The Journal of Veterinary Medical Science

Advance Publication The Journal of Veterinary Medical Science Accepted Date: 2 Aug 2018 J-STAGE Advance Published Date: 15 Aug 2018

1 2 WILDLIFE SCIENCE FULL PAPER 3 4 5 6 Redescription of Subulura (Murisubulura) andersoni (Cobbold, 1876) (Nematoda: Subuluridae) from Bunomys spp. (Rodentia: Muridae) of Sulawesi, Indonesia, with special reference to S. (M.) suzukii and other related species in the adjacent areas 7 8 Kartika DEWI 1), Hideo HASEGAWA 2) and Mitsuhiko ASAKAWA 3) 9 10 11 12 13 14 15 1) Zoology Division (Museum Zoologicum Bogoriense), RC. Biology LIPI, Jl. Raya Jakarta Bogor, Km. 46. Cibinong, West Java 16911, Indonesia. 2) Department of Infectious Disease Control and Department of Biomedicine, Faculty of Medicine, Oita University, Hasama, Yufu, Oita 879-5593, Japan. 3) Department of Pathobiology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069 8501, Japan. 16 17 18 19 20 Correspondence to: Mitsuhiko ASAKAWA. Department of Pathobiology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069 8501, Japan FAX: +81-11-387-5890, Phone: +81-11-388-4758, E-mail: askam@rakuno.ac.jp 21 22 Running head: REDESCRIPTION OF SUBULURA ANDERSONI 23 24 1

25 26 27 28 29 30 31 32 33 ABSTRACT. Subulura (Murisubulura) andersoni (Cobbold, 1876) (Nematoda: Subuluridae) is redescribed in detail using light and scanning electron microscopy based on materials collected from Bunomys chrycosomus and Bunomys andrewsi (Rodentia: Muridae) of Sulawesi, Indonesia, to provide basic reference data for identification. Comparison was made among congeners from adjacent areas, especially Subulura (Murisubulura) suzukii Yagi and Kamiya, 1981 of Japan. Because morphological differences distinguishing S. (M.) andersoni from S. (M.) suzukii and S. (M.) sipiroki were only the thickness of female tail and length of male tail, respectively, further study using DNA sequence analysis is necessary to prove their distinctness. 34 35 KEY WORDS: Bunomys, Indonesia, Murisubulura, SEM, Subulura 36 2

37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Subulura (Murisubulura) andersoni (Cobbold, 1876) (Nematoda: Subuluridae) is widely distributed in the areas ranging from India to Australia [e.g. 2, 3, 8, 10, 17, 19 22]. Although it was first described from a sciurid in India [2], this nematode is known as a common parasite of murine rodents in Indonesia [3]. Some related species have been proposed to this subgenus, namely Subulura (Murisubulura) suzukii Yagi & Kamiya, 1981 and Subulura (Murisubulura) sipiroki Purwaningsih, 2003 from murids of Japan and Indonesia, respectively [12, 27]. Close morphological resemblance of S. (M.) suzukii with S. (M.) andersoni was pointed out previously [4]. After the first description of S. (M.) andersoni by Cobbold [2], Thwaite [25] redescribed it from a squirrel in Ceylon (=Sri Lanka). Thereafter, this redescription is used by many researchers as a reference to identify S. andersoni. However, the redescription was not in detail accompanying only two figures (anterior extremity and caudal end of male). Because there has been no detailed description of S. (M.) andersoni, it has been difficult to compare them to prove validity. In this paper we give full description of S. (M.) andersoni based on specimens collected from Bunomys spp. of Sulawesi, Indonesia, using light and scanning electron microscopy. Examination of S. (M.) suzukii is also made to verify its validity. 54 55 56 57 58 59 60 MATERIALS AND METHODS The following nematode specimens were subjected to morphological observation. Subulura (M.) andersoni 1. Ten females and 10 males collected from Bunomys chrysocomus of Pakuli, Gumbara, Donggala, Central Sulawesi, Indonesia (MZBNa 430) 3

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 2. Eleven females and 10 males collected from Bunomys andrewsi of Sumarorong, West Sulawesi, Indonesia (MZBNa 752) Subulura (M.) suzukii 1. Eight females and 10 males collected from Apodemus speciosus on Shimokoshiki Is., Kagoshima Prefecture, Japan. (Type specimens; preserved in the Department of Veterinary Parasitology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Reg. No. 2410) 2. Five females and 12 males collected from Apodemus speciosus on Goto Island, Nagasaki Prefecture, Japan (MZBNa 753). Subulura (M.) sipiroki 1. Two females and 3 males (type specimens) collected from Leopoldamys sabanus in Sumatra, Indonesia (MZB Na 306, 312) The nematodes were cleared in lactophenol or glycerol-ethanol, mounted on glass slide with lactophenol or 50% glycerol, respectively, and observed using Olympus BX50 microscope equipped with differential interference contrast apparatus. Measurements were made using ocular micrometers and ImageJ v. 1.51m9 software (NIH, USA). Figures were made with the aid of Olympus U-DA drawing tube. For scanning electron microscopy (SEM), the nematodes were post-fixed in glutaraldehyde, dehydrated through an ethanol series and vacuum-dried using TAITEC VC-96N (TAITEC, Koshigaya, Saitama, Japan), at least for 30 min. Dried specimens were then coated with gold at 5 8mA for 5 min in an Eico I-B2 ion coater and observed with a JEOL JSM 5310 LV SEM with accelerate voltage 15kV. 83 84 RESULTS 4

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 Redescription Subulura (Murisubulura) andersoni (Cobbold, 1876) (Figures 1 3) Synonyms: Ascaris andersoni Cobbold, 1876; Latibuccana funambulensis Patwardhan, 1935; Subulura hindi Mirza, 1936. General Medium sized nematode. Anterior portion bending dorsally. Cuticle with numerous fine striations. Cephalic end oval in apical view; four large cephalic papillae present; amphidial openings conspicuous (Figs. 1A and 3B). Mouth complex, lips much reduced, six in number, same in shape and size; each with two tiny cuticular lappets (Fig. 3B and 3C). Buccal cavity longer than wide, thick-walled, with six large teeth on anterior margin, each extending between lappets of lips (Fig. 1A). Anterior portion of pharynx twisted (Fig. 1B and 1D). Esophagus consisting of club-shape corpus widening posteriorly, isthmus as deep constriction and posterior bulb with valve (Fig. 1E and 1F). Esophageal bulb longer than wide. Cervical alae well developed in both sexes, beginning at level of anterior end of short pharynx, ending anterior to esophageal bulb (Figs. 1C, E, F and 3A). Lateral alae absent. Nerve ring surrounding anterior part of corpus; excretory pore posterior to nerve ring (Fig. 1E and 1F). Cervical papillae not observed. Female Vulva not protruded, situated anterior to midbody, dividing worm length ca. 2 : 3 (Fig. 1G). Vagina running anteriorly from vulva, forming valve-like structure and constriction and then recurved posteriorly (Fig. 1G). Uterus in gravid female occupying almost portions extending from esophageal bulb to posterior end of body. Tail long, slender with a terminal spike (Figs. 1H, 1I and 3D). Eggs rounded with thick shell, and containing coiled embryo with cephalic hooklet-like structure at deposition (Fig. 1J). 5

109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 Male Posterior body bent ventrally. Caudal alae absent. Ten pairs and one unpaired caudal papilla present: one (1st) pair lateral to precloacal sucker; two (2nd and 3rd) pairs almost parallel between precloacal sucker and cloaca; two (4th and 5th) pairs closely set, situated antero-laterally to cloaca; one median at anterior lip of cloaca; two (6th and 7th) pairs postcaudal papilla; one (8th) lateral pair and two submedian pairs (9th and 10th) grouped near tip of tail (Figs. 2A, 2B, 3E, 3F and 3H J). Phasmidial pore placed between 8th and 9th papillae (Figs. 2B and 3J). Precloacal sucker narrow, elongated, supported by radiating muscle fibers (Fig. 3B and 3F). Rim of precloacal sucker not clearly defined but ornamented with very fine longitudinal ridges (Fig. 3F, 3G). Tail, curved ventrally, tapered with spike distally (Figs. 2A, 2B and 3E). Spicules equal, alate, with slight subapical constriction, blunt distally (Figs. 2A, 2B, 2E, 3E and 3I). Gubernaculum long, prominent, tongue-like shape, with window-like structure basally (Fig. 2C and D). Measurements are given in Table 1 comparing with previously published data of S. (M.) andersoni, S. (M.) suzukii and S. (M.) spiroki. Remarks. The present species is a typical member of the subgenus Murisubulura because it possesses six simple labial lobes [1, 14]. The original description of S. (M.) andersoni (as Ascaris andersoni) was very short with diagrammatic figures of posterior ends of male and female [2]. The precloacal sucker of male and vulva in female were not described. Later, Thwaite [25] redescribed this species as Subulura but gave measurements of esophagus and position of excretory pore as mixed data of both sexes. As shown in Table 1, S. (M.) andersoni has considerable variations in the measurements. The smallest male was from Sciurus palmarum of India (8.26 mm) and the largest specimen was from Maxomys bartelsii of Java, Indonesia (18.0 mm) [8, 26]. The 6

133 134 135 136 137 138 139 140 variation was also found in the length of gubernaculum, the shortest was from Maxomys bartelsii of Java, Indonesia (126 µm; 0.78% of worm length) and the longest from S. palmarum (180 µm; 2.18% of worm length) [8, 26]. Wiroreno [26] regarded the differences in length of the gubernaculum and position of vulva between S. (M.) andersoni from the squirrels and that collected from the Javanese murines as population variation. Although the previous measurements of S. (M.) andersoni are often fragmental, those of the present specimens from Bunomys spp. generally coincided with them (Table 1). 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 DISCUSSION From Indonesia and Sahul region, three species of Subulura (Murisubulura) have been known from murid rodents: S. (M.) andersoni (Cobbold, 1876), S. (M.) ortleppi (Inglis, 1960) and S. (M.) sipiroki Purwaningsih, 2003. Subulura (M.) andersoni was first described from a squirrel (?Sciurus sp.) in India [2], and later redescribed from a squirrel in Ceylon (=Sri Lanka) by Thwaite [25]. Mirza [8] described Subulura hindi as a new species from a squirrel, Funambulus palmarum (as Sciurus palmarum) in India, but it was subsequently synonymized with S. (M.) andersoni by Maplestone and Bhaduri [6]. Latibuccana funambulensis Patwardhan, 1935 was described as a new genus and new species from a squirrel, Funambulus pennanti, in India [11]. Yamaguti [28] synonymized it with S. (M.) andersoni, whereas Skrjabin et al. [18] retained it as distinct genus but in undetermined family of Subuluroidea. As a murid helminth, S. (M.) andersoni was first demonstrated in Rattus norvegicus (recorded as Mus decumanus) of India [6]. This nematode was then recorded 7

157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 from Leopoldamys siporanus (as Rattus sabanus) and Niviventer fulvescens (as Rattus fulvescens), Berylmys bowersi (as Rattus bowersi) and Niviventer cremoriventer (as Rattus cremoriventer) of Peninsular Malaysia [10, 23]. In Indonesia, this nematode was first recorded from Maxomys bartelsii (as Rattus bartelsii) in Java [26]. Since then, this species has been reported from wide variety of murine rodents in Indonesia, namely, Rattus lugens in Sumatra, Rattus tanezumi on Krakatau Is., M. bartelsii and Rattus tiomanicus in Java, Bunomys chrysocomus, Bunomys penitus, Bunomys prolatus, Rattus hoffmanni, Margaretamys elegans, Maxomys bartelsii; Rattus hoffmanni, Rattus marmosurus, Rattus xanthurus and Rattus sp. in Sulawesi [3]. In Sahul region, it has been recorded from Rattus leucopus, Pogonomys championi and Uromys anak and Uromys caudimaculatus [19 22]. Subulura (M.) ortleppi was first described from Rhabdomys pumilio and Micaelamys namaquensis (as Rattus (Praomys) namaquensis) of South Africa [5], but once recorded from Rattus fuscipes on Pearson Island, Australia [7]. This species is readily distinguished from S. (M.) andersoni in that the cervical alae are extending to the level at posterior end of esophagus [5]. Subulura (M.) sipiroki was described from Leopoldamys sabanus in Sumatra, Indonesia [12]. This species is also almost identical with that of S. (M.) andersoni by SEM and light microscopy, but has a much longer male tail, 0.36 0.39 (0.38) mm long, of which distal half forming a spike (Table 1). Subulura (M.) suzukii was first described from Apodemus speciosus on Shimokoahiki Island, Kagoshima Prefecture, Japan [27]. Since the description, it has been recorded from A. speciosus sporadically in Japanese mainland and its surrounding islets and from Rattus tanezumi (as Rattus rattus) on Yokoate Is., Tokara Archipelago, Japan [4, 15, 16, 24, 27]. In the original description, Yagi and Kamiya [27] compared S. 8

181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 (M.) suzukii with S. (M.) ortleppi, and S. (M.) williaminglisi Quentin, 1965, another species parasitic in African murids [13], but they apparently overlooked that S. (M.) andersoni had been collected from the Indian and Southeast Asian murines. Hasegawa and Izawa [4] pointed out the close resemblance between S. (M.) suzukii and S. (M.) andersoni. In the present study using both light microscopy and SEM, we could not find clear distinguishing characteristics except for the thinner female tail in S. (M.) andersoni. It is interesting that S. (M.) andersoni, S. (M.) suzukii, and S. (M.) sipiroki possess almost identical morphological characteristics. Purwaningsih [12] observed three types of the male tail in S. (M.) andersoni, i.e. without a spike (host: R. tanezumi from Krakatau), with a short spike (R. hoffmanni from Sulawesi and M. bartelsii and R. tiomanicus from Java) and with a long and ventrally-curved spike (B. penitus from Sulawesi). Because it is difficult to prove their distinctness by morphological study only, DNA sequence analysis is expected to prove their distinctness and solve their phylogenetic relationship. There remains concern whether S. (M.) andersoni could parasitize sciurids and murids because detailed comparison between those from the both hosts has not been made. From Eurasian sciurids, some additional Murisubulura species have been described. For example, Subulura (M.) tanjinensis Ming & Zhang, 2010 from Eutamias sibiricus of China resembles S. (M.) andersoni closely in general morphology and measurements. However the authors compared it with only three representatives, namely S. (M.) ortleppi, S. (M.) suzukii and S. (M.) williaminglisi, all parasitic in murids [9]. Interestingly, S. (M.) tanjinensis is clearly distinguished from these three species and also from S. (M.) andersoni by having more simple oral lips [9]. However, such a 9

205 206 207 208 minute feature might not be considered in the earlier studies in which the murine- and scuirid-parasitic worms were judged as conspecific. Further detailed studies on Subulura of sciurids of Indonesia and surrounding areas using advanced techniques are necessary to draw conclusion on this problem. 209 210 211 212 213 214 215 216 ACKNOWLEDGMENTS The SEM observation was funded by the Indonesian government through DIPA Research Center for Biology-LIPI 2018 under project Fauna Jawa (KD). Thank are also rendered to Dr. Ryo Nakao, Hokkaido University, and Dr. Kinpei Yagi, Hokkaido Institute of Public Health, for the loan of the type specimens of S. (M.) suzukii. This study was partly supported by a Grant-in-Aid for Scientific Research (C-26460513) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 10

217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 REFERENCES 1. Chabaud, A. G. 1978. No. 6. Keys to genera of the Superfamilies Cosmocercoidea, Seuratoidea, Heterakoidea and Subuluroidea. pp 1 71. In: CIH Keys to the Nematode Parasites of Vertebrates. (Anderson, R. C., Chabaud, A. G. and Willmott, S. eds.), Commonwealth Agricultural Bureaux, Farnham Royal, Buckinghamshire. 2. Cobbold, T. S. 1876. Notes on Entozoa. Part 4. Proc. Zool. Soc. Lond.: 294 298. 3. Dewi, K. and Purwaningsih, E. 2013. A checklist of nematode parasites from Indonesian murids. Zootaxa 3608: 531 546. 4. Hasegawa, H. and Izawa, M. 1999. Parasitic helminths found in Rattus rattus from Yokoate Island, Tokara Islands, Japan. Biol. Mag., Okinawa 37: 9 14. (In Japanese) 5. Inglis, W. G. 1960. Further observations on the comparative anatomy of the head in the nematode family Subuluridae: with the description of a new species. Proc. Zool. Soc. Lond. 135: 125 136. 6. Maplestone, P. A. and Bhaduri, N. V. 1942. Helminth parasites of certain rats in India. Rec. Indian Museum 44: 201 206. 7. Mawson, P. M. 1971. Pearson Island Expedition 1969. 8. Helminths. Trans. R. Soc. S. Aust. 95: 169 183. 8. Mirza, M. B. 1936. Subulura hindi n. sp. a new nematode parasite of Sciurus palmarum. Proc. Indian Acad. Sci. Section B 3: 125-127. 11

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258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 16. Sakata, K., Takada, Y., Uematsu, Y., Sakai, E., Tateishi, T., Hasegawa, M., Kageyama, M. and Asakawa, M. 2006. The first report of parasitic nematodes obtained from the large Japanese field mice, Apodemus speciosus (Muridae, Rodentia), collected on Izu Islands, Japan, with a brief zoogeographical comment for its nematode fauna. Bull. Biogeogr. Soc. Jpn. 61: 135 139. (In Japanese) 17. Singh, M. and Cheong, C.-H. 1971. On a collection of nematode parasites from Malayan rats. Southeast Asian J. Trop. Med. Public Health 2: 516 522. 18. Skrjabin, K. I., Shikhobalova, N. P. and Lagodovskaya, E. A. 1964. Oxyurata of Animals and Man. Osnovi Nematodologii III, Izdatel stvo Akademii Nauk SSSR, Moscow, Russia. (Translated by Israel Program for Scientific Translations, Jerusalem, 1976, 486pp.) 19. Smales, L. R. 2012. Chapter 6. Helminth parasites of hydromyine rodents from the Island of New Guinea. pp. 99 117. In: Triunveri, A. and Scalise, D. eds., Rodents. Nova Science Publisher, Inc., New York. 20. Smales, L. R. 2013. Nematodes from the caecum and colon of Pogonomys (Muridae: Anisomyini) from Papua New Guinea with the descriptions of a new genus of Oxyuridae (Nematoda: Oxyurida) and a new species of Trichuridae (Nematoda: Enoplida). Zootaxa 3599: 577 587. 21. Smales, L. R. and Spratt, D. M. 2004. Helminth community structure in Rattus leucopus (Gray) (Muridae) from Australia, Papua New Guinea and Papua. Aust. J. Zool. 52: 283 291. 13

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296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 Legends for figures Figure 1. Female of Subulura (Murisubulura) andersoni from Bunomys andrewsi of Sumarorong, West Sulawesi, Indonesia. A. Apical view of cephalic end; B. Cross section through bottom of buccal cavity; C. Cross section through pharynx; D. Cephalic end, lateral view; E. Anterior end, left lateral view; F. Anterior end, ventral view; G. Ovejector, left lateral view; H, I. Posterior end of female, left lateral (H) and ventral (I) views; J. Egg. Figure 2. Male of Subulura (Murisubulura) andersoni from Bunomys andrewsi of Sumarorong, West Sulawesi, Indonesia. A, B. Posterior end, ventral (A) and left lateral (B) views; C, D. Gubernaculum, ventral (C) and right lateral (D) views; E. Distal end of spicules Figure 3. Scanning electron microscopy of Subulura (Murisubulura) andersoni collected from Bunomys chrysocomus of Pakuli, Gumbara, Donggala, Central Sulawesi, Indonesia. A. Anterior end of male, left lateral view; B. Apical view of cephalic end; C. lip, apical view; D. Posterior end of female, ventral view; E. Posterior end of male, ventral view; F. Precloacal sucker of male, ventral view; G. Striations at the lining of precloacal sucker of male; H. Unpaired median papilla on anterior cloacal lip; I. Posterior end (higher magnification), left lateral view; J. Phasmidial pore between 8th and 9th papillae. 315 15

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