Phylogenetic relationships of three species within the family Heligmonellidae (Nematoda; Heligmosomoidea) from Japanese rodents and a lagomorph based on the sequences of ribosomal DNA

Nematodes of the family Heligmonellidae (Heligmosomoidea; Trichostrongylina) reside in the digestive tracts of rodents and lagomorphs. Although this family contains large numbers of genera and species, genetic information on the Heligmonellidae is very limited. We collected and isolated adult worms of three species in Japan that belong to the family Heligmonellidae, namely Heligmonoides speciosus (Konno, 1963) Durette-Desset, 1970 (Hs) from Apodemus argenteus, Orientostrongylus ezoensis Tada, 1975 (Oe) from Rattus norvegicus and Lagostrongylus leporis (Schulz, 1931) (Ll) from Pentalagus furnessi, and sequenced the entire internal transcribed spacer regions, ITS-1 and ITS-2 of ribosomal DNA. ITS-1 of Hs, Oe and Ll was 426, 468 and 449 bp in length, and had a G+C content of about 41, 41 and 37 %, respectively. ITS-2 of Hs, Oe and Ll was 297, 319 and 276 bp in length and had a G+C content of about 38, 40 and 28%, respectively. The data of Hs, Oe and Ll were compared with those of two other known species within the family Heligmonellidae, Calorinensis minutus (Dujardin, 1845) (Cm) and Nippostrogylus brasiliensis (Travassos, 1914) (Nb), and with those of two species of Heligmosomidae (Heligmosomoidea), Heligmosomoides polygyrus bakeri and Ohbayashinema erbaevae. Phylogenetic analysis placed Hs, Oe and Ll in the same clade with Cm and Nb, forming a Heligmonellidae branch in both ITS-1 and ITS-2, separate from the Heligmosomoidea branch. These results demonstrated that the ITS-1 and ITS-2 sequences are useful for differentiating the Seitaro Yamada, Atushi Yoshida, Keiko Yoshida, Takeshi Kuraishi, Shosaku Hattori, Chieko Kai, Yumiko Nagai, Takeshi Sakoda, Masaya Tatara, Shintaro Abe and Shin-ichiro Fukumoto *) 1) Unit of Veterinary Parasitology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan. 2) Amami Laboratory of Injurious Animals, the Institute of Medical Science, the University of Tokyo, Setouchi-cho, Ohshima-gun, Kagoshima, 894-1531, Japan Amami Wildlife Center, Yamato-son, Ohshima-gun, Kagoshima, 894-3104, Japan Received for publication, November 29, 2011; accepted, January 13, 2012 *Corresponding author: Shin-ichiro Fukumoto, Unit of Veterinary Parasitology, Department of Pathobiology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan Phone: +81-11-388-4747. Fax: +81-11-387-5890. E-mail: [email protected] Phylogenetic relationships of Heligmonellidae nematodes 16 group as an experimental model of intestinal namatodiasis, using Nippostrongylus brasiliensis (Heligmonellidae) or the members represented by Heligmosomoides spp. (Heligmosomidae). Genetic classification or phylogenic analysis using sequence comparisons has been applied to the suborder Trichostrongylina. Internal transcribed spacer (ITS) regions of the ribosomal DNA are useful for elucidating the relationships among congeneric species and closely related genera in many eukaryotes as well as parasitic nematode species including Strongylida. The ITS regions of ribosomal DNA are now one of the most widely sequenced DNA regions in parasitic nematodes including Trichostrongylina. However, genetic information reported so far for Heligmonellidae is scant. The ITS sequence of Heligmonellidae nematodes has been reported only for Nippostrongylus brasiliensis (Travassos, 1914) and Carolinensis minutus (Dujardin, 1845). In the present study, we sequenced and analyzed the ITS-1 and ITS-2 regions of Heligmonellidae nematodes collected from the small intestines of mammals in Japan, including Heligmonoides speciosus (Konno, 1958) [major host, Apodemus speciosus], Orientostrongylus ezoensis Tada, 1975 [major host, Rattus norvegicus] belonging to the subfamily Nippostrongylinae, and Lagostrongylus leporis (Schulz, 1931) [major hosts, Lepus spp. and Pentalagus furnessi] of the subfamily Brevistriatinae. These nematodes and their hosts are indigenous to the Japanese Archipelago or in the far eastern areas of Asia and have species-specific host-parasite relationships. We also compared their ITS-1 and ITS-2 sequences with those previously described for two Heligmonellidae species, N. brasiliensis and C. minutus, and for two species of family Heligmosomidae, Heligmosomoides polygyrus Introduction Family Heligmonellidae is one of the bursate parasitic nematode groups belonging to the superfamily Heligmosomoidea Skrjabin and Schikobalova, 1952 (as a tribe; as a family, Durette-Desset and Chabaud, 1977). This family Heligmonellidae constitutes the superfamily Heligmosomoidea with the family Heligmosomidae (originally as a subfamily, Travassos, 1914) Cram, 1927 (suborder Trichostrongylina DuretteDesset and Chabaud, 1993: order Strongylida). These heligmosome nematodes reside in digestive tracts of a wide range of rodent and lagomorph hosts, and many new species and genera are still being reported. Taxonomy of the Heligmonellidae is complex and difficult, not only because of the large number of species involved, but also these nematodes are quite small in size and morphologically very similar. The classification of the suborder Trichostrongylina including the family Heligmonellidae is based on the morphological characteristics of adult worms, specifically, anterior structure, the arrangement of bursal rays and the shape of spicules of male. Also, in this group, the arrangement of longitudinal ridges on the body surface, present in both sexes, is important in identifying the morphological structure as the synlophe. In addition to the above morphological classification, many reports have described systematic analyses of nematode differentiation during their diversification occurring in association with host evolution, as supplementary information to the geographical distribution-based systematic classification of these nematodes and their hosts. Furthermore, many studies have described the members of this heligmosome Heligmonellidae nematode species. This study is the first to describe the ITS-1 and ITS-2 sequences of Hs, Oe and Ll.