Morphological and molecular genetic characterization of three Capillaria spp. (Capillaria anatis, Capillaria pudendotecta, and Capillaria madseni) and Baruscapillaria obsignata (Nematoda: Trichuridae: Capillariinae) in avians

Abstract

Morphological and genetic analyses were performed on four avian species of the subfamily Capillariinae (Nematoda: Trichuridae), i.e., Capillaria anatis from chickens (Gallus gallus domesticus) in Japan and the Philippines, Baruscapillaria obsignata from chickens and captive swans (Cygnus olor and Cygnus atratus) in Japan, Capillaria pudendotecta from captive swans in Japan, and Capillaria madseni from carrion and jungle crows (Corvus corone and Corvus macrorhynchos) in Japan. Although morphometric variations of male and female worms from different hosts and/or localities made the species identification difficult, the 18S ribosomal RNA gene (rDNA) sequences clarified their taxonomic position and phylogenetic relationships. Species of the same genus clustered robustly into a single clade in the phylogenetic tree based on the 18S rDNA, demonstrating to the extent possible the validity of the latest classification of the subfamily following Moravec's rearrangement in 1982. Male worms of C. pudendotecta are described here for the first time.

Integrated taxonomic approaches to seven species of capillariid nematodes (Nematoda: Trichocephalida: Trichinelloidea) in poultry from Japan and Indonesia, with special reference to their 18S rDNA phylogenetic relationships

“…The birds were dissected on the day after euthanasia according to the guidelines for animal experiments outlined by the university, and helminth parasites were collected according to standard procedures (Tamaru et al 2015). The collected parasites were preserved in either 10% neutral-buffered formalin solution or 70% ethanol and were categorized by host organ, parasite sex, and certain morphological characters under a dissection microscope.…”

“…The DNA of male and/or female worms of different species, stored in 70% ethanol, was extracted using an Illustra™ tissue and cells genomicPrep Mini Spin Kit (GE Healthcare UK, Buckinghamshire, UK) according to the manufacturer'sinstructions. PCR amplification of overlapping fragments of 18S rDNA was performed as described previously (Tamaru et al 2015). For some worms, two forward primers, i.e., S.r.18S-SSU22F and S.r.18S-SSU23F, were used instead of the primer NSF573/19 described by Tamaru et al (2015).…”

“…PCR amplification of overlapping fragments of 18S rDNA was performed as described previously (Tamaru et al 2015). For some worms, two forward primers, i.e., S.r.18S-SSU22F and S.r.18S-SSU23F, were used instead of the primer NSF573/19 described by Tamaru et al (2015). Table 1 summarizes all the forward and reverse universal eukaryotic primers employed in the current study.…”

“…The DNA polymerase was Blend Taq-Plus-(TOYOBO, Kita-ku, Osaka, Japan), and PCRs were conducted in a thermal cycler in 20-or 25-μL reactions using the following cyclingprotocol:2minat94°C;followedby40cyclesat94°C f o r3 0s ,6 4o r6 2°Cf o r3 0s ,a n d7 2°Cf o r9 0s ;a n df i n a l extension at 72°C for 7 min. The PCR products were purified using a FastGene Gel/PCR Extraction Kit (NIPPON Genetics Co., Tokyo, Japan) and sequenced directly with the primers for amplification and sequencing as described by Tamaru et al (2015). When direct sequencing was not satisfactory, the purified PCR products were cloned into a plasmid vector, pTA2 (TArget Clone™; TOYOBO), and transformed into Escherichia coli JM109 cells (TOYOBO) according to the manufacturer'si nstructions.…”

“…In an earlier study from our laboratory (Tamaru et al 2015), we performed morphological and molecular genetic characterizations of four avian species of the family Capillariidae and assessed as far as was possible the validity of the latest classification of the family following Moravec'sr e d e f i n i t i o n of its genera in 1982 based on the male caudal end as the most important morphological feature for separating the genera (Moravec 1982;Gibbons 2010). In that study, we characterized three Capillaria spp., i.e., Capillaria anatis (Schrank 1790) Travassos 1915, Capillaria pudendotecta Lubimova 1947, and Capillaria madseni Wakelin, Schmidt et Kuntz 1970, and Baruscapillaria obsignata (Madsen 1945) Moravec 1982 collected from chickens (Gallus gallus domesticus (Linnaeus 1758)), captive swans (Cygnus olor (Gmelin 1789) and Cygnus atratus (Latham 1790)), and carrion and jungle crows (Corvus corone Linnaeus 1758 and Corvus macrorhynchos Wagler 1827) in Japan and the Philippines; and several species of the genera Aonchotheca López-Neyra 1947, Pearsonema Freitas et Mendona 1960 and Eucoleus Dujardin 1845 from mammalian hosts.…”