PHYLOGENETIC ANALYSIS OF THE LUNGWORMS (NEMATODA: METASTRONGYLOIDEA) INFERRED USING NUCLEAR RIBOSOMAL AND MITOCHONDRIAL DNA SEQUENCES.

Phylogenetic relationships among the mammal-parasitic lungworms (Metastrongyloidea) were inferred using small- and large-subunit ribosomal DNA sequences together with 12S ribosomal mtDNA sequences. Maximum parsimony and Bayesian inference methods were used from optimal alignments and those filtered for alignment ambiguity. Analysis of 30 ingroup sequences using ribosomal DNA sequences yielded a single most parsimonious tree. Monophyly of the Metastrongyloidea was supported, but there was no support for monophyly of any of the 7 families as they have been traditionally defined. Parafilaroides decorus, an abursate lungworm of pinnipeds currently classified in the Filaroididae, was nested within a clade containing members of the Pseudaliidae, parasites of cetaceans. The tree also shows clades somewhat resembling the traditional familial divisions of the Metastrongyloidea, but in all groups, paraphyletic relationships were recovered. In a combined analysis of nuclear rDNA and 12S mtDNA, maximum parsimony and Bayesian analyses showed similar patterns to those observed with only nuclear rDNA sequences. Based on the phylogeny, the respiratory tract was inferred to be the ancestral predilection site for Metastrongyloidea, with multiple evolutionary invasions of extrapulmonary sites such as sinuses, circulatory system, and meninges. Similarly, the ancestral host was inferred to be a carnivore with subsequent colonization events into marsupial, rodent, artiodactyl, pinniped, and cetacean hosts.

Morphological and molecular characterization of two species of nematodes (Oxyuridomorpha: Thelastomatoidea: Protrelloididae, Thelastomatidae) parasitic in the cockroach Blaberus discoidalis Serville (Blattaria: Blaberidae) from Cuba.

Protrelleta floridana Chitwood, 1932 and Cranifera cranifera (Chitwood, 1932) (Oxyuridomorpha: Thelastomatoidea: Protrelloididae, Thelastomatidae) are recorded for the first time in Cuba. These nematodes were found to parasitize the cockroach Blaberus discoidalis Serville (Blattaria: Blaberidae), constituting a new host record for them. Both species are redescribed with the aid of scanning electron microscopy and the arrangement of the copulatory papillae of the males of P. floridana is amended. The present specimens coincide in their morphology and most of the measurements with the type populations from USA and the records from Costa Rica, with only minor differences. The molecular phylogeny was inferred by mean of the D2-D3 domain of the 28S rDNA and the Cuban P. floridana and C. cranifera form monophyletic clades with sequences of both taxa from Costa Rica as well as a sequence of C. cranifera from Russia. In the case of C. cranifera its phylogeny and that of its blaberid hosts reveal coevolutionary relationships.

Three new genera and new species of hystrignathid nematodes (Nematoda: Thelastomatoidea) from passalid beetles (Insecta: Passalidae) from Yunnan Province, China with phylogenetic analysis of Hystrignathidae.

Three new genera and three new species of hystrignathid nematodes were collected from the hindgut of passalid beetles in the Yunnan Province, China. Huntia morffei gen. n. et sp. n. from Ceracupes fronticornis (Westwood) differs from related genera in the shape of the stoma, in the cephalic structure, and in the lack of annulation in the cervical region. Parakongonema sinica gen. n. et sp. n. from Aceraius grandis (Burmeister) and C. fronticornis can be easily distinguished from related genera in having the oral opening surrounded by a cuticular ring, the ring being surrounded by a cuticular flap, in the shape of the oesophagus, and in the cephalic structure. Parahystrignathus tongbiguanensis gen. n. et sp. n. from C. fronticornis and A. grandis differs from related genera by having the cervical region armed with alternating rows of spines, with 16 spines in the first row, and by lacking the first cephalic annule. In addition, the 18S and 28S rDNA partial sequences of three new species were obtained. The phylogeny of Hystrignathidae was inferred using partial sequences of the 28S rDNA and revealed patterns that suggest that the shape of the parasite digestive tract may reflect the evolutionary relationships of hystrignathid nematodes.

Morphological and molecular characterization of Buzionema lutgardae n. sp. (Nematoda: Oxyuridomorpha: Thelastomatidae) from the cockroach Byrsotria sp. (Blattaria: Blaberidae) in Cuba.

Buzionema lutgardae n. sp. (Nematoda: Oxyuridomorpha: Thelastomatidae) is described from the cockroach Byrsotria sp. (Blattaria: Blaberidae), endemic to Cuba. Females of B. lutgardae n. sp. are shorter than those of B. validum Kloss, 1966 (16002150 µm vs. 31313378 µm), but the oesophagus is comparatively longer (b = 2.963.77 vs. 4.654.87). The lateral alae of the new species extend from ca. the midpoint of the cylindrical part of the procorpus to the level of the anus in contrast to the base of the basal bulb to the level of the anus in B. validum. The males of B. lutgardae n. sp. are shorter than those of B. validum (780940 µm vs. 11771423 µm) and their lateral alae end at some distance before the cloaca instead the level of the cloaca in B. validum. The phylogeny of B. lutgardae n. sp. is inferred by the D2-D3 domains of the 28S rDNA. B. lutgardae n. sp. and B. validum form a monophyletic clade with strong nodal support, as sister-group of the genus Leidynema Schwenck in Travassos, 1929.

Morphological and molecular characterization of Xyo pseudohystrix Travassos amp; Kloss, 1958 (Nematoda: Oxyuridomorpha: Hystrignathidae) from Odontotaenius disjunctus (Illiger, 1800) (Coleoptera: Passalidae) from USA and discussion on its taxonomic status.

The females of Xyo pseudohystrix Travassos Kloss, 1958 (Nematoda: Oxyuridomorpha: Hystrignathidae) are redescribed and illustrated with the aid of SEM. New features of the cephalic end, arrangement of the cervical spines and genital tract were observed. The taxonomic status of the species is discussed on the basis of discrepancies with the generic diagnosis of Xyo Cobb, 1898. Due to the lack of proper information on the genus the status of incertae sedis is proposed. The identity of the males was confirmed by molecular studies and the morphology of the specimens previously assigned by Christie (1932) as males of Hystrignathus rigidus Leidy, 1858 correspond to the current species. New locality records are given for the states of Georgia and Ohio, USA. The phylogenetic position of the species is inferred on the basis of the D2-D3 segment of the LSU rDNA and SSU rDNA.

On the status of Suifunema peregrinatum (Carreno, 2017) n. comb. (Nematoda: Oxyuridomorpha: Thelastomatidae) and first record of the species for Japan.

Blattophila peregrinata Carreno, 2017 (Nematoda: Oxyuridomorpha: Thelastomatidae) is proposed as a junior synonym of Suifunema peregrinatum (Carreno, 2017) n. comb., based on the revision of the type material of S. caudelli Chitwood, 1932, the type and only species of the genus. The revision permitted the emendation of some features of the original descriptions, namely the position of the nerve ring at level of the corpus instead the isthmus and the female genital tract didelphic-prodelphic instead didelphic-amphidelphic. The males of S. peregrinatum n. comb. are the only ones known for the genus. Thus, an amended generic diagnosis is given, including information on the males. Both S. peregrinatum n. comb. and S. caudelli differ by having a more slender body, a longer tail, and the vulva farther from the anterior end in the former. S. peregrinatum n. comb. is recorded for the first time from Pycnoscelus indicus Fabricius, 1775 (Blattodea: Blaberidae) from Japan. The specimens from Japan are morphologically consistent with other populations of the species from the USA and the Galapagos Islands of Ecuador. Only some morphometrics vary slightly, but these are within the range of the species. The phylogenetic position of the genus among the family Thelastomatidae is discussed, including sequences of the D2-D3 LSU rDNA of specimens from both the type population (Ohio, USA) and Japan.

A new species of Aoruroides Travassos amp; Kloss, 1958 (Nematoda: xyuridomorpha: Thelastomatidae) parasite of the wood-burrowing cockroach Panesthia angustipennis spadica (Shiraki, 1906) (Blattodea: Blaberidae: Panesthiinae) from Japan with comments on the validity of the genus Aoruroides.

Aoruroides chubudaigaku n. sp. (Nematoda: Oxyuridomorpha: Thelastomatidae) is described from the wood-burrowing cockroach Panesthia angustipennis spadica (Shiraki, 1906) (Blattodea: Blaberidae: Panesthiinae) from Aichi prefecture, Japan. Females of A. chubudaigaku n. sp. are similar to A. costaricensis Carreno Tuhela, 2011 by the position of the nerve ring at level of the first third of the isthmus, the body length and the comparative measurements of the oesophagus and tail. They differ by the position of the excretory pore and the vulva. The males of A. chubudaigaku n. sp. are characterized by the absence of ornamentations in the cervical cuticle and the nerve ring located at the posterior third of the corpus. In addition, the males of the new species can be differentiated by the length of the body and the comparative lengths of the oesophagus and the tail. The phylogeny of A. chubudaigaku n. sp. is inferred by the D2-D3 domains of the LSU rDNA and the validity of the genus Aoruroides Travassos Kloss, 1958 is discussed on the basis of both molecular and morphological data. This constitutes the first species of the genus Aoruroides described from the Japanese archipelago.

Pinworms (Nematoda: Thelastomatoidea) from Insects Collected in Mississippi, USA, with description of a new species of Protrellus Cobb, 1920 from the Cockroach Ischnoptera deropeltiformis Brunner von Wattenwyl, 1865 (Blattaria: Ectobiidae).

Thelastomatoid nematodes from the hindguts of several species of insects were collected in 4 localities in Mississippi, U.S.A. Protrellus browni n. sp. is described from Ischnoptera deropeltiformis (Blattaria: Ectobiidae) using light and scanning electron microscopy. This species is distinguished on the basis of its large size as well as its large eggs having one broad end tapering toward the opposite pole and with the presence of small spines on part of the shell. Protrellus aurifluus (Chitwood, 1932) Basir, 1956 was found in Parcoblatta fulvescens (Blattaria: Ectobiidae). Four other species of blattarians sampled were negative for pinworms. Additional nematodes recovered from other hosts included Zonothrix columbianus Adamson Buck 1989 (Thelastomatoidea: Pseudonymidae) from Tropisternus blatchleyi blatchleyi and Tropisternus collaris (Coleoptera: Hydrophilidae), Hystrignathus rigidus Leidy, 1850 and Xyo pseudohystrix Travassos Kloss, 1958 (Thelastomatoidea: Hystrignathidae) from Odontotaenius disjunctus (Coleoptera: Passalidae). These findings include several new host and geographical records.

New species of Hammerschmidtiella Chitwood, 1932, and Blattophila Cobb, 1920, and new geographical records for Severianoia annamensis Van Luc & Spiridonov, 1993 (Nematoda: Oxyurida: Thelastomatoidea) from Cockroaches (Insecta: Blattaria) in Ohio and Florida, U.S.A.

Two new species of thelastomatid nematodes parasitic in the hindgut of cockroaches are described. Hammerschmidtiella keeneyi n. sp. is described from a laboratory colony of Diploptera punctata (Eschscholtz, 1822) from a facility in Ohio, U. S. A. This species is characterized by having females with a short tail and males smaller than those described from other species. The new species also differs from others in the genus by a number of differing measurements that indicate a distinct identity, including esophageal, tail, and egg lengths as well as the relative position of the excretory pore. Blattophila peregrinata n. sp. is described from Periplaneta australasiae (Fabricius, 1775) and Pycnoscelus surinamensis (Linnaeus, 1758) in a greenhouse from Ohio, U.S.A. and from wild P. surinamensis in southern Florida, U.S.A. This species differs from others in the genus by having a posteriorly directed vagina, vulva in the anterior third of the body, no lateral alae in females, and eggs with an operculum. In P. surinamensis from southern Florida, an additional species, Severianoia annamensis Van Luc & Spiridonov, 1993 was found and did not co-parasitize the host with B. peregrinata n. sp. Blattophila peregrinata n. sp. and S. annamensis also occur in the Galápagos Islands, Ecuador, indicating that these have a widespread global range.

Distribution of Thelastomatoid Nematodes (Nematoda: Oxyurida) in Endemic and Introduced Cockroaches on the Galápagos Island Archipelago, Ecuador.

The thelastomatoid pinworm fauna (Nematoda: Oxyurida: Thelastomatoidea) was surveyed in 3 endemic species and 6 introduced species of cockroach hosts (Insecta: Blattaria) in the Galápagos Islands, Ecuador. A total of 658 host specimens were examined from preserved collections that had been collected between 1966 and 2003 from 7 islands in the archipelago. Eight species of pinworms were identified from these cockroach hosts, including the dominant species Cephalobellus ovumglutinosus and a Severianoia sp. as well as Leidynema appendiculata, Hammerschmidtiella diesingi, an unidentified Cephalobellus species resembling Cephalobellus magalhaesi, an unidentified Protrellus species closely resembling Protrellus shamimi, and an undescribed Blattophila sp. Five new host records are identified for C. ovumglutinosus including the endemic Galápagos cockroaches Chorisoneura carpenteri, Ischnoptera snodgrassii, and Ischnoptera santacruzensis. These endemics were also infected with an undescribed Blatticola sp. Other species recorded resemble known pinworms from other hosts around the world. Prevalence between islands and between host species was variable, but total prevalence for individual pinworm species was consistently low (<10%). A single host specimen examined was infected with more than 1 pinworm species; otherwise only a single species was observed in each infected host. At least 1 introduced pinworm species carried to the islands via invasive cockroach hosts was present in endemic host species, but several globally widespread introduced pinworm species were absent from endemic cockroaches. Santa Cruz was inhabited by the greatest number of pinworm species, likely due to a higher rate of invasive host introduction. This survey, the first from this region, showed that the distribution and transmission of pinworms in the Galápagos Islands is complex and may provide future models of invertebrate dispersal and speciation in an ecosystem already rich with examples of evolution.

The systematics and evolution of pinworms (Nematoda: Oxyurida: Thelastomatoidea) from invertebrates.

The nematode order Oxyurida is unique in including species for which definitive host ranges are broad and may include vertebrate or invertebrate hosts. The superfamily Thelastomatoidea is a highly diverse assemblage of oxyurids occurring in cockroaches, diplopods, hydrophilid beetles, passalid beetles, several other coleopteran larvae, mole crickets, and, with few representative species documented, other arthropod hosts. Published research and revision of the Thelastomatoidea, particularly in the 1980s and 1990s, provided several interesting hypotheses on the systematics and evolution of this group. In this review, these hypotheses are examined in the context of recent advances in taxonomy, discovery of additional species diversity and distribution, and preliminary phylogenetic hypotheses that have been proposed. There continues to remain a paucity of phylogenetic data that explore the phylogenetic relationships of the Thelastomatoidea and their relationships to vertebrate-parasitizing pinworms. A combination of mitochondrial and nuclear DNA sequences for representative species across all of the major lineages will be important for more robust phylogenetic hypotheses. Much broader geographical and host taxon sampling is necessary to determine true diversity of the Thelastomatoidea. Modern approaches to species descriptions, such as improvements in light and scanning electron microscopy and the use of molecular approaches to matching male and female nematodes, can also be applied to improve our understanding of the evolution of these fascinating parasites.

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

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

Discovery of an undescribed protostrongylid nematode from the endangered pampas deer (Ozotoceros bezoarticus celer) in Argentina.

Dorsal-spined protostrongylid nematode larvae (Metastrongyloidea: Protostrongylidae) were recovered from the feces of the endangered pampas deer (Ozotocerus bezoarticus celer) in Campos del Tuyú Wildlife Reserve, Bahia Samborombón, Argentina. Partial DNA sequences from the large subunit ribosomal RNA (LSU rRNA) gene and from the second internal transcribed spacer region (ITS2) were amplified, cloned, sequenced, and compared to those of other nematodes. Nucleotide alignment and phylogenetic analysis of the sequences indicate that this protostrongylid nematode is most closely related to Parelaphostrongylus spp. as inferred from the LSU rRNA sequence analysis. Analysis of the ITS2 spacer indicated that the pampas deer protostrongylid is nested in a clade containing Parelaphostrongylus and Elaphostrongylus spp. These sequences differed considerably from those of other protostrongylid nematodes, and were most similar to those of Parelaphostrongylus spp. and Elaphostrongylus spp. in spite of clear variability from both genera. These results suggest that the protostrongylid from pampas deer is an undescribed nematode that likely belongs in the subfamily Elaphostrongylinae.

Phylogenetic identification of Cystoisospora spp. from dogs, cats, and raccoon dogs in Japan.

Cystoisospora spp. from feces in dogs, cats, and raccoon dogs were isolated, sequenced at the small subunit ribosomal RNA gene locus and compared to other Cystoisospora spp. Cystoisospora oocysts from dogs and raccoon dogs were morphologically similar with those of C. ohioensis, and cat isolates were similar with those of C. felis. The sequences from dogs and raccoon dogs, and cats have a homology with C. ohioensis and C. felis, respectively. Phylogenetic analysis of the DNA sequences showed that the dog and raccoon dog isolates were nested in a clade with other Cystoisospora spp. including C. ohioensis, C. belli, and C. orlovi. The cat isolate formed a sister group with C. felis that was a separate clade from the dog and raccoon dog group. We report sequence variation in these Cystoisospora sequences and have identified raccoon dogs as another carnivore host for Cystoisospora spp. infecting dogs.

Characterization of Dictyocaulus species (Nematoda: Trichostrongyloidea) from three species of wild ruminants in northwestern Spain.

Specimens of Dictyocaulus spp. were extracted from the respiratory tracts of 3 ruminant hosts including roe deer (Capreolus capreolus), red deer (Cervus elaphus), and chamois (Rupicapra rupicapra) from wild populations in the province of Le6n, northwestern Spain. The near-complete nuclear small-subunit ribosomal RNA gene, and 2 regions of the large-subunit ribosomal RNA gene, were amplified by PCR and sequenced. The SSU rDNA gene sequences indicated a high level of similarity between the isolate from C elaphus and the published sequences for Dictyocaulus eckerti. SSU rDNA gene sequences were identical in the isolates from C. capreolus and R. rupicapra, and both corresponded to published sequences for D. capreolus. The LSU rDNA gene sequences differed in isolates from the latter 2 hosts, indicating the possible presence of an undescribed Dictyocaulus sp. in R. rupicapra. These results showed that the LSU rDNA gene sequences are useful indicators of genetic and species diversity in species of Dictyocaulus.

A new heligmonellid (Nematoda) from Sylvilagus floridanus (Leporidae) in Costa Rica, with some comments on species of Paraheligmonella Durette-Desset, 1971.

Paraheligmonella kinsellai n. sp. (Nematoda: Heligmonellidae: Heligmonellinae) is described from the small intestine of a cottontail rabbit Sylvilagus floridanus (Allen, 1890) (Leporidae) from Costa Rica. The new species is similar to P. romerolagi (Gibbons and Kumar, 1980), parasitic in Romerolagus diazi from Mexico and to P. lamothei Digiani, Carreño, and Durette-Desset, 2008, parasitic in Sylvilagus floridanus from Costa Rica, by the characters of the synlophe at mid-body, i.e., 14 cuticular ridges and a single axis of orientation inclined at 30 degrees to the sagittal axis in both sexes. The males of the new species are distinguished from those of P. romerolagi by a caudal bursa that is not bell-shaped, a bursal pattern of type 2-3 with a tendency to 2-2-1, and a genital cone that is not hypertrophied. They are also distinguished from the males of P. lamothei by having the same bursal pattern in both lobes (in P. lamothei the pattern is 2-2-1 on the right lobe and 2-3, with a tendency to 2-2-1, on the left lobe), rays 3 are much longer than rays 2, rays 2 and 3 diverging more proximally, and rays 8 are longer than the dorsal ray. The females of the new species also differ from those of P. lamothei by the shape of the posterior extremity (long and strongly curved vs. short and nearly straight) and by the synlophe at the ovejector level (ridge no. 1' hypertrophied and remaining ridges reduced vs. ventral ridges hypertrophied and dorso-lateral ridges reduced).

Mermithid nematode infection in a colony of blue-winged grasshoppers (Tropidacris collaris).

A die-off occurred in a captive colony of blue-winged grasshoppers (Tropidacris collaris) at the Toronto Zoo. One fourth of the colony died within a year due to infection with worms initially mistaken for nematomorphs but later identified as nematodes belonging to the Mermithidae, genus Mermis. Mortality persisted and the grasshopper population dwindled over the following years. Mermithid larvae developed in the hemocoel of the insects until they eventually emerged from a hollowed-out exoskeleton. Circumstantial evidence suggests that the parasites were introduced with raspberry browse that was grown on site and contaminated with mermithid eggs.

Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms.

The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.

Babesia conradae, sp. Nov., a small canine Babesia identified in California.

Small piroplasms as a cause of canine babesiosis have usually been identified as Babesia gibsoni. Recent genetic studies suggested that small piroplasms are more likely comprised of at least three genotypically distinct species. In southern California, canine babesiosis caused by a small piroplasm has been documented since 1990. Morphological characteristics of this parasite include a small (0.3-3.0 microm) intraerythrocytic merozoite stage with predominantly ring, piriform, tetrad, amoeboid, or anaplasmoid forms. Transmission electron microscopic images of merozoites demonstrate the presence of an apical complex consisting of an inner subplasmalemmal membrane and rhoptries. Based on phylogenetic analyses of the 18S rRNA and the ITS-2 genes, the Californian small piroplasm isolate is more closely related to piroplasm isolates from wildlife and humans in the western United States than it is to B. gibsoni. Molecular and morphologic evidence supports naming the small piroplasm from southern California as a distinct species, Babesia conradae.