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Protistology 4 (4), 357-360 (2006/7)
Protistology
Nematocystis indica sp. n. (Protozoa, Apicomplexa, Eugregarinida), an endoparasitic monocystid gregarine from the seminal vesicles of an Indian earthworm Amynthas diffringens Baird (Annelida, Oligochaeta)
Probir K. Bandyopadhyay, Partha Mallik and Amlan Kumar Mitra
Parasitology Laboratory, Department of Zoology, University of Kalyani, West Bengal, India
Summary
During an ongoing biotic survey of the gregarine parasites of the earthworms of West Bengal, India, a new species of the genus Nematocystis Hesse, 1909 was obtained from the seminal vesicles of the earthworm Amynthas diffringens Baird. The new species is a perfect nematode-like organism, solitary with several bulges and constrictions, especially in mature forms. Its average size is 2773.3 + 30.6 ^m. Nucleus elongated, measuring on the average 46.2 + 14.8 ^m. Extreme ends rounded. Gametocysts rounded to ovoid, measuring on the average 88.2 ^m in diameter. Oocysts navicular, average size 17.9 + 11.7 ^m.
Key words: gregarines, parasite, Nematocystis indica sp. n., seminal vesicles, earthworm, Amynthas diffringens, India
Introduction
Levine (1977) listed 27 species of Nematocystis Hesse, 1909. After that, one species was added to this genus by Segun (1978), five species by Pradhan and Dasgupta (1980), two species by Roychoudhury and
Haldar (1984) and two species by Bandyopadhyay and Mitra (2005). Out of these 37 Nematocystis species, 13 species are from West Bengal, India. The present paper deals with a yet another species of Nematocystis obtained from the seminal vesicles of the earthworm Amynthas diffringens Baird.
© 2007 by Russia, Protistology
358 • Probir K. Bandyopadhyay et al.
Material and Methods
Biotic survey of gregarine parasites in oligochaetes was performed in Midnapore East district, West Bengal, India. Earthworms Amynthas diffringens Baird were collected and taken to the laboratory. They were dissected while alive and their seminal vesicles were carefully removed. These were placed on clean glass with a drop of 0.6% NaCl solution. A thin film of seminal fluid was drawn out on a slide covered with a cover slip for examination of living individuals under a phase contrast microscope. The content of the seminal vesicles was semidried and fixed in Schaudin's fluid (20 min). The fixed smears were stored in 70% ethyl alcohol for removal of mercuric chloride. The slides were then passed through a descending series of alcohols (5 min each) and placed in distilled water. These were transferred to a 3% iron alum solution (overnight) and stained with Heidenhain's haematoxylin solution (20 min). Differentiation was done with 1% iron alum solution under the low power objective lens of the light microscope. The slides were washed thoroughly, dehydrated in an ascending series of alcohol, cleared in xylene and mounted in Canada balsam. Camera lucida drawings of different stages of gregarines were made, and photomicrographs were taken with the help of an Olympus phase contrast microscope and Olympus camera. All measurements are in micrometers (^m). Description of plane shapes follows the guidelines of Clopton (2004).
Results and Discussion
Nematocystis indica sp. n. (Figs 1-6)
Phylum: Apicomplexa Levine, 1977
Order: Eugregarinida Leger, 1900
Family: Monocystidae Butschli, 1882
Subfamily: Monocystinae Bhatia, 1930
Length of body (LB): 1969.9-3139.6 (2773.3 ± 404.1); width of body (WB): 10.4-45.8 (30.6 ± 2.3); length of nucleus (LN): 37.4-58.2 (46.2 ± 6.9); width of nucleus (WN): 8.3-20.8 (14.8 ± 3.6); diameter of gametocyst (DG): 83.2-91.5 (88.2 ± 3.3); length of oocyst (LO): 16.2-19.3 (17.9 ± 1.2); width of oocyst (WO): 10.8-12.3 (11.7 ± 0.7); LB : WB = 90 : 1; LB : LN = 60 :1l; WB : WN = 2 : 1; LB : DG = 31 : 1; DG : WB = 2.8 : 1; LB : LO = 154.6 : 1; WB : WO = 2.6 : 1 (Number of individuals measured: 41).
With the characters of the genus Nematocystis Hesse, 1909, as given by Levine (1977); "Gamonts large, cylindroid, nematoid, often with mucron at anterior end, solitary, oocysts biconical".
Gamont solitary, elongated, ribbon-like, having parallel sides and 1-4 constrictions along the length of the body in mature forms. Both ends of gamont rounded. Ectoplasm very thin (1-2^m). Endoplasm with very few vacuoles and with numerous granules of various size. Distribution of granules not uniform. Nucleus is shifted towards one end of body. Nucleus very elongated, occasionally remaining within body constrictions. Nucleoplasm diffused at one end. Holdfast organ not observed. Gametocyst with two almost equal gametocytes loosely associated with each other. Oocysts biconical.
The presence of large and long solitary nematoid gamont and biconical oocysts justifies the inclusion of the present form into the genus Nematocystis Hesse, 1909. To date, 37 Nematocystis species have been described. Since the present species has very peculiar characters (elongated, ribbon-like thin body with almost parallel sides, ending in a V-shaped tapering at both nds, several constrictions along the length of the body, elongated nucleus diffused at one end), it should be compared with only a few similar species, i.e., N. bunmii Segun, 1978; N. levinei Pradhan et Dasgupta, 1980; N. gardenica Bandyopadhyay et Mitra, 2005 and N. kalyaniensis Bandyopadhyay et Mitra, 2005. The length of the gamont of the new species is greater than that of N. burnii (388-900 + 6-30 ^m), N. gardenica (110-1320 +11-18 ^m) and N. kalyaniensis (246-1232 + 7-13 ^m) but does not show much difference with the length of N. levinei (2523.04 + 107.3 ^m). The trophozoites of N. bunmii are pointed anteriorly, gradually increasing in width toward the middle and tapering toward the pointed posterior pole (Segun, 1978), but the gamonts of the new species are ribbonlike with almost parallel borders with no bulging anywhere and with more than one constriction along the length of the body. The two ends of the trophozoites of N. bunmii are pointed, those in the new species are rounded. The nucleus in N. bunmii is subspherical but that of the new species is elongated. The oocysts of N. burnii contain bipolar plugs, but no plugs have been observed in the species under discussion. The new species also differs from N. gardenica and N. kalyanien-sis in the shape of the gamonts and oocysts. The gamonts of N. gardenica are almost straight with sharply pointed tips and do not have any constrictions. But the gamonts of the new species are ribbon-like, much longer and with several constrictions along the body. The gamonts of N. kalyaniensis are much smaller, with swellings at the anterior and posterior body ends; one swelling harbours a rather large spherical nucleus. But in the species under discussion gamonts are elongated with rounded ends, with no swellings at both the ends. Moreover, constrictions are absent in N. kalyaniensis.
Protistology ■ 359
Figs 1-6. Photomicrographs of different stages of Nematocystis indica sp. n. from the seminal vesicles of Amynthas diffriengens. 1 - A mature trophozoite; 2-4 - different portions of gamonts showing constrictions and shape of the nucleus; 5 - a gametocyst; 6 - oocysts. Scale bars: 1 - 1000 ^m, 5 - 100 ^m, 6 - 15 ^m.
The oocyst shape in the new species is also different species shows some degree of similarity with the latter
from that in N. gardenica and N. kalyaniensis. The new two species as to body dimensions, but the shape of
36g
gamonts is obviously different. The ends of the gamonts of N. levinei are blunt, while in the species under discussion, rounded. In N. levinei one end of the gamont is broader than the other and the body has fine longitudinal striations converging at both ends. Striations are absent in the new species.
Considering all these differences, we propose that the gregarine studied here is a new species and designate it as Nematocystis indica sp. n.
Taxonomic summary
Type host: Amynthas diffringens Baird.
Symbiotype: Host AD/ NG/02/2005 deposited in the Museum of the Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India.
Infection site: Seminal vesicles.
Type locality: Midnapore East district, West Bengal, India.
Prevalance: 7 out of 44 (15.9 %).
Type material: Holotype, slide NI/08/2005, and paratypes, slides NG/01/2005, NG/05/2005, NG/06/ 2005 are in the collection of the Parasitology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India.
Etymology: This species was named after our motherland India.
References
Bandyopadhyay P.K. and Mitra A.K. 2005. Observation on two new species of Nematocystis Hesse, 1909 (Protozoa: Monocystidae) from earthworms (Annelida: Oligochaeta) ofWest Bengal, India. Animal Biology. 55, 133-139.
Clopton R.E. 2004. Standard nomenclature and metrics of plane shapes for use in gregarine taxonomy. Comp. Parasitol. 71, 130- 140.
Levine N.D. 1977. Revision and checklist of the species of the aseptate gregarine family Monocystidae. Folia Parasitol. 24, 1-24.
Roychoudhury U.S. and Haldar D.P 1984. Studies in aseptate gregarines from earthworms ofWest Bengal: Two new species of Nematocystis and one new species of Stomatophora. J. Beng. Nat. Hist. Soc. (N.S.). 3, 1727.
Segun A.O. 1978. Monocystid gregarine parasites of Nigerian earthworms. J. Protozool., 25,157-162.
Pradhan D. and Dasgupta B. 1980. Record of some new gregarines in earthworms from the hill areas of Darjeeling district-I. North Bengal Univ. Rev. (Sci. Tech.). 1, 135-139.
Address for correspondence: Probir K. Bandyopadhyay. Parasitology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India. E-mail: [email protected]
Editorial responsibility: Andrew Dobrovolskij
