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.

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.

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.

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.

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.

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.

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.

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.

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.

Molecular identification of a new species of Rhigonema (Nematoda: Rhigonematidae) and phylogenetic relationships within the infraorder Rhigonematomorpha.

BACKGROUND: The infraorder Rhigonematomorpha comprises a group of obligate parasitic nematodes of millipedes (Arthropoda: Diplopoda). The current species identification of Rhigonematomorpha nematodes remains mainly based on morphological features, with molecular-based identification still in its infancy. Also, current knowledge of the phylogeny of Rhigonematomorpha is far from comprehensive. METHODS: The morphology of Rhigonematomorpha nematodes belonging to the genus Rhigonema, collected from the millipede Spirobolus bungii Brandt (Diplopoda: Spirobolida) in China, was studied in detail using light and scanning electron microscopy. Five different genetic markers, including the nuclear small ribosomal subunit (18S), internal transcribed spacer (ITS) and large ribosomal subunit (28S) regions and the mitochondrial cox1 and cox2 genes of these Rhigonematomorpha nematodes collected from China and Rhigonema naylae collected from Japan were sequenced and analyzed using Bayesian inference (BI) and Assemble Species by Automatic Partitioning (ASAP) methods. Phylogenetic analyses that included the most comprehensive taxa sampling of Rhigonematomorpha to date were also performed based on the 18S + 28S genes using maximum likelihood (ML) and BI methods. RESULTS: The specimens of Rhigonema collected from S. bungii in China were identified as a new species, Rhigonema sinense n. sp. Striking variability in tail morphology was observed among individuals of R. sinense n. sp. ASAP analyses based on the 28S, ITS, cox1 and cox2 sequences supported the species partition of R. sinense n. sp. and R. naylae, but showed no evidence that the different morphotypes of R. sinense n. sp. represent distinct genetic lineages. BI analyses also indicated that R. sinense n. sp. represents a separated species from R. naylae based on the cox1 and cox2 genes, but showed that R. naylae nested in samples of R. sinense n. sp. based on the ITS and 28S data. Phylogenetic results showed that the representatives of Rhigonematomorpha formed two large clades. The monophyly of the families Carnoyidae and Ichthyocephalidae and the genus Rhigonema was rejected. The representatives of the family Ransomnematidae clustered together with the family Hethidae with strong support. CONCLUSIONS: A new species of Rhigonematomorpha, R. sinense n. sp. is described based on morphological and molecular evidence. ASAP analyses using 28S, ITS, cox1 and cox2 data indicate the striking variability in tail morphology of R. sinense n. sp. as intraspecific variation, and also suggest that partial 28S, ITS, cox1 and cox2 markers are effective for molecular identification of Rhigonematomorpha nematodes. The phylogenetic results support the traditional classification of Rhigonematomorpha into the two superfamilies Rhigonematoidea and Ransomnematoidea, and indicate that the families Carnoyidae and Ichthyocephalidae and the genus Rhigonema are non-monophyletic. The present phylogeny strongly supports resurrection of the family Brumptaemiliidae, and also indicates that the family Ransomnematidae is sister to the family Hethidae.

Morphology, molecular characterization and phylogeny of Bolbosoma nipponicum Yamaguti, 1939 (Acanthocephala: Polymorphidae), a potential zoonotic parasite of human acanthocephaliasis.

Human acanthocephaliasis is a rare parasitic zoonosis mainly caused by acanthocephalans belonging to the genera Acanthocephalus, Bolbosoma, Corynosoma, Macracanthorhynchus, and Moniliformis. In the present paper, the juveniles of Bolbosoma nipponicum Yamaguti, 1939 collected from the northern fur seal Callorhinus ursinus (Linnaeus) (Mammalia: Carnivora) in Alaska, USA were precisely identified based on morphological characters and genetic data. Their detailed morphology was studied using light and, for the first time, scanning electron microscopy. The molecular characterization of the nuclear genes [small ribosomal subunit (18S) and large ribosomal subunit (28S)] and the mitochondrial cytochrome c oxidase subunit 1 (cox1) sequence data of B. nipponicum are provided for the first time. Moreover, in order to clarify the phylogenetic relationships of the genus Bolbosoma and the other genera in the family Polymorphidae, phylogenetic analyses were performed integrating different nuclear (18S + ITS+28S) and mitochondrial (cox1) sequence data using maximum likelihood (ML) and Bayesian inference (BI). The phylogenetic results showed that Bolbosoma has a sister relationship with Corynosoma, and also revealed that Southwellina is sister to Ibirhynchus + Hexaglandula. Our molecular phylogeny also indicated a possible host-switch pattern during the evolution of the polymorphid acanthocephalans. The ancestors of polymorphid acanthocephalans seem to have originally parasitized fish-eating waterfowl in continental habitats, then extended to fish-eating marine birds in brackish water and marine habitats, and finally, opportunistically infected the marine mammals.

Morphology and molecular characterization of Acanthogyrus (Acanthosentis) bilaspurensis Chowhan, Gupta & Khera, 1987 (Acanthocephala: Gyracanthocephala: Quadrigyridae) from the common carp Cyprinus carpio Linnaeus (Cypriniformes: Cyprinidae) in Pakistan.

Acanthogyrus (Acanthosentis) bilaspurensis Chowhan, Gupta & Khera, 1987 is a poorly known acanthocephalan species reported from the reba carp Cirrhinus reba (Hamilton) (Cypriniformes: Cyprinidae) in India. In the present study, the detailed morphology of A. (Acanthosentis) bilaspurensis were studied using light microscopy and, for the first time, scanning electron microscopy (SEM), based on newly collected specimens from the common carp Cyprinus carpio Linnaeus (Cypriniformes: Cyprinidae) in Pakistan. The SEM observations revealed the outer shell of eggs with numerous remarkable protuberances and the gonopore of female located at a discoid protrusion surrounded by some very small spines. This is the first time that A. (Acanthosentis) bilaspurensis was reported in Pakistan, and the common carp represents a new host for this species. In addition, the molecular characterization of the 18S and 28S rDNA of A. (Acanthosentis) bilaspurensis is provided for the first time. There was no intraspecific genetic variation detected in the 18S and 28S regions between different individuals of A. (Acanthosentis) bilaspurensis, but high level of interspecific nucleotide divergence was found in the 18S (4.15-16.7%) and 28S regions (3.60-15.4%). Phylogenetic results based on the 18S, 28S and 18S + 28S sequence data, respectively, all revealed the Quadrigyridae, Pallisentinae and the genus Neoechinorhynchus are not monophyletic groups, and the genera Acanthogyrus and Pallisentis have far relationship. The systematic status of the genus Acanthogyrus and some species of Neoechinorhynchus need further clarify based on different mtDNA and nuclear DNA data including broader representatives of the class Eoacanthocephala.

Morphology, genetic characterization and molecular phylogeny of the poorly known nematode parasite Cissophyllus leytensis Tubangui & Villaamil, 1933 (Nematoda: Ascaridida) from the Philippine sailfin lizard Hydrosaurus pustulatus (Eschscholtz, 1829) (Reptilia: Squamata).

BACKGROUND: The genus Cissophyllus (Cosmocercoidea: Kathlaniidae) is a rare group of nematodes parasitic in turtles and lizards. To date, only four species have been reported in Asia and North America. However, most of them are inadequately described. The species Cissophyllus leytensis has never been reported since it was originally described by Tubangui and Villaamil in 1933 from the Philippine sailfin lizard Hydrosaurus pustulatus (Eschscholtz, 1829) (Reptilia: Squamata). Furthermore, the systematic status of Cissophyllus/Cissophyllinae in the family Kathlaniidae of the superfamily Cosmocercoidea remains under debate. METHODS: The detailed morphology of C. leytensis was studied using light microscopy (LM) and, for the first time, scanning electron microscopy (SEM), based on newly collected specimens from the type host H. pustulatus. Six different genetic markers, including nuclear sequences [small ribosomal subunit (18S), internal transcribed spacer (ITS) and large ribosomal subunit (28S)], plus mitochondrial genes [cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2) and 12S small subunit ribosomal RNA gene] of C. leytensis were sequenced. Additionally, in order to test the validity of the subfamily Cissophyllinae and clarify the phylogenetic relationships of Cissophyllus and the other genera in the family Kathlaniidae, phylogenetic analyses based on 18S + 28S and ITS sequence data were performed using maximum likelihood (ML) and Bayesian inference (BI) analyses, respectively. RESULTS: Our observations using LM and SEM revealed some previously unreported morphological features, necessitating the redescription of this poorly known species. The presence of remarkable morphological variation in the isthmus and the position of excretory pore among different individuals was found. Molecular analysis showed no intraspecific nucleotide divergence detected in the 18S, ITS, 28S, cox2 and 12S regions among different individuals of C. leytensis, but a low level of intraspecific genetic variation was found in the cox1 (0.52%). Our phylogenetic results showed the representatives of the Cosmocercoidea divided into four large clades (Cosmocerca + Aplectana + Cosmocercoides representing the family Cosmocercidae, Cruzia representing the subfamily Cruzinae of Kathlaniidae, Falcaustra + Cissophyllus + Megalobatrachonema representing the subfamily Kathlaniinae of Kathlaniidae, and Orientatractis + Rondonia representing the family Atractidae). The genus Cissophyllus clustered together with the genus Megalobatrachonema in both the ML and BI trees using ITS sequence data, but displayed a sister relationship to the genus Falcaustra in the ML tree and to the genera Falcaustra + Megalobatrachonema in the BI tree using 18S + 28S sequence data. CONCLUSIONS: Molecular phylogenetic results further confirmed that the family Kathlaniidae is not a monophyletic group. The subfamily Cruziinae should be moved from the hitherto-defined family Kathlaniidae and elevated as a separate family Cruziidae. The present phylogenetic results also negated the validity of the subfamily Cissophyllinae and supported the genus Cissophyllus assigned in the subfamily Kathlaniinae. Molecular analysis indicated that the morphological variation in the isthmus and position of excretory pore among different individuals should be considered as intraspecific variation. Moreover, some characters important for the specific diagnosis of C. leytensis are reported for the first time: the number of acuminate denticles (lamellae) on each lip, the chitinized pharynx with three flabellate pharyngeal plates, the presence of single medioventral precloacal papilla and the detailed morphology of caudal papillae. The present study is only the second record of C. leytensis.

Ultrasound-guided paravertebral nerve block anesthesia on the stress response and hemodynamics among lung cancer patients.

BACKGROUND: Thoracic surgery for radical resection of lung tumor requires deep anesthesia which can lead to an adverse inflammatory response, loss of hemodynamic stability, and decreased immune function. Herein, we evaluated the feasibility and benefits of ultrasound-guided paravertebral nerve block anesthesia, in combination with general anesthesia, for thoracic surgery for lung cancer. The block was performed by diffusion of anesthetic drugs along the paravertebral space to achieve unilateral multi-segment intercostal nerve and dorsal branch nerve block. AIM: To evaluate the application of ultrasound-guided paravertebral nerve block anesthesia for lung cancer surgery to inform practice. METHODS: The analysis was based on 140 patients who underwent thoracic surgery for lung cancer at our hospital between January 2018 and May 2020. Patients were randomly allocated to the peripheral + general anesthesia (observation) group (n = 74) or to the general anesthesia (control) group (n = 66). Patients in the observation group received ultrasound-guided paravertebral nerve block anesthesia combined with general anesthesia, with those in the control group receiving an epidural block combined with general anesthesia. Measured outcomes included the operative and anesthesia times, as well as the mean arterial pressure (MAP), heart rate (HR), and blood oxygen saturation (SpO2) measured before surgery, 15 min after anesthesia (T1), after intubation, 5 min after skin incision, and before extubation (T4). RESULTS: The dose of intra-operative use of remifentanil and propofol and the postoperative use of sufentanil was lower in the observation group (1.48 ± 0.43 mg, 760.50 ± 92.28 mg, and 72.50 ± 16.62 mg, respectively) than control group (P < 0.05). At the four time points of measurement (T1 through T4), MAP and HR values were higher in the observation than control group (MAP, 90.20 ± 9.15 mmHg, 85.50 ± 7.22 mmHg, 88.59 ± 8.15 mmHg, and 90.02 ± 10.02 mmHg, respectively; and HR, 72.39 ± 8.22 beats/min, 69.03 ± 9.03 beats/min, 70.12 ± 8.11 beats/min, and 71.24 ± 9.01 beats/min, respectively; P < 0.05). There was no difference in SpO2 between the two groups (P > 0.05). Postoperative levels of epinephrine, norepinephrine, and dopamine used were significantly lower in the observation than control group (210.20 ± 40.41 pg/mL, 230.30 ± 65.58 pg/mL, and 54.49 ± 13.32 pg/mL, respectively; P < 0.05). Similarly, the postoperative tumor necrosis factor-α and interleukin-6 levels were lower in the observation (2.43 ± 0.44 pg/mL and 170.03 ± 35.54 pg/mL, respectively) than control group (P < 0.05). There was no significant difference in the incidence of adverse reactions between the two groups (P > 0.05). CONCLUSION: Ultrasound-guided paravertebral nerve block anesthesia improved the stress and hemodynamic response in patients undergoing thoracic surgery for lung cancer, with no increase in the rate of adverse events.

Morphology, genetic characterization and phylogeny of Aplectana dayaoshanensis n. sp. (Nematoda: Ascaridida) from frogs.

Cosmocercoid nematodes are common parasites in the digestive tract of amphibians. However, our knowledge of the species diversity, genetic data and molecular phylogeny of the superfamily Cosmocercoidea are far from being well understood. In the present study, large numbers of cosmocercoid nematodes were collected from the fine-spined frog Sylvirana spinulosa (Smith) (Anura: Ranidae) and the white-spotted thigh tree-frog Polypedates megacephalus (Hallowell) (Anura: Rhacophoridae) in Guangxi Province, China. Integrated morphological and genetic evidence reveals these nematode specimens to be a new species of the genus Aplectana, A. dayaoshanensis n. sp. (Cosmocercoidea: Cosmocercidae). The molecular characterization of small ribosomal DNA (18S), internal transcribed spacer (ITS) and large ribosomal DNA (28S) of A. dayaoshanensis n. sp., together with the 28S of A. chamaeleonis (Baylis, 1929) (collected from Hyperolius kivuensis Ahl in Rwanda), were reported for the first time. Moreover, phylogenetic analyses using maximum likelihood (ML) inference based on 18S + 28S and ITS sequence data, respectively, both supported the family Cosmocercidae to be a monophyletic group and the family Kathlaniidae to be a paraphyletic group. Our phylogenetic results rejected the monophyly of the genus Aplectana. The present results contribute to the knowledge of the species diversity and genetic data of cosmocercoid nematodes, and preliminarily revealed the phylogenetic relationships of the major families and some genera in the Cosmocercoidea.

[Preparation of Sulfidated Copper-Iron Bimetallic Composited Material and Its Mechanism for Chromium Removal].

Sulfidated copper-iron bimetallic particles (S-Fe-Cu) were prepared by sulfidation of copper-iron bimetallic particles (Fe-Cu) obtained by the replacement reaction in the liquid phase. The influencing factors of S-Fe-Cu in removing Cr(Ⅵ) in water were determined. BET, SEM-EDX, and XPS were used to analyze the superficial structure and mineralogy of S-Fe-Cu. Combined with batch experiments, the mechanisms of Cr removal were analyzed. The results showed that FeSx was successfully loaded on the surface of S-Fe-Cu, and the optimum S/Fe molar ratio and Cu/Fe mass ratio in theory were 0.056 and 0.025, respectively. Compared with Fe-Cu, the specific surface area of S-Fe-Cu increased by 2.1 times, and the Cr removal efficiency increased by 6.1 times under a pH of 5. A high Cr removal efficiency was maintained under alkaline conditions. Meanwhile, chloride ions could penetrate the passivation layer of iron-based material, which was beneficial to the direct oxidation of Fe0 to produce Fe(Ⅱ) and advance the performance of S-Fe-Cu for Cr removal. The XPS results showed that the removal mechanism of Cr(Ⅵ) in water included adsorption, reduction, and coprecipitation.

Morphological and genetic characterization of Porrocaecum angusticolle (Molin, 1860) (Nematoda: Ascaridomorpha) from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic.

Porrocaecum angusticolle is a nematode species mainly parasitic in the birds of Accipitriformes and Strigiformes. However, some aspects of the morphology of P. angusticolle remain insufficiently known. In the present study, the detailed morphology of P. angusticolle was studied using light and, for the first time, scanning electron microscopy, based on newly collected specimens from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic. Some previously unreported morphological features of taxonomic significance were observed. The nuclear and mitochondrial DNA markers, including partial large ribosomal DNA (28S), complete internal transcribed spacer (ITS-1 + 5.8S + ITS-2), cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2) of P. angusticolle were sequenced for molecular identification of this species. There was no intraspecific genetic variation detected in the 28S and ITS regions among different individuals of P. angusticolle, but low level of intraspecific nucleotide divergence was found in the cox1 (0.26-0.78%) and cox2 regions (1.0%). The 28S and cox2 of P. angusticolle were sequenced for the first time. Our molecular evidence supported the validity of both P. angusticolle and P. depressum. The newly obtained genetic data are helpful for further studies of DNA-based taxonomy, population genetics and phylogeny of the genus of Porrocaecum.

Description of a new species of Aplectana (Nematoda: Ascaridomorpha: Cosmocercidae) using an integrative approach and preliminary phylogenetic study of Cosmocercidae and related taxa.

BACKGROUND: Nematodes of the family Cosmocercidae (Ascaridomorpha: Cosmocercoidea) are mainly parasitic in the digestive tract of various amphibians and reptiles worldwide. However, our knowledge of the molecular phylogeny of the Cosmocercidae is still far from comprehensive. The phylogenetic relationships between Cosmocercidae and the other two families, Atractidae and Kathlaniidae, in the superfamily Cosmocercoidea are still under debate. Moreover, the systematic position of some genera within Cosmocercidae remains unclear. METHODS: Nematodes collected from Polypedates megacephalus (Hallowell) (Anura: Rhacophoridae) were identified using morphological (light and scanning electron microscopy) and molecular methods [sequencing the small ribosomal DNA (18S), internal transcribed spacer 1 (ITS-1), large ribosomal DNA (28S) and mitochondrial cytochrome c oxidase subunit 1 (cox1) target regions]. Phylogenetic analyses of cosmocercoid nematodes using 18S + 28S sequence data were performed to clarify the phylogenetic relationships of the Cosmocercidae, Atractidae and Kathlaniidae in the Cosmocercoidea and the systematic position of the genus Aplectana in Cosmocercidae. RESULTS: Morphological and genetic evidence supported the hypothesis that the nematode specimens collected from P. megacephalus represent a new species of Aplectana (Cosmocercoidea: Cosmocercidae). Our phylogenetic results revealed that the Cosmocercidae is a monophyletic group, but not the basal group in Cosmocercoidea as in the traditional classification. The Kathlaniidae is a paraphyletic group because the subfamily Cruziinae within Kathlaniidae (including only the genus Cruzia) formed a seperate lineage. Phylogenetic analyses also showed that the genus Aplectana has a closer relationship to the genus Cosmocerca in Cosmocercidae. CONCLUSIONS: Our phylogenetic results suggested that the subfamily Cruziinae should be moved from the hitherto-defined family Kathlaniidae and elevated as a separate family, and the genus Cosmocerca is closely related to the genus Aplectana in the family Cosmocercidae. The present study provided a basic molecular phylogenetic framework for the superfamily Cosmocercoidea based on 18S + 28S sequence data for the first time to our knowledge. Moreover, a new species, A. xishuangbannaensis n. sp., was described using integrative approach.

Morphology, genetic characterization and molecular phylogeny of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) from the endangered Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae).

BACKGROUND: The Tibetan antelope Pantholops hodgsonii (Abel) (Artiodactyla: Bovidae) is an endangered species of mammal endemic to the Qinghai-Tibetan Plateau. Parasites and parasitic diseases are considered to be important threats in the conservation of the Tibetan antelope. However, our present knowledge of the composition of the parasites of the Tibetan antelope remains limited. METHODS: Large numbers of nematode parasites were collected from a dead Tibetan antelope. The morphology of these nematode specimens was observed using light and scanning electron microscopy. The nuclear and mitochondrial DNA sequences, i.e. small subunit ribosomal DNA (18S), large subunit ribosomal DNA (28S), internal transcribed spacer (ITS) and cytochrome c oxidase subunit 1 (cox1), were amplified and sequenced for molecular identification. Moreover, phylogenetic analyses were performed using maximum likelihood (ML) inference based on 28S and 18S + 28S + cox1 sequence data, respectively, in order to clarify the systematic status of these nematodes. RESULTS: Integrated morphological and genetic evidence reveals these nematode specimens to be a new species of pinworm Skrjabinema longicaudatum (Oxyurida: Oxyuridae). There was no intraspecific nucleotide variation between different individuals of S. longicaudatum n. sp. in the partial 18S, 28S, ITS and cox1 sequences. However, a high level of nucleotide divergence was revealed between the new species and its congeners in 28S (8.36%) and ITS (20.3-23.7%) regions, respectively. Molecular phylogenetic results suggest that the genus Skrjabinema should belong to the subfamily Oxyurinae (Oxyuroidea: Oxyuridae), instead of the subfamily Syphaciidae or Skrjabinemiinae in the traditional classification, as it formed a sister relationship to the genus Oxyuris. CONCLUSIONS: A new species of pinworm Skrjabinema longicaudatum n. sp. (Oxyurida: Oxyuridae) is described. Skrjabinema longicaudatum n. sp. represents the first species of Oxyurida (pinworm) and the fourth nematode species reported from the Tibetan antelope. Our results contribute to the knowledge of the species diversity of parasites from the Tibetan antelope, and clarify the systematic position of the genus Skrjabinema.