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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, 50603 Kuala Lumpur, Malaysiab Department of Parasitology, Graduate School of Medicine, Osaka City University, Abeno-ku, Osaka 545‐8585, Japanc Parasitologie comparée, UMR 7205, Muséum National d'Histoire Naturelle, 75231 Paris, Franced Hikiiwa Park Center, Tanabe, Wakayama 646‐0051, Japane Department of Environmental Health Science, Faculty of Medicine, Kochi University, Nankoku, Kochi 783‐8505, Japanf Laboratory Animal Center, Graduate School of Medicine, Osaka City University, Osaka 545‐8585, Japang The Kyoto University Museum, Yoshida Honmachi, Sakyo-ku, Kyoto 606‐8501, Japanh Research Promotion Project, Oita University, Oita 879‐5593, Japani Department of Infectious Disease Control, Faculty of Medicine, Oita University, Oita 879‐5593, Japan

a b s t r a c ta r t i c l e i n f o

Article history:Received 30 January 2012Received in revised form 9 August 2012Accepted 15 August 2012Available online 25 August 2012

Keywords:Acanthocheilonema delicata n. sp.Dermal microfilariaEndemicityFilarioidMeles anakumaMolecular identificationWolbachia

Acanthocheilonema delicata n. sp. (Filarioidea: Onchocercidae: Onchocercinae) is described based on adultfilarioids and microfilariae obtained from subcutaneous connective tissues and skin, respectively, of Japanesebadgers (Meles anakuma) inWakayama Prefecture, Japan. No endemic species of the genus had been found inJapan. 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 distinguishedfrom its congeners by morphologic characteristics such as body length, body width, esophagus length, spiculelength, and the length of microfilariae. Based on the molecular data of the mitochondrial cytochrome coxidase subunit 1 (cox1) gene, A. delicata n. sp. was included in the clade of the genus Acanthocheilonemabut 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 mam-mals on the Japanese islands is characterized by a high level of endemicity.

© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Cobbold created the genus Acanthocheilonema with one species,Acanthocheilonema dracunculoides, from specimens collected fromthe aardwolf (Proteles cristatus: Hyaenidae) in South Africa [1]. Thegenus consists of 14 species which have a wide range of mammalianhosts (Carnivora, Macroscelidea, Rodentia, Pholidota, Edentata, andMarsupialia) from the major continents of the world.

Although a high degree of endemicity appears to characterize thespecies among several genera of filarioids obtained from mammalianhosts in Japan [2–7], no endemic species have yet been found in thegenus Acanthocheilonema. The widespread species recorded in Japanare: A. reconditum (Grassi, 1889) from dogs in Okinawa [8]; A. odendhali(Perry, 1967) and A. spirocauda (Leidy, 1858) from Pinnipedia in thecoasts of Hokkaido [9,10].

Acanthocheilonema reconditum may be a zoonotic agent as shownrecently in Australia [11], similar to several filarial species from dogs,

the most common being Dirofilaria immitis (Leidy, 1856) and Dirofilariarepens Railliet and Henry, 1911 [12,13], and Onchocerca lupi Rodonaja,1967 with a single case from Turkey [14]. Onchocerca dewittei japonicaUni, Bain and Takaoka, 2001 fromwild boar was identified as a zoonoticagent in Japan [15–17]. Because of the potential of onchocercid wormsto infest humans, research endeavors on the fauna of filarial parasitesand on their vectors are needed for public health [18].

In this article, we describe a new species of Acanthocheilonemabased on adult filarioids and the microfilariae collected from Japanesebadgers and show its phylogenetic relationships by an analysis of theDNA sequences of its mitochondrial cytochrome c oxidase subunit 1(cox1) gene. We also indicate the absence of the endosymbiont bacte-ria Wolbachia in the new species, because Wolbachia have biologicaland evolutional relationships to the Onchocercidae, as well as patho-logic 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.

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⁎ Corresponding author. Tel.: +60 17 6131725; fax: +60 3 7967 4173.E-mail address: unishigehiko@um.edu.my (S. Uni).

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Twenty-five animals were found in road-kill between December 2005and May 2010 in the vicinity of Tanabe City, Wakayama Prefecture,Japan. The carcasses were shipped refrigerated to a laboratory forfilarial examination one or two days after being found.

For the detection of microfilariae, skin snips were taken from theface, ears, neck, back, limbs, and tail of each animal by the methodsdescribed by Uni et al. [20]. Blood films were made from each badgerand stained with 3% Giemsa's solution in 0.1 M phosphate butter(pH 6.8). For the detection of adult worms, skin and subcutaneousconnective tissues of the whole animal were inspected under astereomicroscope.

Adult worms detected were placed in 2% formalin in saline solu-tion and the specimens were cleared in lactophenol for morphologicstudy. Drawings of the parasites were done by the use of a cameralucida. For histologic examinations, the midbody of a female parasitewas embedded in paraffin by routine methods and the sectionswere stained with hematoxylin and eosin. Scanning electron micros-copy (SEM) of the male worms was prepared as described elsewhere[4]. The body length of adult worms, the length of ovejector, and thelength of area rugosa were measured in millimeters; the other di-mensions in micrometers. Esophagus ratio (%) was determined asthe ratio of the esophagus length to the body length. In the male,the caudal pairs of papillae were tentatively numbered on the basisof Chabaud and Petter [21]. The authorities of the new species areUni and Bain.

For molecular analysis, three female worms were frozen at −20 °Candwere used to examine the nucleotide sequences of themitochondrialcytochrome c oxidase subunit 1 (cox1) of these females as describedin Agatsuma et al. [22]. DNA extraction and PCR amplification were car-ried out according to the methods in our previous study. The primersused for the cox1 mitochondrial region were COIinfF (5′-TGA TTG GTGGTT TTG GTA A-3′) and COIintR (5′-ATA AGT ACG AGT ATC AATATC-3′) [22].

The cox1 sequences of the following 14 species from the GenBankwere used in this analysis: Acanthocheilonema delicata n. sp. (accessionnumber, tentatively registered);A. reconditum;A. viteae (Krepkogorskaya,1933) [23]; Cercopithifilaria bulboidea Uni and Bain, 2001 [4]; C. crassaUni, Bain and Takaoka, 2002 [20]; C. minuta Uni and Bain, 2001 [4];C. multicauda Uni and Bain, 2001 [4]; C. shohoi Uni, Suzuki and Katsumi,1998 [24]; C. tumidicervicata Uni and Bain, 2001 [4]; C. japonica (Uni,1983) [25]; Mansonella (Cutifilaria) perforata Uni, Bain and Takaoka,2004 [5];O. dewittei japonica;O. skrjabiniRukhlyadev, 1961 [3]; and Filariamartis Gmelin, 1790.

Pairwise distances in amino acid sequences of the cox1 geneamong the 14 species mentioned above were obtained using theJones–Taylor–Thornton (JTT) matrix-based model. The rate of varia-tion among sites was modeled with a gamma distribution (MAGA 5)[26]. All positions containing gaps, missing data, or both gaps andmissing data were eliminated. There were a total of 191 positions inthe final dataset.

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 themethods described by Kramer et al. [27]. Briefly, one female specimenof A. delicata n. sp. fixed in 4% paraformaldehyde was embeddedin paraffin. Sections (4 μm thick) were made and placed on Silane(3-aminpropyltriethoxysilane)-coated glass slides. A rabbit polyclonalantiserum raised against the Wolbachia surface protein (WSP) ofthe endobacteria from Brugia pahangi (Buckley and Edeson, 1956)was used (1:2000) to stain sections of A. delicata n. sp. Sectionsof Litomosoides sigmodontis Chandler, 1931 were used as a positivecontrol. Negative controls were done by omitting the primary anti-body. 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 16SrDNA.

3. Results

3.1. Description of Acanthocheilonema delicata Uni & Bain, n. sp.

Small, thin, and delicate filarioids. Females long, twice as long asmales (Table 1). Anterior extremity slightly bulbous, bearing two setsof four papillae and amphids (Fig. 1A-B). Buccal cavity distinct; buccalcapsule with thick buccal ring (Fig. 1C–E). Esophagus divided intoshort anterior muscular portion and long, broad posterior glandularportion (Fig. 1A). Deirids at level of junction of muscular and glandularportions of esophagus (Fig. 1F–G). Caudal extremity of females andmales equipped with three conical lappets (detailed description infemale, microfilaria, and male sections).

Female (19 complete specimens and 17 fragments; Fig. 1A–N): fe-male worms with body swelling at level posterior to vulva (Fig. 1A, *).Cephalic end trapezoidal in most of specimens in lateral view. Headpapillae (Fig. 1B): anterior set of four external labial papillae andposterior set of four cephalic papillae; en face view, both sets in twosquares; buccal cavity narrow with wall composed of two parts(Fig. 1D): cuticle of anterior part of head invaginated, widened nearposterior buccal ring, and flattened on it; buccal ring as wide asesophagus apex. Length of buccal cavity 6 (holotype; range: 6–11)μmand length of buccal ring 2.5 (2.5)μm. Slits of amphids anterior to papil-lae (Fig. 1E, arrow).

Nerve ring at middle level of anterior esophagus (Fig. 1A). Deiridswith short point 320 and 325 (320–380)μm from head (Fig. 1F–G).Intermediary flat piece at junction of anterior and posterior parts ofesophagus in some worms. Intestine narrower than glandular esoph-agus. Vulva, transverse slit at anterior level of glandular esophagus(Fig. 1A and H). Vagina with complex lumen; length/width: 70/70(70–100/65–80)μm (Fig. 1H). In vagina, transverse flattened shorttube, chamber directed posteriorly and lined with thick epithelialcells, narrow tube directed anteriorly, and beginning of ovejector di-rected posteriorly. Long, thin, and straight ovejector 2.1–3.4 mmlong (Fig. 1I). Uteri narrow, straight, and opisthodelphic with aligneddeveloping embryos.

At midbody (Fig. 1J), lateral cuticular thickening (*) and lateralchord (LC) 40 μm wide. Single, lateral, deiridlike structure near levelof anus, extending outwards from left lateral chord (Fig. 1K, arrow).Ovary apices rounded, 340 and 710 (340–3000)μm from tail end(Fig. 1L, *). Tail dorsally at level of anus. Three cuticular terminal lap-pets ventrally (Fig. 1M–N); each lappet conical with terminal smallnipple; at caudal end, two lateral extensions and shorter dorsal oneof subcuticular tissue of body (Fig. 1M). Phasmids subterminal atbase of lateral lappets (Fig. 1N).

Microfilaria (Fig. 1O–Q): Microfilariae from uterus unsheathed.Relatively short and thick body (Table 1, Fig. 1O); left cephalic hookshort (Fig. 1P); head round with slight neck (Fig. 1Q), distinctly inmedian view; cephalic space 5 μm long; column of body nuclei thin-ner than body width; nerve ring 37 μm and excretory pore 60 μmfrom head; tail 32 μm long. Tail attenuated; terminal end not sharp;anucleated terminal part 12 μm long. Microfilariae in skin snips ofhost animals tightly and spirally coiled in general.

Male (9 complete specimens and 2 fragments; Fig. 2): Anteriorpart with slightly bulbous cervix (Fig. 2A). Lateral thickening and lat-eral 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 posteriorto nerve ring (Fig. 2A, arrow; D, arrows; E); both deirids 320(280–320)μm from head. Esophagointestinal junction in Fig. 2F. Tes-tis apex bent, 240 (215–275) μm posterior to esophagointestinaljunction (Fig. 2G).

Area rugosa extending from anterior border 1.93 (1.2–2.2)mmfrom tail end to posterior border 0.48 (0.25–0.85)mm from tail end,being composed of transverse bands (160–222 bands) with longitudi-nal rods in band (Fig. 2H). Posterior region of body spirally coiled

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(Fig. 2I–J); caudal alae present. Right spicule short with narrowhandle (Fig. 2K–L); posterior half spoon-shaped with membranousedges; delicate dorsal crests in terminal third of spoon (Fig. 2L, *);at tip, membranous triangular small flap. Left spicule thin with han-dle, long twisted intermediary piece (50–60 μm), long lamina withrod-like, round posterior end (Fig. 2I–J; M–N). Ratio of left spiculeto right spicule 2.5–2.9.

Caudal papillae (Fig. 2I; O–R): regular arrangement consisted offour pairs of precloacal papillae numbered 1 to 4; two post-cloacalpairs (pairs 5 and 6) almost on same transverse line posteriorlyclose to cloacal aperture; pair 7 close to this line; pairs 8 and 9 nearcaudal end (pair 8 ventral; pair 9 lateral). Papillae of pairs 1 and 7inconstant: one of papillae of pair 7 rarely at mid-length of tail (Fig. 2I).Phasmids subterminal at base of lateral lappets. Tail end narrow, withthree cuticular lappets (Fig. 2Q–R).

On transverse section of midbody of female, thicker cuticle atlateral fields than that of median field; 6–9 muscular cells perquadrant (Fig. 3). In SEM pictures of male worms, deirid withsmall projection (0.7 μm long, Fig. 4); distance between cuticu-lar striations at midbody 0.5 μm (Fig. 5); area rugosa: longitudi-nal rods with 2 μm high in transverse band and distancebetween bands 3 μm (Fig. 6); and three lappets at posteriorend, 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 thepresent filarioid in the blood films of the badgers examined.

Type locality: Mountainside, Nakaheji-cho, Tanabe City, WakayamaPrefecture, Japan.

Collection dates: Type specimens on 7–8 May 2009.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; paratypesfemales (N40, N43) and males (N22, N30), Collection number ofMNHN, 91YU. Paratypes females (N4, N7) and males (N2, N10) inthe 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 ofA. delicata n. sp. in Japanese badgers

Adult specimens of A. delicata n. sp. (47worms) were collected fromonly one badger (ID no. A15): 36 females and 11 males. Thirty-eightspecimens were found in the subcutaneous connective tissues in the

Table 1Measurements of adults and microfilariae of Acanthocheilonema delicata n. sp. and other species of the genus.

A. delicata n. sp. A. dracunculoidesCobbold, 1870

A. filaria(Kou, 1958)

A. mansonbahri(Nelson, 1961)

A. mephitis(Webster andBeauregard,1964)

A. odendhali(Perry, 1967)

Female N37 (n=10)Body length 24 (22–38) [25] 38.5–45.2 36.2–48.1 42.5–63.5 130–155 100–150Body width at midbody 110 (85–140) [100] 220–280 410–490 155–260 320–368 348–415Nerve ring from head 200 (175–260) [209] 200–220 136–167 200–210 400–480 241–335Total esophagus length 3000 (3000–4125) [3517] 1940–2500 4728–5449 1170–1270 3360–4790 1782–2197Esophagus ratios* 12.5 (9.5–17.0) 4.9 12.9 2.5 2.5 1.7Muscular esophagus length 360 (360–490) [412] 340–400 278–319 345–450 680–830 456–670Vulva from head 600 (580–890) [650] 1280–1600 1043–1216 670–980 1704–2860 918–1434Tail 210 (167–250) [194] 240–395 187–220 245–280 400–504 214–322Number of terminal lappets 3 3** 2 lobes and 2 lappets 3 3 3MicrofilariaBody length 164 (153–180) [169] 121–218** 202*** 237–262 186–218.5 231–249Body width 7 (7–9) [8] 4.5–5.2** 3.3*** 4.8–6.3 3.0–3.8 3.5Male N46 (n=10)Body length 14 (10–16) [13] 21.7–24.0 23.1–26.5 24–30 49–65 46–64Body width 60 (60–85) [69] 140–160 160–220 130–150 160–200 228–281Nerve ring from head 200 (170–210) [189] 200–240 146–153 – 312–384 335, 362Total esophagus length 3100 (2520–3350) [2838] 1960–2170 4421–4861 1020–1200 2400–3316 1890–2251Esophagus ratios* 22.1 (17.8–23.9) 9.0 19.0 4.1 4.8 4.1Muscular esophagus length 375 (320–390) [353] 360–410 241–331 320–450 560–640 516–697Right spicule length 80 (75–103) [85] 120–148 105–119 95–135 160–165.3 214–241Left spicule length 233 (218–265) [242] 320–340 340–495 250–415 340–369 536–643Tail 113 (113–162) [128] 140–180 208 135–210 200–275 147–245Number of terminal lappets 3 3** 3 3 3 3Host animal(s) Meles anakuma

(Mustelidae)Proteles cristatusand spotted hyaena(Hyaenidae), anddogs (Canidae)

Manis pentadactyla(Manidae)

Pedetes surdasterlarvalis (Pedetidae)

Mephitis mephitis(Musteridae)

Zalophus californianuscalifornianus(Otariidae)

Parasitic location(s) ofadult worms

Subcutaneousconnective tissue

Peritoneal cavity Blood vessels Subcutaneousconnective tissue

Subcutaneoustissue

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.

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anterior part of the body and the thoracic limbs of the host animal; ninespecimens were found in the posterior part of the body and the pelviclimbs. As the animal had less adipose tissue in the early summer, thesmall 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) fromwhich skin snips were taken (Table 2). The highestdetection rate of the microfilariae was obtained in the skin of themidback; the largest number of microfilariae was 160 microfilariae/cm2

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 adultworms. We dissected some ticks on the host animals, but no filariallarvae were found.

3.4. Molecular analyses

To examine the taxonomic position of A. delicata n. sp., DNA se-quence from the cox1 gene (621 bp) was determined andmultiple se-quence alignment analyses were carried out by the use of amino acidsequences. Pairwise divergences among the 14 species of filarioidswere calculated, taking into account amino acid sequences of cox1(Table 3). The intra-genus distances obtained were 0.147, 0.139, and0.121 for Onchocerca, Cercopithifilaria, and Acanthocheilonema, respec-tively. Likewise, the inter-genus distances were 0.217, 0.222, and 0.231,

between Onchocerca and Cercopithifilaria, between Onchocerca andAcanthocheilonema, and betweenCercopithifilaria andAcanthocheilonema,respectively. Overall mean distance among all of the species used in thisstudy was 0.236. The result of alignment analyses showed that theamino acid sequence of A. delicata n. sp. was close to those of two speciesof Acanthocheilonema, but mean genetic distance was 0.231 amongthe three species of Acanthocheilonema and the value fell within thoseamong seven separate species of Cercopithifilaria as well as betweentwo species of Onchocerca (Table 3).

TheNJ tree showed thatA. delicatan. sp.was positionedwithin a clus-ter with two congeneric species, A. reconditum and A. viteae, indicatingthese to be monophyletic (Fig. 8). A similar topology was obtained intheML tree at the generic level, although positions of the species slightlydiffered within the genus Cercopithifilaria (Fig. 9).

3.5. Wolbachia screening

Sections of a female A. delicata n. sp. were negative for theWolbachiain the intestine, hypodermis (lateral chords), and muscular cells byimmunohistologic staining. PCR analysis was also negative.

3.6. Comparisons with related Acanthocheilonema species

The present new species belongs to the genus Acanthocheilonemaon the basis of the following diagnostic characteristics: the buccal

A. pachycephalum(Ortlepp, 1961)

A. pricei(Vaz andPereira, 1934)

A. procyonis(Price, 1955)

A. reconditum(Grassi, 1889)

A. sabanicolaeEberhard andCampo-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 20.7–25.5 25–30.6 81.5 155, 143 49–72 29–33280–360 231–250 200–240 146–168 61–78 230 660 230–350 250240 243, 304 280 231 123–133 – 330, 380 250–280 2601500–2000 1500,1660 2900 2040–2340 978–1263 2290******* 1300, 1800 1370–1570 3000–35503.4 2.0 8.3 9.6 4.0 2.8 0.8 2.6 10.3375 510, 569 520 380–440 761 490******* 550 370–420 5301270–1360 1290, 1450 1100 680–920 518–608 2500 1200, 1300 1000–1400 910375–430 347, 432 400 180–300 123–129 500 260, 250 320–470 3303 3 4 3 3 – 3 3 3

200–220***** 154–176 120–149****** 270 112–131 – 266–302******** 180–200********* 270–3184***** 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 10.9–15.3 40 87, 93 38–41 18.6–20180 124–148 117–135****** 92–100 42–44 160 400, 330 155–165 145220 243, 219 370 200 104–123 – 340 270–290 –

1520 1000, 1090 3780 1860–2040 817–1026 2290******* 1900, 2000 1600–1660 2580–28706.8 4.5 15.2 20.0 7.0 5.7 2.1 4.2 13.8330 337–365 570 340–400 624 490******* 580 480–490 –

170 134–151 150 92–104 40–48 170 210, 270 140–145 150390 359–376 360 220–300 110–146 360 470 355–370 370265 160–180 145 80–145 85–108 270 230, 200 280 2803 or 4 pairs 3 4 3 3 3 4 3 3Poecilogale albinucha,Ictonyx striatus(Mustelidae)

Didelphismarsupialis(Didelphidae)

Procyonlotor lotor(Procyonidae)

Crocuta crocuta andHyaena hyaena(Hyaenidae), dogs,and jackals (Canidae)

Dasypussabanicola(Dasypodidae)

Suricata suricatta(Herpestidae)

Phoca vitulinaconcolor(Phocidae)

Merioneslibycus(Muridae)

Elephantulus rozeti(Macroscelididae)

Ducts of liver Subcutaneoustissues

Subcutaneousconnectivetissue

Subcutaneousconnective tissue

Subcutaneoustissues

Adipose tissuesurroundingintestine

Pulmonary arteryand right ventricle

Subcutaneoustissue

Subdermal tissue andabdominal cavity

Probably blood – Skin Blood Skin – Blood******** Blood –

South Africa Brazil USA Italy, India, USA,Kenya, Japan

Venezuela South Africa The coast ofMaine, USA

Iran North Africa

[32]; *****[41] [42] [43]; ******[44] [45] [46] *******[47] [48];********[61] [50]; *********[23],**********[62]

[51,52]

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capsule with a distinct posterior ring, the esophagus withwell-developed glandular part, and the tail with caudal lappets inboth sexes [30–32].

Acanthocheilonema delicata n. sp. and 14 other congeneric species arelisted (Table 1). The specimens herein described have been first discov-ered from Japanese badgers (M. anakuma) among Mustelidae.

A

100

BC

D

E

F

G

H

P

Q

JLC

LK

M NI

O

2050

5050

50

1010

200

20

20

20

20 20

20

2020

Fig. 1. Acanthocheilonema delicata n. sp., females andmicrofilariae. 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. Enlargeddeirid. H. Vaginawith body swelling. I. Ovejector. J.Midbody, lateral view. Twouteriwith embryos and microfilariae. LC: lateral chord. * Lateral cuticular thickening. K. Posterior part. Single, lateral, deiridlike structure near level of anus (arrow), ventral view. L. Posteriorpart, 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, medianview. Bars, micrometers.

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7

4567

89

1

2

3

B

A

E

H

IK

ML

J

O

P

Q

R

C

D

50

50

50

20 20

20

F

20

G

20

20 10

20

2010

10

50

N

50

50

10

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, rightlateral 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, ventralview. 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.

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Comparisons of A. delicata n. sp. with 14 known specieswere done on thebody length, esophagus length, esophagus ratios, body width, spiculelength, and the length ofmicrofilariae. Distinctive characteristics are indi-cated below.

Adult worms of A. dracunculoides Cobbold, 1870 are at least twiceas thick as those of A. delicata n. sp.; the microfilariae with a filamen-tous 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 esoph-agus among the genus; one pair of ventral subterminal lappets andtwo 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 headwith neck, a particularly short esophagus, and a larger axial lappet thanthe two lateral ones [37]. Females ofA.mephitis (Webster and Beauregard,1964) have a body five times as large with thick conical, straight tailthan that of A. delicata n. sp. [38]. Adult worms of A. odendhali havegreater body and the left spicule has a bifurcated tip [39,40]; themicrofilariae have long cephalic hook and filamentous tail [40].

In A. pachycephalum (Ortlepp, 1961) three small caudal points arefound in the female; three or four round tubercles on the tail end ofthe 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 specimenswith a high esophagus ratio, but the tail end is equipped with fourlappets in both sexes [43,44]. Microfilariae of A. procyonis and A. delicatan. sp. have a tiny cephalic hook and a short nonfilamentous tail.Acanthocheilonema reconditum together with A. delicata n. sp. has thesmallest adult worms in the genus, but the microfilariae are longerthan A. delicata n. sp. [45] and have a filamentous tail [8].

Acanthocheilonema sabanicolae Eberhard and Campo-Aasen, 1986distinctly differs from A. delicata n. sp. and other congeners, becausethe anterior esophagus is longer than the posterior esophagus [46].Adult worms of A. setariosa (Mönnig, 1926) have a large and thickbody; the esophagus ratios are low (the sex of these worms was notindicated) [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 threesalient conical lappets in our specimens. Adult worms of A. viteaehave a short esophagus and the male has a long left spicule [50].Adult worms of A. weissi (Seurat, 1914) have a short and thick body[51]; the microfilariae have a long body with a conspicuous cephalichook and a filamentous tail [52].

Regarding parasitic locations of adult worms and microfilariae ofAcanthocheilonema species in the host animals (Table 1), adult wormsof eight species including A. delicata n. sp. occur in the subcutaneousconnective tissues and the remaining species occur in various othertissues; microfilariae of eight species occur in the blood and onlythree species (A. delicata n. sp., A. procyonis, and A. sabanicolae) occurin the skin.

Therefore, we conclude that A. delicata n. sp. found from the Japanesebadgers can be distinguished from all the 14 Acanthocheilonema specieson the basis of morphologic characteristics and parasitic locations inthe host animals.

DNA sequences of the cox1 gene of A. delicata n. sp. indicated thatthe present new species was included in the clade of the genusAcanthocheilonema but distinguishable from two other congenericspecies, A. viteae and A. reconditum.

4. Discussion

Species of Cercopithifilaria and Onchocerca were also found to bemonophyletic, and clustered together; the cluster of these two generawas 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 wasfound to be basal to all of these filarioids. These findings obtained by

themolecular analysis established the genus Acanthocheilonema definedby morphologic characteristics [53].

Regarding the vectors, the louse-fly, fleas, and lice have beenrecorded to harbor Acanthocheilonema species [34,37,45]. In the pres-ent study, we did not find any potential vectors of this filarioid.Wolbachia endosymbionts were not detected in A. delicata n. sp. byimmunohistologic examination and PCR screening. The result is similarto the previous findings that the endosymbionts are not present inA. viteae and A. reconditum [19]. The loss of Wolbachia is likely to haveoccurred in the evolution of an ancestral species of Acanthocheilonemain Onchocercinae, because Wolbachia-like gene sequences were incor-porated in the filarial nuclear genomes of the endobacteria-free A. viteae[54]. Many species of the other filarioids in the subfamily possess theendosymbionts, with which they have coevolved since one infectionevent [29].

Chabaud and Bain [55] have suggested that the Acanthocheilonemalineage originated in Africa and expanded to the Oriental and Holarcticregions, and later to South America. From the present study, A. delicatan. sp. from Japanese badgers is closely similar to A. procyonis fromraccoons in the North America. The morphologic similarity has beenfound between Mansonella (Mansonella) akitensis (Uni, 1983) frombears 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 foundfrom bears in Japan and North America [25]. These findings suggestthat the geographic expansion of filarioids has occurred by means ofthe migration of host animals from Eurasia towards North Americathrough the Bering Land Bridge [57].

Eurasian badgers are divided into four species according to theirmitochondrial DNA as well as morphologic characteristics: Meles melesin Europe, an unspecified species in Southwest Asia, M. leucurus inNorth and East Asia, and M. anakuma in Japan [58,59]. Suzuki [60]suggested that half of more than 100 species of mammals recordedfrom the Japanese islands are endemic andmaintain their own lineages.

We have examined 20 species of filarioids in five genera(Acanthocheilonema, Cercopithifilaria, Loxodontofilaria, Mansonella, andOnchocerca) from endemic mammals such as badgers, bears, boars,serows, and sika deer living on the Japanese Archipelago, and have todate found 15 new species (with A. delicata n. sp.) and one new subspe-cies; four other species (A. reconditum, A. spirocauda, A. odendhali, andO. skrjabini) were not endemic [3,4,7]. It is likely that the fauna offilarioids from mammals on the Japanese islands is characterized by ahigh level of endemicity.

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.

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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 2Distribution 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 NDA5 ND − ND + + ND − −A6 +++ +++ +++ +++ +++ +++ ++ +A7 − + ++ +++ +++ − + +A8 +++ + ++ +++ ++ + ++ ++A10 ND + + + + − − −A12 + + ND ++ +++ ++ +++ +A15⁎ +++ +++ +++ +++ +++ +++ ++ ++A16 + + +++ + + + + −A17 +++ ++ +++ ++ ++ + + +A18 + + +++ +++ +++ + ++ ++A19 − − − − + − − −A20 + + +++ + +++ + + +A21 + + − − − − + +Total 9 11 10 11 13 8 10 9Detection rate (9/11) (11/14) (10/12) (11/13) (13/14) (8/12) (10/13) (9/13)

Number of microfilariae: (−) not found; (+) 1–9; (++) 10–19; (+++) 20 or more. ND: not done.⁎ Adult worms were found.

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Acknowledgements

We are grateful to Academician Dr. Yong Hoi Sen, Senior fellowof Academy of Sciences Malaysia, and Professor Emeritus, Universityof Malaya, who has warmly encouraged us in our research efforts.We heartily thank Professors Dr. Mohd Sofian Bin Azirun, Dean, theFaculty of Science, University of Malaya; Dr. Rosli Bin Hashim, Head,

the Institute of Biological Sciences, Faculty of Science, University ofMalaya; and Dr. Rosli Bin Ramli of the same institute, who likewisehave supported our study. We thank Mr. T. Kenko, the Central Labora-tory, Osaka City University Medical School for the histologic prepara-tion and Mr. H. Nakagawa of the same laboratory for SEMexamination of the parasites. We thank Mr. J. L. Yohay for readingthe manuscript.

Table 3Pairwise distance in amino acid sequences of the cox1 gene among 14 species of filarioids.

Species 1 2 3 4 5 6 7 8 9 10 11 12 13

1. A. delicata n. sp. (JQ289993) –

2. A. reconditum JF461456 0.102 –

3. A. viteae AJ272117 0.144 0.118 –

4. O. dewittei japonica AB18872 0.220 0.213 0.249 –

5. O. skrjabini AM74269 0.225 0.177 0.249 0.147 –

6. C. bulboidea AB178834 0.240 0.203 0.244 0.247 0.228 –

7. C. minuta AB178846 0.227 0.196 0.248 0.208 0.205 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 –

10. C. shohoi AB178850 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 0.220 0.191 0.099 0.100 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 0.203 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 0.420 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.

C. minuta AB178846 C. tumidicervicata AB178852

C. shohoi AB178850 C. bulboidea AB178834 C. crassa AB178840

C. multicauda AB178848 C. japonica AM749262

Cercopithifilaria

O. dewittei japonica AB18872 O. skrjabini AM74269

Onchocerca

A. reconditum JF461456 A. viteae AJ272117

A. delicata n. sp. (JQ289993)Acanthocheilonema

M. (Cutifilaria) perforata AM749265 F. martis AJ544880

82

74

62

66

61

69

0.05

C. bulboidea AB178834 C. crassa AB178840 C. shohoi AB178850

C. minuta AB178846 C. tumidicervicata AB178852

C. multicauda AB178848 C. japonica AM749262

Cercopithifilaria

O. dewittei japonica AB18872 O. skrjabini AM74269

Onchocerca

A. reconditum JF461456 A. viteae AJ272117

A. delicata n. sp. (JQ289993)

Acanthocheilonema

M. (Cutifilaria) perforata AM749265 F. martis AJ544880

70

64

6058

0.05

Figs. 8–9. Taxonomic position of A. delicata n. sp. with two other species of Acanthocheilonema. Trees are constructed by the JTTmatrix-basedmodel on the basis of amino acid sequencesof the cox1 gene among 14 species, usingMEGA5. 8. TheNJ tree (upper). 9. TheML tree (lower).A: Acanthocheilonema, C: Cercopithifilaria, F: Filaria,M:Mansonella, andO:Onchocerca. Bars,the number of changes inferred as having occurred along each branch.

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