Origins, phylogenetic relationships and host-parasite interactions of Troglotrematoidea since the cretaceous.

In the current study, we raise the issue concerning origins and historical relationships of the trematodes from the families Troglotrematidae and Paragonimidae using phylogenetic analysis and molecular-clock method for estimating evolutionary rates. For the first time we provided 28S rRNA gene fragment (1764 bp) for the type species Troglotrema acutum - zoonotic trematodes that cause cranial lesions (troglotremiasis) in mustelid and canid mammals of the Central Europe, Iberian Peninsula, and North-West Caucasus. Molecular genetic analysis revealed that T. acutum belongs to the monophyletic family Troglotrematidae sister with the family Paragonimidae. The family Troglotrematidae includes five genera: Nanophyetus, Troglotrema, Skrjabinophyetus, Nephrotrema, and Macroorchis; and the family Paragonimidae is monotypic including the only genus Paragonimus. We recover the superfamily Troglotrematoidea for these two families. Divergence of the common ancestor of the superfamily Troglotrematoidea (common troglotrematoid ancestor) likely occurred during the Cretaceous period of the Mesozoic Era and potentially originated in the Asiatic region. The lineage of the family Troglotrematidae is much closer to the common troglotrematoid ancestor than the species of the family Paragonimidae. The radiation time of the common troglotrematoid ancestor (126 Ma, the Early Cretaceous), and formation of the families Troglotrematidae and Paragonimidae (96 Ma and 73 Ma respectively, the Late Cretaceous) corresponds to the time of settling in East Asia by many species of mammaliaforms (about 130-70 Ma).

Molecular identification of morphologically similar microcercous cercariae of two trematode families, Paragonimidae and Troglotrematidae, concurrently found in the same snail species of the subfamily Triculinae.

Microcercous cercariae possess a very short tail and are produced by digenean species of several families including medically important species, such as members of the genera Paragonimus Braun, 1899, Nanophyetus Chapin, 1927 and Troglotrema Odhner, 1914. During our survey of cercariae of Paragonimus spp. in Vietnam, we found microcercous cercariae from ten (0.29%) out of 3,400 snails of Triculinae gen. sp. 2. They were morphologically and molecularly analysed for species identification. The molecular analysis, based on ITS2 sequences, revealed two distinct species: four specimens were identical to Paragonimus proliferus Hsia et Chen, 1964 (Paragonimidae Dollfus, 1939), and the other six specimens were closest to members of the family Troglotrematidae Odhner, 1914 and were temporarily named Troglotrematidae gen. sp. Morphologically, cercariae of the two species found in this study are similar to each other in their gross characteristics but can be distinguished from one another by subtle morphological details. The cercaria of P. proliferus has an I-shaped excretory bladder and does not have mucous gland cells. In contrast, that of Troglotrematidae gen. sp. has a Y-shaped excretory bladder and mucous gland cells. Besides, the redia of P. proliferus is elongate with a short intestine and contains 5-6 cercariae whereas that of Troglotrematidae gen. sp. is more round with a longer intestine and harbours 3-4 cercariae. Our results have shown the importance of the shape of the excretory bladder and the presence/absence of mucous gland cells of the cercaria as well as the shape and size of the redia, and its intestinal length as valuable taxonomic characters of intramolluscan trematode larvae. In addition, the finding of similar microcercous cercariae of different species in the same snail species suggests that careful attention to morphological details is required in the differentiation of Paragonimus cercariae and those of closely related species.

Digeneans of northern fur seals Callorhinus ursinus (Pinnipedia: Otariidae) from five subpopulations on St. Paul Island, Alaska.

A parasitological survey of 651 northern fur seals Callorhinus ursinus L. from five subpopulations was conducted on St. Paul Island, Alaska, during July-August 2012-2014. Digenean trematodes were found in 210 of 651 fur seals with a total prevalence of 32.3%. Intensity of infection varied from 1 to 1540 parasites with mean intensity 18.4 ± 111.1 SD and median intensity of 2 specimens per host. Significant differences in prevalence and intensity of infection in northern fur seals between separate rookeries was not observed (Mann-Whitney test; p > 0.05). Four species of digeneans belonging to the families Heterophyidae (Apophallus zalophi Price, 1932, Phocitrema fusiforme Goto and Ozaki, 1930, and Galactosomum ubelakeri (Dailey, 1969)) and Troglotrematidae (Nanophyetus salmincola (Chapin, 1926)) were found. Nanophyetus salmincola is reported from C. ursinus for the first time. We obtained partial 28S rDNA sequences for all digenean species and conducted molecular phylogenetic analysis to demonstrate their phylogenetic relationships.

Long-Distance Travellers: Phylogeography of a Generalist Parasite, Pholeter gastrophilus, from Cetaceans.

We studied the phylogeography and historical demography of the most generalist digenean from cetaceans, Pholeter gastrophilus, exploring the effects of isolation by distance, ecological barriers and hosts' dispersal ability on the population structure of this parasite. The ITS2 rDNA, and the mitochondrial COI and ND1 from 68 individual parasites were analysed. Worms were collected from seven oceanic and coastal cetacean species from the south western Atlantic (SWA), central eastern Atlantic, north eastern Atlantic (NEA), and Mediterranean Sea. Pholeter gastrophilus was considered a single lineage because reciprocal monophyly was not detected in the ML cladogram of all individuals, and sequence variability was <1% for mtDNA and 0% for ITS2. These results rule out a recent suggestion that P. gastrophilus would actually be a cryptic-species complex. The genetic cohesion of P. gastrophilus could rely on the extensive exploitation of wide-ranging and highly mobile cetaceans, with a putative secondary role, if any, of intermediate hosts. Unique haplotypes were detected in SWA and NEA, and an AMOVA revealed significant population structure associated to the genetic variation in these regions. The Equator possibly acts as a significant geographical barrier for cetacean movements, possibly limiting gene flow between northern and southern populations of P. gastrophilus. A partial Mantel tests revealed that the significant isolation of NEA populations resulted from geographic clustering. Apparently, the limited mobility of cetaceans used by P. gastrophilus as definitive hosts in this region, coupled with oceanographic barriers and a patchy distribution of potential intermediate hosts could contribute to significant ecological isolation of P. gastrophilus in NEA. Rather unexpectedly, no genetic differentiation was found in the Mediterranean samples of this parasite. Historical demographic analyses suggested a recent population expansion of P. gastrophilus in the Atlantic Ocean, perhaps linked to initial association and subsequent spreading in cetaceans.

Genetic divergence of human pathogens Nanophyetus spp. (Trematoda: Troglotrematidae) on the opposite sides of the Pacific Rim.

Human and animal nanophyetiasis is caused by intestinal flukes belonging to the genus Nanophyetus distributed on both North American and Eurasian coasts of Northern Pacific. In spite of the wide geographical distribution and medical and veterinary importance of these flukes, the intra-generic taxonomy of Nanophyetus spp. remains unresolved. The two most widely distributed nominal species, Nanophyetus salmincola and Nanophyetus schikhobalowi, both parasitizing humans and carnivorous mammals, were described from North America and eastern Eurasia, respectively. However, due to their high morphological similarity their interrelationships remained unclear and taxonomic status unstable. In this study, we explored genetic diversity of Nanophyetus spp. from the Southern Russian Far East in comparison with that of samples from North America based on the sequence variation of the nuclear ribosomal gene family (18S, internal transcribed spacers, ITS1-5·8S-ITS2 and 28S). High levels of genetic divergence in each rDNA region (nucleotide substitutions, indels, alterations in the secondary structures of the ITS1 and ITS2 transcripts) as well as results of phylogenetic analysis provided strong support for the status of N. salmincola and N. schikhobalowi as independent species.

A molecular perspective on the genera Paragonimus Braun, Euparagonimus Chen and Pagumogonimus Chen.

The status of the genera Euparagonimus Chen, 1963 and Pagumogonimus Chen, 1963 relative to Paragonimus Braun, 1899 was investigated using DNA sequences from the mitochondrial cytochrome c oxidase subunit I (CO1) gene (partial) and the nuclear ribosomal DNA second internal transcribed spacer (ITS2). In the phylogenetic trees constructed, the genus Pagumogonimus is clearly not monophyletic and therefore not a natural taxon. Indeed, the type species of Pagumogonimus, P. skrjabini from China, is very closely related to Paragonimus miyazakii from Japan. The status of Euparagonimus is less obvious. Euparagonimus cenocopiosus lies distant from other lungflukes included in the analysis. It can be placed as sister to Paragonimus in some analyses and falls within the genus in others. A recently published morphological study placed E. cenocopiosus within the genus Paragonimus and probably this is where it should remain.