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.

Subulura (Murisubulura) andersoni (Cobbold, 1876) (Nematoda: Subuluridae) is redescribed in detail using light and scanning electron microscopy based on materials collected from Bunomys chrysocomus 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.

Subulura (Murisubulura) andersoni (Cobbold, 1876) (Nematoda: Subuluridae) is widely distributed in the areas ranging from India to Australia [2, 3, 8, 10, 17, 19,20,21,22]. 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 and S. (M.) sipiroki is also made to verify their validity.

MATERIALS AND METHODS

The following nematode specimens were subjected to morphological observation.

Subulura (M.) andersoni

Ten females and 10 males collected from Bunomys chrysocomus of Pakuli, Gumbara, Donggala, Central Sulawesi, Indonesia (MZBNa 430).

Eleven females and 10 males collected from Bunomys andrewsi of Sumarorong, West Sulawesi, Indonesia (MZBNa 752).

Subulura (M.) suzukii

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).

Five females and 12 males collected from A. speciosus on Goto Is., Nagasaki Prefecture, Japan (MZBNa 753).

Subulura (M.) sipiroki

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, U.S.A.). 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 5–8 mA for 5 min in an Eico I-B2 ion coater and observed with a JEOL JSM 5310 LV SEM with accelerate voltage 15 kV.

RESULTS

Redescription

Subulura (Murisubulura) andersoni (Cobbold, 1876) (Figs. 1, 2, 3)

Fig. 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.

Fig. 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.

Fig. 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.

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-shaped 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, 1E, 1F 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).

Male. Posterior body bent ventrally. Caudal alae absent. Ten pairs and one unpaired caudal papillae 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 postcloacal 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 and 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 2D).

Measurements are given in Table 1 comparing with previously published data of S. (M.) andersoni, S. (M.) suzukii and S. (M.) sipiroki.

Table 1.

Comparison of measurements among Subulura (Murisubulura) andersoni, Subulura (Murisubulura) suzukii and Subulura (Murisubulura) sipiroki (in μm unless otherwise stated)

Species		Subulura (Murisubulura) andersoni							Subulura (Murisubulura) suzukii			Subulura(Murisubulura) sipiroki
Host		Squirrel	Squirrel	Funambulus pennanti	Sciurus palmarum	Maxomys bartelsii	Bunomys andrewsi	Bunomys chrysocomus	Apodemus speciosus			Leopoldamys sabanus
Locality		India	Sri Lanka	India	India	Java, Indonesia	West Sulawesi, Indonesia	Central Sulawesi, Indonesia	Shimokoshiki Is., Japan		Goto Is., Japan	Sumatra, Indonesia
Source		Cobbold [2]	Thwaite [25]	Patwardhan [11]a)	Mirza [8]b)	Wiroreno [26]	Present study	Present study	Yagi & Kamiya [27]	Present study	Present study	Present study
Female							n=11	n=10		n=8	n=5	n=3
	Worm length, mm	19.05	15.9–23.1	20.0–22.5	17.01–23.37	27.0–36.2	17.9–22.7 (19.1)c)	16.0–19.5 (17.4)	16.7–29.9	16.9–17.8 (17.3)	17.4–31.0 (24.5)	24.8–26.0 (25.7)
	Worm width, mm	1.02	0.4–0.53	0.48–0.50	0.49–0.52	0.49–0.56	0.40–0.51 (0.45)	0.41–0.55 (0.48)	0.433–0.974	0.50–0.56 (0.53)	0.56–0.87 (0.71)	0.47–0.51 (0.49)
	Buccal depth			59	{50}d)		53–63 (60)	50–60 (55)		54–58 (56)	68–85 (79)	50–58 (54)
	Pharynx length						65–80 (71)	57–75 (65)		50–63 (57)	90–105 (95)	60–65 (62)
	Esophageal corpus length, mm						1.23–1.39 (1.33)	1.20–1.38 (1.31)		1.23–1.33 (1.29)	1.34–1.65 (1.48)	1.30–1.42 (1.36)
	Esophageal corpus width						140–175 (157)	135–163 (146)		135–165 (155)	160–230 (196)	155–172 (162)
	Esophageal bulb length		{240–287}	250–290			200–235 (223)	205–240 (226)		210–250 (233)	265–330 (291)	208–250 (232)
	Esophageal bulb width		{207–276}	180			200–255 (226)	190–250 (222)		210–285 (231)	235–370 (295)	250–275 (251)
	Total esophagus lengthe), mm		{1.68–2.4}	1.94–2.10			1.53–1.69 (1.63)	1.50–1.68 (1.61)	1.60–2.00	1.56–1.65 (1.61)	1.71–2.07 (1.87)	1.57–1.72 (1.66)
	Nerve ringf)						260–340 (313)	250–310 (295)	272–390	290–335 (307)	315–370 (340)	300–310 (313)
	Excretory pore f)		{460–560}				420–550 (472)	420–490 (461)	466–575	416–497 (460)	495–600 (545)	460–490 (477)
	Vulva, mm f)		6.5–8.0	2/5g)	5.69	6.5–11.9	6.77–8.61 (7.49)	6.70–7.93 (7.09)	6.80–11.64	6.53–7.48 (7.02)	7.01–13.00 (10.21)	8.24–9.25 (8.78)
	Tail length, mm		up to 1.13		1.25		1.20–1.74 (1.39)	1.09–1.25 (1.10)	0.964–1.640	0.92–1.04 (0.99)	0.82–1.29 (1.07)	1.23–1.46 (1.34)
	Egg length	51–64	78	87	70		55–70 (63.4)	57–73 (66.8)	80–94	63–80 (72.1)	70–102 (85.0)	57–75 (66)
	Egg width		60	61	60		45–59 (50.4)	47–58 (53.7)	58–67	48–60 (54.6)	58–85 (68.0)	47–56 (53)
Male							n=10	n=10		n=10	n=12	n=2
	Worm length, mm	12.7	13.5–14.9	12.5	8.26–13.06	13.5–18.0	9.3–12.8 (11.7)	10.3–12.3 (11.2)	9.6–13.2	10.0–12.2 (10.7)	11.5–16.7 (13.8)	16.2–17.1 (16.7)
	Worm width, mm		0.36–0.43	0.32	0.24–0.41	0.32–042	0.30–0.46 (0.37)	0.31–0.39 (0.34)	0.43–0.54	0.35–0.50 (0.40)	0.43–0.58 (0.50)	0.38–0.46 (0.42)
	Buccal depth				{50}		48–55 (52)	40–50 (44)		45–53 (48)	55–68 (62)	42–45 (43)
	Pharynx length						63–70 (64)	45–50 (49)		38–53 (45)	58–80 (73)	44–46 (45)
	Esophageal corpus length, mm						1.05–1.22 (1.12)	1.06–1.20 (1.12)		0.97–1.19 (1.10)	1.09–1.32 (1.22)	1.14–1.20 (1.17)
	Esophageal corpus width						105–155 (132)	110–150 (128)		115–157 (139)	128–175 (154)	110–120 (115)
	Esophageal bulb length		{240–287}	230	240		163–225 (192)	177–210 (191)		190–240 (208)	200–265 (240)	191–200 (195)
	Esophageal bulb width		{207–276}	170	190		143–200 (177)	170–210 (185)		175–225 (194)	200–255 (227)	182–190 (186)
	Total esophagus length e), mm		{1.68–2.4}	1.74	1.72		1.30–1.48 (1.38)	1.29–1.43 (1.36)	1.35–1.66	1.22–1.48 (1.37)	1.38–1.65 (1.53)	1.38–1.44 (1.41)
	Nerve ring f)						275–329 (299)	270–332 (300)	259–366	246–350 (292)	242–340 (307)	285–290 (287)
	Excretory pore f)		{460–560}				385–509 (450)	390–470 (417)	406–496	375–441 (424)	415–555 (516)	390–410 (400)
	Spicule length, mm		0.85–1.0	0.9	0.91		0.68–0.86 (0.79)	0.89–1.02 (0.95)		0.85–1.12 (1.01)	1.11–1.39 (1.26)	0.94–0.96 (0.95)
	Gubernaculum length		155–175		180	126–140	130–166 (150)	140–165 (153)	132–180	140–165 (151)	172–203 (182)	141–153 (147)
	Suckerh), mm		0.5–0.62i)		0.924		0.61–0.88 (0.78)	0.69–0.80 (0.74)	0.634–1.050	0.67–0.86 (0.74)	0.86–1.11 (0.97)	0.92–0.95 (0.93)
	Tail length, mm		0.25–0.3		0.244		0.23–0.30 (0.27)	0.21– 0.29 (0.25)		0.22–0.26 (0.24)	0.22–0.29 (0.26)	0.36–0.39 (0.38)

a) Species was recorded as Latibuccana funambulensis. b) Species was recorded as Subulura hindi. c) Range followed by mean in parenthesis. d) { }: Data of both sexes were mixed or sex was not mentioned. e) Combined length of pharynx, corpus, isthmus and bulb. f) Distance from anterior extremity. g) Of worm length from anterior extremity. h) Distance from caudal extremity. i) Distance from cloacal aperture.

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 variation was also found in the length of gubernaculum, the shortest was from M. 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).

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 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 M. 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, B. chrysocomus, Bunomys penitus, Bunomys prolatus, Margaretamys elegans, Rattus hoffmanni, Rattus marmosurus, Rattus xanthurus and Rattus sp. in Sulawesi [3]. In Sahul region, it has been recorded from Rattus leucopus, Pogonomys championi, Uromys anak and Uromys caudimaculatus [19,20,21,22].

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 L. sabanus in Sumatra, Indonesia [12]. This species is also almost identical with 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 A. speciosus on Shimokoshiki Is., 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. (M.) suzukii with S. (M.) ortleppi, and S. (M.) williaminglisi Quentin, 1965, another species parasitic in African murids [13], but they presumably overlooked the fact 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 validity by morphological study only, DNA sequence analysis is expected to prove their validity 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 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.