GENETIC AND TRAIT VARIABILITY OF GYRINICOLA REVEALS THE EXISTENCE OF AT LEAST FOUR SPECIES WITHIN THE UNITED STATES.

The tadpole-dwelling pinworm, Gyrinicola batrachiensis (Walton, 1929) Adamson, 1981 was recognized as the sole representative of the genus across Canada and the United States. However, evaluation of the morphology of these parasites across their range revealed considerable morphological variability that suggested diagnosable morphotypes. These morphotypes were associated with different species of anurans, several of which occurred in sympatry. Herein we use an extensive geographic sampling across the United States to obtain the morphotypes, screen their genetic diversity, and analyze this information using an integrative approach. We reconstructed their phylogeny using nuclear ribosomal partial genes 18S and 28S, ITS1, 5.8S, and ITS2, as well as 5 mitochondrial genes generated with Next-Generation sequencing technology. This phylogeny reveals 3 well-resolved lineages, which upon the use of a statistical approach (bPTP [Bayesian implementation of the Poisson tree processes]) supports the delimitation of 4 distinct groups equivalent to species. These putative species groups were tested using morphological characteristics paired with a MANOVA and canonical variate analysis. Results suggest that at least 4 species of Gyrinicola are present within North America, resulting in the resurrection of G. armatus (Walton, 1933) and the description of 2 new species.

Molecular data aids pinworm diagnosis in night monkeys (Aotus spp., Primates: Aotidae) with the resurrection of a Trypanoxyuris species (Nematoda: Oxyuridae).

Neotropical primates (Platyrrhines) are commonly parasitized by pinworm nematodes of the genus Trypanoxyuris Vevers, 1923. The taxonomic identity of Trypanoxyuris sampled in night monkeys (Aotus Iliger) has been rather controversial. Two species have been described, namely T. microon (Linstow, 1907) and T. interlabiata (Sandosham, 1950). The latter was synonymized with T. microon considering that the observed morphological differences corresponded to different developmental stages of the nematode rather than to differences between both species. Here, we used an integrative taxonomy approach, based on morphological and molecular data along with host identity, in order to assess the validity of both species. Our results evidenced that these different morphotypes correspond to different and reciprocally monophyletic groups; thus, we propose the resurrection of T. interlabiata. We redescribe both pinworm species using specimens sampled in Aotus monkeys from Colombia and discuss the advantages of combining molecular and morphological data to uncover pinworm diversity, and to understand the potential forces determining the diversification process in pinworms from platyrrhine primates.

MOLECULAR PHYLOGENETIC ANALYSIS OF GYRINICOLA BATRACHIENSIS (NEMATODA: OXYUROIDEA) LENDS SUPPORT TO THE MONOPHYLY OF THE GENUS AND THE RESURRECTION OF GYRINICOLINAE.

Gyrinicola Yamaguti, 1938, includes 6 species of oxyurid found within the intestinal tract of numerous, larval, anuran species in Europe, Asia, South America, and North America. The systematic placement and hierarchical treatment of the genus has shifted at least 5 times since its discovery; the group was first considered as its own family (Gyrinicolidae), then treated as a subfamily (Gyrinicolinae) of Cosmocercidae, then as a member of the Pharyngodonidae, followed by movement back to the Cosmocercidae, and finally a recent proposal suggested the resurrection of the Gyrinicolidae. Species in the genus vary widely in the morphology of the uterine tract, a characteristic often used to indicate membership in the genus, as it is tied to the reproductive mode. However, until recently very few genetic data were available to aid in the placement of this unique group of worms, and before this study to the best of our knowledge none existed for the North American species. To examine the monophyly and placement of the Gyrinicola we sampled populations of Gyrinicola batrachiensis across North America and screened them for genetic diversity using nuclear markers 18S and 28S. Phylogenies suggest at least 3 clades exist among the nematodes from North America and that these clades, alongside Gyrinicola japonica, form a well-supported group within Oxyuroidea. Further representation of Pharyngodonidae from other vertebrate classes may help clarify the relationship of this historical grouping to other members of the Oxyuroidea.

Skrjabinodon castillensis n. sp. (Nematoda:Pharyngodonidae) from the Homonota horrida and H.darwinii (Squamata: Phyllodactylidae) from Argentina and key for the Neotropical species of the genus Skrjabinodon.

Skrjabinodon castillensis n. sp. is described and illustrated here, based on specimens found in the large intestines of Homonota horrida (province San Juan) and Homonota darwinii (province Neuquén) (Squamata: Phyllodactylidae) from Argentina. The new species is assigned to Skrjabinodon based lateral alae present in males, absent in females. Lateral alae beginning midway between lips and nerve ring and ending just posterior to first pair of caudal papillae. Females with vulva near esophageal bulb. In males, caudal alae absent, paired caudal papillae present. Skrjabinodon castillensis n. sp. represents the 9th species from the Neotropical realm. The new species differs from all other species assigned to Skrjabinodon by morphology of tail filament and number of tail filament spines. Skrjabinodon castillensis n. sp. is the only species of this genus known from Argentina. A key to the species of Skrjabinodon in the Neotropical realm is provided.

Parapharyngodon hispidus n. sp. (Nematoda: Pharyngodonidae) in Tropidurus hispidus (Spix) (Squamata: Tropiduridae) from Caatinga Biome of the Vale do São Francisco, state of Pernambuco, Brazil with a key for the Neotropical species of the genus Parapharyngodon Chatterji.

One species of Parapharyngodon Chatterji (Nematoda: Pharyngodonidae), parasitizing the large intestine of Tropidurus hispidus (Spix) (Squamata: Tropiduridae) in Caatinga biome from Brazil is described and illustrated . This new species differs from a part of its congeners by a set of characters in males: the presence of four pairs of caudal papillae, unpaired post cloacal papilla absent, and smooth anterior cloacal lip. Among the Parapharyngodon species with four pairs caudal papillae, unpaired post clocal papilla absent, and smooth cloacal lips, only P. hispidus n. sp. and P. jairaipurii Rizvi Bursey (Oriental realm), features the same characteristics including sharp pointed tip of spicule, stout terminus or spike of tail of females, and punctate ornamentation of the eggshell. However, these two species are different by the size of spicules (spicules of P. jairaipuri are around 55% of the size spicules of P. hispidus n. sp.); and the arrangement of the caudal papillae (two pairs of adcloacal papillae in P. jaraipuri which are absent in P. hispidus n. sp.). Parapharyngodon hispidus n. sp. is the 55th species assigned to the genus and the 11th species of this genus described in Neotropical hosts.

Batracholandros salamandrae (Oxyuroidea: Pharyngodonidae) in Endemic Salamanders (Amphibia: Plethodontidae) of the Trans-Mexican Volcanic Belt: Host Range Wide Distribution or Cryptic Species Complex?

Salamanders of the tribe Bolitoglossini Hallowell are a highly diversified group of amphibians, and their helminth parasite fauna has been scarcely studied. Some species of plethodontid salamanders distributed along the Trans-Mexican Volcanic Belt, in central Mexico, were sampled, and their helminth parasites were recovered for taxonomic identification. Specimens of a pharyngodonid nematode from 2 species of bolitoglossines of the genus Pseudoeurycea Taylor were morphologically identified as Batracholandros salamandrae (Schad, 1960) Petter and Quentin, 1976. These specimens were studied in further detail through light and scanning electron microscopy and were sequenced for 2 ribosomal genes and 1 mitochondrial gene to test the hypothesis of whether B. salamandrae is a species widely distributed in salamanders across the Nearctic biogeographic region, or if it represents a cryptic species complex. Our molecular results revealed that these specimens consisted of 2 genetic lineages in concordance with host species, although with slight morphological differences among specimens in each of them. A thorough study, including the generation of molecular data from individuals from other areas of North America, and the examination of type specimens, is required to test the reliability of these morphological differences and to corroborate the species identity of the 2 genetic lineages.

Oxyuricassis ekstromi n. sp. (Oxyurida: Pharyngodonidae) from Lasiancistrus saetiger (Siluriform: Loricariidae) from the Eastern Amazon.

Oxyuricassis ekstromi n. sp. is the third species of the genus described from the intestine of Lasiancistrus saetiger Armbruster in Brazil. This species can be easily differentiated from its congeners by the presence of a tooth-like sclerotized piece that occupies half of the stoma and absence of spine projections on the tail of both males and females. Also, males of the new species lack caudal alae present in Oxyuricassis coronatus and Oxyuricassis hexaspinatus. Additionally, we observed a different number and pattern of the distribution of cloacal papilla; males of O. coronatus and O. hexaspinatus have 1 pair of pre-cloacal and 2 pairs of post-cloacal papillae; while the new species has 1 pre-cloacal and 3 post-cloacal pairs of papillae.

Morphological description and phylogenetic assessment of 28S rRNA for Thelandros chalcidiae sp. nov. from Chalcides ocellatus.

Thelandros is a genus of oxyurid nematodes which parasitize both omnivorous and herbivorous hosts. Thelandros chalcidiae sp. nov. is a new taxa described from the large intestine of the ocellated skink, Chalcides ocellatus, from the South Sinai Desert in Egypt. The recovered parasite species was examined at both morphological and molecular levels in order to determine the exact taxonomic position within Pharyngodonidae family. The current pharyngodonid species is characterized by a mouth opening bounded by three bilobed lips; male worms are characterized by the presence of two pairs of cloacal papillae (pre- and adcloacal) and one single caudal papillae (postcloacal) and caudal alae in an auricular form and females with post-equatorial vulva, amphidelphic ovary in which anterior ovary extended to level of excretory pore and posterior ovary extended posteriorly up anal opening. The recovered nematodes were compared with other known species from different hosts and it was found to be morphologically different from them. Molecular characterization based on the partial 28S rRNA nuclear ribosomal gene sequence showed sequence identities ≥ 83.15% with taxa under family Pharyngodonidae, 74.84-87.37% with Oxyuridae, 80.54% with Heteroxynematidae, and 75.98-77.72% with Thelastomatidea. Phylogenetic analysis showed that parasite sequence in conjunction with existing data facilitate placement of this species within Oxyurida. The present species is deeply embedded in genus Thelandros with close relationships to previously described Thelandros sp. and T. galloti in same taxon. This study highlighted importance of combining morphological and genetic data with taxonomy in pharyngodonid species.

Morphological redescription and phylogenetic assessment of Spauligodon aspiculus (Oxyuroidea: Pharyngodonidae) infecting the white-spotted gecko, Tarentola annularis (Squamata: Gekkonidae).

Thirty white-spotted geckos, Tarentola annularis, from the South Sinai desert in Egypt, were examined for helminth parasites. Spauligodon aspiculus was observed to infect 19 geckos with 63.33% as a prevalence of parasitic infection. The present nematode species is separated from congeners by morphological and metrical characteristics such as lateral alae, aspinose filamentous tail, and no spicule, and three pairs of caudal papillae with posterior pair excluded from envelopment by the caudal alae in the male worms, and knobbed eggs, and postbulbar vulva in females. It compared morphometrically with other Spauligodon species described previously and showed few differences in measurements. Molecular characterization based on the partial 28S rRNA nuclear ribosomal gene sequence showed that there was a close identity, up to 72%, with other sequences retrieved from GenBank. Phylogenetic analysis showed that the parasite sequence in conjunction with existing data facilitates the investigation of the placement of this pharyngodonid species within Oxyuridae. The present species is deeply embedded in the genus Spauligodon with close relationships to previously described Spauligodon nicolauensis (gb| JN619349.1, and JF829243.1) as more related sister taxa. This study highlights the importance of combining genetic and morphological data with taxonomy in pharyngodonid species.

Redescription of Gyrinicola japonica, a Tadpole-Endoparasitic Nematode from Japan, with Resurrection of the Family Gyrinicolidae (Nematoda: Oxyurina).

The taxonomic account of the tadpole-parasitic nematode Gyrinicola japonica Yamaguti, 1938, which is the type species of the genus, was reassessed based on syntypes and newly-collected specimens from the type locality. Our redescription of G. japonica addresses the erroneous original description of a spicule in this nematode, and emends the diagnosis of the species. Additionally, molecular phylogenetic trees based on nuclear 18S and 28S rDNA sequences revealed that G. japonica forms a distinctive lineage within the suborder Oxyurina, and this tadpole-specialist is phylogenetically close to the lizard-parasitic nematodes that belong to the family Pharyngodonidae. The results of morphological examination with the aid of molecular phylogenetic trees highlight the systematic uniqueness of this tadpole-parasitic group within Oxyurina, and Gyrinicolidae is accordingly resurrected as a distinctive oxyurinan family, with redefinition of the family and the genus Gyrinicola.

A new species of Parapharyngodon Chatterji, 1933 (Oxyuroidea: Pharyngodonidae), parasitic in Osteocephalus taurinus (Anura: Hylidae) from Brazil.

Parapharyngodon politoedi n. sp. is described here, based on specimens found in the large intestines of Osteocephalus taurinus from the Caxiuanã National Forest, State of Pará, Brazil. The new species is assigned to Parapharyngodon based on the presence of non-embryonated eggs with sub-terminal opercula, when in the ovijector. Parapharyngodon politoedi belongs to a group of species with three pairs of cloacal papillae and differs from its congeners by morphometric aspects, such as the length of the spicule, and the combination of the following morphological characters: ovaries never encircling the oesophagus, tail shape in females, cloacal lips, sharply pointed spicule and presence of genital cone. This is the second species of nematode reported to parasitize O. taurinus and the eleventh species of Parapharyngodon from hylids in the Neotropical region.

The importance of integrative approaches in nematode taxonomy: the validity of Parapharyngodon and Thelandros as distinct genera.

Despite the advances of molecular tools, new nematode species are still described mainly based on morphological characters. Parapharyngodon and Thelandros are two genera of oxyurids with unclear related taxonomic histories. Here we use morphological characters (linear measurements and categorical variables) and genetic information (18S rRNA, 28S rRNA and COI partial gene sequences) to confirm the relationships between representatives of these two genera and to determine whether they can be discriminated morphologically. Genetic results confirm the existence of two main clades, mostly congruent with Parapharyngodon and Thelandros genera but with several discordances. Thelandros is polyphyletic, with two of the species analysed (T. filiformis and T. tinerfensis) being part of the Thelandros clade, but with a third one (T. galloti) falling within the Parapharyngodon clade. Regarding the Parapharyngodon clade, P. cubensis, P. scleratus and Parapharyngodon sp. from Mexico form congruent lineages, while most P. echinatus samples cluster in another group, with one exception. Interestingly, P. micipsae samples are scattered across the Parapharyngodon clade, suggesting that they were misidentified or rather represent alternative morphotypes of other species. Morphological analysis identified the length of the tail, number of caudal papillae, position of the nerve ring, presence of caudal alae and length of the lateral alae as reliable characters to distinguish between Parapharyngodon and Thelandros genetic clades. Our study highlights the current taxonomic inconsistency in these groups, mainly derived from the exclusive use of morphological data. As such, we advocate for the routine implementation of molecular data in nematode taxonomic studies.

Cholinergic signaling plasticity maintains viscerosensory responses during Aspiculuris tetraptera infection in mice small intestine.

Intestinal parasites alter gastrointestinal (GI) functions like the cholinergic function. Aspiculuris tetraptera is a pinworm frequently observed in laboratory facilities, which infests the mice cecum and proximal colon. However, little is known about the impact of this infection on the GI sensitivity. Here, we investigated possible changes in spontaneous mesenteric nerve activity and on the mechanosensitivity function of worm-free regions of naturally infected mice with A. tetraptera. Infection increased the basal firing of mesenteric afferent nerves in jejunum. Our findings indicate that nicotinic but not muscarinic receptors, similarly affect spontaneous nerve firing in control and infected animals; these axons are mainly vagal. No difference between groups was observed on spontaneous activity after nicotinic receptor inhibition. However, and contrary to the control group, during infection, the muscarinic signaling was shown to be elevated during mechanosensory experiments. In conclusion, we showed for the first time that alterations induced by infection of the basal afferent activity were independent of the cholinergic function but changes in mechanosensitivity were mediated by muscarinic, but not nicotinic, receptors and specifically by high threshold nerve fibers (activated above 20mmHg), known to play a role in nociception. These plastic changes within the muscarinic signaling would function as a compensatory mechanism to maintain a full mechanosensory response and the excitability of nociceptors during infection. These changes indicate that pinworm colonic infection can target other tissues away from the colon.

Oxyuricassis coronatus n. gen. n. sp. and O. hexaspinatus n. sp. (Oxyurida: Pharyngodonidae): Parasites of Lasiancistrus saetiger (Siluriformes: Loricariidae) in Freshwater Rivers of the Brazilian Amazon.

A new nematode genus is described from intestines of Lasiancistrus saetiger Ambruster from freshwater rivers in the Brazilian Amazon. Oxyuricassis n. gen. is characterized by a cephalic cone, small buccal cavity, long esophagus with a well-developed isthmus, and a conical tail with spines. Two new species are described: Oxyuricassis coronatus n. sp. is characterized by the absence of lateral alae, a conical tail with truncated extremity with spine-like sclerotized projections around the tail tip, and caudal alae in males expanded. Oxyuricassis hexaspinatus n. sp. is characterized by lateral alae, a conical tail with tapered extremity, with 2 pairs of proximal spines at the posterior extremity of the tail, 1 dorsal pair and 1 ventral pair, plus a pair of lateral spines that vary in position between sexes, and male of O. hexaspinatus has narrow caudal alae. Both species were found co-parasitizing all of the analyzed host specimens.

New species of Parapharyngodon (Nematoda: Pharyngodonidae) in Phymaturus spp. (Iguania: Liolaemidae) from Argentina.

Parapharyngodon sanjuanensis sp. nov. (Nematoda: Pharyngodonidae) from the large intestines of Phymaturus punae and Phymaturus williamsi (Squamata: Liolaemidae) from province of San Juan, Argentina, is described and illustrated. Parapharyngodon sanjuanensis sp. nov. is the 54th species assigned to the genus and the 8th from the Neotropical region. It differs from other species in the genus in that males possess 8 caudal papillae, 6 of which are large and pedunculate, 2 are small, almost inconspicuous; anterior lip echinate, posterior lip bilobate; females possess prominent vulva and short stiff tail spike.

A new species of Spauligodon (Nematoda; Oxyuroidea; Pharyngodonide) and other Helminths in Ptychozoon Kuhli (Squamata: Gekkonidae) from East Malaysia.

Spauligodon geckonis sp. nov. from the intestines of Ptychozoon kuhli (Gekkonidae) from East Malaysia is described and illustrated. Spauligodon geckonis sp. nov. represents the 52nd species assigned to the genus and the third species from the Oriental Region. The new species is separated from congeners by the unique combination of aspinose filamentous tail and spicule in the male and spinose filamentous tail and fusiform, two knobbed eggs in the female. Gravid individuals 3 species of Nematoda, Meteterakis singaporensis, Physalopteroides grismeri, and Skrjabinelazia machidai, as well as larvae assignable to the Ascaridae were also found.

Morphology of the oxyurid nematodes Trypanoxyuris (T.) cacajao n. sp. and T. (T.) ucayalii n. sp. from the red uakari monkey Cacajao calvus ucayalii in the Peruvian Amazon.

Cacajao calvus ucayalii (Thomas, 1928) (Primates: Pitheciidae), a subspecies endemic to the Peruvian Amazon, occurs in patchy and sometimes isolated populations in north-eastern Peru and is in a vulnerable situation, mainly due to habitat loss and hunting. This rareness and remote distribution means that, until now, parasitical studies have been limited. Based on optical and scanning electron microscopy of specimens of both sexes, we report two new species of Trypanoxyuris pinworms occurring in the large intestine of the Peruvian red uakari, namely Trypanoxyuris (Trypanoxyuris) cacajao and Trypanoxyuris (Trypanoxyuris) ucayalii. Both species showed a distinct morphology of the lips and cephalic structure. Sexual dimorphism in the lateral alae was observed in both male and the female worms, with ventral ornamentation being shown in the oesophageal teeth. The finding of these new pinworm species highlights the possibility of discovering other species.

A new species of Parapharyngodon (Nematoda: Pharyngodonidae) infecting Dermatonotus muelleri (Anura: Microhylidae) from Caatinga, Northeastern Brazil.

Parapharyngodon silvoi n. sp. (Nematoda: Pharyngodonidae) is described from the large and small intestine of the Muller's termite frog Dermatonotus muelleri (Boettger, 1885) from the biome Caatinga, Exu municipality, Pernambuco State, Brazil, Dermatonotus muelleri is a fossorial species with a specialized termite diet, and feeding and reproductive behavior occurring only during the wet season. The new species is distinguished from other species of the genus Parapharyngodon by showing ovary not coiled around the esophagus, morphology of anterior cloacal lip, spicule size and number of caudal papillae.

Syphacia (Syphacia) maxomyos sp. n. (Nematoda: Oxyuridae) from Maxomys spp. (Rodentia: Muridae) from Sulawesi and Sumatra, Indonesia.

The present report describes Syphacia (Syphacia) maxomyos sp. n. (Nematoda: Oxyuridae) from two species of spiny rats, Maxomys musschenbroekii from Sulawesi and M. whiteheadi from Sumatra. It is characterized by a cephalic plate extending laterally with dorsoventral constriction and stumpy eggs with an operculum rim reaching pole. It is readily distinguishable by the former feature from all of hitherto known representatives of this genus in Indonesia, but it resembles parasites in Murini and Hydromyni rodents in continental Asia and Sahul. This is the first Syphacia species distributed in both the Sunda Shelf and Sulawesi with the exception of Syphacia muris, a cosmopolitan pinworm found in rodents of the of genus Rattus. It is surmised that S. maxomyos is specific to Maxomys and that it was introduced to Sulawesi by dispersal of some Maxomys from the Sunda Shelf.

Trypanoxyuris atelis and T. atelophora (Nematoda: Oxyuridae) in wild spider monkeys (Ateles geoffroyi) in tropical rain forest in Mexico: Morphological and molecular evidence.

Two species of pinworms, Trypanoxyuris atelis and Trypanoxyuris atelophora were collected from the black-handed spider monkey (Ateles geoffroyi) in several localities across southeastern Mexico, representing the first record for both species in Mexican primates. Identification of pinworm species was based on morphological and molecular data. These pinworms are distinguished from other congeners, and from each other, by the buccal structure, the lateral alae, and the morphology of the oesophagus. Phylogenetic analyses based on sequences of the mitochondrial cytochrome c oxidase subunit 1 gene placed T. atelis as the sister species of Trypanoxyuris minutus, a parasite of the howler monkey Alouatta palliata, and T. atelophora as the sister species of T. microon, a parasite of the night monkey, Aotus azarae. These relationships were supported with high posterior probability values by Bayesian inference. Comparisons of additional pinworm taxa from Neotropical primates are needed to assess oxyurid diversity, and to better understand the evolutionary relationships among these nematodes and their primate hosts.