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PARINT-031; No of Pages Parasitology International xxx (12) xxx xxx Contents lists available at SciVerse ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint Acanthocheilonema delicata n. sp. (Nematoda: Filarioidea) from Japanese badgers (Meles anakuma): Description, molecular identification, and Wolbachia screening Shigehiko Uni a,b,, Odile Bain c, Kazuo Suzuki d, Takeshi Agatsuma e, Masashi Harada f, Masaharu Motokawa g, Coralie Martin c, Emilie Lefoulon c, Masako Fukuda h,i, Hiroyuki Takaoka a,i a Institute of Biological Sciences, Faculty of Science, University of Malaya, 603 Kuala Lumpur, Malaysia b Department of Parasitology, Graduate School of Medicine, Osaka City University, Abeno-ku, Osaka 545 8585, Japan c Parasitologie comparée, UMR 75, Muséum National d'histoire Naturelle, 75231 Paris, France d Hikiiwa Park Center, Tanabe, Wakayama 646 0051, Japan e Department of Environmental Health Science, Faculty of Medicine, Kochi University, Nankoku, Kochi 783 85, Japan f Laboratory Animal Center, Graduate School of Medicine, Osaka City University, Osaka 545 8585, Japan g The Kyoto University Museum, Yoshida Honmachi, Sakyo-ku, Kyoto 606 81, Japan h Research Promotion Project, Oita University, Oita 879 5593, Japan i Department of Infectious Disease Control, Faculty of Medicine, Oita University, Oita 879 5593, Japan article info abstract Article history: Received 30 January 12 Received in revised form 9 August 12 Accepted 15 August 12 Available online xxxx Keywords: Acanthocheilonema delicata n. sp. Dermal microfilaria Endemicity Filarioid Meles anakuma Molecular identification Wolbachia Acanthocheilonema delicata n. sp. (Filarioidea: Onchocercidae: Onchocercinae) is described based on adult filarioids and microfilariae obtained from subcutaneous connective tissues and skin, respectively, of Japanese badgers (Meles anakuma) in Wakayama Prefecture, Japan. No endemic species of the genus had been found in Japan. Recently, some filarioids (e.g., Acanthocheilonema reconditum, Dirofilaria spp., and Onchocerca spp.) have come to light as causative agents of zoonosis worldwide. The new species was readily distinguished from its congeners by morphologic characteristics such as body length, body width, esophagus length, spicule length, and the length of microfilariae. Based on the molecular data of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, A. delicata n. sp. was included in the clade of the genus Acanthocheilonema but differed from two other congeneric species available for study, A. viteae and A. reconditum. Acanthocheilonema delicata n. sp. did not harbor Wolbachia. It is likely that the fauna of filarioids from mammals on the Japanese islands is characterized by a high level of endemicity. 12 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Cobbold created the genus Acanthocheilonema with one species, Acanthocheilonema dracunculoides, from specimens collected from the aardwolf (Proteles cristatus: Hyaenidae) in South Africa [1]. The genus consists of 14 species which have a wide range of mammalian hosts (Carnivora, Macroscelidea, Rodentia, Pholidota, Edentata, and Marsupialia) from the major continents of the world. Although a high degree of endemicity appears to characterize the species among several genera of filarioids obtained from mammalian hosts in Japan [2 7], no endemic species have yet been found in the genus Acanthocheilonema. The widespread species recorded in Japan are: A. reconditum (Grassi, 1889) from dogs in Okinawa [8]; A. odendhali (Perry, 1967) and A. spirocauda (Leidy, 1858) from Pinnipedia in the coasts of Hokkaido [9,]. Acanthocheilonema reconditum may be a zoonotic agent as shown recently in Australia [11], similar to several filarial species from dogs, Corresponding author. Tel.: +60 17 6131725; fax: +60 3 7967 4173. E-mail address: unishigehiko@um.edu.my (S. Uni). the most common being Dirofilaria immitis (Leidy, 1856) and Dirofilaria repens Railliet and Henry, 1911 [12,13], and Onchocerca lupi Rodonaja, 1967 with a single case from Turkey [14]. Onchocerca dewittei japonica Uni, Bain and Takaoka, 01 from wild boar was identified as a zoonotic agent in Japan [15 17]. Because of the potential of onchocercid worms to infest humans, research endeavors on the fauna of filarial parasites and on their vectors are needed for public health [18]. In this article, we describe a new species of Acanthocheilonema based on adult filarioids and the microfilariae collected from Japanese badgers and show its phylogenetic relationships by an analysis of the DNA sequences of its mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. We also indicate the absence of the endosymbiont bacteria Wolbachia in the new species, because Wolbachia have biological and evolutional relationships to the Onchocercidae, as well as pathologic significance to humans [19]. 2. Materials and methods The host animals examined for filarioids were Japanese badgers (Meles anakuma Temminck, 1844), an endemic animal of Japan. 1383-5769/$ see front matter 12 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/.16/j.parint.12.08.004 molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

2 S. Uni et al. / Parasitology International xxx (12) xxx xxx Table 1 Measurements of adults and microfilariae of Acanthocheilonema delicata n. sp. and other species of the genus. A. delicata n. sp. A. dracunculoides Cobbold, 1870 A. filaria (Kou, 1958) A. mansonbahri (Nelson, 1961) A. mephitis (Webster and Beauregard, 1964) A. odendhali (Perry, 1967) Female N37 (n=) Body length 24 (22 38) [25] 38.5 45.2 36.2 48.1 42.5 63.5 130 155 0 1 Body width at midbody 1 (85 140) [0] 2 280 4 490 155 260 3 368 348 415 Nerve ring from head 0 (175 260) [9] 0 2 136 167 0 2 400 480 241 335 Total esophagus length 3000 (3000 4125) [3517] 1940 20 4728 5449 1170 1270 3360 4790 1782 2197 Esophagus ratios* 12.5 (9.5 17.0) 4.9 12.9 2.5 2.5 1.7 Muscular esophagus length 360 (360 490) [412] 340 400 278 319 345 4 680 830 456 670 Vulva from head 600 (580 890) [6] 1280 1600 43 1216 670 980 1704 2860 918 1434 Tail 2 (167 2) [194] 240 395 187 2 245 280 400 4 214 322 Number of terminal lappets 3 3** 2 lobes and 2 lappets 3 3 3 Microfilaria Body length 164 (153 180) [169] 121 218** 2*** 237 262 186 218.5 231 249 Body width 7 (7 9) [8] 4.5 5.2** 3.3*** 4.8 6.3 3.0 3.8 3.5 Male N46 (n=) Body length 14 ( 16) [13] 21.7 24.0 23.1 26.5 24 30 49 65 46 64 Body width 60 (60 85) [69] 140 160 160 2 130 1 160 0 228 281 Nerve ring from head 0 (170 2) [189] 0 240 146 153 312 384 335, 362 Total esophagus length 30 (25 33) [2838] 1960 2170 4421 4861 2400 3316 1890 2251 Esophagus ratios* 22.1 (17.8 23.9) 9.0 19.0 4.1 4.8 4.1 Muscular esophagus length 375 (3 390) [353] 360 4 241 331 3 4 560 640 516 697 Right spicule length 80 (75 3) [85] 1 148 5 119 95 135 160 165.3 214 241 Left spicule length 233 (218 265) [242] 3 340 340 495 2 415 340 369 536 643 Tail 113 (113 162) [128] 140 180 8 135 2 0 275 147 245 Number of terminal lappets 3 3** 3 3 3 3 Host animal(s) Meles anakuma (Mustelidae) Manis pentadactyla (Manidae) Pedetes surdaster larvalis (Pedetidae) Mephitis mephitis (Musteridae) Parasitic location(s) of adult worms connective tissue Proteles cristatus and spotted hyaena (Hyaenidae), and dogs (Canidae) Peritoneal cavity Blood vessels connective tissue tissue Zalophus californianus californianus (Otariidae) Intermuscular fascia Parasitic location of microfilariae Skin Blood Blood Blood Blood**** Locality(ies) Japan Kenya China Kenya Canada California, USA References (Present study) **[33]; [34] [35]; ***[36] [37] [38] [39]; ****[40] Holotype (N37) of A. delicata n. sp. is presented first, followed by range and then mean. Unit: See text. Allotype (N46). Not reported. * Esophagus length/body length (%). ******* Sex of the worm was not indicated. Other asterisks: Cited references. Twenty-five animals were found in road-kill between December 05 and May in the vicinity of Tanabe City, Wakayama Prefecture, Japan. The carcasses were shipped refrigerated to a laboratory for filarial examination one or two days after being found. For the detection of microfilariae, skin snips were taken from the face, ears, neck, back, limbs, and tail of each animal by the methods described by Uni et al. []. Blood films were made from each badger and stained with 3% Giemsa's solution in 0.1 M phosphate butter (ph 6.8). For the detection of adult worms, skin and subcutaneous connective tissues of the whole animal were inspected under a stereomicroscope. Adult worms detected were placed in 2% formalin in saline solution and the specimens were cleared in lactophenol for morphologic study. Drawings of the parasites were done by the use of a camera lucida. For histologic examinations, the midbody of a female parasite was embedded in paraffin by routine methods and the sections were stained with hematoxylin and eosin. Scanning electron microscopy (SEM) of the male worms was prepared as described elsewhere [4]. The body length of adult worms, the length of ovejector, and the length of area rugosa were measured in millimeters; the other dimensions in micrometers. Esophagus ratio (%) was determined as the ratio of the esophagus length to the body length. In the male, the caudal pairs of papillae were tentatively numbered on the basis of Chabaud and Petter [21]. The authorities of the new species are Uni and Bain. For molecular analysis, three female worms were frozen at C andwereusedtoexaminethenucleotidesequencesofthemitochondrial cytochrome c oxidase subunit 1 (cox1) of these females as described in Agatsuma et al. [22]. DNA extraction and PCR amplification were carried out according to the methods in our previous study. The primers used for the cox1 mitochondrial region were COIinfF (5 -TGA TTG GTG GTT TTG GTA A-3 ) andcoiintr(5 -ATAAGTACGAGTATCAATATC-3 ) [22]. The cox1 sequences of the following 14 species from the GenBank were used in this analysis: Acanthocheilonema delicata n. sp. (accession number, tentatively registered); A. reconditum; A. viteae (Krepkogorskaya, 1933) [23]; Cercopithifilaria bulboidea Uni and Bain, 01 [4]; C. crassa Uni, Bain and Takaoka, 02 []; C. minuta Uni and Bain, 01 [4]; C. multicauda Uni and Bain, 01 [4]; C. shohoi Uni, Suzuki and Katsumi, 1998 [24]; C. tumidicervicata Uni and Bain, 01 [4]; C. japonica (Uni, 1983) [25]; Mansonella (Cutifilaria) perforata Uni, Bain and Takaoka, 04 [5]; O. dewittei japonica; O. skrjabini Rukhlyadev, 1961 [3]; and Filaria martis Gmelin, 1790. Pairwise distances in amino acid sequences of the cox1 gene among the 14 species mentioned above were obtained using the Jones Taylor Thornton (JTT) matrix-based model. The rate of variation among sites was modeled with a gamma distribution (MAGA 5) [26]. All positions containing gaps, missing data, or both gaps and missing data were eliminated. There were a total of 191 positions in the final dataset. molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

S. Uni et al. / Parasitology International xxx (12) xxx xxx 3 A. pachycephalum (Ortlepp, 1961) A. pricei (Vaz and Pereira, 1934) A. procyonis (Price, 1955) A. reconditum (Grassi, 1889) A. sabanicolae Eberhard and Campo-Aasen, 1986 A. setariosa (Mönnig, 1926) A. spirocauda (Leidy, 1858) A. viteae (Krepkogorskaja, 1933) A. weissi (Seurat, 1914) 43 52 72 34.8 37.8.7 25.5 25 30.6 81.5 155, 143 49 72 29 33 280 360 231 2 0 240 146 168 61 78 230 660 230 3 2 240 243, 304 280 231 123 133 330, 380 2 280 260 00,1660 2900 40 2340 978 1263 2290******* 1300, 1800 1370 1570 3000 35 3.4 2.0 8.3 9.6 4.0 2.8 0.8 2.6.3 375 5, 569 5 380 440 761 490******* 5 370 4 530 1270 1360 1290, 14 10 680 9 518 608 20, 1300 00 1400 9 375 430 347, 432 400 180 300 123 129 0 260, 2 3 470 330 3 3 4 3 3 3 3 3 0 2***** 154 176 1 149****** 270 112 131 266 302******** 180 0********* 270 318 4***** 3.8 5.8 3.6 4.9****** 4.5 3 5 4 4.5 4.5********** 5 5.5 22 22 26 24.2 26.0 9.3 17.1.9 15.3 40 87, 93 38 41 18.6 180 124 148 117 135****** 92 0 42 44 160 400, 330 155 165 145 2 243, 219 370 0 4 123 340 270 290 15 00, 90 3780 1860 40 817 26 2290******* 1900, 00 1600 1660 2580 2870 6.8 4.5 15.2.0 7.0 5.7 2.1 4.2 13.8 330 337 365 570 340 400 624 490******* 580 480 490 170 134 151 1 92 4 40 48 170 2, 270 140 145 1 390 359 376 360 2 300 1 146 360 470 355 370 370 265 160 180 145 80 145 85 8 270 230, 0 280 280 3 or 4 pairs 3 4 3 3 3 4 3 3 Poecilogale albinucha, Ictonyx striatus (Mustelidae) Ducts of liver Didelphis marsupialis (Didelphidae) tissues Procyon lotor lotor (Procyonidae) connective tissue Crocuta crocuta and Hyaena hyaena (Hyaenidae), dogs, and jackals (Canidae) connective tissue Dasypus sabanicola (Dasypodidae) tissues Suricata suricatta (Herpestidae) Adipose tissue surrounding intestine Phoca vitulina concolor (Phocidae) Pulmonary artery and right ventricle Meriones libycus (Muridae) tissue Elephantulus rozeti (Macroscelididae) Subdermal tissue and abdominal cavity Probably blood Skin Blood Skin Blood******** Blood South Africa Brazil USA Italy, India, USA, Venezuela South Africa The coast of Iran North Africa Kenya, Japan Maine, USA [32]; *****[41] [42] [43]; ******[44] [45] [46] *******[47] [48];********[61] []; *********[23], **********[62] [51,52] Phylogenetic analyses were done using the neighbor-joining (NJ) and maximum-likelihood (ML) methods in MEGA 5 (ver. 5) [26]. Immunologic staining for Wolbachia was done according to the methods described by Kramer et al. [27]. Briefly, one female specimen of A. delicata n. sp. fixed in 4% paraformaldehyde was embedded in paraffin. Sections (4 μm thick) were made and placed on Silane (3-aminpropyltriethoxysilane)-coated glass slides. A rabbit polyclonal antiserum raised against the Wolbachia surface protein (WSP) of the endobacteria from Brugia pahangi (Buckley and Edeson, 1956) was used (1:00) to stain sections of A. delicata n. sp. Sections of Litomosoides sigmodontis Chandler, 1931 were used as a positive control. Negative controls were done by omitting the primary antibody. PCR screening for Wolbachia of two females of A. delicata n. sp. was conducted following the methods described by Casiraghi et al. [28] and Ferri et al. [29], using general Wolbachia primers for 16S rdna. 3. Results 3.1. Description of Acanthocheilonema delicata Uni & Bain, n. sp. Small, thin, and delicate filarioids. Females long, twice as long as males (Table 1). Anterior extremity slightly bulbous, bearing two sets of four papillae and amphids (Fig. 1A-B). Buccal cavity distinct; buccal capsule with thick buccal ring (Fig. 1C E). Esophagus divided into short anterior muscular portion and long, broad posterior glandular portion (Fig. 1A). Deirids at level of junction of muscular and glandular portions of esophagus (Fig. 1F G). Caudal extremity of females and males equipped with three conical lappets (detailed description in female, microfilaria, and male sections). Female (19 complete specimens and 17 fragments; Fig. 1A N): female worms with body swelling at level posterior to vulva (Fig. 1A, *). Cephalic end trapezoidal in most of specimens in lateral view. Head papillae (Fig. 1B): anterior set of four external labial papillae and posterior set of four cephalic papillae; en face view, both sets in two squares; buccal cavity narrow with wall composed of two parts (Fig. 1D): cuticle of anterior part of head invaginated, widened near posterior buccal ring, and flattened on it; buccal ring as wide as esophagus apex. Length of buccal cavity 6 (holotype; range: 6 11)μm and length of buccal ring 2.5 (2.5)μm. Slits of amphids anterior to papillae (Fig. 1E, arrow). Nerve ring at middle level of anterior esophagus (Fig. 1A). Deirids with short point 3 and 325 (3 380)μm from head (Fig. 1F G). Intermediary flat piece at junction of anterior and posterior parts of esophagus in some worms. Intestine narrower than glandular esophagus. Vulva, transverse slit at anterior level of glandular esophagus (Fig. 1A and H). Vagina with complex lumen; length/width: 70/70 (70 0/65 80) μm (Fig. 1H). In vagina, transverse flattened short tube, chamber directed posteriorly and lined with thick epithelial cells, narrow tube directed anteriorly, and beginning of ovejector molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

S. Uni et al. / Parasitology International xxx (12) xxx xxx 4 D C B E G F 0 H J A LC Q O K N I M 0 P L Fig. 1. Acanthocheilonema delicata n. sp., females and microfilariae. A. Anterior part, left lateral view. * Body swelling. B. Head, apical view. C. Head, median view. D. Buccal capsule. E. Head, lateral view. Amphid (arrow). F. Deirids (arrows) posterior to the nerve ring, median view. G. Enlarged deirid. H. Vagina with body swelling. I. Ovejector. J. Midbody, lateral view. Two uteri with embryos and microfilariae. LC: lateral chord. * Lateral cuticular thickening. K. Posterior part. Single, lateral, deiridlike structure near level of anus (arrow), ventral view. L. Posterior part, left lateral view. * Ovary. M. Posterior end, ventral view. N. Posterior end, right lateral view. O. Microfilaria, right lateral view. P. Anterior part, left lateral view. Q. Anterior part, median view. Bars, micrometers. directed posteriorly. Long, thin, and straight ovejector 2.1 3.4 mm long (Fig. 1I). Uteri narrow, straight, and opisthodelphic with aligned developing embryos. At midbody (Fig. 1J), lateral cuticular thickening (*) and lateral chord (LC) 40 μm wide. Single, lateral, deiridlike structure near level of anus, extending outwards from left lateral chord (Fig. 1K, arrow). molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

S. Uni et al. / Parasitology International xxx (12) xxx xxx 5 B C D E A H J 1 2 3 4 5 6 7 P Q F G 7 I K L M N 8 9 O R Fig. 2. Acanthocheilonema delicata n. sp., males. A. Anterior part, lateral view. * Lateral thickening (center) and lateral chord with both dotted areas. Deirid (arrow). B. Head, median view. C. Head, lateral view. D. Deirids (arrows), median view. E. Enlarged deirid. F. Esophagointestinal junction. G. Testis apex. H. Area rugosa, ventral view. I. Posterior part. J. Posterior part, right lateral view. K. Right spicule, lateral view. L. Posterior part of the right spicule, lateral view. * Ridges. M. Left spicule, ventral view. N. Left spicule, right lateral view. O. Posterior part, ventral view. Numbers indicate the pair of caudal papaillae. P. Cloacal region with different arrangement of the caudal papillae. Q. Posterior end, ventral view. R. Posterior end, left lateral view. Bars, micrometers. molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

6 S. Uni et al. / Parasitology International xxx (12) xxx xxx Ovary apices rounded, 340 and 7 (340 3000) μm from tail end (Fig. 1L, *). Tail dorsally at level of anus. Three cuticular terminal lappets ventrally (Fig. 1M N); each lappet conical with terminal small nipple; at caudal end, two lateral extensions and shorter dorsal one of subcuticular tissue of body (Fig. 1M). Phasmids subterminal at base of lateral lappets (Fig. 1N). Microfilaria (Fig. 1O Q): Microfilariae from uterus unsheathed. Relatively short and thick body (Table 1, Fig. 1O); left cephalic hook short (Fig. 1P); head round with slight neck (Fig. 1Q), distinctly in median view; cephalic space 5 μm long; column of body nuclei thinner than body width; nerve ring 37 μm and excretory pore 60 μm from head; tail 32 μm long. Tail attenuated; terminal end not sharp; anucleated terminal part 12 μm long. Microfilariae in skin snips of host animals tightly and spirally coiled in general. Male (9 complete specimens and 2 fragments; Fig. 2): Anterior part with slightly bulbous cervix (Fig. 2A). Lateral thickening and lateral chord near nerve ring; width of lateral chords 12 (allotype; range: 12 14)μm (Fig. 2A, *). Length of buccal cavity 8 (6 8)μm; length of buccal ring 2.5 (2.0 2.5)μm (Fig. 2B C). Deirid posterior to nerve ring (Fig. 2A, arrow; D, arrows; E); both deirids 3 (280 3)μm from head. Esophagointestinal junction in Fig. 2F. Testis apex bent, 240 (215 275) μm posterior to esophagointestinal junction (Fig. 2G). Area rugosa extending from anterior border 1.93 (1.2 2.2) mm from tail end to posterior border 0.48 (0.25 0.85) mm from tail end, being composed of transverse bands (160 222 bands) with longitudinal rods in band (Fig. 2H). Posterior region of body spirally coiled (Fig. 2I J); caudal alae present. Right spicule short with narrow handle (Fig. 2K L); posterior half spoon-shaped with membranous edges; delicate dorsal crests in terminal third of spoon (Fig. 2L, *); at tip, membranous triangular small flap. Left spicule thin with handle, long twisted intermediary piece ( 60 μm), long lamina with rod-like, round posterior end (Fig. 2I J; M N). Ratio of left spicule to right spicule 2.5 2.9. Caudal papillae (Fig. 2I; O R): regular arrangement consisted of four pairs of precloacal papillae numbered 1 to 4; two post-cloacal pairs (pairs 5 and 6) almost on same transverse line posteriorly close to cloacal aperture; pair 7 close to this line; pairs 8 and 9 near caudal end (pair 8 ventral; pair 9 lateral). Papillae of pairs 1 and 7 inconstant: one of papillae of pair 7 rarely at mid-length of tail (Fig. 2I). Phasmids subterminal at base of lateral lappets. Tail end narrow, with three cuticular lappets (Fig. 2Q R). On transverse section of midbody of female, thicker cuticle at lateral fields than that of median field; 6 9 muscular cells per quadrant (Fig. 3). In SEM pictures of male worms, deirid with small projection (0.7 μm long, Fig. 4); distance between cuticular striations at midbody 0.5 μm (Fig. 5); area rugosa: longitudinal rods with 2 μm high in transverse band and distance between bands 3 μm (Fig. 6); and three lappets at posterior end, with phasmidial pore at base of lateral lappet (Fig. 7, arrow). 3.2. Taxonomic summary Host: Meles anakuma Temminck, 1844 (Japanese badger), Mustelidae, Carnivora. Habitat: Adult worms in the subcutaneous connective tissues. Microfilariae in the skin, mainly of the back. No microfilariae of the present filarioid in the blood films of the badgers examined. Type locality: Mountainside, Nakaheji-cho, Tanabe City, Wakayama Prefecture, Japan. Collection dates: Type specimens on 7 8 May 09. Specimens deposited: Holotype (female, N37), Collection number of the Museum National d'histoire Naturelle (MNHN), Paris, 91YU; allotype (male, N46), Collection number of MNHN, 91YU; paratypes females (N40, N43) and males (N22, N30), Collection number of MNHN, 91YU. Paratypes females (N4, N7) and males (N2, N) in Fig. 3. Histologic section of a female midbody of A. delicata n. sp. I: Intestine. Mf: Microfilaria. U: Uterus. * The cuticular thickening at the lateral field. Bar, micrometers. the Kyoto University Museum, Kyoto, Japan. Fragments of specimens (AG1-3) used for DNA analysis at Kochi University. 3.3. Prevalence and distribution of adult worms and microfilariae of A. delicata n. sp. in Japanese badgers Adult specimens of A. delicata n. sp. (47 worms) were collected from only one badger (ID no. A15): 36 females and 11 males. Thirty-eight specimens were found in the subcutaneous connective tissues in the anterior part of the body and the thoracic limbs of the host animal; nine specimens were found in the posterior part of the body and the pelvic limbs. As the animal had less adipose tissue in the early summer, the small adult worms were found under the stereomicroscope. Microfilariae of A. delicata n. sp. were found in 14 (56%) of 25 badgers (ID no. A1 A25) from which skin snips were taken(table 2). The highest detection rate of the microfilariae was obtained in the skin of the midback; the largest number of microfilariae was 160 microfilariae/cm 2 in the skin of the neck of the badger (ID no. A15). The host animal (ID no. A15) that harbored this filarioid was very thin and showed no apparent lesions in the tissues near the adult worms. We dissected some ticks on the host animals, but no filarial larvae were found. 3.4. Molecular analyses To examine the taxonomic position of A. delicata n. sp., DNA sequence from the cox1 gene (621 bp) was determined and multiple sequence alignment analyses were carried out by the use of amino acid sequences. Pairwise divergences among the 14 species of filarioids were calculated, taking into account amino acid sequences of cox1 (Table 3). The intra-genus distances obtained were 0.147, 0.139, and 0.121 for Onchocerca, Cercopithifilaria, and Acanthocheilonema, respectively. Likewise, the inter-genus distances were 0.217, 0.222, and 0.231, between Onchocerca and Cercopithifilaria, between Onchocerca and Acanthocheilonema, and between Cercopithifilaria and Acanthocheilonema, respectively. Overall mean distance among all of the species used in this study was 0.236. The result of alignment analyses showed that the amino acid sequence of A. delicata n. sp. was close to those of two species of Acanthocheilonema, but mean genetic distance was 0.231 among the three species of Acanthocheilonema and the value fell within those among seven separate species of Cercopithifilaria as well as between two species of Onchocerca (Table 3). molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

S. Uni et al. / Parasitology International xxx (12) xxx xxx 7 Figs. 4 7. SEM of males of A. delicata n. sp. 4. Deirid. 5. Striations of the midbody. 6. Area rugosa. 7. Lappets of the tail end. Phasmidial pore (arrow). Bars, 1 μm. Table 2 Distribution of microfilariae of Acanthocheilonema delicata n. sp. in skin snips of Japanese badgers with such microfilariae. Badger ID no. Face Ears Neck Thoracic limbs Midback Abdomen Pelvic limbs Tail A2 ND + ND + ND ND ND A5 ND ND + + ND A6 +++ +++ +++ +++ +++ +++ ++ + A7 + ++ +++ +++ + + A8 +++ + ++ +++ ++ + ++ ++ A ND + + + + A12 + + ND ++ +++ ++ +++ + A15 +++ +++ +++ +++ +++ +++ ++ ++ A16 + + +++ + + + + A17 +++ ++ +++ ++ ++ + + + A18 + + +++ +++ +++ + ++ ++ A19 + A + + +++ + +++ + + + A21 + + + + Total 9 11 11 13 8 9 Detection rate (9/11) (11/14) (/12) (11/13) (13/14) (8/12) (/13) (9/13) Number of microfilariae: ( ) not found; (+) 1 9; (++) 19; (+++) or more. ND: not done. Adult worms were found. molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

8 S. Uni et al. / Parasitology International xxx (12) xxx xxx Table 3 Pairwise distance in amino acid sequences of the cox1 gene among 14 species of filarioids. Species 1 2 3 4 5 6 7 8 9 11 12 13 1. A. delicata n. sp. (JQ289993) 2. A. reconditum JF461456 0.2 3. A. viteae AJ272117 0.144 0.118 4. O. dewittei japonica AB18872 0.2 0.213 0.249 5. O. skrjabini AM74269 0.225 0.177 0.249 0.147 6. C. bulboidea AB178834 0.240 0.3 0.244 0.247 0.228 7. C. minuta AB178846 0.227 0.196 0.248 0.8 0.5 0.112 8. C. tumidicervicata AB178852 0.224 0.197 0.238 0.254 0.166 0.138 0.079 9. C. multicauda AB178848 0.270 0.223 0.291 0.225 0.152 0.117 0.130 0.116. C. shohoi AB1788 0.249 0.231 0.291 0.309 0.229 0.142 0.127 0.134 0.142 11. C. crassa AB178840 0.218 0.182 0.233 0.2 0.191 0.099 0.0 0.130 0.149 0.152 12. C. japonica AM749262 0.218 0.188 0.231 0.233 0.173 0.176 0.195 0.170 0.156 0.3 0.151 13. M. (Cutifilaria) perforata AM749265 0.224 0.232 0.283 0.271 0.305 0.254 0.283 0.288 0.300 0.286 0.296 0.260 14. F. martis AJ544880 0.401 0.351 0.424 0.4 0.431 0.415 0.456 0.459 0.429 0.406 0.454 0.457 0.425 A: Acanthocheilonema, C: Cercopithifilaria, F: Filaria, M: Mansonella, and O: Onchocerca. The NJ tree showed that A. delicata n. sp. was positioned within a cluster with two congeneric species, A. reconditum and A. viteae, indicating these to be monophyletic (Fig. 8). A similar topology was obtained in the ML tree at the generic level, although positions of the species slightly differed within the genus Cercopithifilaria (Fig. 9). 3.5. Wolbachia screening Sections of a female A. delicata n.sp.were negative for thewolbachia in the intestine, hypodermis (lateral chords), and muscular cells by immunohistologic staining. PCR analysis was also negative. 69 66 62 74 82 61 C. minuta AB178846 C. tumidicervicata AB178852 C. shohoi AB1788 C. bulboidea AB178834 Cercopithifilaria C. crassa AB178840 C. multicauda AB178848 C. japonica AM749262 O. dewittei japonica AB18872 Onchocerca O. skrjabini AM74269 A. reconditum JF461456 A. viteae AJ272117 Acanthocheilonema A. delicata n. sp. (JQ289993) M. (Cutifilaria) perforata AM749265 F. martis AJ544880 0.05 70 58 C. bulboidea AB178834 C. crassa AB178840 C. shohoi AB1788 C. minuta AB178846 Cercopithifilaria 60 C. tumidicervicata AB178852 C. multicauda AB178848 C. japonica AM749262 64 O. dewittei japonica AB18872 O. skrjabini AM74269 Onchocerca A. reconditum JF461456 A. viteae AJ272117 Acanthocheilonema A. delicata n. sp. (JQ289993) M. (Cutifilaria) perforata AM749265 F. martis AJ544880 0.05 Figs. 8 9. Taxonomic position of A. delicata n. sp. with two other species of Acanthocheilonema. Trees are constructed by the JTT matrix-based model on the basis of amino acid sequences of the cox1 gene among 14 species, using MEGA 5. 8. The NJ tree (upper). 9. The ML tree (lower). A: Acanthocheilonema, C: Cercopithifilaria, F: Filaria, M: Mansonella, and O: Onchocerca. Bars, the number of changes inferred as having occurred along each branch. molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

S. Uni et al. / Parasitology International xxx (12) xxx xxx 9 3.6. Comparisons with related Acanthocheilonema species The present new species belongs to the genus Acanthocheilonema on the basis of the following diagnostic characteristics: the buccal capsule with a distinct posterior ring, the esophagus with well-developed glandular part, and the tail with caudal lappets in both sexes [30 32]. Acanthocheilonema delicata n. sp. and 14 other congeneric species are listed (Table 1). The specimens herein described have been first discovered from Japanese badgers (M. anakuma) amongmustelidae.comparisons of A. delicata n. sp. with 14 known species were done on the body length, esophagus length, esophagus ratios, body width, spicule length, and the length of microfilariae. Distinctive characteristics are indicated below. Adult worms of A. dracunculoides Cobbold, 1870 are at least twice as thick as those of A. delicata n. sp.; the microfilariae with a filamentous tail, whereas the microfilariae of A. delicata n. sp. lack a sharp tail [33,34]. Adult worms of A. filaria (Kou, 1958) have the longest esophagus among the genus; one pair of ventral subterminal lappets and two small lobes in the female tail end [35]. Microfilariae of A. filaria (=D. fausti) have a filamentous tail [36]. Adult worms of A. mansonbahri (Nelson, 1961) have a round head with neck, a particularly short esophagus, and a larger axial lappet than the two lateral ones [37]. Females of A. mephitis (Webster and Beauregard, 1964) have a body five times as large with thick conical, straight tail than that of A. delicata n. sp. [38]. Adult worms of A. odendhali have greater body and the left spicule has a bifurcated tip [39,40]; the microfilariae have long cephalic hook and filamentous tail [40]. In A. pachycephalum (Ortlepp, 1961) three small caudal points are found in the female; three or four round tubercles on the tail end of the male [32,41]. Adult worms of A. pricei (Vaz and Pereira, 1934) have a short esophagus [42]. Acanthcheilonema procyonis (Price, 1955) is similar to our specimens with a high esophagus ratio, but the tail end is equipped with four lappets in both sexes [43,44].Microfilariae of A. procyonis and A. delicata n. sp. have a tiny cephalic hook and a short nonfilamentous tail. Acanthocheilonema reconditum together with A. delicata n. sp. has the smallest adult worms in the genus, but the microfilariae are longer than A. delicata n. sp. [45] and have a filamentous tail [8]. Acanthocheilonema sabanicolae Eberhard and Campo-Aasen, 1986 distinctly differs from A. delicata n. sp. and other congeners, because the anterior esophagus is longer than the posterior esophagus [46]. Adult worms of A. setariosa (Mönnig, 1926) have a large and thick body; the esophagus ratios are low (the sex of these worms was not indicated) [47]. The tail in the male of A. spirocauda terminates in four tubercles [48] and has been examined in detail by SEM [49] vis-à-vis three salient conical lappets in our specimens. Adult worms of A. viteae have a short esophagus and the male has a long left spicule []. Adult worms of A. weissi (Seurat, 1914) have a short and thick body [51]; the microfilariae have a long body with a conspicuous cephalic hook and a filamentous tail [52]. Regarding parasitic locations of adult worms and microfilariae of Acanthocheilonema species in the host animals (Table 1), adult worms of eight species including A. delicata n. sp. occur in the subcutaneous connective tissues and the remaining species occur in various other tissues; microfilariae of eight species occur in the blood and only three species (A. delicata n. sp., A. procyonis, and A. sabanicolae) occur in the skin. Therefore, we conclude that A. delicata n. sp. found from the Japanese badgers can be distinguished from all the 14 Acanthocheilonema species on the basis of morphologic characteristics and parasitic locations in the host animals. DNA sequences of the cox1 gene of A. delicata n. sp. indicated that the present new species was included in the clade of the genus Acanthocheilonema but distinguishable from two other congeneric species, A. viteae and A. reconditum. 4. Discussion Species of Cercopithifilaria and Onchocerca were also found to be monophyletic, and clustered together; the cluster of these two genera was grouped with that of Acanthocheilonema (Figs. 8 and 9). Mansonella (C.) perforata was basal to the cluster consisting of three genera (Cercopithifilaria, Onchocerca, and Acanthocheilonema). F. martis was found to be basal to all of these filarioids. These findings obtained by the molecular analysis established the genus Acanthocheilonema defined by morphologic characteristics [53]. Regarding the vectors, the louse-fly, fleas, and lice have been recorded to harbor Acanthocheilonema species [34,37,45]. In the present study, we did not find any potential vectors of this filarioid. Wolbachia endosymbionts were not detected in A. delicata n. sp. by immunohistologic examination and PCR screening. The result is similar to the previous findings that the endosymbionts are not present in A. viteae and A. reconditum [19]. The loss of Wolbachia is likely to have occurred in the evolution of an ancestral species of Acanthocheilonema in Onchocercinae, because Wolbachia-like gene sequences were incorporated in the filarial nuclear genomes of the endobacteria-free A. viteae [54]. Many species of the other filarioids in the subfamily possess the endosymbionts, with which they have coevolved since one infection event [29]. Chabaud and Bain [55] have suggested that the Acanthocheilonema lineage originated in Africa and expanded to the Oriental and Holarctic regions, and later to South America. From the present study, A. delicata n. sp. from Japanese badgers is closely similar to A. procyonis from raccoons in the North America. The morphologic similarity has been found between Mansonella (Mansonella) akitensis (Uni, 1983) from bears in Japan and two congeneric American species, M. (M.) interstitium (Price, 1962) from the gray squirrel and M. (M.) llewellyni (Price, 1962) from the raccoon [25,56]. Dirofilaria ursi Yamaguti, 1941 has been found from bears in Japan and North America [25]. These findings suggest that the geographic expansion of filarioids has occurred by means of the migration of host animals from Eurasia towards North America through the Bering Land Bridge [57]. Eurasian badgers are divided into four species according to their mitochondrial DNA as well as morphologic characteristics: Meles meles in Europe, an unspecified species in Southwest Asia, M. leucurus in North and East Asia, and M. anakuma in Japan [58,59]. Suzuki [60] suggested that half of more than 0 species of mammals recorded from the Japanese islands are endemic and maintain their own lineages. We have examined species of filarioids in five genera (Acanthocheilonema, Cercopithifilaria, Loxodontofilaria, Mansonella, and Onchocerca) from endemic mammals such as badgers, bears, boars, serows, and sika deer living on the Japanese Archipelago, and have to date found 15 new species (with A. delicata n. sp.) and one new subspecies; four other species (A. reconditum, A. spirocauda, A. odendhali, and O. skrjabini) were not endemic [3,4,7]. It is likely that the fauna of filarioids from mammals on the Japanese islands is characterized by a high level of endemicity. Acknowledgements We are grateful to Academician Dr. Yong Hoi Sen, Senior fellow of Academy of Sciences Malaysia, and Professor Emeritus, University of Malaya, who has warmly encouraged us in our research efforts. We heartily thank Professors Dr. Mohd Sofian Bin Azirun, Dean, the Faculty of Science, University of Malaya; Dr. Rosli Bin Hashim, Head, the Institute of Biological Sciences, Faculty of Science, University of Malaya; and Dr. Rosli Bin Ramli of the same institute, who likewise have supported our study. We thank Mr. T. Kenko, the Central Laboratory, Osaka City University Medical School for the histologic preparation and Mr. H. Nakagawa of the same laboratory for SEM examination of the parasites. We thank Mr. J. L. Yohay for reading the manuscript. molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004

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Wallingford Oxon UK: CABI Publishing, CAB International; 00. p. 494-6. [62] Sonin MD. Filariata of Animals and man and diseases caused by them, Part 3, Filariidae, Onchocercinae. Fundamentals of Nematology, vol. 24. Moscow: Nauka Publishers; 1975 (Translated from Russian, published for the US Department of Agriculture, and the National Science Foundation, Washington, D.C., by Amerind Publishing Co., Pvt. Ltd., New Delhi, 1985. p. 273 8). molecular..., Parasitology International (12), http://dx.doi.org/.16/j.parint.12.08.004