J. Helminthol. Soc. Wash. 63(1), 1996, pp. 47-50 Icosiella intani sp. n. (Filarioidea: Onchocercidae), a Parasite of Rana cancrivora from South Kalimantan, Indonesia 1 PURNOMO AND MlCHAEL J. BANGS2 U.S. Naval Medical Research Unit No. 2, Box 3, APO AP 96520-8132, U.S.A. ABSTRACT: Icosiella intani sp. n. (Filarioidea: Onchocercidae) is described from the muscles of the frog, Rana cancrivora, Gravenhorst, 1829, in South Kalimantan, Indonesia. Icosiella intani is distinguished by a microfilarial body length (121 nm) longer than that of the 7 known species in the genus and 4-7 tail nuclei in a single row. This species has a relatively short body length, a nonfiliform left spicule with its shaft about the same size as the "blade," and a right spicule with a lanceolate terminus abruptly pointed. This is the first report of Icosiella from Indonesia and represents the eighth described species within this genus, with all hosts in the order Anura. KEY WORDS: Filarioidea, Onchocercidae, Icosiella intani sp. n., Rana cancrivora, Kalimantan, Indonesia, morphology. In February 1979, during a biomedical survey blunt. Posterior end conical and blunt for female, by the U.S. Naval Medical Research Unit No. 2 and protuberant in male (Figs. 1, 2). Anterior (Detachment) and the Indonesian Department extremity with 4 pair submedian, 2 pair median of Health in Tanah Intan, South Kalimantan, labial cephalic papillae (spines) and 2 lateral am- Indonesia (3°20'S, 115°02'E) investigating zoo- phids (Fig. 3). Buccal cavity absent. Esophagus notic filariasis, filariid worms were found in the divided into anterior muscular and posterior connective tissue of the hind leg muscle of a frog, glandular portions, the latter being much longer Rana cancrivora Gravenhorst, 1829. Subsequent and wider. Nerve ring at posterior half of mus- examination of these specimens revealed they cular region of esophagus. Cuticle with finely were a new species within the genus Icosiella transverse striations. Caudal alae and area rugosa described herein. absent. Anus subterminal. MALE (based on 2 mature specimens): Body Materials and Methods 9.9 (8.8-11.0) mm by 73 urn (65-80) at level of Adult worms were removed from the connective tis- head; width increasing posteriad, 138 (125-150) sue of leg muscle, relaxed in 0.6% saline solution, fixed at level of nerve ring, 165 (155-175) at level of in hot 70% ethanol, and preserved in 70% ethanol/5% esophageal-intestinal junction, and 190 (180- glycerin. All specimens were examined using a tem- 200) at level of midbody, gradually decreasing porary lactophenol wet mount technique (Partono et al., 1977). Microfilariae were obtained from blood and posteriad and bulging at tail end, 115(110-120) thick blood smears processed and stained with Giemsa at level of cloaca. Esophagus (Fig. 4) 4,015 (4,010- diluted 1:15 in pH 7.2 buffer for 15 min. Drawings 4,020), distinctly divided into anterior muscular (Figs. 1-9) were made with the aid of a camera lucida. portion 525 (420-630) by 50 and posterior glan- All measurements are expressed as means followed by the range in parentheses and are given as length by dular portion 3,350 (3,100-3,600) by 90 (80- width in micrometers (/^m) unless otherwise indicated. 100). Muscular to glandular esophageal length ratio 1:4.9-8.5. Nerve ring 345 (310-380) from Results cephalic end. Caudal tail flexed ventrally 65 (55- Icosiella intani sp. n. 75). The 2 spicules unequal in length and mark- (Figs. 1-9) edly dissimilar in appearance (Fig. 5). Larger (left) spicule 400 (380—420), composed of two sec- DESCRIPTION: Adult worms small and fili- tions: a slender tubular shaft 245 (240-250) with form, yellow to white when fixed. Anterior end a proximal cup-shaped portion and a distal blade portion 155 (140-170) ending in a lanceolate- like tip. Smaller (right) spicule 103 (102-104); 1 Reprint requests: Publications Office, U.S. Naval Medical Research Unit No. 2, Box 3, APO AP 96520- with a short proximal portion divided into 2 8132, USA. apparent segments with unevenly thickened edg- 2 Address for correspondence: Uniformed Services es and a longer portion wide distally, narrowing University of the Health Sciences, Department of Pre- slightly, then widening and tapering abruptly to ventive Medicine and Biometrics, 4301 Jones Bridge Road, Bethesda, Maryland 20814-4799, e-mail: a thickened edged lanceolate point. The dorsal [email protected]. edge of the longer portion is markedly thicker. 47 Copyright © 2011, The Helminthological Society of Washington 48 JOURNAL OF THE HELMINTHOLOGICAL SOCIETY OF WASHINGTON, 63(1), JAN 1996 Copyright © 2011, The Helminthological Society of Washington ICOSIELLA INTANI SP. N. FROM RAN A CANCRIVORA 49 Spicule ratio 4.0 (3.7-4.1): 1. Gubernaculum and National Parasite Collection, Beltsville, Mary- caudal papillae absent. land 20705. FEMALE (based on 4 gravid specimens): Body ETYMOLOGY: The specimens were obtained 29.0 (24.6-38.2) mm by 98 /urn (90-100) at level from a region of South Kalimantan purported to of head; width increasing posteriad, 183 (170- be rich in alluvial diamonds. Intan, translated 200) at level of nerve ring, 280 (240-310) at level from Indonesian, means diamond. of vulval opening, 310 (280-330) at level of Discussion esophageal-intestinal junction, 330 (300-350) at midbody, gradually decreasing to 110 (80-150) The monogeneric subfamily Icosiellinae An- at anal opening. Esophagus 4,500 (4,210-4,930), derson, 1958, has been found in amphibia from anterior muscular region 460 (380-590) by 54 Japan (Yamaguti, 1941; Hayashi, 1960), Malay- (50-60) and posterior glandular region 4,040 sia (Yuen, 1962; Bain and Purnomo, 1984), Vi- (3,750-4,340) by 200 (190-210). Muscular to etnam (Walton, 1935), and the Philippines glandular esophageal length ratio 1:6.4-11.4. (Schmidt and Kuntz, 1969). Johnston (1967) re- Nerve ring 300 (260-360) from cephalic end. corded Icosiella for the first time in Papua New Muscular vulva, opening as a transverse slit, just Guinea. Of the 7 previously reported species of posterior to muscular-glandular esophageal Icosiella Seurat, 1917 (Bain and Purnomo, 1984), junction, 1,070 (1,000-1,360) from cephalic end I. intani represents the first report of this genus (Fig. 6). Vagina directed anteriorly, and flexing in an amphibian captured in Indonesia. and looping posteriad, before receiving bifurcat- Icosiella intani adults can be differentiated eas- ed uterus a varying distance below vulva. Uteri ily from all previously described species by stan- paired, loosely entwined, joining oviduct and ex- dard morphometric criteria. Specifically, adults tending anteriorly to within 900 (710-1,100) from are much smaller in length than adults of Ico- cephalic end; posterior coil extending to within siella sasai Hayashi, 1960. The spicule ratio is 510 (200-700) from tip of tail. Tail 50 (30-90) 4:1, whereas in Icosiella neglecta (Diesing, 1851) with blunt rounded end (Figs. 7, 8). Viviparous. Seurat, 1917, Icosiella kobayasii Yamaguti, 1941, MICROFILARIA (based upon 25 specimens): Icosiella innominata Yuen, 1962, and Icosiella Body slender, ensheathed, and 121 (105-127) papuensis Johnston, 1967, it is 3:1 or less. Ico- in length. Sheath much longer than body and siella hoogstraali Schmidt and Kuntz, 1969, has unstained with Giemsa. Width at head level 3.7 an enormous spicule ratio, 123:1. Icosiella lau- (3-5), nerve ring 5.1 (4-6), excretory pore 5.0, renti Bain and Purnomo, 1984, has a total esoph- anal pore 3.7 (3-4), and last tail nucleus 2.2 (2- ageal length only half that of I. intani. Most char- 3). Tail 4.5 (4-6) from last nucleus to posterior acteristic, Icosiella intani microfilariae differ from tip, tail nuclei 4-7 in single row (Fig. 9). all other species by having a longer body length. HOST: Mangrove frog, Rana cancrivora Icosiella has been described from 4 genera of Gravenhorst, 1829. frogs; Rana, Discoglossus, Babina, and Cornufer. SITE IN HOST: Connective tissue of hind leg Based on geographic proximity and host species muscle. (Rana cancrivora), I. innominata and /. intani LOCALITY: Indonesia, South Kalimantan might presumably share the most characters in (Borneo), Tanah Intan rubber estate (3°20'S, common. However, as noted, the microfilaria of 115°02'E). I. intani are significantly larger, more slender, DATE OF COLLECTION: February 1979. and sheathed. The male spicule ratio differs SPECIMENS DEPOSITED: USNPC No. 81170. enough to separate the 2. In particular, the larger Holotype male, allotype female in 70% ethanol/ (left) spicule is longer, falling out of the size range 5% glycerin, and 1 blood slide microfilariae (syn- (300-370) given for 7. innominata (Yuen, 1962). types), Giemsa stained, are deposited in the U.S. Additionally, the smaller (right) spicule is divid- Figures 1-9. Adults and microfilaria of Icosiella intani sp. n. 1. Adult male, macroscopic. 2. Adult female, macroscopic. 3. En face view of female showing arrangement of 4 pair submedian, 2 pair median labial cephalic papillae and 2 lateral amphids. 4. Male, ventral view showing esophagus and spicules. 5. Caudal end, lateral view, of male showing left and right spicules and cloaca. 6. Anterior region, lateral view, of female. 7. Caudal end, ventral view, of female. 8. Caudal end, lateral view, of female. 9. Sheathed microfilaria from thick blood smear. Scale bar for Figures 1, 2, 4 in millimeters (mm). Scale bar for Figures 5-9 in micrometers (nm)- Copyright © 2011, The Helminthological Society of Washington 50 JOURNAL OF THE HELMINTHOLOGICAL SOCIETY OF WASHINGTON, 63(1), JAN 1996 ed into 2 distinct segments proximally and tapers and 3M161102BS13.AD410. The opinions and abruptly to a point, markedly different in ap- assertions contained herein are those of the au- pearance from I. innominata. However, because thors and do not purport to reflect those of the these apparent differences are based on only a U.S. Naval Service or the Indonesian Ministry few (7) mature male specimens by Yuen and our of Health. The authors thank Mr. Sukaeri Sarbini description (2 specimens), caution should be ex- for assistance in the specimen collections. ercised in attributing strong significance to these Literature Cited measurements of spicule length and ratio be- cause biological variability is difficult to assess. Bain, O., and Purnomo. 1984. Description d'Icosiella In fact, Yuen (1962) devised a taxonomic key to laurenti n. sp., Filaire de Ranidae en Malaise et the 4 known Icosiella species at that time, using hypothese sur 1'evolution des Icosiellinae. Bulletin the length of the glandular esophagus (not spic- Museum National d'Histoire Naturelle, Paris 4C ser., 6, 1:31-36. ules) as a diagnostic character. The length range Desportes, C. 1941. Nouvelles recherches sur la mor- for female /. infant (3.75-4.34 mm) exceeds that phologic et sur 1'evolution d'Icosiella neglecta of/, innominata (2.9-3.6 mm) but is below that (Diesing, 1851), filarie commune de la grenouille verte. Annales de Parasitologie Humaine et Com- of/, neglecta (5.3 mm). Until more information paree 18:46-66. on species distribution and additional material Hayashi, S. 1960. Description of a new frog filaria, become available, some doubt will remain con- Icosiella sasai n. sp. with redescription of an allied cerning the diagnostic significance of certain species, Icosiella kobayashii Yamaguti, 1941. Jap- characters currently separating species of Ico- anese Journal of Experimental Medicine 30:1-12. Johnston, M. R. L. 1967. Icosiella papuensis n. sp. siella. and Ochoterenella papuensis n. sp. (Nematoda: Fi- The vector of/, infant is unknown. Desportes larioidea), from a New Guinea frog, Cornufer pa- (1941) implicated a biting midge (Ceratopogon- puensis. Journal of Helminthology 41:41-54. idae) and sand fly (Psychodidae), both nemato- Partono, F., Purnomo, D. T. Dennis, S. Atmosoedjono, S. Oemijati, and J. H. Cross. 1977. Brugia timori ceran flys, as the probable biological vectors of sp. n. (Nematoda: Filarioidea) from Flores Island, /. neglecta based on successful larval develop- Indonesia. Journal of Parasitology 63:540-546. ment in the insect flight muscles. To our knowl- Schmidt, G. D., and R. E. Kuntz. 1969. Nematoda edge this is the only evidence of vector incrim- parasites of Oceanica. VI. Foleylla confusa sp. nov., Icosiella hoogstraali sp. nov. (Filarioidea), and ination within this genus of filaria. Unfortunate- other species from Philippine amphibians. Para- ly, few investigations have explored the host- sitology 59:885-889. vector-parasite relationship between ambibians Walton, A. C. 1935. The Nematoda as parasites of and hematophagous insects. Amphibia, 2. Journal of Parasitology 21:27-50. Yamaguti, S. 1941. Studies on the helminth fauna of Acknowledgments Japan. Part 34. Amphibian nematodes II. Japa- nese Journal of Zoology 9:397^08. This study was supported by the Naval Med- Yuen, P. H. 1962. On a new species of Icosiella Seu- ical Research and Development Command, Navy rat, 1917 from Malaya, with a key of the species Department, for Work Units ZF51.524-009-0085 of Icosiella. Journal of Helminthology 36:237-242. Copyright © 2011, The Helminthological Society of Washington