Deep in the systematics of Camallanidae (Nematoda): using integrative taxonomy to better understand the phylogeny and consistency of diagnostic traits.

Due to conflicts between classic and molecular systematics of Camallanidae, different data types were used for the first time, to better understand the evolutionary history and taxa consistency within this family. Genetic [18S and 28S rDNA; cytochrome c oxidase subunit I (COI) mtDNA], morphological and life history traits were used to infer phylogenies using Bayesian inference, reconstructed from separated and concatenated datasets. The consistency of tree and morphological traits was evaluated using the consistency index. Characters were mapped on the trees and the phylogenetic informativeness of genetic markers was estimated. Phylogenetic informativeness of 18S provided better resolution for outer nodes, COI for inners and 28S had an intermediate profile. New sequences for two camallanid species were obtained. Phylogenies of genetic and concatenated data largely agreed, showing more divergence in the COI dataset, due to its higher mutation rate vs stable morphology for diagnosing higher taxa. No genus sustained monophyly. The lack of autapomorphy and phylogenetic proximity supported the partition of Batrachocamallanus as synonym of Procamallanus and Spirocamallanus, which should not be considered as subgenera. Although traits of buccal capsule, male tail, habitat, host and biogeographic were highly consistent, intrinsic patterns varied according to different taxa assemblages. Morphological systematics of Camallanidae, based on buccal capsule, is artificial for certain taxa.

An integrative approach assesses the intraspecific variations of Procamallanus (Spirocamallanus) inopinatus, a common parasite in Neotropical freshwater fishes, and the phylogenetic patterns of Camallanidae.

Integrative taxonomy was used to evaluate two component populations of Procamallanus (Spirocamallanus) inopinatus in Brazil and the phylogeny Camallanidae. Parasite populations were collected in the characiform Anostomoides passionis from River Xingu (Amazon basin) and Megaleporinus elongatus from River Miranda (Paraguay basin). Morphology was analysed using light and scanning electron microscopy (SEM). Genetic characterization was based on partial sequences of the 18S and 28S rDNA, and COI mtDNA. Phylogenies were based on 18S and COI due to data availability. Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (PTP) and *BEAST were used for species delimitation and validation. SEM revealed for the first time the presence of minute denticles and pore-like structures surrounding the oral opening, phasmids in females and confirmed other important morphological aspects. Statistical comparison between the two-component populations indicated morphometric variations, especially among males. The different component population of P. (S.) inopinatus showed variable morphometry, but uniform morphology and were validated as conspecific by the GMYC, PTP and *BEAST. Some camallanid sequences in GenBank have incorrect taxonomic labelling. Host, environment and geographic aspects seem to be related to some lineages within Camallanidae; however, their real phylogenetic meanings are still unclear.

Morphological and genetic characterization of Pterygodermatites (Paucipectines) zygodontomis (Nematoda: Rictulariidae) from Necromys lasiurus (Rodentia: Sigmodontinae) from Uberlândia, Brazil.

Pterygodermatites (Paucipectines) zygodontomis, a nematode parasite of the small intestine of the rodent Necromys lasiurus, from Uberlândia, Minas Gerais state, Brazil, was analysed by light and scanning electron microscopy. Additionally, phylogenies were inferred from the mitochondrially encoded cytochrome c oxidase I gene (MT-CO1). Details of the helminth surface, such as the oral aperture, cephalic papillae, papillae in the posterior region of the body and longitudinal cuticular elements represented by spine-like projections and fans are presented, adding new taxonomic details. Molecular phylogenetic analysis, based on the MT-CO1, demonstrated that P. (P.) zygodontomis and Pterygodermatites (Paucipectines) jaegerskioldi form a unique evolutionary unit in accordance with the subgenus Paucipectines and corroborated their occurrence in cricetid and didelphid hosts.

Morphological and molecular characterization of Paraleptus chiloscyllii Yin & Zhang, 1983 (Nematoda: Physalopteridae) from the brownbanded bambooshark Chiloscyllium punctatum Müller & Henle (Elasmobranchii: Orectolobiformes).

Paraleptus (Spirurida: Physalopteridae) is a small genus of nematodes, parasitic in fishes, most species of which are inadequately described. Genetic data for these congeners have not been reported. The detailed morphology of P. chiloscyllii was studied using light and scanning electron microscopy, based on newly collected specimens from the brownbanded bambooshark C. punctatum Müller & Henle (Elasmobranchii: Orectolobiformes) in the Taiwan Strait. Some previously unreported morphological features of taxonomic significance were observed, i.e., pseudolabium with two sublateral rows of 6-7 small spines, 7-8 small spines on each lower rim between pseudolabia, deirids not distally bifurcated, vulva with remarkable protruding lip, presence of 1 pair medio-ventral precloacal papillae and 1 pair of discoid protrusions of postcloacal lip in male. The specimens described by González-Solís & Ali's (2015) as P. chiloscyllii from the Arabian carpetshark C. arabicum off Iraq are considered a new species, for which the name P. moraveci n. sp. is proposed. The genetic characterization of the partial small (18S) and large (28S) ribosomal DNA, and the partial mitochondrial cytochrome c oxidase subunit 1 (cox1) of P. chiloscyllii are provided for the first time. There was no intraspecific nucleotide divergence detected in the 18S and 28S regions among different individuals of P. chiloscyllii, but a low level of intraspecific genetic variation was found in the cox1 (0.62-0.92%). The present genetic data is very important for molecular identification, and will be valuable for further invertigantions on the population genetics and phylogeny of this group.

Prevalence of Blatticola blattae (Thelastomatidae) in German cockroaches Blattella germanica in Japan.

A survey for nematode infection in German cockroaches captured in restaurants in various areas of Japan as well as the laboratory-bred colony was carried out. The nematodes were then identified morphologically and molecularly. Of the 320 German cockroaches collected at 79 restaurants in 26 prefectures in Japan, 66.6% (213/320) were found to be parasitized by a single species of pinworm in the hindgut. The mean number of pinworms per cockroach was less than 1.6. Of the three laboratory-bred lines of cockroaches examined, 2 lines (NIID and NK) were found to be infected with a single species of nematode. The prevalence was 93.0% (40/43) and 84.8% (39/46), respectively. The other laboratory line (WAT) was found to be free of the nematode infection. The nematode detected in this study was identified as Blatticola blattae. This is the first report of B. blattae infection in German cockroaches in Japan. Our study showed that B. blattae is distributed all over Japan together with its host Blattella germanica. An experimental infection with B. blattae in nematode-free cockroach by contaminating the rearing environment with infected-cockroach feces showed that once the environment of the cockroach is contaminated with B. blattae eggs, the pinworm infection could spread easily.

Sequencing of the Complete Mitochondrial Genome of Pingus sinensis (Spirurina: Quimperiidae): Gene Arrangements and Phylogenetic Implications.

Despite several decades of intensive research on spirurine nematodes, molecular data on some of the main lineages are still absent, which makes taxonomic classification insufficiently resolved. In the present study, we sequenced the first complete mitogenome for the family Quimperiidae, belonging to P. sinensis (Spirurina: Quimperiidae), a parasite living in the intestines of snakehead (Ophiocephalus argus). The circular mitogenome is 13,874 bp long, and it contains the standard nematode gene set: 22 transfer RNAs, 2 ribosomal RNAs and 12 protein-coding genes. There are also two long non-coding regions (NCR), in addition to only 8 other intergenic regions, ranging in size from 1 to 58 bp. To investigate its phylogenetic position and study the relationships among other available Spirurina, we performed the phylogenetic analysis using Bayesian inference and maximum likelihood approaches by concatenating the nucleotide sequences of all 36 genes on a dataset containing all available mitogenomes of the suborder Spirurina from NCBI and compared with gene order phylogenies using the MLGO program. Both supported the closer relationship of Ascaridoidea to Seuratoidea than to Spiruroidea. Pingus formed a sister-group with the Cucullanus genus. The results provide a new insights into the relationships within Spirurina.

Description and Molecular Differentiation of a New Falcaustra (Nematode: Kathlaniidae) from the Indochinese Water Dragon, Physignathus cocincinus (Squamata: Agamidae) in North-central Vietnam.

Falcaustra vietnamensis n. sp. is described from the small intestine of Physignathus cocincinus from north-central Vietnam. The new species is characterized by the large male worms (20.2-28.8 mm in length and 557-724 µm in width) relative to known members of the genus, 2 sharply pointed alate spicules of equal length (1,128-1,256 µm in length), gubernaculum including 2 separate pieces, 1 ventral with a pointed distal end and 1 dorsal with a blunt distal end (164-192 µm and 155-172 µm in length, respectively), and 12 pairs of caudal papillae. Female worms are larger than male worms (24.2-34.1 mm in length and 532-735 µm in width), with the vulva situated in the posterior half of body, and elliptical eggs, 60-70 µm long by 42-47 µm wide. Falcaustra vietnamensis n. sp. represents the 38th species assigned to the genus and the third species recorded from a lizard host in the Oriental biogeographical region. Partial sequences of the 18S ribosomal RNA gene (rDNA), internal transcribed spacer regions (ITS), and cytochrome c oxidase subunit 1 (COI) are provided for the new species. The molecular phylogenetic position of the genus Falcaustra is briefly discussed.

Description and Molecular Differentiation of a New Skrjabinoptera (Nematode: Physalopteridae) from Eutropis macularia (Sauria: Scincidae) in North-Central Vietnam.

Skrjabinoptera vietnamensis n. sp. is described from specimens recovered from the stomach of Eutropis macularia in north-central Vietnam. The new species is characterized by the medium-sized male worms (6.7-8.7 mm in length and 154-182 µm in width) relative to known members of the genus, 2 pointed spicules of unequal length (87-112 µm and 56-72 µm in length), and 10 pairs of caudal papillae. Female worms are larger than male worms (10.7-18.4 mm in length and 264-411 µm in width), with the vulva situated in the anterior part, and embryonated, elliptical eggs, 35-46 µm long by 20-24 µm wide. Skrjabinoptera vietnamensis n. sp. represents the ninth species assigned to the genus and the first species recorded from the Oriental region. Partial sequences of the 18S ribosomal RNA gene (rDNA), and cytochrome c oxidase subunit 1 (COI) are provided for the new species. The molecular phylogenetic position of the genus Skrjabinoptera is briefly discussed.

Development of conventional multiplex PCR method for discrimination between Dispharynx nasuta and Cheilospirura hamulosa (Nematoda: Acuariidae) parasitizing poultry.

The poultry infections caused by Dispharynx nasuta and Cheilospirura hamulosa nematodes are difficult to be diagnosed by fecal examination because of their egg similarity. In this study, we analyzed DNA sequences of nuclear ribosomal 18S-ITS1-5.8S-ITS2-28S region of D. nasuta and C. hamulosa and developed conventional multiplex PCR method using species-specific primers for discriminating between the two species. The method amplified 455-bp and 319-bp fragments specific to D. nasuta and C. hamulosa, respectively, and did not produce them against the other chicken nematode species, Ascaridia galli, Oxyspirura mansoni, Heterakis gallinarum, Heterakis beramporia, and Heterakis indica, suggesting that the multiplex PCR is sensitive and available for species diagnosis.

DIPLOTRIAENA OBTUSA (NEMATODA: DIPLOTRIAENIDAE) FROM BARN SWALLOWS (HIRUNDO RUSTICA) AND CLIFF SWALLOWS (PETROCHELIDON PYRRHONOTA) COLLECTED DURING MORTALITY EVENTS IN THE UPPER MIDWEST, USA.

Several mortality events involving barn swallows (Hirundo rustica) and cliff swallows (Petrochelidon pyrrhonota) were reported in the Upper Midwestern states in 2017 and 2018. Barn swallow mortality followed unseasonal cold snaps, with the primary cause of death being emaciation with concurrent air sac nematodiasis. Lesions in cliff swallows were consistent with blunt force trauma from suspected car impacts. Examination of air sac nematodes from both bird species revealed morphological characters consistent with Diplotriaena obtusa. Sequence analysis of the partial 18S rRNA gene indicated the samples clustered with other species in the genus Diplotriaena. These nematodes provide a link between morphological specimens and DNA sequence data for D. obtusa.

Molecular Phylogeny of Thelastomatoidea (Nematoda) with the Description of a New Genus and Two New Species of Hystrignathidae Associated with Bess Beetles (Coleoptera: Passalidae) from Oaxaca, Mexico.

Bess beetles (Passalidae) display important roles in forestall ecosystems, particularly in energy extraction from dead wood. These organisms maintain complex biological interactions with their gut symbiotic communities, including bacteria, protists, and metazoans. Very little is known about symbionts since most of the species of Passalidae haven't been studied from a parasitological point of view. Here we describe a new genus and 2 new species of nematodes of the family Hystrignathidae associated with 2 beetle species of the tribe Proculini collected in the State of Oaxaca, Mexico. Tuhmai garciaprietoi n. gen., n. sp., found in Vindex agnoscendus is characterized by the presence of an unarmed cervical cuticle, a subcylindrical procorpus and a conspicuous isthmus, a monodelphic-prodelphic reproductive system, and a short subulate tail. Urbanonema osorioi n. sp., found in Verres hageni mainly differs from other species of Urbanonema by the number and disposition of cervical spines, as well as by a subulate tail. For each new taxon, we describe the external and internal morphology, and we generated molecular data (nuclear ribosomal DNA) to place the new taxa in a phylogenetic context.

Additional Morphological Features and Molecular Data of Paracamallanus cyathopharynx (Nematoda: Camallanidae) Infecting Clarias gariepinus (Actinopterygii: Clariidae) in Kenya.

This study provides additional taxonomic features based on scanning electron microscopy (SEM) and molecular data for Paracamallanus cyathopharynx ( Baylis, 1923 ) (Nematoda: Camallanidae). Parasites were collected from the posterior end of the intestine of cultured freshwater Sharptooth catfish Clarias gariepinus (Burchell, 1822) from Kibos fish farm, Kisumu County, Kenya, from December 2017 to March 2018. Additional taxonomic features recorded for P. cyathopharynx include the occurrence of 4-5 equal length digitate processes on the caudal end of the female, 4 processes (2 smaller and 2 larger) on the male caudal end, and a description of the shape of the distal tip of the right spicule. The study provides SEM images of the exposed sclerotized buccal capsule. This gives more information on the tridents, the sclerotized plate that extends laterally from the buccal capsule, and the narrow isthmus separating the anterior buccal capsule from the posterior. The prevalence, intensity, mean intensity, and mean abundance was 52.91%, 2-38, 12.37 and 6.54, respectively. 18S rDNA fragments were amplified, sequenced, and compared to other camallanid taxa, and 18S data confirmed the identity of the newly obtained sequences (MN396556) as that of P. cyathopharynx, being identical to sequence DQ813445 from Tanzania. This represents the first geographical record of P. cyathopharynx in Kenya.

A New Species of Triumphalisnema (Nematoda: Coronostomatoidea) Parasite of Proculejus hirtus (Coleoptera: Passalidae) from Mexico, and the Position of the Family in the Oxyuridomorpha.

A new species of the nematode Triumphalisnema Kloss, 1962 (Oxyuridomorpha), is described from the wood beetle Proculejus hirtus Truqui from the mountain mesophilic forest in Hidalgo State, Mexico. Triumphalisnema zuuei n. sp. is distinguished from the other 4 congeners species by the presence of an expanded cervical ring, well-developed lateral alae, an obtuse cauda with a short and bifurcated caudal appendage, series of cuticular folds at ventral and dorsal body surface from excretory pore level to anal region, and ellipsoidal eggs ornamented with numerous small mushroom-like structures over the surface. The phylogenetic position of the new species is inferred based on a Maximum Likelihood and Bayesian Inference analysis of partial sequences of 18S SSU rRNA. The phylogenetic analysis showed that Triumphalisnema zuuei n. sp., the only representative of the Traklosiidae in our study, is closely related to Coynema poeyi and species of Longior, Hystrignathus, and Lepidonema, all of them members of Hystrignathidae. These relationships are supported by high support values. The present study increases to 5 the number of species assigned to Triumphalisnema, all of them parasites of Passalidae. Additionally, a taxonomic key to the species of the genus is provided.

Morphology and Molecular Characterization of Parabronema smithii (Cobbold, 1882) (Nematoda: Habronematidae) from Wild Asian Elephant (Elephas maximus maximus) of Sri Lanka.

PURPOSE: The aim of the present study was to carry out a detailed study of morphological features and to determine the phylogenetic position of Parabronema smithii (Cobbold, 1882) found in wild elephants in Sri Lanka. METHODS: Adult worms were collected from stomach ulcers at postmortem examination of wild elephants in the Udawalawe National Park, Sri Lanka. The detailed morphology of P. smithii was studied using light microscopy and, for the first time, scanning electron microscopy. Fifteen morphological characteristics were investigated. The phylogenetic analysis was conducted using the second internal transcribed spacer region (ITS2), and portions of the large subunit ribosomal DNA (28S) and cytochrome c oxidase subunit 1 (cox1). Furthermore, the present study provides a comparison of morphology and morphometrics of Parabronema species that occur in different hosts. CONCLUSION: Parabronema smithii isolated from wild elephants exhibited the key morphological features. Phylogenetic analysis of selected genes revealed that P. smithii is closely associated with P. skrjabini and Habronema spp. Findings of the present study enhance our understanding of the biology and taxonomy of P. smithii in wild elephant in Sri Lanka and will contribute to future phylogeographic studies.

Phylogenetic Relationships of Spiruromorph Nematodes (Spirurina: Spiruromorpha) In North American Freshwater Fishes.

Nematodes are common in the parasite communities of North American freshwater fishes, and the majority of them belong to 1 conventional order, Spirurida Chitwood, 1933. Within the Spirurida, the superfamilies Habronematoidea Chitwood and Wehr, 1932 and Thelazioidea Sobolev, 1949 have undergone considerable diversification. The dominant families of these 2 superfamilies, Cystidicolidae Skrjabin 1946 and Rhabdochonidae Railliet, 1916, respectively, are particularly common, widely distributed, and diverse, especially in North America, yet their phylogenetic relationships remain largely unexplored. In this study, we use near complete sequences of the 18S rRNA genes ( SSU rDNA) from species in 6 genera ( Capillospirura Skrjabin, 1924, Cystidicola Fischer, 1798, Salmonema Moravec, Santos and Brasil-Sato, 2008, Rhabdochona Railliet, 1916, Spinitectus Fourment, 1883, and a putative new cystidicolid in mooneyes, Hiodontidae), along with a species of Hedruris Nitzsch, 1812 from newts as a surrogate for the fish parasite Hedruris tiara VanCleave and Mueller, 1932, to explore their phylogenetic relationships. These sequences, together with available sequences from a range of other nematodes, including fish nematodes in other groups (Camallanoidea and 'Seuratoidea'), were analyzed using Bayesian inference and maximum likelihood. The results from both analyses indicate, for the first time, support for the close relationships of the sturgeon parasite Capillospirura with Ascarophis van Beneden, 1871 and Cystidicola; the relationship of the cystidicolid from Hiodontidae with Salmonema of salmonid fishes; the monophyly of the 2 dominant spiruridan genera of fishes, Rhabdochona and Spinitectus; and for previous relationships among Nearctic Spinitectus spp. The results also indicate a closer relationship of Rhabdochona and Spinitectus than is suggested by their conventional classification and reject the monophyly of Habronematoidea, Thelazioidea, and Cystidicolidae. Hedruridae appears to be an early branching lineage of spirurins. Finally, the pattern of association between the fish parasites in this study and their hosts indicates, with few exceptions, ecologically driven diversification events involving host shifting not related to the phylogenetic relationships of their hosts.

Evolutionary relationships of Spirurina (Nematoda: Chromadorea: Rhabditida) with special emphasis on dracunculoid nematodes inferred from SSU rRNA gene sequences.

The analysis of 26 new small subunit rRNA sequences obtained from helminths that primarily parasitize fishes sampled from five continents provided well-supported trees, allowing us to study the phylogenetic relationships among spirurid nematodes. The analyses have shown that Dracunculoidea is a paraphyletic taxon and Anguillicolidae and Gnathostomatidae constitute the basal branch of the suborder Spirurina. The genera Philometra and Philometroides appear to be paraphyletic, while on the higher taxonomic level, good correlation between the morphology-based system and molecular data was observed. Neither co-evolution of the studied helminths with their hosts, nor phylogeographic pattern, are apparent in our dataset.