Ramon A. Carreno

Molecular phylogeny of clade III nematodes reveals multiple origins of tissue parasitism

Molecular phylogenetic analyses of 113 taxa representing Ascaridida, Rhigonematida, Spirurida and Oxyurida were used to infer a more comprehensive phylogenetic hypothesis for representatives of clade III. The posterior probability of multiple alignment sites was used to exclude or weight characters, yielding datasets that were analysed using maximum parsimony, likelihood, and Bayesian inference methods. Phylogenetic results were robust to differences among inference methods for most high-level taxonomic groups, but some clades were sensitive to treatments of characters reflecting differences in alignment ambiguity. Taxa representing Camallanoidea, Oxyurida, Physalopteroidea, Raphidascarididae, and Skrjabillanidae were monophyletic in all 9 analyses whereas Ascaridida, Ascarididae, Anisakidae, Cosmocercoidea, Habronematoidea, Heterocheilidae, Philometridae, Rhigonematida and Thelazioidea were never monophyletic. Some clades recovered in all trees such as Dracunculoidea and Spirurina included the vast majority of their sampled species, but were non-monophyletic due to the consistent behaviour of one or few rogue taxa. Similarly, 102 of 103 clade III taxa were strongly supported as monophyletic, yet clade III was paraphyletic due to the grouping of Truttaedacnitis truttae with the outgroups. Mapping of host habitat revealed that tissue-dwelling localization of nematode adults has evolved independently at least 3 times, and relationships among Spirurina and Camallanina often reflected tissue predilection rather than taxonomy.

Molecular phylogenetics and diagnosis of soil and clinical isolates of Halicephalobus gingivalis (Nematoda: Cephalobina: Panagrolaimoidea), an opportunistic pathogen of horses

Phylogenetic relationships among six isolates of Halicephalobus gingivalis (Stefanski, 1954), a species with pathogenic potential in horses and humans, were evaluated using DNA sequences from the nuclear large-subunit ribosomal RNA (LSU rDNA) gene. Sequences from nematodes obtained from in vitro cultures (soil or clinical sources), or isolated from infected horse tissues, were compared. Gene sequences from a fatal equine clinical case from southern California and a free-living isolate recovered from southern California soil showed no fixed differences. Sequences from isolates representing two fatal equine cases from North America, one from Ontario, Canada and another from Tennessee also showed no fixed differences. In contrast, two equine cases from Tennessee had 18 fixed differences for this LSU region, the greatest observed among isolates from horses. Phylogenetic analysis of six Halicephalobus sequences and four outgroup taxa by maximum parsimony yielded one tree with five well-supported clades. This phylogeny did not group isolates of Halicephalobus strictly by region of geographic isolation or source of sample, and depicted one clinical and one soil isolate as sister taxa. These results confirm that free-living environmental isolates are potential sources of infection for horses. The phylogeny also reveals that diverse isolates can cause infections in horses within a relatively limited geographic region, and conversely that genetically similar sister taxa can be recovered from geographically distant localities. PCR primers that selectively amplify Halicephalobus DNA were designed and tested based on comparison of closely related nematodes as inferred from phylogenetic analysis.

PHYLOGENETIC ANALYSIS OF THE METASTRONGYLOIDEA (NEMATODA: STRONGYLIDA) INFERRED FROM RIBOSOMAL RNA GENE SEQUENCES

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.

Phylogeny of Rhigonematomorpha based on the complete mitochondrial genome of Rhigonema thysanophora (Nematoda: Chromadorea)

We determined the complete mitochondrial genome sequence of Rhigonema thysanophora, the first representative of Rhigonematomorpha, and used this sequence along with 57 other nematode species for phylogenetic analyses. The R. thysanophora mtDNA is 15 015 bp and identical to all other chromadorean nematode mtDNAs published to date in that it contains 36 genes (lacking atp8) encoded in the same direction. Phylogenetic analyses of nucleotide and amino acid sequence data for the 12 protein‐coding genes recovered Rhigonematomorpha as the sister group to the heterakoid species, Ascaridia columbae (Ascaridomorpha). The organization of R. thysanophora mtDNA resembles the most common pattern for the Rhabditomorpha+Ascaridomorpha+Diplogasteromorpha clade in gene order, but with some substantial gene rearrangements. This similarity in gene order is in agreement with the sequence‐based analyses that indicate a close relationship between Rhigonematomorpha and Rhabditomorpha+Ascaridomorpha+Diplogasteromorpha. These results are consistent with certain analyses of nuclear SSU rDNA for R. thysanophora and some earlier classification systems that asserted phylogenetic affinity between Rhigonematomorpha and Ascaridomorpha, but inconsistent with morphology‐based phylogenetic hypotheses that suggested a close (taxonomic) relationship between rhigonematomorphs and oxyuridomorphs (pinworms). These observations must be tempered by noting that few rhigonematomorph species have been sequenced and included in phylogenetic analyses, and preliminary studies based on SSU rDNA suggest the group is not monophyletic. Additional mitochondrial genome sequences of rhigonematids are needed to characterize their phylogenetic relationships within Chromadorea, and to increase understanding of mitochondrial genome evolution.

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.

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.

Characterization of Dictyocaulus Species (Nematoda: Trichostrongyloidea) from Three Species of Wild Ruminants in Northwestern Spain

Abstract 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 León, 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 SPECIES OF SKRJABINGYLUS PETROV, 1927 (NEMATODA: METASTRONGYLOIDEA) FROM THE FRONTAL SINUSES OF THE HOODED SKUNK, MEPHITIS MACROURA (MUSTELIDAE)

Skrjabingylus santaceciliae n. sp. is described based on specimens from the frontal sinuses of a hooded skunk, Mephitis macroura, collected from the Area de Conservación Guanacaste, Costa Rica. Skrjabingylus santaceciliae n. sp. differs from the other 5 species in the genus in having pointed spicule tips that lack a rounded or lobed formation and by lacking a prominent distal projection at the tail tip. Morphometric comparisons show that S. santaceciliae n. sp. is much smaller than the only other valid species from Mephitis, Skrjabingylus chitwoodorum Hill, 1939. Likewise, morphometric comparisons also distinguish S. santaceciliae n. sp. from other described Skrjabingylus species.

Description of a New Species of Thelastoma Leidy, 1849 (Nematoda: Oxyurida: Thelastomatidae) from the Millipede Euryurus leachii (Gray, 1832) in Ohio, U.S.A.

Abstract Thelastoma krausi n. sp. is described based on specimens from the hindgut of Euryurus leachii (Gray, 1832) collected in central Ohio, U.S.A. Females of this species have the excretory pore located at the point of the esophageal bulb and have a longer tail than that of several other species. In comparison to species that also share these characters, no other species shares similar anatomical dimensions. In most similar species, the female esophageal length is longer than that of T. krausi n. sp. The female tail length is also longer than that of many similar species. The males of T. krausi n. sp. lack lateral alae and have an arrangement of papillae around the genital cone, including only a single postcloacal papilla, which varies from that of other described species.

The Systematics and Evolution of Pinworms (Nematoda: Oxyurida: Thelastomatoidea) from Invertebrates

Abstract: 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.