageal length). Our SEM observations also revealed the presence of ca. 64–76 small conical denticles on each lip. In addition, Angusticaecum wuyiensis Wang, 1981, collected from Rana schmackeri Boettger (Amphibia: Anura) from Wuyi Mountain in Fujian Province, China was considered as a new synonym of S. numidica. The ITS and cox1 sequences of S. numidica were also sequenced for the first time and there is no nucleotide variability detected in both regions. The present supplementary morphological and molecular data (especially the ITS and cox1 sequences) obtained herein is extremely important and useful to determine the morphological variability, population genetics and phylogenetic position of S. numidica in the future.

Species of Cosmocerca Diesing, 1861 (Ascaridomorpha: Cosmocercoidea), are common nematode parasites of amphibians. In the present study, a new species of Cosmocerca, namely C. simile n. sp., was described using light and scanning electron microscopy, and sequencing different nuclear and mitochondrial genetic markers (i.e. small ribosomal DNA (18S), large ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1)). Cosmocerca simile n. sp. differs from its congeners based on body size, morphology and number of plectanes, relative length of spicules and gubernaculum and spicules to total body length and morphology and length of tail. Molecular analysis showed no nucleotide polymorphisms among different individuals of the new species regarding nuclear DNA. Very low intraspecific nucleotide variation (0.52-0.78%) was detected in cox1 mtDNA. In contrast, the level of interspecific nucleotide variation between C. simile n. sp. and its congeners were distinctly higher (2.74-18.1% in the partial ITS region and 10.2-13.5% in the partial cox1 region, respectively) than that of intraspecific variation. Phylogenetic analyses using maximum likelihood (ML) inference based on the partial ITS and cox1 sequence data both supported the new species to be a member of the genus Cosmocerca, and formed a sister relationship to C. japonica. The newly obtained genetic data are important for further studies of DNA-based taxonomy, population genetics and phylogenetics of the Cosmocercoidea.

Species of Megalobatrachonema Yamaguti, 1941 (Ascaridida: Cosmocercoidea) are important nematode parasites in amphibians and reptiles. However, the phylogenetic relationship of its included two subgenera Megalobatrachonema and Chabaudgolvania remains unclear. In the present study, a new species of Megalobatrachonema, M. (Chabaudgolvania) wangi sp. nov., was described based on the specimens collected from the lesser spiny frog Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura) in China. The ribosomal [large ribosomal DNA (28S) and internal transcribed spacer (ITS1–5.8S-ITS2)] and mitochondrial [12S small subunit ribosomal DNA and cytochrome c oxidase subunit 1 (cox1)] target regions of the new species and M. (Chabaudgolvania) terdentatum, together with the 12S region of M. (Megalobatrachonema) hainanensis, were amplified and sequenced for molecular identification and phylogeny. Moreover, in order to clarify the systematic position of the new species and the phylogenetic relationship of the two subgenera Megalobatrachonema and Chabaudgolvania, phylogenetic analyses based on 28S + ITS1–5.8S-ITS2 + 12S sequence data were performed using maximum likelihood (ML) inference and Bayesian inference (BI). The molecular phylogenetic results conflicted with the current classification and challenged the validity of the subgenus Chabaudgolvania, that should be a synonym of the subgenus Megalobatrachonema. The presence or absence of valves in the oesophageal bulb as a key criterion for delimitation of the two subgenera Megalobatrachonema and Chabaudgolvania seems to be unreliable.

Subulura chinensis Schwartz, 1926 is a hitherto poorly known nematode species. The morphology of S. chinensis has not been sufficiently well described. In addition, the molecular data from species of the Subuluroidea are extremely limited. The detailed morphology of S. chinensis was studied using light microscopy and, for the first time, scanning electron microscopy, based on newly collected specimens from the little owl Athene noctua (Scopoli) (Strigiformes: Strigidae) in China. The internal transcribed spacer (ITS) of ribosomal DNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) target regions of S. chinensis were first amplified by polymerase chain reaction (PCR), then sequenced and analysed for the molecular identification of this species. Our SEM observations showed for the fist time the detailed morphology of the cephalic extremity, precloacal pseudo-sucker, caudal papillae, gubernaculum, phasmids and vulva of S. chinensis, and also determined the presence of a small, single medio-ventral precloacal papilla in the male. Moreover, we detected the presence of 0.08–0.40% and 0–1.30% nucleotide divergence among different individuals of S. chinensis in the ITS and cox1 regions, respectively. The supplementary morphological characters and genetic data will be very useful for the diagnosis of this poorly known species.

Marine fishes represent the important components of the diet in the coastal areas of China and they are also natural hosts of various parasites. However, to date, little is known about the occurrence of ascaridoid parasites in the frequently consumed marine fishes in China. In order to determine the presence of ascaridoid parasites in the frequently consumed marine fishes in the coastal town Huizhou, Guangdong Province, China, 211 fish representing 45 species caught from the South China Sea (off Daya Gulf) were examined. Five species of ascaridoid nematodes at different developmental stages were detected in the marine fishes examined herein, including third-stage larva of Anisakis typica (Diesing, 1860), third and fourth-stage larvae of Hysterothylacium sp. IV-A of Shamsi, Gasser & Beveridge, 2013, adult and third-stage larvae of Hysterothylacium zhoushanense Li, Liu & Zhang, 2014, adults and third-stage larvae of Raphidascaris lophii (Wu, 1949) and adults of Raphidascaris longispicula Li, Liu & Zhang, 2012. The overall prevalence of infection is 18.0%. Of them, Hysterothylacium sp. IV-A with the highest prevalence (17.5%) and intensity (mean=14.6) of infection was the predominant species. The prevalence and intensity of A. typica were very low (1/211 of marine fish infected with an intensity of one parasite per fish). The morphological and molecular characterization of all nematode species was provided. A cladistic analysis based on ITS sequence was constructed in order to determine the phylogenetic relationships of these ascaridoid parasites obtained herein. The present study provided important information on the occurrence and diagnosis of ascaridoid nematodes in the commercially important marine fishes from the South China Sea. The low level of infection and the species composition of ascaridoid nematodes seem to indicate the presence of low risk of human anisakidosis when local population consumed these marine fishes examined herein.

A new species of nematode,Orientatractis longicaudatan. sp. is described from the intestine ofIndotestudo elongata(Blyth) (Testudinidae) from Zoo of Tianjin, Tianjin, China. The new species can be easily distinguished from its congers by having longer tail, by the length of gubernaculum and left spicule, and by the numbers of caudal papillae. This is first species ofOrientatractisPetter, 1966 reported in China. A key to the species ofOrientatractisis provided.

A catalogue, based on both examined specimens and the published literature, of all the ascaridoid nematodes recorded in China is presented. A total of 95 recognised species, representing 26 genera in five families, are reported. Detailed information on the type-host, type-locality, original reference, synonyms, annotated subsequent references of taxonomic importance, other host records, site of infection, location of type-specimens and distribution are listed for each recognised species. Additional comments on the taxonomic status of some species are also given. Moreover, some nomenclatural changes are proposed: (i) Toxascaris selenarctis Wang, 1965 and T. ailuri Wu, He & Hu, 1987 are placed in synonymy with Baylisascaris transfuga (Rudolphi, 1819); (ii) Raphidascaris lophii Wang & Wu, 1991 is a secondary homonym of R. lophii (Wu, 1949) and a replacement name, R. wangi nom. nov., is proposed for the former species; (iii) Aliascaris aetoplatea Luo, 2001 is transferred to Terranova Leiper & Atkinson, 1914, as T. aetoplatea (Luo, 2001) n. comb., and should be considered a species inquirenda; (iv) Ophidascaris orientalis (Wang, 1965) is resurrected as a valid species; (v) Phocascaris longispiculum Wang & Wu, 1991 and Ophidascaris agkistrodontis Wang, 1979 are treated as incertae sedis; and (vi) Hysterothylacium sauridae Li, Xu & Zhang, 2008 is listed as a nomen nudum.

The starry batfish Halieutaea stellata (Vahl) is a small, benthic fish found in Indo-West Pacific Oceans. However, our present knowledge of the helminth parasites of this fish is still fragmentary. In this study, a total of 29 fish collected from the East and South China Sea were examined to determine the prevalence, intensity and species composition of helminth parasites in H. stellata. Using morphological and molecular approaches, four species of nematodes were found parasitic in this fish host, including the adults and fourth-stage larvae of Raphidascaroides nipponensis Yamaguti 1941; adults and third-stage larvae of Raphidascaris lophii (Wu 1949), third- and fourth-stage larvae of Hysterothylacium larval type IV-A of Shamsi, Gasser & Beveridge 2013 and third-stage larvae of Hysterothylacium amoyense (Hsu 1993). Halieutaea stellata represents a new host record for the three last-named nematodes. Raphidascaroides nipponensis with the highest prevalence (82.5%) and intensity (mean = 13.5) of infection was considered as the dominant parasite species in H. stellata. The detailed morphology of the different developmental stages of the four nematode species was studied using light and scanning electron microscopy. All nematode species were also genetically characterized by sequencing and analysing the internal transcribed spacer (ITS) of the ribosomal DNA. This study provides further data on the occurrence of nematode parasites in H. stellata and also contributes to facilitate an accurate and rapid diagnosis of the infection by these little-known nematodes.

A new cosmocercid species, Cosmocercoides qingtianensis sp. n., collected from the intestine of the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) is described using integrated approaches, including light and scanning electron microscopy, and sequencing and analyzing the ribosomal [small ribosomal DNA (18S) and internal transcribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit 1 (cox1)] target regions, respectively. The new species can be distinguished from its congeners by the combination of the following morphological characters, including the large body size, the presence of lateral alae and somatic papillae in both sexes, the length of spicules, the particular morphology and length of gubernaculum, the number, arrangement and morphology of caudal rosettes, the presence of large medioventral precloacal papilla and the long tail. Our molecular analysis revealed the level of intraspecific genetic variation of C. qingtianensis sp. n. distinctly lower than that of the interspecific genetic variation in the ITS and cox1 regions. However, there are some overlaps in the range of intra- and interspecific 18S sequence divergence between the new species and some closely related species. The results of molecular analysis supported the validity of the new species based on the morphological observations. The 18S, ITS, and cox1 regions of C. pulcher collected from Bufo japonicus formosus in Japan were also sequenced and analyzed. The results showed a low level of intraspecific genetic variation in 18S and ITS regions (0-0.12% and 0-0.23% nucleotide differences, respectively), but a relatively high level of intraspecific genetic variation in cox1 region (0.78-4.69% nucleotide differences). In addition, it seems more powerful and practical to use the cox1 region as a genetic marker for the accurate identification and differentiation of species of Cosmocercoides than the 18S and ITS regions, especially for the closely related species.

To obtain microRNA (miRNA) profile and clarify their biological function in tumorigenic Sca-1(+) CD34(+) cells during carcinogenesis of lung adenocarcinoma. After intranasal infection with recombinant Adeno-Cre viruses (AdV-Cre), lung adenocarcinoma was identified pathologically in Lox-stop-lox Kras (LSL-Kras) G12D mice. Sca-1(+) CD34(+) cells were sorted by flow cytometry and tested for tumor-initiating ability, self-renewal and tumorigenicity. MiRNA profiles were obtained using microarray and further confirmed by real-time RT-PCR (qRT-PCR). MiRNA functions were predicted bioinformatically, and miR-294 function was verified to explore its role in tumor migration and invasion. Lung adenocarcinoma was induced in LSL-Kras G12D mice within 30 days. In vivo, the tumorigenicity of Sca-1(+) CD34(+) cells was 25 times stronger than Sca-1(-) CD34(-) cells. During tumorigenesis of lung adenocarcinoma, the expression of 145 miRNAs in Sca-1(+) CD34(+) cells increased and 72 miRNAs decreased (P < 0.01). Four successively up-regulated miRNAs (miR-15a*, miR-203, miR-294 and miR-295*) and three successively down-regulated ones (miR-19b, miR-483 and miR-615-5p) were identified. Among them, miR-294 could constitutively bind to 3'-UTR of matrix metalloproteinase 3 (MMP3), and down-regulate MMP3 protein expression. MiR-294 also significantly inhibited migration and invasion of Lewis lung cancer cells. MiRNAs are characteristically expressed in tumor-initiating Sca-1(+) CD34(+) cells of lung adenocarcinoma, and may play important roles during the carcinogenesis of lung adenocarcinoma.

Ascaridoids are among the commonest groups of zooparasitic nematodes (roundworms) and occur in the alimentary canal of all major vertebrate groups, including humans. They have an extremely high diversity and are of major socio-economic importance. However, their evolutionary history remains poorly known. Herein, we performed a comprehensive phylogenetic analysis of the Ascaridoidea. Our results divided the Ascaridoidea into six monophyletic major clades, i.e., the Heterocheilidae, Acanthocheilidae, Anisakidae, Ascarididae, Toxocaridae, and Raphidascarididae, among which the Heterocheilidae, rather than the Acanthocheilidae, represents the sister clade to the remaining ascaridoids. The phylogeny was calibrated using an approach that involves time priors from fossils of the co-evolving hosts, and dates the common ancestor of the Ascaridoidea back to the Early Carboniferous (approximately 360.47-325.27 Ma). The divergence dates and ancestral host types indicated by our study suggest that members of the Ascaridoidea first parasitized terrestrial tetrapods, and subsequently, extended their host range to elasmobranchs and teleosts. We also propose that the fundamental terrestrial-aquatic switches of these nematodes were affected by changes in sea-level during the Triassic to the Early Cretaceous.

Heliconema hainanensis sp. nov. collected from Uroconger lepturus (Richardson) (Anguilliformes: Congridae), Muraenesox cinereus (Forsskål) and Congresox talabonoides (Bleeker) (Anguilliformes: Muraenesocidae) in the South China Sea was described using light and scanning electron microscopy. The new species differs from its congeners by the following morphology: pseudolabia, the number and arrangement of caudal papillae (4 pairs of pedunculate precloacal papillae arranged in 2 groups of 2 and 2 pairs and 6 pairs of pedunculate postcloacal papillae arranged in 4 groups of 1, 2, 1 and 2 pairs), the length of spicules [left spicule 0.51-0.69 mm, right spicule 0.20-0.27 mm, spicule (right:left) ratio 1:2.20-2.69] and the morphology of the female tail tip. In addition, specimens of the new species collected from the three different hosts and specimens of an unidentified species of Heliconema collected from U. lepturus were characterised using molecular methods by sequencing the internal transcribed spacer (ITS) of ribosomal DNA. Analyses and comparison of the ITS sequence of H. hainanensis sp. nov. with Heliconema sp. support the validity of the new species based on morphological observations. An identification key to the species of Heliconema is also provided.

Durettenema guangdongense gen. et sp. nov. is described from Hipposideros larvatus (Horsfield) (Chiroptera: Rhinolophidae) in Guangdong Province, China. The new genus differs from the other genera of subfamily Molineinae in the structure of the synlophe, the absence of lateral alae, the arrangement of the bursa rays, the shape of the spicules, the female tail and the presence of gubernaculum. Meanwhile, considering the morphological characters of Macielia rhinolophi Yin, 1980, including the pattern of the bursa ray, the shape of the spicules, and the female tail, this species should be transferred to the genus Durettenema, as D. rhinolophi (Yin, 1980) comb. nov., which can be distinguished from D. guangdongense in the shape of the ovejector. In addition, the ITS-1 sequences of D. guangdongense were also analysed, these sequence added new data for the molecular diagnosis of trichostrongylid nematodes.

Habronematid nematodes were collected from the stomachs of donkeys, Equus asinus L., in the Tarim Basin, Xinjiang, China. After examination by light and scanning electron microscopy, Habronema muscae (Carter, 1861) and H. majus (Creplin, 1849) were identified. The morphology of our specimens representing H. muscae (Carter, 1861) agreed well with previous redescriptions in the shape of the lateral lips, origin of the lateral alae, ratio of left and right spicules, and number and arrangement of caudal papillae. However, H. majus (Creplin, 1849) differs from H. microstoma (Schneider, 1866) in the arrangement of the caudal papillae in the male. Moreover, molecular analysis also showed interspecific differences of 26.2-28.2% in ITS2 and 8.6-8.9% in cox1 between H. majus and H. microstoma, a divergence much higher than the known intraspecific variation of Habronema spp. (6.6-8.7% in ITS2; 0.2-2.2% in cox1). The results indicate that both H. microstoma (Schneider, 1866) and H. majus (Creplin, 1849) are valid species.

Longicollum pagrosomi Yamaguti, 1935 (Acanthocephala: Pomphorhynchidae) collected from the barred knifejaw Oplegnathus fasciatus (Temminck & Schlegel) (Perciformes: Oplegnathidae) in the East China Sea (off Zhoushan Islands) was studied using light and scanning electron microscopy. The SEM observations revealed for the first time the presence of about 28 well-developed sensory papillae arranged in a circle on the copulatory bursa. In addition, L. pagrosomi was characterised using molecular methods by sequencing of the internal transcribed spacer (ITS) of the ribosomal DNA based on the newly collected material. Longicollum pagrosomi is the first species of the genus with the ITS region sequenced for the purpose of species identification. These new morphological and molecular data contributed to a reliable and accurate specific identification and differentiation of species.

Hysterothylacium zhoushanensis sp. nov. collected from the intestine of the flatfish Pseudorhombus oligodon (Bleeker) (Pleuronectiformes: Paralichthyidae) in the East China Sea is described and illustrated by light and scanning electron microscopy. The new species can be easily distinguished from its congeners by the presence of remarkable lateral alae, the very short intestinal caecum, the unusually long ventricular appendix (ratio of intestinal caecum to ventricular appendix 1:8.74-23.8), the short spicules (0.58-0.81 mm long, representing 1.70-2.08 % of body length) and the number and arrangement of male caudal papillae (35-42 pairs in total, arranged as 26-32 pairs of precloacal, two pairs of paracloacal and six to eight pairs of postcloacal). In addition, the adults and the putative third-stage larvae identified morphologically of the new species are characterised by sequencing and analysing the internal transcribed spacer (ITS) of the ribosomal DNA. The result reveals that they are homogeneous genetically, and all belong to the same species. Molecular analysis by comparing the ITS gene of H. zhoushanensis sp. nov. with these species of Hysterothylacium available in GenBank also seem to support the validity of the new species based on the morphological observation.

Goeziine nematodes identified as Goezia nankingensis Hsü, 1933 were collected from the Chinese paddlefish Psephurus gladius (Martens) (Acipenseriformes: Polyodontidae), a critically endangered freshwater fish, from the Yangtze River in China. Their examination, using light microscopy and, for the first time, scanning electron microscopy, revealed some erroneous and previously unreported morphological features, necessitating the redescription of this poorly known species. Goezia nankingensis is easily distinguished from its congeners by the morphology and arrangement of its cuticular spines, the short intestinal caecum and long ventricular appendix (ratio of intestinal caecum to ventricular appendix 1:10.0-18.3), the short spicules (0.34-0.49 mm long, representing 6.09-10.1% of the body length), the number and arrangement of male caudal papillae [14-16 pairs in total, approximately arranged: precloacal 7-10 pairs, paracloacal 2 pairs and postcloacal 4-6 pairs (one pair double)], and the presence of a particular medio-ventral, precloacal papilla in the male.

Nematodes of the family Anisakidae parasitize fish, mammals, birds and reptiles, with the larval stages of some species causing severe clinical disease in humans. Therefore, the accurate identification of anisakid nematodes is a key component of disease surveillance and control. An epidemiological survey of 123 fishes comprising eight different species from the Yellow Sea in China revealed that more than 25% of fish were infected with the larvae of anisakids, 200 third-stage larvae (L3s) were collected from fish and then subjected to morphological and molecular study. Larvae identified as Anisakis type I (n=197) and Hysterothylacium sp. (n=3), based on morphological criteria were characterized genetically by mutation scanning, followed by targeted sequencing of the first and second internal transcribed spacers of nuclear ribosomal DNA. Comparison of the sequences obtained from a subset of 27 specimens with those available in public gene databases showed that all samples identified morphologically as Anisakis type I (n=197) were Anisakis pegreffii, whereas those identified as Hysterothylacium sp. (n=3) were Hysterothylacium aduncum. The approach used herein was an effective means of matching incompletely identifiable larval nematodes with identifiable reference sequences, and provides a basis for exploring the composition of populations of anisakid larvae in fish as well as their ecology, particularly their life cycles and transmission patterns.

Ophidascaris excavata Hsu & Hoeppli, 1931 is a poorly known ascaridid parasite reported from the short-tailed pit viper Gloydius brevicaudus (Stejneger) (Reptilia: Viperidae) in China. In the present paper, the detailed morphology of this nematode was studied using light and scanning electron microscopy (SEM) based on newly collected material. The results revealed several important, but previously unreported, morphological features, including the presence of one pair of small, finger-like prolongations on each lip, narrow cervical alae beginning well posterior to the base of the ventrolateral lips and the second pair of postcloacal ventro-lateral papillae being double; in addition, there is no intestinal caecum. These supplementary morphological and morphometric data, especially the detailed morphological features obtained herein under SEM, would help us to understand the relationships of O. excavata with its congeners and enable us to diagnose this species more accurately.

Porrocaecum parvum n. sp. is described from the grey-faced buzzard Butastur indicus (Gmelin) (Accipitriformes: Accipitridae) in China. The new species differs from its congeners in having well-developed cervical alae, small interlabia and very short intestinal caecum (0.34 mm long, representing 11.9% of oesophageal length) and in the number and arrangement of the caudal papillae (29 pairs in total, arranged as follows: 21 pairs precloacal, single double pair paracloacal and seven pairs postcloacal) and in the morphology of the male tail. In addition, Porrocaecum reticulatum (Linstow, 1899), collected from the purple heron Ardea purpurea L., the grey heron A. cinerea L. and the little egret Egretta garzetta (L.) (Pelecaniformes: Ardeidae) in China, was also studied using light and, for the first time, scanning electron microscopy. Previously unreported and erroneous morphological features of taxonomic significance are revealed, including the presence of narrow cervical alae, single pair of small, submedial pores and single, short medial ditch on each lip, interlabia with very pointed anterior prolongation, single medio-ventral precloacal papilla on anterior cloacal lip and double paracloacal papillae slightly posterior to cloaca.

Ascaridia galli (Schrank, 1788) is a common parasite of various galliform birds worldwide. Although A. galli has been extensively studied by many author, knowledge of the morphology of this species in detail is still insufficient. In the present paper, the detailed morphology of A. galli was further studied using light and scanning electron microscopy, based on specimens collected from the endangered green peafowl Pavo muticus Linnaeus (Galliformes: Phasianidae) in China. The results revealed some erroneous and previously unreported morphological features, including the lips lacking real denticles, the lateral alae beginning at some distance posterior to the base of the ventrolateral lips and the caudal papillae with 4 different morphotypes. The present morphological and morphometric data complement previous descriptions and enable us to recognize this species more precisely.

Ophidascaris wangi sp. n. collected from the king rat snake Elaphe carinata (Gunther) (Serpentes: Colubridae) in China is described using both light and scanning electron microscopy. The new species differs from its congeners in the presence of narrow lateral alae originating a short distance posterior to the base of the ventrolateral lips, its relatively long oesophagus (3.57-4.54 mm long, representing 6.6-7.6% of body length), its short spicules (1.89-2.14 mm long, representing 3.9-4.3% of body length), the number and arrangement of caudal papillae (49-57 pairs in total, arranged as follows: 43-51 pairs precloacal, 2 pairs joined paracloacal and 4 pairs postcloacal), the presence of a particular papilliform medioventral, postcloacal ornamentation and the morphology of the eggs and tip of the female tail. In addition, Ophidascaris najae (Gedoelst, 1916), collected from the king cobra Ophiophagus hannah Cantor (Serpentes: Elapidae) in China, is also redescribed. The morphology of the cervical papillae, labial denticles and phasmids of the female is described for the first time.

Philometra lagocephali Moravec et Justine, 2008 was redescribed using light and scanning electron microscopy based on the new material collected from the lunartail puffer Lagocephalus lunaris (Bloch et Schneider) (Tetraodontiformes: Tetradontidae) in the South China Sea. Our specimens are identical with the original description in the body size of gravid female, the width of cephalic and caudal end, the number and arrangement of cephalic papillae, the relative length of the oesophagus to body length, the size of ventriculus, the morphology of oesophageal bulb and posterior end, and the body size of larvae. In addition, specimens of P. lagocephali were also characterized using molecular methods by sequencing and analysing the small ribosomal DNA (18S rDNA) and mitochondrial cytochrome c coxidase subunit 1 (cox1) target regions. Phylogenetic analyses of genus Philometra base on 18S rDNA sequences was made using Mega 5.2. The results confirmed P. lagocephali is a valid species. It’s the first molecular study of this species. These new morphological and molecular data contributed to a more precise and rapid diagnosis of this hitherto poorly known philometrid nematode.

Toxascaris leonina (Linstow, 1902) is a most common intestinal parasite of various animals in Felidae and Canidae. In the present paper, light and scanning electron microscopy (SEM) were used to study the morphological aspects of adult worms of this nematode in detail, based on the material collected from Panthera leo (Linnaeus) and Felis lynx (Linnaeus) (Carnivora: Felidae) in China. The results showed that there were some morphometric differences between the present material and the previous studies, including the body size, the width and length of cervical alae, the number of denticles on each lip and the tail length of the female. Previously unreported morphological features were also revealed. These supplementary morphological and morphometric data contributed to a more accurate identification of this worldwide distributed ascarid nematode.

A new cucullanid nematode, Dichelyne (Dichelyne) breviculus n. sp., collected from the intestine of the goatee croaker Dendrophysa russelii (Cuvier) (Perciformes: Sciaenidae), the burrowing goby Trypauchen vagina (Bloch & Schneider) and the tropical sand goby Acentrogobius caninus (Valenciennes) (Perciformes: Gobiidae) in the South China Sea, is described using both light and scanning electron microscopy. The new species differs from its congeners in the size of body (2.16–2.96 mm in male), the position of the excretory pore and deirids, the length of the spicules (0.90–1.32 mm, representing 32.4–51.9% of body length), the arrangement of the caudal papillae and the morphology of the tail. In addition, in order to primarily assess the validity of the new species genetically, the specimens of D. breviculus n. sp. collected from the three different hosts were also characterised using molecular methods by sequencing and analysing ribosomal [small ribosomal subunit (18S rDNA) and internal transcribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit 1 (cox1)] target regions. The molecular analyses support the validity of the new species based on the morphological observations.

The Chinese alligator Alligator sinensis Fauvel (Reptilia: Crocodylia) is considered as one of the most critically endangered species of the 23 extant crocodiles. However, our knowledge of the helminth parasites of this rare animal is completely lacking. During a helminthological survey of reptiles in China, we found a new ascaridoid nematode, Dujardinascaris gigantea sp. n. from A. sinensis. The morphology of D. gigantea sp. n. was studied using light and scanning electron microscopy. The new species was also characterised using molecular methods by sequencing and analysing the small ribosomal DNA (18S) and the second internal transcribed spacer (ITS-2).

A new species of ascaridoid nematode, Hysterothylacium gibsoni sp. nov., is described based on specimens collected from the intestine of the slender lizardfish Saurida elongata (Temminck et Schlegel) (Aulopiformes: Synodontidae) in the Yellow Sea, China. The new species differs from its congeners by its small body size (12.8–13.2 mm), the absence of cervical alae, a very short intestinal caecum (representing 8.86–9.52% of oesophageal length) and a long ventricular appendix (intestinal caecum to ventricular appendix ratio 1:15.3–20.0), short spicules (0.38–0.41 mm, representing 2.97–3.11% of body length), the number and arrangement of the caudal papillae (25–28 pairs arranged as follows: 18–22 pairs precloacal, 3 pairs paracloacal, and 3–4 pairs postcloacal). In addition, Hysterothylacium tetrapteri (Bruce et Cannon, 1989) is also redescribed based on the material collected from the striped marlin Kajikia audax (Philippi) (Perciformes: Istiophoridae) in the South China Sea.

Cucullanus hainanensis sp. nov., collected from Muraenichthys gymnopterus (Bleeker) (Anguilliformes: Ophichthidae) in the South China Sea, was described using both light and scanning electron microscopy. The new species can be readily distinguished from its congeners by the large pseudobuccal capsule, the position of excretory pore and deirids, the length of spicules (0.64–0.76 mm, 5.84–6.67% of body length) and gubernaculum (0.21–0.24 mm), the number and arrangement of caudal papillae and the particular morphology of cloacal region in male. The new species was also characterized using molecular methods by sequencing and analysing the small subunit (18S) and the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA). In addition, Cucullanus muraenesocis (Yin et Zhang, 1983) was regarded a homonym of C. muraenesocis Yamaguti, 1961, and a new name, Cucullanus wangi nom. nov. was given to it.

Cucullanid nematodes identified morphologically as Dichelyne (Cucullanellus) pleuronectidis (Yamaguti, 1935) were collected from the ridged-eye flounder Pleuronichthys cornutus (Temminck & Schlegel) (Pleuronectiformes: Pleuronectidae) in the East China Sea. Their examination using light microscopy and, for the first time, scanning electron microscopy, revealed several important, but previously unreported morphological features and the presence of remarkable morphological differences in the intestinal caecum and deirids among some individuals. Consequently, specimens of D. pleuronectidis were characterised using molecular methods by sequencing and analysing the internal transcribed spacer (ITS) of the ribosomal DNA to test whether the present material with broad range of morphological variability, represents a complex of sibling species or a single species. The results of molecular analyses proved that the differences in the intestinal ceacum and deirids should be considered as intraspecific variation and that the nematode material collected from P. cornutus in the East China Sea represented a single species, D. pleuronectidis. These new morphological and genetic data contributed to an accurate diagnosis of this hitherto insufficiently known nematode and also indicated that a more rigorous study based on morphological and genetic data with broader representation of the Cucullanidae is required to assess whether the traditionally used diagnostic character of absence or presence of intestinal ceacum is of generic importance in distinguishing Dichelyne and Cucullanus.

Gnathostomatid nematodes identified morphologically as Spiroxys japonica Morishita, 1926 were collected from the dark-spotted frog Pelophylax nigromaculatus (Hallowell) (Amphibia: Ranidae) in China. Light and scanning electron microscopy were used to study the morphology of this species in detail. Previously unreported morphological features are revealed and others corrected. In addition, adult nematodes of S. japonica collected from P. nigromaculatus and Spiroxys hanzaki Hasegawa, Miyata & Doi, 1998 collected from Andrias japonicus (Temminck) (Caudata: Cryptobranchidae) in China and Japan, respectively, and the third-stage larva of S. japonica collected from Lithobates catesbeianus (Shaw) (Anura: Ranidae) in Japan, were characterised using molecular methods by sequencing and analysing ribosomal [large ribosomal DNA (18S) and internal transcribed space] and mitochondrial [cytochrome c oxidase subunit 1] target regions, respectively. The new morphological and genetic data contributes to a more accurate diagnosis of this hitherto little known nematode genus.

To construct a lentiviral expression vector of the PIAS-NY gene, and establish a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.PIAS-NY was synthesized, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU. After digestion and sequencing, pGC-FU-PIAS-NY, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells. Then the lentiviral particles were used to transfect the mouse spermatocyte-derived cells. The expression of the PIAS-NY protein was detected by Western blot.We successfully constructed the lentiviral expression vector pGC-FU-PIAS-NY and established a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.The construction of the lentiviral expression vector pGC-FU-PIAS-NY and the obtainment of stably transfected mouse spermatocyte-derived cells have paved the way for further studies on the roles of the PIAS-NY gene in spermatogenesis.

Seven species of Cylicocyclus Ihle, 1922 (Nematoda: Strongylidae) were collected from donkeys from Henan Province, China. Five samples of each species were selected for sequencing. Sixteen different internal transcribed spacer (ITS) sequences representing the seven species of Cylicocyclus were obtained. Sequence differences in the first internal transcribed spacer (ITS-1) among species was lower than that of the second internal transcribed spacer (ITS-2). Phylogenetic analyses were conducted using the combined ITS-1 and ITS-2 data sets from the present study and using reference sequences from the GenBank database. The MP and ML trees were similar in topology. The phylogenetic trees were divided into two clades. Clade I included 8 species of Cylicocyclus; within this group, Cylicocyclus leptostomus (Kotlan, 1920) is nested between different samples of Cylicocyclus ashworthi (LeRoux, 1924), suggesting C. ashworthi may represent a species complex. Clade II included Cylicocyclus elongatus (Looss, 1900) and Cylicocyclus ultrajectinus (Ihle, 1920); however, these two species always clustered with the comparative species (Petrovinema poculatum (Looss, 1900) and Poteriostomum imparidentatum Quiel, 1919), suggesting that C. elongatus and C. ultrajectinus represent members of other genera.

Contracaecum spasskii Mozgovoi, 1950, collected from the great crested grebe Podiceps cristatus (Linnaeus) (Podicipediformes: Podicipedidae), is redescribed using both light and, for the first time, scanning electron microscopy. Contracaecum spasskii differs from its congeners by having marked transverse cuticular annulations, the length of the oesophagus and spicules, the ratio between the intestinal caecum and the ventricular appendix, the number and arrangement of male caudal papillae, and especially by the particular morphology of the lips and interlabia. Some previously unreported morphological features of C. spasskii are also revealed and others corrected. Contracaecum rudolphii Hartwich, 1964 (sensu lato) is also redescribed based on the specimens collected from the great cormorant Phalacrocorax carbo sinensis (Blumenbach) (Pelecaniformes: Phalacrocoracidae) from China. Based on the geographical perspective, the present Chinese material may represent the species C. rudolphii B.

Mawsonascaris parva sp. nov. collected from the Arabian whipray Himantura randalli Last, Manjaji-Matsumoto et Moore (Rajiformes: Dasyatidae) off the coast of Iraq is described and illustrated using light and scanning electron microscopy. The new species differs from its congeners by its small body size (8.11–18.2 mm long), the absence of an intestinal caecum, the slightly unequal spicules (left and right spicule 1.08–2.50 mm and 0.98–2.01 mm long, respectively, representing 12.1–13.7% and 10.1–12.1% of body length), the number and arrangement of male caudal papillae (13–14 pairs in total, arranged as: 7–8 pairs of precloacal, 1 pair of double paracloacal and 5 pairs of postcloacal) and the presence of a single, median precloacal papilla. Thus, a total of six species of Mawsonascaris are now known and this is the first species of this genus recorded from species of Himantura.

Hysterothylacium simile n. sp., collected from the Japanese seabass Lateolabrax japonicus (Cuvier) (Perciformes: Lateolabracidae) in the Bohai Sea off China, is described using both light and scanning electron microscopy. The new species differs from its congeners in the presence of narrow lateral alae originating a short distance posterior to the base of the ventrolateral lips, a long intestinal caecum (60.4-79.1% of oesophageal length) and a relatively short ventricular appendix (intestinal caecum to ventricular appendix ratio 1:0.58-0.85), long spicules (2.11-2.99 mm, 4.25-7.83% of body length), the number and arrangement of the caudal papillae (32-36 pairs arranged as follows: 27-31 pairs precloacal, 1 pair paracloacal, and 4-5 pairs postcloacal with the second or third pair double) and the presence of a particular midventral precloacal papilla. Specimens originally identified as Contracaecum paralichthydis Yamaguti, 1941 [now H. paralichthydis (Yamaguti, 1941)] by Xu (1957), collected from the yellow striped flounder Pseudopleuronectes herzensteini (Jordan & Snyder) (Pleuronectiformes: Pleuronectidae) in the Yellow Sea off China, were also re-examined. Their morphology clearly revealed they belong to H. aduncum (Rudolphi, 1802), which is also redescribed based on Xu's material. In addition, the morphological variation of caudal papillae in H. aduncum from P. herzensteini was compared, using scanning electron microscopy, with specimens collected from another three fish hosts, Lophius litulon (Jordan) (Lophiiformes: Lophiidae), Scomberomorus niphonius (Cuvier) (Perciformes: Scombridae) and Cleisthenes herzensteini (Schmidt) (Pleuronectiformes: Pleuronectidae), from the Yellow Sea off China.

The little known ascaridoid nematode Raphidascaris (Ichthyascaris) lophii (Wu, 1949) is redescribed and illustrated based on newly collected specimens from the five different marine fishes: Lophius litulon (Jordan) (Lophiiformes: Lophiidae), Lophiomus setigerus (Vahl) (Lophiiformes: Lophiidae), Antennarius hispidus (Bloch et Schneider) (Lophiiformes: Antennaridae), Zeus faber Linnaeus (Zeiformes: Zeidae) and Ostichthys japonicus (Cuvier) (Beryciformes: Holocentridae) from the East and South China Sea. This species differs from all congeners in the subgenus Ichthyascaris by the length of the ventricular appendix (0.52–0.98 mm long), the number and arrangement of caudal papillae (26–32 pairs of precloacal, 3–4 pairs of paracloacal and 8–11 pairs of postcloacal) and the length of the spicules (0.49–0.88 mm long, representing 3.08–4.70% of body length). In addition, nematodes collected from these five different fishes have been characterized using molecular methods by sequencing and analysing the internal transcribed spacer (ITS) of ribosomal DNA. No variation in size and nucleotide polymorphisms is detected within the target sequence among all samples analysed. These data contribute to facilitate an accurate diagnosis of this poorly known nematode. An identification key to the species of the subgenus Ichthyascaris is also provided.

Aspiculuris tianjinensis sp. nov. recovered from the intestine of Clethrionomys rufocanus from Tianjin, China is described and illustrated using light microscopy and scanning electron microscopy. The new species differs from congeners in the shape of the cervical alae, and the number and arrangement of caudal papillae.

To screen and identify PIAS2-interacting proteins from the mouse spermatogonial cDNA library using the yeast two-hybrid system, and to investigate the action mechanism of PIAS2 in spermatogenesis.With pGBKT7-PIAS2 as a bait plasmid, the positive clones interacting with pGBKT7-PIAS2 were screened from the mouse spermatogonial cDNA library, the inserted fragments were sequenced and underwent bioinformatic analysis, and their interaction was verified using the yeast two-hybrid system.Through screening, sequencing, homology analysis and yeast two-hybrid verification, we obtained 8 different candidate proteins interacting with PIAS2, including Cyfip2, Psmb3, Nmel, nischarin, Ints10, Nsun5, Gnb211 and Ndufaf3.Eight different genes were successfully obtained using the yeast two-hybrid system, and their encoding proteins interacted with PIAS2, which might be related with male fertility regulation. Our findings have offered some new clues to the action mechanism of PIAS2 in spermatogenesis.

Ascaridoid nematodes identified as Pseudanisakis rajae (Yamaguti, 1941) were collected from the skates Bathyraja smirnovi (Soldatov & Pavlenko), Okamejei kenojei (Muller & Henle) and Raja pulchra Liu (Rajiformes: Rajidae) in the Yellow Sea and Taiwan Strait off the coast of China. Their examination using light microscopy and, for the first time, scanning electron microscopy revealed erroneous and previously unreported morphological features, necessitating the redescription of this little known species. In addition, specimens of P. rajae collected from the three different hosts were characterised using molecular methods by sequencing and analysing the internal transcribed spacer (ITS) of the ribosomal DNA. These new morphological and molecular data enabled an updated diagnosis of this nematode and the presentation of an identification key to the species of Pseudanisakis Layman & Borovkova, 1926.

A new anisakid nematode, Raphidascaris (Ichthyascaris) longispicula sp. nov., is described from the intestine and stomach of the marine fish Uroconger lepturus (Richardson) (Anguilliformes: Congridae) from the South China Sea (Guangdong and Hainan Province, respectively). The new species differs from all congeners in the subgenus Ichthyascaris by the short ventricular appendix (0.36–0.49 mm long), the number and arrangement of caudal papillae (25–28 pairs of preanal, 1–2 pairs of paranal, and 6–8 pairs of postanal), and the very long spicules (1.13–1.32 mm long, representing 9.34–10.3% of body length). The sequences of the internal transcribed spacer of ribosomal DNA of R. (I.) longispicula sp. nov. are analyzed and compared with the closely related nematode sequences. Molecular analyses seem to support the validity of this new nematode species based on morphological observation.

Larval nematodes were collected from marine fishes from the Yellow Sea, China. Specimens (n=1731) of Anisakis type I from 311 fishes (representing 40 species) were each identified based on morphological characters. From the genomic DNA from individual specimens, a region of nuclear ribosomal DNA was amplified by PCR, followed by digestion with restriction endonuclease HinfI, TaqI or HhaI. Subsequently, the ITS-1 and ITS-2 regions of selected samples were sequenced. The results revealed three species of Anisakis, namely Anisakis pegreffii (n=1709), A. typica (n=3) and a genotype (n=19) proposed, also based on comparison with previous studies, to be a "hybrid" between A. pegreffii and A. simplex sensu stricto. Thus, A. pegreffii was the dominant species, accounting for 98.7% of the total number of specimens examined herein. This is the first report of A. typica and the "hybrid" genotype from fishes from the Yellow Sea. This study provides important basic information on Anisakis in this region and suggests that the genus Anisakis has substantial host and geographical distributions.

In the present study, we have extended earlier taxonomic, biochemical and experimental investigations to characterize two species of Taenia from carnivores in Kenya by use of the sequences of a variable domain (D1) of nuclear ribosomal DNA and the cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 genes of mitochondrial DNA. Emphasis was placed on the characterization of Taenia madoquae from the silver-backed jackal (Canis mesomelas) and Taenia regis from the lion (Panthera leo), given the previous absence of any DNA sequence data for them, and on assessing their genetic relationships with socioeconomically important taeniids. The study showed that T. regis was genetically most closely related to T. hydatigena, and T. madoquae to T. serialis, T. multiceps or T. saginata. The present findings provide a stimulus for future work on the systematic relationships and epidemiology of lesser-known taeniid cestodes in Africa and other continents, employing mitochondrial sequence data sets.

The expansion segments or divergent (D) domains in the large subunit (LSU) of the ribosomal DNA have been suggested as genetic markers for taxonomic and/or phylogenetic studies of parasites. In the present study, we assessed the degree of sequence variation in the D3 domain and flanking core regions of the LSU for 21 species of equine strongyles (Strongylida: Strongylidae) and determined which positions in the secondary structure of the LSU were associated with the nucleotide alterations. No intraspecific sequence variation was detected in 17 species, for which multiple individual worms were available. Mutations in sequence among species were detected at 19 nucleotide positions, most of which were located in the D3 domain. Fifteen alterations were transitions, three were transversions and one represented a site of multiple mutations. In relation to the secondary structure element of D3, 26% of these mutations were located in unpaired regions (i.e., end of loops, or in bulges of helices) and thus did not appear to alter the pairing arrangement in the helices of the secondary structure. Many of the other mutations represented partial or complete compensatory base pair changes. The magnitude of interspecific nucleotide variation in the D3 domain (0-4%) was considerably less than that recorded for some other nematode groups (enoplids and thelastomatoids), indicating that this region alone is of limited value for taxonomic and phylogenetic studies for strongyles of equids but is interesting in relation to the evolution of ribosomal DNA.

International audience; OBJECTIVE: To construct and express DNA plasmid of Trichinella spiralis. METHODS: The BamHI and HindIII enzyme sites and KOZAK sequences were introduced at both ends of Ts87 gene by PCR. Ts87 gene was ligated into pcDNA3.1(+) vector with T4 ligase. The recombinant plasmid pcDNA3.1/Ts87 and plasmid pcDNA3.1 were transfected into an eukaryotic cell line COS7 through Lipofectamine, respectively. The BALB/c mice were immunized with the purified plasmid DNA pcDNA3.1/Ts87 through two routes: intramuscular injection and gene-gun injection. RESULTS AND CONCLUSION: The pcDNA3.1/Ts87 was expressed both in COS7 and in the BALB/c mice.

To investigate Hysterothylacium aduncum (Anisakidae) infection in marine fishes from Bohai Sea and Yellow Sea.Nematodes were collected from the digestive tract of fishes, fixed with hot 4% formalin and preserved in 70% ethanol for study. The specimens were cleared in lactophenol for light microscopical examination, and properly treated for scanning electron microscopy (SEM).Among the fishes examined, 14 out of 93 species (15.1%) were found infected by H. aduncum, with a higher prevalence in the fish of Lophius litulon (66.7%), Scomberomorus niphonius (47.5%), and Gadus macrocephalus (33.3%). H. aduncum infection was first recorded in elasmobranch-Raja smirnovi. Morphological differences of H. aduncum were observed, including the width of lateral alae and the length of intestinal caecum.H. aduncum in fishes of Bohai Sea and Yellow Sea in China may be a complex species, and its high prevalence in some fishes reminds the risk of anisakiasis by eating raw fishes.