Morphology and ASAP analysis of the important zoonotic nematode parasite Baylisascaris procyonis (Stefahski and Zarnowski, 1951), with molecular phylogenetic relationships of Baylisascaris species (Nematoda: Ascaridida).

Species of Baylisascaris (Nematoda: Ascarididae) are of great veterinary and zoonotic significance, owing to cause Baylisascariosis or Baylisascariasis in wildlife, captive animals and humans. However, the phylogenetic relationships of the current 10 Baylisascaris species remain unclear. Moreover, our current knowledge of the detailed morphology and morphometrics of the important zoonotic species B. procyonis is still insufficient. The taxonomical status of B. procyonis and B. columnaris remains under debate. In the present study, the detailed morphology of B. procyonis was studied using light and scanning electron microscopy based on newly collected specimens from the raccoon Procyon lotor (Linnaeus) in China. The results of the ASAP analysis and Bayesian inference (BI) using the 28S, ITS, cox1 and cox2 genetic markers did not support that B. procyonis and B. columnaris represent two distinct species. Integrative morphological and molecular assessment challenged the validity of B. procyonis, and suggested that B. procyonis seems to represent a synonym of B. columnaris. Molecular phylogenetic results indicated that the species of Baylisascaris were grouped into 4 clades according to their host specificity. The present study provided new insights into the taxonomic status of B. procyonis and preliminarily clarified the phylogenetic relationships of Baylisascaris species.

Characterization of the complete mitochondrial genomes of the zoonotic parasites Bolbosoma nipponicum and Corynosoma villosum (Acanthocephala: Polymorphida) and the molecular phylogeny of the order Polymorphida.

Acanthocephalans of the order Polymorphida mainly parasitic in birds and mammals, are of veterinary, medical and economic importance. However, the evolutionary relationships of its 3 families (Centrorhynchidae, Polymorphidae and Plagiorhynchidae) remain under debate. Additionally, some species of Polymorphida (i.e. Bolbosoma spp. and Corynosoma spp.) are recognized as zoonotic parasites, associated with human acanthocephaliasis, but the mitochondrial genomes for representatives of Bolbosoma and Corynosoma have not been reported so far. In the present study, the complete mitochondrial genomes B. nipponicum and C. villosum (Acanthocephala: Polymorphidae) are reported for the first time, which are 14 296 and 14 241 bp in length, respectively, and both contain 36 genes [including 12 PCGs, 22 tRNA genes and 2 rRNA genes] and 2 non-coding regions (NCR1 and NCR2). The gene arrangement of some tRNAs in the mitogenomes of B. nipponicum and C. villosum differs from that found in all other acanthocephalans, except Polymorphus minutus. Phylogenetic results based on concatenated amino acid (AA) sequences of the 12 protein-coding genes (PCGs) strongly supported that the family Polymorphidae is a sister to the Centrorhynchidae rather than the Plagiorhynchidae, and also confirmed the sister relationship of the genera Bolbosoma and Corynosoma in the Polymorphidae based on the mitogenomic data for the first time. Our present findings further clarified the phylogenetic relationships of the 3 families Plagiorhynchidae, Centrorhynchidae and Polymorphidae, enriched the mitogenome data of the phylum Acanthocephala (especially the order Polymorphida), and provided the resource of genetic data for diagnosing these 2 pathogenic parasites of human acanthocephaliasis.

Morphology, genetic characterization and phylogeny of Moniliformis tupaia n. sp. (Acanthocephala: Moniliformidae) from the northern tree shrew Tupaia belangeri chinensis Anderson (Mammalia: Scandentia).

A new species of Moniliformis, M. tupaia n. sp. is described using integrated morphological methods (light and scanning electron microscopy) and molecular techniques (sequencing and analysing the nuclear 18S, ITS, 28S regions and mitochondrial cox1 and cox2 genes), based on specimens collected from the intestine of the northern tree shrew Tupaia belangeri chinensis Anderson (Scandentia: Tupaiidae) in China. Phylogenetic analyses show that M. tupaia n. sp. is a sister to M. moniliformis in the genus Moniliformis, and also challenge the systematic status of Nephridiacanthus major. Moniliformis tupaia n. sp. represents the third Moniliformis species reported from China.

Further description of the poorly known zooparasitic nematode Abbreviata baltazardi Chabaud, 1953 (Spirurida: Physalopteridae).

Abbreviata baltazardi Chabaud, 1953 (Nematoda: Physalopteridae) is a hitherto poorly known parasitic nematode species reported from Phrynocephalus helioscopus Pallas (Squamata: Agamidae) in Iran. The current knowledge on the morphology of A. baltazardi is still very limited. In the present study, the detailed morphology of A. baltazardi was studied using light microscopy and, for the first time, scanning electron microscopy, based on newly collected specimens from the yarkand toad-headed agama P. axillaris Blanford Pallas (Squamata: Agamidae) in China. Some erroneous or previously unreported morphological features of A. baltazardi were observed using SEM, which include the presence of one large semicircular protrusion and 20-30 denticles on each pseudolabium, the absence of precloacal medioventral papilla in some individuals and the presence of 4-5 postcloacal medioventral papillae in males. SEM observations also clearly showed the detailed morphology of deirids, cloacal ornamentation, caudal papillae, vulva and egg. Abbreviata baltazardi represents the first species of Abbreviata Travassos, 1920 reported in China.

Further studies on the morphology of Baylisascaris transfuga (Rudolphi, 1819) (Nematoda: Ascaridomorpha: Ascarididae) from the polar bear Ursus maritimus Phipps (Carnivora: Ursidae).

Baylisascaris transfuga (Rudolphi, 1819) is a common parasitic nematode in the digestive tract of various species of bears worldwide, with great veterinary significance. However, our present knowledge on the morphology of B. transfuga remains insufficient. In the present study, the detailed morphology of B. transfuga was studied using light and scanning electron microscopy (SEM), based on specimens collected from the polar bear Ursus maritimus Phipps (Carnivora: Ursidae) in the Shijiazhuang Zoo, China. The results revealed some morphological and morphometric variation between the present specimens and some of those from previous studies, including oesophageal length of female, number and morphology of postcloacal papillae and morphology of tail of males. Present SEM observations clearly showed the detailed morphology of lips, cervical alae, cloacal ornamentation, precloacal medioventral papilla, phasmids and tail tip. These supplementary morphological and morphometric data enable us to identify this ascaridid nematode more accurately.

Integrated evidence reveals a new species of Cosmocerca (Ascaridomorpha: Cosmocercoidea) from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura).

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.

Morphological and genetic characterisation of Centrorhynchus clitorideus (Meyer, 1931) (Acanthocephala: Centrorhynchidae) from the little owl Athene noctua (Scopoli) (Strigiformes: Strigidae) in Pakistan.

Centrorhynchus Lühe, 1911 is a large genus of acanthocephalans mainly parasitic in various strigiform and falconiform birds. Some species of Centrorhynchus have not been adequately described. Here, the detailed morphology of C. clitorideus (Meyer, 1931) was studied using light and, for the first time, scanning electron microscopy, based on newly collected specimens from the little owl Athene noctua (Scopoli) (Strigiformes: Strigidae) in Pakistan. Partial sequences of the 18S and 28S nuclear ribosomal RNA genes and the mitochondrial cytochrome c oxidase subunit 1 (cox1) of C. clitorideus were generated for the first time. No nucleotide variation was detected for the partial 18S and 28S regions, but 3.30% of intraspecific nucleotide divergence was found for the cox1 gene. Phylogenetic analyses based on 28S and 18S sequence data showed that C. clitorideus formed a sister relationship with Centrorhynchus sp. MGV-2005 or Centrorhynchus sp. MGV-2005 + C. microcephalus (Bravo-Hollis, 1947), respectively.

Molecular Phylogeny and Dating Reveal a Terrestrial Origin in the Early Carboniferous for Ascaridoid Nematodes.

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.

Remarkable morphological variation in the proboscis of Neorhadinorhynchus nudus (Harada, 1938) (Acanthocephala: Echinorhynchida).

The acanthocephalans are characterized by a retractible proboscis, armed with rows of recurved hooks, which serves as the primary organ for attachment of the adult worm to the intestinal wall of the vertebrate definitive host. Whilst there is a considerable variation in the size, shape and armature of the proboscis across the phylum, intraspecific variation is generally regarded to be minimal. Consequently, subtle differences in proboscis morphology are often used to delimit congeneric species. In this study, striking variability in proboscis morphology was observed among individuals of Neorhadinorhynchus nudus (Harada, 1938) collected from the frigate tuna Auxis thazard Lacépède (Perciformes: Scombridae) in the South China Sea. Based on the length of the proboscis, and number of hooks per longitudinal row, these specimens of N. nudus were readily grouped into three distinct morphotypes, which might be considered separate taxa under the morphospecies concept. However, analysis of nuclear and mitochondrial DNA sequences revealed a level of nucleotide divergence typical of an intraspecific comparison. Moreover, the three morphotypes do not represent three separate genetic lineages. The surprising, and previously undocumented level of intraspecific variation in proboscis morphology found in the present study, underscores the need to use molecular markers for delimiting acanthocephalan species.

Morphological and molecular evidence for a new species of the genus Cosmocercoides Wilkie, 1930 (Ascaridida: Cosmocercidae) from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura).

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.

Neoascarophis sinensis n. sp. (Nematoda: Cystidicolidae), a parasite of Conger myriaster (Brevoort) (Anguilliformes: Congridae) in Chinese waters.

Neoascarophis sinensis n. sp. collected from the whitespotted conger Conger myriaster (Brevoort) (Anguilliformes: Congridae) in the Yellow and East China Seas, is described using both light and scanning electron microscopy. The new species is characterised mainly by the body size (8.5-10.5 mm in the males, 9.5-14.0 mm in the females), the location of the vulva (near equatorial region of the body), the non-bifurcate deirids, the lengths of the vestibule (40-50 µm in the males, 30-60 µm in the females) and glandular oesophagus (2.5-3.1 mm in the males, 3.1-3.5 mm in the females) and the morphology and length of the spicules (left spicule 400-410 µm, right spicule 130-150 µm). Neoascarophis sinensis n. sp. is the first species of Neoascarophis Machida, 1976 reported from the anguilliform fish and is also the only species of this genus found in the Chinese waters.

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.

Morphology, complete mitochondrial genome, and molecular phylogeny of Rhabdias macrocephalum n. sp. (Nematoda: Rhabdiasidae) from Diploderma splendidum (Reptilia: Agamidae).

Species of the genus Rhabdias Stiles & Hassall, 1905 are common parasitic nematodes occurring in the lungs of amphibians and reptiles worldwide. In the present study, Rhabdias macrocephalum n. sp. is described using integrated morphological methods (light and scanning electron microscopy) and molecular approaches (sequencing of the nuclear 28S and ITS regions, and mitochondrial cox1, cox2, and 12S genes) based on specimens collected from the green striped tree dragon Diploderma splendidum (Barbour & Dunn) (Reptilia: Agamidae) in China. The complete mitochondrial genome of R. macrocephalum n. sp. was sequenced and annotated: it is 14,819 bp in length, including 12 protein coding genes (missing atp8), 22 tRNA genes, 2 rRNA genes and three non-coding regions. The gene arrangement of R. macrocephalum n. sp. is different from all of the currently available mitogenomes of nematodes and represents a novel type of mitochondrial gene arrangement reported in Nematoda. Molecular phylogenetic results based on the ITS + 28S data support the monophyly of Entomelas, Pneumonema, Serpentirhabdias, and Rhabdias, and showed R. macrocephalum n. sp. forming a most basal lineage in Rhabdias.

Title: Morphologie, génome mitochondrial complet et phylogénie moléculaire de Rhabdias macrocephalum n. sp. (Nematoda : Rhabdiasidae) de Diploderma splendidum (Reptilia : Agamidae). Abstract: Les espèces du genre Rhabdias Stiles & Hassall, 1905 sont des nématodes parasites courants présents dans les poumons des amphibiens et des reptiles du monde entier. Dans cette étude, Rhabdias macrocephalum n. sp. est décrit à l'aide de méthodes morphologiques intégrées (microscopie optique et électronique à balayage) et d'approches moléculaires (séquençage des régions nucléaires 28S et ITS et des gènes mitochondriaux cox1, cox2 et 12S) basées sur des spécimens collectés chez le lézard Diploderma splendidum (Barbour & Dunn) (Reptilia : Agamidae) de Chine. Le génome mitochondrial complet de R. macrocephalum n. sp. a été séquencé et annoté : il a une longueur de 14 819 pb, dont 12 gènes codants pour des protéines (atp8 manquant), 22 gènes d'ARNt, 2 gènes d'ARNr et trois régions non codantes. L'arrangement génétique de R. macrocephalum n. sp. est différent de tous les mitogénomes de nématodes actuellement disponibles et représente un nouveau type d'arrangement de gènes mitochondriaux signalé chez les nématodes. Les résultats phylogénétiques moléculaires basés sur les données ITS + 28S ont soutenu la monophylie d'Entomelas, Pneumonema, Serpentirhabdias et Rhabdias, et ont montré que R. macrocephalum n. sp. forme la lignée la plus basale chez Rhabdias.

Novel gene arrangement in the mitochondrial genome of Aspersentis megarhynchus (Acanthocephala, Echinorhynchida, Heteracanthocephalidae), and its phylogenetic implications.

The Heteracanthocephalidae Petrochenko, 1956 is a rare family of acanthocephalans mainly parasitic in fishes. The pattern of mitogenomic evolution of the Heteracanthocephalidae is still unknown, and the phylogenetic relationships of the Heteracanthocephalidae with the other 14 families within the order Echinorhynchida remain unclear. In the present study, the complete mitochondrial genome of Aspersentis megarhynchus (von Linstow, 1892) Golvan, 1960 was sequenced and annotated for the first time, which represents the first mitogenomic data for the genus Aspersentis and also for the family Heteracanthocephalidae. The mitogenome of A. megarhynchus has 14,661 bp and includes 36 genes, containing 12 protein-coding genes (PCGs) (missing atp8), 22 tRNA genes, and 2 ribosomal RNAs (rrnS and rrnL), plus two non-coding regions. Comparative mitochondrial genomic analysis revealed that the presence of translocations of several tRNA genes (trnV, trnE, and trnT) and the gene arrangement in the mitogenome of A. megarhynchus represents a new type in Acanthocephala. Moreover, the mitogenomic phylogenetic results based on concatenated amino acid sequences of 12 protein-coding genes strongly supported the validity of the Heteracanthocephalidae and suggested close affinity between the Heteracanthocephalidae and Echinorhynchidae in the order Echinorhynchida.

Title: Nouvel arrangement de gènes dans le génome mitochondrial d'Aspersentis megarhynchus (Acanthocephala, Echinorhynchida, Heteracanthocephalidae) et ses implications phylogénétiques. Abstract: Les Heteracanthocephalidae Petrochenko, 1956 sont une famille rare d'acanthocéphales principalement parasites de poissons. Le schéma d'évolution mitogénomique des Heteracanthocephalidae est encore inconnu, et les relations phylogénétiques des Heteracanthocephalidae avec les 14 autres familles de l'ordre Echinorhynchida restent floues. Dans la présente étude, le génome mitochondrial complet d'Aspersentis megarhynchus (von Linstow, 1892) Golvan, 1960 a été séquencé et annoté pour la première fois, ce qui représente les premières données mitogénomiques pour le genre Aspersentis et également pour la famille Heteracanthocephalidae. Le mitogénome d'A. megarhynchus compte 14 661 pb et comprend 36 gènes, contenant 12 gènes codant pour des protéines (atp8 manquant), 22 gènes d'ARNt et 2 ARN ribosomiques (rrnS et rrnL), plus deux régions non codantes. L'analyse génomique mitochondriale comparative a révélé que la présence de translocations de plusieurs gènes d'ARNt (trnV, trnE et trnT) et la disposition des gènes dans le mitogénome d'A. megarhynchus représentent un nouveau type chez les Acanthocephala. De plus, les résultats phylogénétiques mitogénomiques basés sur des séquences concaténées d'acides aminés de 12 gènes codant pour des protéines soutiennent fortement la validité des Heteracanthocephalidae et suggèrent une affinité étroite entre les Heteracanthocephalidae et les Echinorhynchidae dans l'ordre des Echinorhynchida.

Molecular phylogeny of the family Rhabdiasidae (Nematoda: Rhabditida), with morphology, genetic characterization and mitochondrial genomes of Rhabdias kafunata and R. bufonis.

BACKGROUND: The family Rhabdiasidae (Nematoda: Rhabditida) is a globally distributed group of nematode parasites, with over 110 species parasitic mainly in amphibians and reptiles. However, the systematic position of the family Rhabdiasidae in the order Rhabditida remains unsolved, and the evolutionary relationships among its genera are still unclear. Moreover, the present knowledge of the mitochondrial genomes of rhabdiasids remains limited. METHODS: Two rhabdiasid species: Rhabdias kafunata Sata, Takeuchi & Nakano, 2020 and R. bufonis (Schrank, 1788) collected from the Asiatic toad Bufo gargarizans Cantor (Amphibia: Anura) in China, were identified based on morphology (light and scanning electron microscopy) and molecular characterization (sequencing of the nuclear 28S and ITS regions and mitochondrial cox1 and 12S genes). The complete mitochondrial genomes of R. kafunata and R. bufonis were also sequenced and annotated for the first time. Moreover, phylogenetic analyses based on the amino acid sequences of 12 protein-coding genes (PCGs) of the mitochondrial genomes were performed to clarify the systematic position of the family Rhabdiasidae in the order Rhabditida using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic analyses based on the 28S + ITS sequences, were also inferred to assess the evolutionary relationships among the genera within Rhabdiasidae. RESULTS: The detailed morphology of the cephalic structures, vulva and eggs in R. kafunata and R. bufonis was revealed using scanning electron microscopy (SEM) for the first time. The characterization of 28S and ITS regions of R. kafunata was reported for the first time. The mitogenomes of R. kafunata and R. bufonis are 15,437 bp and 15,128 bp long, respectively, and both contain 36 genes, including 12 PCGs (missing atp8). Comparative mitogenomics revealed that the gene arrangement of R. kafunata and R. bufonis is different from all of the currently available mitogenomes of nematodes. Phylogenetic analyses based on the ITS + 28S data showed Neoentomelas and Kurilonema as sister lineages, and supported the monophyly of Entomelas, Pneumonema, Serpentirhabdias and Rhabdias. Mitochondrial phylogenomic results supported Rhabdiasidae as a member of the superfamily Rhabditoidea in the suborder Rhabditina, and its occurrance as sister to the family Rhabditidae. CONCLUSIONS: The complete mitochondrial genome of R. kafunata and R. bufonis were reported for the first time, and two new gene arrangements of mitogenomes in Nematoda were revealed. Mitogenomic phylogenetic results indicated that the family Rhabdiasidae is a member of Rhabditoidea in Rhabditina, and is closely related to Rhabditidae. Molecular phylogenies based on the ITS + 28S sequence data supported the validity of Kurilonema, and showed that Kurilonema is sister to Neoentomelas. The present phylogenetic results also indicated that the ancestors of rhabdiasids seem to have initially infected reptiles, then spreading to amphibians.

Molecular data reveal a new species of Seuratascaris Sprent, 1985 (Nematoda: Ascaridoidea) from Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura).

The genus Seuratascaris Sprent, 1985 is a group of obligate nematode parasites of amphibians. In the present study, a new species of Seuratascaris, S. physalis sp. n. was described using light and scanning electron microscopy based on specimens collected from Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura) in China. The new species differs from S. numidica (Seurat, 1917) by the cuticle of the cervical region distinctly inflated to form a cephalic vesicle-like structure and the absence of single medio-ventral precloacal papilla. The molecular characterization of the nuclear large ribosomal DNA (28S) and internal transcribed spacer (ITS) and the mitochondrial cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2) and 12S small subunit ribosomal RNA gene of S. physalis sp. n., together with the 28S, cox2 and 12S of S. numidica are provided for the first time. Molecular analysis revealed the presence of high level of interspecific genetic variation between the two species in the ITS (5.50%), cox1 (13.3%), cox2 (10.6%) and 12S regions (10.5%), which strongly supported that S. physalis sp. n. represented a different species from S. numidica. Angusticaecum ranae Wang, Zhao & Chen, 1978 reported from the frog Quasipaa spinosa (David) (Anura: Dicroglossidae) in China was transferred into the genus Seuratascaris as S. ranae (Wang, Zhao & Chen, 1978) comb. n. based on the morphology of lips and the presence of very short and robust spicules without alae and small numbers of precloacal papillae. The present study provided useful genetic data for molecular identification of species of Seuratascaris and provides the foundation for being able to determine if S. numidica represents a species complex of some sibling species or a single species.

Morphology and genetic characterization of Physaloptera sibirica Petrow & Gorbunov, 1931 (Spirurida: Physalopteridae), from the hog-badger Arctonyx collaris Cuvier (Carnivora: Mustelidae), with molecular phylogeny of Physalopteridae.

BACKGROUND: Nematodes of the family Physalopteridae (Spirurida: Physalopteroidea) commonly parasitize the alimentary canal of all major vertebrate groups. However, many physalopterid species are not adequately described, especially regarding the detailed morphology of the cephalic end. The current genetic database for Physaloptera species is still very limited, which seriously hampers molecular-based species identification. Additionally, the systematic status of some genera and the evolutionary relationships of the subfamilies in the Physalopteridae remain under debate. METHODS: New morphological data for Physaloptera sibirica was gathered using light and scanning electron microscopy based on newly collected specimens from the hog badger Arctonyx collaris Cuvier (Carnivora: Mustelidae) in China. Six different genetic markers, including nuclear small ribosomal DNA (18S), large ribosomal DNA (28S) and internal transcribed spacer (ITS), mitochondrial cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2), and the 12S small subunit ribosomal RNA gene of P. sibirica were sequenced and analyzed for the first time to our knowledge. Additionally, to construct a basic molecular phylogenetic framework for the Physalopteridae, phylogenetic analyses were performed based on the cox1 and 18S + cox1 genes using maximum likelihood (ML) and Bayesian inference (BI) methods. RESULTS: Scanning electron microscopy (SEM) observation displayed the details of the cephalic structures, deirids, excretory pore, caudal papillae, vulva, phasmids and egg of P. sibirica for the first time to our knowledge. Pairwise comparison of the sequences obtained for P. sibirica did not reveal intraspecific divergence regarding the 18S, 28S, cox1 and 12S genetic markers and a low level of divergence in the ITS (0.16%) and cox2 (2.39%) regions. Maximum likelihood and Bayesian inference analyses showed that the representatives of Physalopteridae formed two major clades (species of Physalopterinae + Thubunaeinae parasitic in terrestrial vertebrates and Proleptinae only occurring in marine or freshwater fishes). Turgida turgida was found nested among representatives of Physaloptera. Physaloptera sibirica clustered together with P. rara. Physalopteroides sp. (Thubunaeinae) formed a sister relationship to the physalopterine Abbreviata caucasica. CONCLUSIONS: Physaloptera sibirica was redescribed, which is the fourth nematode parasite reported from the hog badger A. collaris, and A. collaris represents a new host for P. sibirica. The phylogenetic results challenged the validity of the subfamily Thubunaeinae and of the genus Turgida and supported dividing the family Physalopteridae into two subfamilies, Physalopterinae and Proleptinae. However, we do not make any immediate systematic changes in the Physalopteridae, because a more rigorous study with broader representation of the Physalopteridae is required. These present findings contribute to morphologically identifying P. sibirica more accurately and provide new insights into the systematics of the Physalopteridae.

Mitogenomic phylogenies suggest the resurrection of the subfamily Porrocaecinae and provide insights into the systematics of the superfamily Ascaridoidea (Nematoda: Ascaridomorpha), with the description of a new species of Porrocaecum.

BACKGROUND: The family Toxocaridae is a group of zooparasitic nematodes of veterinary, medical and economic significance. However, the evolutionary relationship of Porrocaecum and Toxocara, both genera currently classified in Toxocaridae, and the monophyly of the Toxocaridae remain under debate. Moreover, the validity of the subgenus Laymanicaecum in the genus Porrocaecum is open to question. Due to the scarcity of an available genetic database, molecular identification of Porrocaecum nematodes is still in its infancy. METHODS: A number of Porrocaecum nematodes collected from the Eurasian marsh harrier Circus aeruginosus (Linnaeus) (Falconiformes: Accipitridae) in the Czech Republic were identified using integrated morphological methods (light and scanning electron microscopy) and molecular techniques (sequencing and analyzing the nuclear 18S, 28S and ITS regions). The complete mitochondrial genomes of the collected nematode specimens and of Porrocaecum (Laymanicaecum) reticulatum (Linstow, 1899) were sequenced and annotated for the first time. Phylogenetic analyses of ascaridoid nematodes based on the amino acid sequences of 12 protein-coding genes of mitochondrial genomes were performed using maximum likelihood and Bayesian inference. RESULTS: A new species of Porrocaecum, named P. moraveci n. sp., is described based on the morphological and genetic evidence. The mitogenomes of P. moraveci n. sp. and P. reticulatum both contain 36 genes and are 14,517 and 14,210 bp in length, respectively. Comparative mitogenomics revealed that P. moraveci n. sp. represents the first known species with three non-coding regions and that P. reticulatum has the lowest overall A + T content in the mitogenomes of ascaridoid nematodes tested to date. Phylogenetic analyses showed the representatives of Toxocara clustered together with species of the family Ascarididae rather than with Porrocaecum and that P. moraveci n. sp. is a sister to P. reticulatum. CONCLUSIONS: The characterization of the complete mitochondrial genomes of P. moraveci n. sp. and P. reticulatum is reported for the first time. Mitogenomic phylogeny analyses indicated that the family Toxocaridae is non-monophyletic and that the genera Porrocaecum and Toxocara do not have an affinity. The validity of the subgenus Laymanicaecum in Porrocaecum was also rejected. Our results suggest that: (i) Toxocaridae should be degraded to a subfamily of the Ascarididae that includes only the genus Toxocara; and (ii) the subfamily Porrocaecinae should be resurrected to include only the genus Porrocaecum. The present study enriches the database of ascaridoid mitogenomes and provides a new insight into the systematics of the superfamily Ascaridoidea.

Phylomitogenomic Analyses Provided Further Evidence for the Resurrection of the Family Pseudoacanthocephalidae (Acanthocephala: Echinorhynchida).

The phylum Acanthocephala is an important monophyletic group of parasites, with adults parasitic in the digestive tracts of all major vertebrate groups. Acanthocephalans are of veterinary, medical, and economic importance due to their ability to cause disease in domestic animals, wildlife, and humans. However, the current genetic data for acanthocephalans are sparse, both in terms of the proportion of taxa surveyed and the number of genes sequenced. Consequently, the basic molecular phylogenetic framework for the phylum is still incomplete. In the present study, we reported the first complete mitochondrial genome from a representative of the family Pseudoacanthocephalidae Petrochenko, 1956. The mitogenome of Pseudoacanthocephalus bufonis (Shipley, 1903) is 14,056 bp in length, contains 36 genes (12 protein-coding genes (PCGs) (lacking atp8), 22 tRNA genes, and 2 rRNA genes (rrnL and rrnS)) and two non-coding regions (NCR1 and NCR2), and displayed the highest GC-skew in the order Echinorhynchida. Phylogenetic results of maximum likelihood (ML) and Bayesian inference (BI) using the amino acid sequences of 12 protein-coding genes in different models provided further evidence for the resurrection of the family Pseudoacanthocephalidae and also supported that the order Echinorhynchida is paraphyletic. A monophyletic clade comprising P. bufonis and Cavisoma magnum suggests a close affinity between Pseudoacanthocephalidae and Cavisomatidae. Our phylogenetic analyses also showed that Polymorphidae has a closer relationship with Centrorhynchidae than Plagiorhynchidae in the monophyletic order Polymorphida.

Native species Maxvachonia chabaudi Mawson, 1972 (Nematoda: Cosmocercoidea) found in the invasive marine toad Rhinella marina (Linnaeus) (Anura: Bufonidae) in Australia.

The genus Maxvachonia Chabaud et Brygoo, 1960 (Ascaridomorpha: Cosmocercidae) is a poorly known group of parasitic nematodes. Species of Maxvachonia are native to Madagascar-Australo-Papuan Region, where they are known to parasitise frogs, snakes and skinks. Unfortunately, most of Maxvachonia species have been inadequately described. In the present study, we report the native species Maxvachonia chabaudi Mawson, 1972 from the intestine of the invasive marine toad Rhinella marina (Linnaeus) in Australia for the first time. We speculate that the marine toads infected with M. chabaudi are likely related to their eating skinks or the similarity in diet/habitat/ecology between the toad and the skinks. The detailed morphology of M. chabaudi was studied using light microscopy and, for the first time, scanning electron microscopy, based on the newly collected specimens. Some characters important for the specific diagnosis of M. chabaudi are reported for the first time, including each lip with distinct inner flanges, the location of vulva varying from anterior to posterior of the oesophageal bulb and the presence of single medio-ventral precloacal papilla. An identification key to the species of Maxvachonia is provided.