A New Species of Cosmocercoides (Ascaridida: Cosmocercidae) Parasitic in Tree Frogs from Southern Amazonia.

Cosmocercoides meridionalis n. sp. is described from the large and small intestines of 5 species of tree frogs belonging to the families Hylidae and Phyllomedusidae collected from 2 localities in Southern Amazonia. The new species is allocated to the genus Cosmocercoides Wilkie, 1930, due to the presence of papillae in rosettes, which are complex caudal papillae surrounded by punctuations. Cosmocercoides meridionalis n. sp. differs from its congeners by a combination of the following characters: the absence of the gubernaculum, the total length of the female (twice the size of the male), and the presence of 26 rosette papillae with a unique arrangement and distribution pattern: 11 pre-cloacal pairs, 1 ad-cloacal pair, and 1 post-cloacal pair. This is the first species of the genus described from the Amazonia region.

Molecular phylogeny of Megalobatrachonema (Nematoda: Ascaridida), with description of a new species based on morphological and molecular evidence.

Species of MegalobatrachonemaYamaguti, 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.

Redescription of Cruzia americana Maplestone, 1930 (Nematoda: Kathlaniidae) a parasite of Didelphis virginiana (Kerr) (Mammalia: Didelphidae) in the USA.

Cruzia americana Maplestone, 1930 is a common nematode parasite of opossums in North America. However, our present knowledge on the morphology of this species is still limited and its validity is uncertain. In the present study, specimens of C. americana collected from the type-host Didelphis virginiana (Kerr) (Mammalia: Didelphidae) in the USA were studied using light microscopy and, for the first time, scanning electron microscopy (SEM). The SEM observations revealed the internal structures of the pharynx of C. americana and the presence of pharyngeal lamellae rather than pharyngeal teeth/tooth-like structures. In addition, the detailed morphology of the lips, vulva, eggs and phasmids in females and the caudal papillae in males are also reported for the first time.

Morphological and Genetic Characterization of the Poorly Known Species Subulura chinensis Schwartz, 1926 (Nematoda: Ascaridida) from Athene noctua (Scopoli) (Strigiformes: Strigidae).

INTRODUCTION: 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. METHODS: 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. RESULTS: 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.

Redescription of three species of nematodes (Nematoda) parasitising fishes in the USA, with a key to the species of Dichelyne Jägerskiöld, 1902 parasitic in freshwater and brackish-water fishes of North America.

Three species of parasitic nematodes are redescribed based on light and scanning electron microscopical (SEM) examinations of newly collected specimens in fishes of South Carolina, USA: Dichelyne (Cucullanellus) bullocki Stromberg & Crites, 1972 from Fundulus heteroclitus (Linnaeus) (Fundulidae); Dichelyne (Dichelyne) diplocaecum Chandler, 1935 from Ictalurus furcatus (Valenciennes) (Ictaluridae); and Hysterothylacium pelagicum Deardorff & Overstreet, 1982 from Coryphaena hippurus Linnaeus (Coryphaenidae). For the first time, intraspecific variations in the number of intestinal caeca were observed in D. bullocki, as well as previously unknown males and gravid females of D. diplocaecum are described; this enabled to synonymise D. mexicanus Caspeta-Mandujano, Moravec & Salgado-Maldonado, 1999 with D. diplocaecum. Unlike most congeneric species, H. pelagicum has no double postanal papillae, as confirmed by SEM. A key to the species of Dichelyne Jägerskiöld, 1902 parasitic in freshwater and brackish-water fishes in North America is provided.

Typhlophoros kwenae n. sp. (Nematoda: Ascaridida: Heterocheilidae), a gastric parasite from the Nile crocodile Crocodylus niloticus Laurenti (Reptilia: Crocodylidae) in South Africa.

Based on light and scanning electron microscopical observations, Typhlophoros kwenae n. sp. (Heterocheilidae), a new nematode parasite, is described from the stomach of the Nile crocodile Crocodylus niloticus Laurenti in South Africa. In having three lips with well-developed posterior prolongations and prominent interlabial longitudinal cuticular ridges, four pairs of precloacal papillae and complex spicules divided into handle and broad alate blade in males, as well as the position of the vulva near mid-body in females, the specimens conform to the generic diagnosis of Typhlophoros von Linstow, 1906. They can, however, be distinguished from the two previously described congeners, T. lamellaris von Linstow, 1906 and T. spratti Sprent, 1999, by the number of complete interlabial ridges, the length of spicules in males and the position of the vulva as well as the length of the tail in females. This is the first record of the genus Typhlophoros from the Afrotropical Realm.

Ingwenascaris n. g. (Nematoda: Ascaridida: Heterocheilidae) established for I. sprenti n. sp. and I. assymmetrica (Ortlepp, 1932) n. comb., parasites of African crocodiles, and an identification key to the genera of the Heterocheilidae.

Ingwenascaris n. g. (Nematoda: Heterocheilidae) is established to accommodate Ingwenascaris sprenti n. g., n. sp., described from the stomach of Crocodylus niloticus Laurenti (Reptilia: Crocodylidae) in South Africa, based on light and scanning electron microscopy studies of its morphology. The new genus can be distinguished from other heterocheilid genera through a combination of its characters, including the pronounced asymmetry of each subventral lip due to an alate ventral margin and a non-alate margin facing the dorsal lip, the presence of continuous ridges of triangular denticles along the free labial margins, the lack of interlocking processes or a rostral plate, interlabia being indistinct or represented by small lateral interlabia between the dorsal and ventral lips only, the absence of prominent interlabial longitudinal cuticular ridges, the presence of lateral alae that are fused with the subventral lips, the presence of lateral caudal alae in both sexes, spicules of males that are composed of handle and alate blade, the presence of a gubernaculum, the number and arrangement of male caudal papillae and the position of the vulva near the anterior and middle third of the body in females. Ingwenascaris sprenti n. g., n. sp. represents the sixth heterocheilid genus parasitising African crocodilians. Trispiculascaris assymmetrica (Ortlepp, 1932) (syn. Porrocaecum assymmetricum Ortlepp, 1932) from a Central African crocodile is transferred to the new genus as I. assymmetrica (Ortlepp, 1932) n. comb. The genus Trispiculascaris Skrjabin, 1916 is considered a genus incertae sedis. An identification key to the genera of the family Heterocheilidae is presented.

Two new species of nematode parasites, Cucullanus epinepheli sp. n. (Cucullanidae) and Procamallanus (Spirocamallanus) sinespinis sp. n. (Camallanidae), from marine serranid and haemulid fishes off New Caledonia.

Based on light and scanning electron microscopical studies, two new species of parasitic nematodes are described from marine perciform fishes off New Caledonia: Cucullanus epinepheli sp. n. (Cucullanidae) from the intestine of the brownspotted grouper Epinephelus chlorostigma (Valenciennes) (Serranidae) and Procamallanus (Spirocamallanus) sinespinis sp. n. from the intestine of the silver grunt Pomadasys argenteus (Forsskål) (Haemulidae). Cucullanus epinepheli sp. n. differs from its congeners mainly in possessing a unique structure of the anterior, elevated cloacal lip with a large posterior outgrowth covering the cloacal aperture and in the presence of cervical alae and two small preanal papillae on the median dome-shaped precloacal elevation. This is the second known nominal species of this genus parasitising fishes of the family Serranidae and the second representative of Cucullanus Müller, 1777 recorded from fishes in New Caledonian waters. Procamallanus (Spirocamallanus) sinespinis sp. n. is mainly characterised by 10-12 spiral ridges in the buccal capsule, the presence of wide caudal alae, three pairs of pedunculate preanal papillae, two unequally long spicules (465-525 µm and 218-231 µm) and by the tail tip with a knob-like structure in the male, and the broad, rounded tail with a terminal digit-like protrusion without cuticular spikes in the female. This is the fifth nominal species of the subgenus Spirocamallanus Olsen, 1952 reported from fishes in New Caledonian waters.

Morphological variability, ultrastructure and molecular characterisation of Hysterothylacium reliquens (Norris & Overstreet, 1975) (Nematoda: Raphidascarididae) from the oriental sole Brachirus orientalis (Bloch & Schneider) (Pleuronectiformes: Soleidae).

Hysterothylacium reliquens (Norris & Overstreet, 1975), a common ascaridoid nematode parasitic in the marine fishes in the Indian and Atlantic Oceans, has been reported to exhibit remarkable morphological variability in the lengths of body, oesophagus and spicules, and the number and arrangement of postcloacal papillae. In order to determine whether H. reliquens with a broad range of morphological variability in the above-mentioned respects, represents a complex of sibling species or a single species, and to evaluate if the specimens of H. reliquens collected from the Indian and Atlantic Oceans are the same species, numbers of H. reliquens collected from the oriental sole Brachirus orientalis (Bloch & Schneider) (Pleuronectiformes: Soleidae) in the Arabian Gulf (off Iraq) were characterised using molecular approaches by sequencing and analysing the ribosomal [large ribosomal DNA (28S) and internal transcribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit 2 (cox2)] target regions, respectively. The results of molecular analysis strongly supported that: (i) the broad ranges of morphological variability in the above-mentioned respects in the newly collected specimens of H. reliquens, should be considered as intraspecific variation and the nematode material collected from B. orientalis in the Arabian Gulf represented a single species; (ii) the specimens of H. reliquens collected from the Indian and Atlantic Oceans belong to the same species, but they possibly represent different geographical populations. Moreover, the detailed morphology of the labial papillae, lateral alae, excretory pore, distal end of spicules, midventral precloacal papilla, phasmids and caudal papillae was revealed by SEM for the first time. The molecular and morphological data of H. reliquens obtained herein contribute to a more accurate diagnosis of this little known ascaridoid nematode.

Morphological and molecular characterization of Cucullanus bourdini Petter et Le Bel, 1992 (Ascaridida: Cucullanidae) from the yellowback fusilier Caesio xanthonota Bleeker (Perciformes: Caesionidae).

The detailed morphology of Cucullanus bourdini Petter et Le Bel, 1992 was further studied using both light and scanning electron microscopy, based on specimens collected from the yellowback fusilier Caesio xanthonota Bleeker (Perciformes: Caesionidae) from off the Taiwan Strait. In addition, C. bourdini was characterized genetically for the first time using molecular approaches by sequencing and analysing ribosomal [small ribosomal subunit (18S rDNA) and internal transcribed spacer 2 (ITS-2)] and mitochondrial [cytochrome c oxidase subunit 1 (cox1)] target regions. The molecular analysis of C. bourdini displayed no nucleotide differences in the 18S sequences, but detected 0.20-2.82% and 0.52-1.04% nucleotide divergence in the ITS-2 and cox1 regions, respectively. The new morphological and genetic data obtained herein would enable us to identify this hitherto little known species more rapidly and accurately.

Morphological and molecular genetic diversity of Strongyluris calotis (Nematoda: Ascaridida: Heterakidae) in South East and East Asian lizards.

Strongyluris calotis is a heterakid nematode in the large intestine of agamid lizards (Reptilia: Sauria: Agamidae) from the Oriental Region. The standard light microscopic definition of the species counts the "caudal papillae" as 10 pairs on male worms. However, previous work from our group using scanning electron microscopy (SEM) on the heterakid from agamid lizards in Japan, Taiwan, and Singapore revealed that this counting contained a pair of phasmids and that two pairs of postcloacal papillae were completely fused to form a pair of united papillae, thus resulting in "10 pairs." In the present study, we examined S. calotis specimens from the Emma Gray's forest lizard, Calotes emma (Agamidae), living in the plain forest at low altitude, and the Vietnam false bloodsucker, Pseudocalotes brevipes (Agamidae), living in the mountainous forest at high altitude in the northern part of Vietnam. Using SEM, the arrangement of caudal papillae in male worms from an Emma Gray's forest lizard was found to be comparable to classical S. calotis specimens from agamid lizards collected in Japan, Taiwan, and Singapore. However, male worms from Vietnam false bloodsuckers did not have a pair of united papillae but had 10 pairs of independent caudal papillae with a pair of phasmids. Molecular genetic analyses of the ribosomal RNA gene (rDNA) of worms of the classical S. calotis morphotype from Japan and Singapore and two S. calotis morphotypes from Vietnam demonstrated absolutely identical nucleotide sequences of partial 18S rDNA (at least 1764 base pairs (bp)) and 5.8S rDNA (158 bp). However, intraspecific differences were detected in other regions of the rDNA, related to the geographical distribution of hosts regardless of morphotype: 97.8-98.5 % identity (443-446 bp/453 bp) in the internal transcribed spacer (ITS)-1 region, 96.6-98.0 % identity (425-431 bp/440 bp) in the ITS-2 region, and 99.6-99.7 % identity (1149-1151 bp/1154 bp) in the 28S rDNA. Thus, in the future, taxonomic relationships of S. calotis distributed widely in the Oriental Region as well as other nominal Oriental Strongyluris spp., currently six in number, need to be extensively explored based on molecular genetic analyses in addition to intensive morphological characterization.

Morphological study of Ophidascaris excavata Hsü & Hoeppli, 1931 (Ascaridida: Ascarididae) from Gloydius brevicaudus (Stejneger) (Reptilia: Viperidae).

Ophidascaris excavata Hsü & 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.

A Morphological and Molecular Study of Spectatus spectatus (Kathlaniidae), Including Redescription of the Species and Amendment of Genus Diagnosis.

Spectatus spectatus Travassos, 1923 (Nematoda: Kathlaniidae) found in the intestine of Piaractus mesopotamicus (Holmberg, 1887) (Characiformes: Serrasalmidae) from the River Miranda, Mato Grosso do Sul, Brazil is redescribed based on morphological evaluation of newly collected material and examination of type and voucher specimens from the Coleção Helmintológica do Instituto Oswaldo Cruz. The following characteristics previously unreported or insufficiently described were observed: morphology of lips, presence of lamellae-like supplementary lips, presence of pharynx and cuticular ring surrounding the oral opening associated with a complex cuticular apparatus anterior to the pharynx, the number and arrangement of caudal papillae (13 pairs plus 1 unpaired), and the position of nerve ring. Since S. spectatus is the type species of Spectatus, the diagnosis of this Neotropical genus is amended. Synonymy of Chabaudinema Díaz-Ungría, 1968 with Spectatus, first proposed in 1980 by Baker, is supported by the present data. Molecular data that include the first sequence of the SSU rDNA for any species of Spectatus indicate a basal position of S. spectatus within Cosmocercoidea, forming a distant lineage from that comprising 2 species of Falcaustra Lane, 1915. This separate position of S. spectatus supports validity of the genus.

Dujardinascaris gigantea sp. n. (Nematoda: Ascaridida) from the critically endangered crocodile Alligator sinensis Fauvel (Reptilia: Crocodylia).

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).

Subulura halli (Ascaridida: Subuluridae) from the endangered great bustard Otis tarda Linnaeus (Aves: Gruiformes) in China.

Subulurid nematodes identified as Subulura halli Barreto, 1918 were collected from the endangered bird Otis tarda Linnaeus (Gruiformes: Otididae) in China. A detailed redescription of the hitherto poorly known species is presented using both light and, for the first time, scanning electron microscopy. Previously unreported and erroneous morphological features of taxonomic significance are revealed. This species can be readily distinguished from its congeners by the relatively long oesophagus (1.47-1.92 mm long, representing 10.6-16.9% of body length), the number and arrangement of male caudal papillae (11 pairs in total, arranged as five pairs of precloacal and six pairs of postcloacal papillae), the equal length of spicules (1.35-1.52 mm long, representing 10.7-13.7% of body length) and the presence of a small medioventral, precloacal papilla in the male.

A new species of Cucullanus Müller, 1777 (Nematoda: Cucullanidae) parasitic in the grey triggerfish Balistes capriscus Gmelin (Osteichthyes: Balistidae) off Rio de Janeiro, Brazil.

Cucullanus brevicaudatus n. sp. (Cucullanidae) is described from the intestine of Balistes capriscus Gmelin (Balistidae) off Rio de Janeiro, Brazil. The new species can be differentiated from its congeners in the small body length, the shape of the gubernaculum, the particular size and morphology of the tail in both males and females, the arrangement of the caudal papillae, the narrow oesophageal base, and the eggs with rugged shells. In addition, the combination of some features, i.e. number of caudal papillae, spicule length, oesophagus/body length ratio, host family and geographical distribution, can help to easily distinguish the new species from other cucullanids. Considering these features, C. brevicaudatus differs from the species assigned to Dichelyne Törnquist, 1931 which exhibit morphological proximity with Cucullanus Müller, 1777. Regarding the life-cycle of cucullanid nematodes, available evidence suggests that some species are primarily heteroxenous using invertebrates (i.e. crustaceans, polychaetes) as intermediate hosts, but in other a histotrophic phase in the definitive host replaces the intermediate host.

Dichelyne (Cucullanellus) tornquisti n. sp. (Nematoda: Cucullanidae) from corocoro grunt, Orthopristis ruber (Cuvier, 1830) (Perciformes: Haemulidae) from Southeastern Brazil.

Dichelyne (Cucullanellus) tornquisti n. sp. is described from the intestine of the marine fish Orthopristis ruber (Haemulidae) of Rio de Janeiro, Brazil. The new species differs from the congeners by the location of the deirids (same level) and the excretory pore (posterior) in relation to esophagus-intestinal junction and by papillae 5 to 9 not forming a subventral line. It is the first Dichelyne species described from fish family Haemulidae in the Brazilian coastal zone.

A simple and efficient method to observe internal structures of helminths by scanning electron microscopy.

Morphological studies of helminths by scanning electron microscopy are generally limited to the external topography of the organisms. In this work, we present a simple technique using ethanol as a cryoprotectant without postfixation in osmium tetroxide that allows for observation of the inner organization of helminths and preserves cellular structures. We tested the technique in three helminths: Echinostoma paraensei, Cruzia tentaculata, and Hassalstrongylus epsilon. The results show that this technique could potentially be applied to study the morphology, ultrastructure, and taxonomy of helminths.

Aspidodera kinsellai n. sp. (Nematoda: Heterakoidea) from nine-banded armadillos in Middle America with notes on phylogeny and host-parasite biogeography.

Aspidodera kinsellai n. sp. (Heterakoidea: Aspidoderidae) from the 9-banded armadillo, Dasypus novemcinctus , is herein described. This nematode occurs from Costa Rica north through central Mexico where it can be found causing co-infections with Aspidodera sogandaresi . Aspidodera kinsellai n. sp. can be discriminated from this and all other species in the family based on 3 key features, including (1) conspicuous lateral grooves with no lateral alae starting immediately after the hood and terminating at the cloacal/anal region; (2) long hoods in both male (360 µm) and female (401 µm), and (3) a relatively long (152 µm) terminal spine or terminus that gradually tapers to a point from the last pair of papillae. This is the 18th recognized species of the family and the 3rd in the genus present outside of South America. A phylogenetic analysis of the species in the genus with the use of the mitochondrial partial genes cytochrome C oxidase subunit 1 (cox1), the ribosomal large subunit (rrnL), and the internal transcriber spacer (ITS) shows that 2 species of Aspidodera may have entered into North America from the south via 2 independent events.

Strongyluris amazonicus n. sp. (Nematoda: Heterakidae): a parasite of Tropidurus oreadicus from the Brazilian Amazon.

Tropidurus oreadicus Rodrigues 1987 is a recently described species, thus there are no reports of helminthes parasites for this species. In this study, a morphological characterisation was performed of a nematode species parasite of the large intestine of T. oreadicus captured in an urban area. This urban area is similar to the estuarine dale from Guamá river and the "igarapés" of Belém, Pará State, Brazil. Morphological analysis suggested that the parasite is a new species of nematode, based mainly on the number and distribution pattern of the caudal papillae in males, which is unique for this species. In the present work, we describe for the first time the structure, number, and disposition of the cervical papillae. The morphological data were supported by scanning electron microscopy, which served as an important tool for distinguishing these nematodes from other species of the genus.