LIST OF PUBLISHED PAPERS

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1 PUBLISHED PAPERS

2 LIST OF PUBLISHED PAPERS 1. Sarkar S. and Bandyopadhyay, P.K. (2013). A checklist of the species under the genus Monocystis von Stein,1848 (Apicomplexa: Sporozoa:Monocystidae) described from oligochate hosts. Zootaxa (4): (ISSN: ) 2. Sarkar S., Bandyopadhyay, P.K. and Göçmen B. (2012). Description of Rhynchocystis silvae sp. nov (Apicomplexa: Eugregarinida) from Metaphire peguana Rosa(1890) of Dhaka, Bangladesh. Turkiye Parazitoloji Dergisi. 36: Sutapa Sarkar and P. K. Bandyopadhyay (2013). An annotated list of protozoan parasites belonging to the genus Monocystis Von Stein, 1848 (Apicomplexa:Monocystidae) described from Oligochaete hosts. Rec. Zool. Surv. India. 113 (Part-3): Sarkar S. and Bandyopadhyay, P.K. (2012). Studies on the biodiversity of aseptate gregarine parasite from the oligochaete host of Bangladesh. Bangladesh Journal of Zoology. 40(1): Sarkar. S, Bandyopadhyay, P.K, and Mitra A.K. (2012). On the occurrence of a gregarine parasite (Apicomplexa: Eugregarinorida) from the seminal vesicles of earthworms (Annelida: Oligochaeta) of Bangladesh. Journal of Parasitic Diseases (Springer). 36(1): Sarkar S. and Bandyopadhyay, P.K. (2014). Monocystis apareshae a new gregarine parasite (Protozoa: Apicomplexa: Monocystidae) of the Oligochaete host of Bangladesh. Modern Parasitology Sarkar, S., Bandyopadhyay, P.K. (2011). First record of a Gregarine from an earthworm of Bangladesh. Proceedings of 22 nd National Congress of Parasitology (ISBN: ). 8. Sutapa Sarkar and P.K. Bandyopadhyay (2013). A new species of Monocystis Von Stein, 1848 (Apicomplexa; Conoidacida; Eugregarinida) from an Earthworm Host Bangladesh. Proceeding of the National Conference of Challenges in Biodiversity and Resource Management (ISBN: ). 301

3 Zootaxa 0000 (0): Copyright 2013 Magnolia Press Article ISSN (print edition) ZOOTAXA ISSN (online edition) A checklist of the species under the genus Monocystis Von Stein, 1848 (Apicomplexa: Sporozoa: Monocystidae) described from oligochaete hosts SUTAPA SARKAR & PROBIR K. BANDYOPADHYAY 1 Parasitology Laboratory, Department of Zoology, University of Kalyani, Kalyani , West Bengal, India 1 Corresponding author. prabir0432@hotmail.com Abstract A checklist of 24 species of the genus Monocystis is presented and includes the salient features of each species, type host and site of infection. Key words: Annotated list, protozoa, aseptate gregarines, Monocystis Introduction Gregarines represent an extremely large, diverse and highly abundant group of early branching Apicomplexan parasites that inhabit the intestine, coeloms and reproductive vesicles of aquatic and terrestrial invertebrates. Most of the important apicomplexans fall into five main groups. Perhaps the most primitive of these are the gregarines, with about 250 genera and 1650 species have described so far (Levine 1976, 1977, 1988; Clopton 2000; Hausmann et al. 2003). The majority of apicomplexan protozoa infect a wide range of animals from molluscs to mammals. Many of them cause diseases of medical and veterinary importance and represent a significant economic burden and global health care challenge. There are two major groups of gregarines, Aseptate and Septate. In aseptate gregarines (acephaline), the trophozoite has a single compartment, but in the Septate group (cephaline), there are several compartments by endoplasmic septa. Arthropods tend to harbour Septate gregarines, but aseptate gregarines are frequently found in seminal vesicles and coelomic cavity of earthworms. The majority of the gregarines reported so far are septate gregarines from insects. Monocystid gregarines are parasitic and are frequently found in seminal vesicles and coelomic cavities of terrestrial oligochaetes. Gregarines are important from an evolutionary perspective because of their suspected early divergence within the Apicomplexa. The genus Monocystis was established by Von Stein in Monocystis is chiefly coelozoic or lumen dwelling protozoan of invertebrates, especially arthropods and annelids, are considered as aseptate gregarine. The genus Monocystis Von Stein, 1848, is characterized by having no mucron, ovoid gamonts, short or elongated body, solitary, biconical oocysts, and being symmetrical (Levine, 1988). Only few reports are available concerning biodiversity among Monocystis species based on morphological characterization. Levine (1988) listed 74 Monocystis species under the genus Monocystidae. There is no systematic checklist with description of Monocystis species. In view of this it is considered useful to compile a further checklist to include later additional species. The checklist with description of the different Monocystis species is described below including the infested organ, type host, type locality and the original reference of the species. Accepted by A. D. Wright: 19 Aug. 2013; published:?? Month

4 1) Monocystis amynthae Bandyopadhyay et al. 2006d Infested organ. Seminal vesicles Type host. Amynthas hawayanus Type locality. Darjeeling, West Bengal, India Description. The gamont of the species is characterized by having an elongated body with broad anterior end, separated from the narrow posterior end by a prominent constriction. It measures (66.0±1.3) µm (37.0±2.8) µm. The gametocysts are oval-shaped, measuring (58.0±2.1) µm. The oocysts are navicular, measuring (10.5±1.1) µm (5.5±1.1) µm. 2) Monocystis apporectodae Bandyopadhyay et al. 2006b Infested organ. Seminal vesicles Type host. Apporectodae trapizoides Type locality. Bankura, West Bengal, India Description. Ribbon-like organisms with one or more prominent constrictions especially in some mature forms and measuring with (203.0±5.0) µm (46.0±1.5) µm. The extreme ends are pointed. Gametocysts ovoid and measure (113.0±1.1) µm (83.0±1.1) µm. Oocysts are navicular in shape. The length of the oocysts ranges from µm and the width, from µm. 3) Monoystis arabindae Bandyopadhyay et al Infested organ. Seminal vesicles Type host. Eutyphoeus incommodus Type locality. West Midnapur, West Bengal, India Description. Gamonts of Monocystis arabindae is a bi-lobular structure due to presence of a constriction at the middle. The mucron is indistinct. The gamonts measure (137.1±5.2) µm (49.3±2.6) µm. The gametocystsare ellipsoidal, measuring (89.1) µm (76.2) µm. The oocysts biconical in shape, measuring (23.5) µm (11.9) µm. 4) Monocystis arcuata Boldt, 1910 Infested organ. Seminal vesicles Type host. Lumbricus castaneus, L. rubellus and Eisenia foetida Type locality. The United Kingdom Description. Elongated curved, bow shaped characterized with a small hemispherical projection and measures µm µm. Nucleus ovoid and varies from 6 5 µm to 28 9µm. 5) Monocystis ayeshae Sarkar & Bandyopadhyay, 2011 Infested organ. Seminal vesicles Type host. Metaphire posthuma Type locality. Satkhira, Bangladesh Description. Trophozoites are oval, measuring (95.21±1.98) µm (53.40±3.24) µm. Nucleus ovoid and measuring (16.02±1.91) µm. Gametocyst rounded and measuring (136.83±4.04) µm (99.26±1.98) µm. Oocyst navicular and measures (11.78±2.17) µm (6.81±1.75) µm. 2 Zootaxa 0000 (0) 2013 Magnolia Press SARKAR & BANDYOPADHYAY

5 6) Monocyctis beddardi Ghosh, 1923 Infested organ. Seminal vesicles Type host. Eutyphoeus nicholsoni Type locality. Calcutta, Lucknow, India. Description. Trophozoite fusiform, size µm µm. Nucleus spherical, gametocyst spherical and measures µm in diameter. Oocyst navicular and 12.5 µm in length. 7) Monocystis bengalensis Ghosh, 1923 Infested organ. Seminal vesicles Type host. Pheritima posthuma Type locality. Calcutta, West Bengal, India Description. Trophozoite elongated, club shaped and measuring µm µm. Gametocyst rounded or ovoid and measures µm in diameter. Oocyst not stated. 8) Monocystis clubae Bandyopadhyay et al. 2006a Infested organ. Seminal vesicles Type host. Lampito mauritii Type locality. Kalyani, Nadia, India Description. Club-shaped gamont having a tail- like structure at the posterior end and measures (160.0±24.5) µm (64.0±6.5) µm. Gametocysts are rounded to ovoid and measures (104.0±1.3) µm (96.0±1.9) µm in diameter. Oocysts are navicular and measures (10.4± 0.3) µm (7.6±0.2) µm. 9) Monocystis constricta Bandyopadhyay et al. 2009b Infested organ. Seminal vesicles Type host. Eutyphoeus quaripapillatus Type locality. Calcutta, West Bengal, India Description. Gamonts are elongated, ovoid, have a hood like structure at the anterior end and measure (188.1± 2.1) µm (72.3 ± 1.1) µm. Nucleus measures (17.3±1.9) µm (18.2±2.1) µm. Gametocysts are ellipsoidal and measure (111.2 ± 2.1) (82.6 ± 3.6) µm. Prominent syzygy was apparent. Oocysts are navicular, measuring (18.1 ± 3.2) (11.9 ± 1.1) µm. 10) Monocystis darjeelingensis Bandyopadhyay &Mitra, 2005 Infested organ. Seminal vesicles Type host. Amynthus robusta Type locality. Senchal, Darjeeling, India Description. Gamonts of M. darjeelingensis are cylindroids in shape: (231.0 ± 2.0) µm (98.0±1.8) µm. Nucleus spheroid or ovoid with (31.0±0.5) µm (27.0±0.5). Spherical gametocyst measures (156.0±0.4) µm; navicular oocyst measures (17.0 ±1.0) (8.2 ±0.5) µm. CHECKLIST OF MONOCYSTIS DESCRIBED FROM OLIGOCHAETE HOSTS Zootaxa 0000 (0) 2013 Magnolia Press 3

6 11) Monocystis elongatum Bandyopadhyay et al Infested organ. Seminal vesicles Type host. Perionyx excavatus Type locality. Murshidabad, West Bengal, India Description. Gamonts are elongated and curved at the anterior and gradually tapering towards the posterior end. Gamont measures (310.0±2.6) µm (52.0±3.1) µm. Nucleus rounded to ovoid measuring (19.4±1.7) µm (16.3±1.8) µm. Gametocysts are unique shaped with two equal sized gametocytes and measuring (82.0±1.9) µm (72.5±2.7) µm. Oocysts are Biconical, measuring (12.3±3.1) µm (9.4±1.1) µm. 12) Monocystis lampitae Bhowmik, et al Infested organ. Seminal vesicles Type host. Lampito mauritii Type locality. Bagdaha, West Bengal, India Description. Gamont Ovoid and measures (120.0±18.1) µm (57.0±10.4) µm. Nucleus rounded, measures (14.1±3.3)µm. Gametocyst ovoid and measures (38.6±4.0)µm. Oocyst biconical, measuring (17.2±1.0) µm (8.6±0.6) µm. 13) Monocystis lalbagensis Bandyopadhyay et al Infested organ. Seminal vesicles Type host. Metaphire posthuma Type locality. Lalbag, Murshidabad, West Bengal, India Description. Aseptate gregarine, cone shaped, measuring µm in length and µm in width. Nucleus spherical and measures µm Gametocyst measures µm µm. Oocyst biconical in shape with more or less rounded extremities. It measures µm µm. 14) Monocystis levinei Bandyopadhyay et al Infested organ. Seminal vesicles Type host. Eutyphoeus incommodus Type locality. Kalyani, Nadia, West Bengal, India Description. Gamonts are elongated in shape, measuring (163.0±1.5) µm (29.0±1.1) µm with a prominent tail like protrusion at the posterior end. Rounded nucleus measuring (22.3±7.3) µm (26.4±2.6) µm. Gametocysts are ovoid in shape ranging from (152.0±0.4) µm, whereas oocysts are navicular in shape, ranging in size from (10.4±1.0) µm (6.9±0.5) µm. 15) Monocystis lloydi Ghosh, 1923 emend. Loubatieres, 1955 Infested organ. Seminal vesicles Type host. Pheretima posthuma Type locality. Calcutta, West Bengal, India Description. Trophozoite rounded or ovoid, length 100 µm, gametocyst spherical or ovoid and 84 µm in diameter. 4 Zootaxa 0000 (0) 2013 Magnolia Press SARKAR & BANDYOPADHYAY

7 16) Monocystis megascolexae Kar, 1946 Infested organ. Seminal vesicles Type host. Megascloex sp. Type locality. Calcutta, West Bengal, India Description. Trophozoite measures µm µm with variable form, elongated, one end rounded, the other end pointed. Nucleus spherical and measures 25 µm in diameter. Gametocyst spherical and 135 µm in diameter in diameter. Oocysts spindle shaped with blunt end measuring µm. 17) Monocystis metaphire Bandyopadhyay et al. 2006c Infested organ. Seminal vesicles Type host. Metaphire houlleti Type locality. Madhyamgram, North 24 Parganas, West Bengal, India Description. The species is characterized by having bean-shaped gamonts measuring (119.0±16.0) µm (66.0±8.0) µm. The anterior end of the gamont is always wider than the posterior end. The mucron is always present at the wider end. The occurrence of syzygy (end to end, caudo-frontal) is a very rare feature which has been observed in the life cycle of the species. The gametocyst is ovoid consisting of two unequal gamonts, measuring (93.0±6.0) µm. Oocysts are navicular, measuring (9.0±1.1) µm (5.5±1.9) µm. 18) Monocystis nadiensis Bandyopadhyay &Biswas, 2002 Infested organ. Seminal vesicles Type host. Metaphire posthuma Type locality. Fulia, Nadia, West Bengal, India Description. The gamont is elongated, flattened and balloon shaped with distinct pellicle. It measures µm µm. Nucleus rounded, measuring µm (13.05±1.59) in diameter. Gametocyst ovoidal. The large gametocysts measure (105.9±7.66) µm (105.9±7.66) µm and small gametocysts µm µm. Oocyst biconical, measuring (8.9±0.717) µm (4.41±0.254) µm. 19) Monocystis nidata Boisson, 1957 Infested organ. Seminal vesicle Type host. Pontodrilus ephippiger Type locality. India and China Description. Intrablastophorical, small cell like, measures µm µm. Nucleus spherical with 50 µm in diameter. Cyst spherical, measuring 3 4 µm (diameter is not given). Gametes are not described. Spores navicular, 9 µm in length. 20) Monocystis pheretimi Bhatia &Chatterjee, 1925 Infested organ. Seminal vesicles, Coelom Type host. Pheretima posthuma Type locality. India Description. Variable, spherical, ovoidal, ellipsoidal or dumb bell shaped, measures up to µm. Nucleus spherical or ovoidal. Gametocyst spherical and measures 80 µm. Oocyst not stated. CHECKLIST OF MONOCYSTIS DESCRIBED FROM OLIGOCHAETE HOSTS Zootaxa 0000 (0) 2013 Magnolia Press 5

8 21) Monocystis pontodrili Subbarao et al Infested organ. Seminal vesicles, coelom, body muscle Type host. Pontodrilus bermudensis Type locality. Lalbag, Murshidabad, West Bengal, India Description. Elongated or cylindrical, µm Mucron present. Cyst also embedded in body muscle, size µm. Gamets are isogamous, in diameter. Spores boat shaped, µm. 22) Monocystis ranaghatensis Bandyopadhyay &Mitra, 2005 Infested organ. Seminal vesicles Type host. Eutyphoeus waltoni Type locality. Ranaghat, Nadia, West Bengal, India Description. Trophozoites of M. ranaghatensis are napiform, having a nearly spheroid to ellipsoid or ovoid body, (124.0±11.0) µm (62.0±1.9) µm with a prominent tail like structure at the posterior end and ellipsoidal nucleus, (12.0±0.4) µm (10.0±1.5) µm. Gametocysts are egg-shaped measuring (105.0±0.9) µm Oocysts are biconical measuring (9.3±1.1) µm (4.5±1.3) µm. 23) Monocystis senchalensis Pradhan & Dasgupta, 1982 Infested organ. Seminal vesicles Type host. Apporectodae trapezoides Type locality. Senchal, Darjeeling, West Bengal, India Description. Elongated, cylindrical, both ends blunt but middle portion widest. Trophozoite measures µm µm. Nucleus spherical or ovoid, measures µm. Oocyst biconical with µm. Gametocyst not stated. 24) Monocystis septum Bandyopadhyay et al. 2009a Infested organ. Seminal vesicles Type host. Eutyphoeus orientalis Type locality. Murshidabad, West Bengal, India Description. The unique feature of the gamonts is the presence of a constriction at the anterior body end that tends to divide the cell into two parts, similarly to septate gregarines. The gamonts measure (90.4±4.2) µm in length and (78.2±0.9) µm in width. Nucleus rounded to ovoid, measuring (14.3±1.7) µm (9.4±1.1) µm.gametocyst consists of two gametocytes of unequal size and peculiar shape: one is axe-like, the other semicircular. The gametocyst measures (64.2±1.2) µm in length and (62.4±1.8) µm in width. Bi-conical oocysts measure (8.2±0.9) µm in length and (6.1±2.1) µm in width. Conclusion In this checklist, 24 species belonging to the genus Monocystis Von Stein, 1848 have been included. The 24 species have been described from different hosts and different geographical distribution, including the site of infection. It is hoped that this paper will throw some light on the systematics and evolution of the genus Monocystis. 6 Zootaxa 0000 (0) 2013 Magnolia Press SARKAR & BANDYOPADHYAY

9 Acknowledgments One of the authors (SS) is thankful to the authorities of the University of Kalyani for extending their permission to work at the Parasitology Laboratory of the Department of Zoology, University of Kalyani, Nadia, Kalyani , West Bengal, India and Zoological Survey of India (ZSI), Calcutta for identification of the host specimens. References Bandyopadhyay, P.K., Roychoudhuri, U.S. & Biswas, G. (2001) Observation on an endoparasite (Apicomplexa: Sporozoa) from the earthworm of West Bengal, India. Uttar Pradesh Journal of Zoology, 21 (1), Bandyopadhyay, P.K. & Biswas, G. (2002) Studies on an endoparasite. Monocystis nadiensis sp. n. from the earthworm of Nadia, West Bengal, India. Transactions of the Zoological Society of East India, 6 (1), Bandyopadhyay, P.K. & Mitra, A.K. (2005) Observations on two new species of Monocystis Stein, 1848 (Protozoa: Apicomplexa: Monocystidae) Monocystis darjeelingensis sp. n. and M. ranaghatensis sp. n. from earthworms (Annelida: Oligochaeta) of West Bengal, India. Animal Biology, 55, Bandyopadhyay, P.K., Bhowmik, B. &Mitra, A.K. (2005) Observations on a new species of Monocystis Stein, 1848 (Protozoa: Apicomplexa: Monocystidae) Monocystis levinei sp. nov. from an Inidan earthworm (Annelida : Oligochaeta) Eutyphoeus incommodus. Zootaxa, 1016, Bandyopadhyay, P.K., Mitra, A.K. & Mallik, P. (2006a) Biology of Monocystis clubae sp. nov. (Apicomplexa: Eugregarinida) from an Indian earthworm Lampito mauritii (Annelida: Oligochaeta) of India. Zootaxa, 1120, Bandyopadhyay, P.K., Mallik, P., Göçmen, B. & Mitra, A.K. (2006b) Monocystis apporectodae sp. nov. (Protoza: Apicomplexa: Eugregarinida), from an Indian earthworm Apporectodea trapezoides. Acta Parasitologica Turcica,30 (1), Bandyopadhyay, P.K., Mallik, P., Göçmen, B. &Mitra, A.K. (2006c) Monocystis metaphirae sp. nov. (Protista: Apicomplexa Monocystidae) from the earthworm Metaphire houlleti (Perrier). Acta Parasitologica Turcica, 30 (1), Bandyopadhyay, P.K., Göçmen, B., Bhowmik, B. &Mitra, A.K. (2006d) A new species of Monocystis Stein, 1848 (Protista: Apicomplexa: Eugregarinida) from the Indian earthworm, Amynthas hawyanaus Rosa, 1891 (Annelida: Oligochaeta). Acta Parasitologica Turcica, 30, Bandyopadhyay, P.K., Mallik, P. & Mitra, A.K. (2007) Observations on Monocystis arabindae n. sp. and Nematocystis majumdari n. sp. (Protozoa: Apicomplexa: Monocystidae) from Seminal Vesicles of an Earthworm Eutyphoeus incommodus (Beddard) from West Bengal, India. Acta Protozoologica, 46, Bandyopadhyay, P.K., Chakraborty, A., Mitra, A.K. & Göçmen, B. (2008) Monocystis elongatum n. sp. (Protoza: Apicomplexa: Monocystidae) from seminal vesicles of the earthworm Perionyx excavatus (Perrier) (Annelida: Oligochaeta) in West Bengal, India. North-Western Journal of Zoology, 4, Bandyopadhyay, P.K., Chakraborty, A. & Mitra, A.K. (2009a) Monocystis septum n. sp. (Protozoa: Apicomplexa: Monocystidae) from seminal vesicles of earthworms (Annelida: Oligochaeta) in West Bengal, India. Protistology, 6 (1), Bandyopadhyay, P.K., Mitra, A.K. & Göçmen, B. (2009b) Observation on Monocystis constricta (Protozoa; Apicomplexa; Monocystidae) from an Indian Earthworm, Eutyphoeus quaripapillatus Michelsen, Türkiye Parazitoloji Derğisi, 33 (3), Bhatia, B.L. & Chatterjee, C.B. (1925) On some more gregarine parasites of Indian earthworm. Archiv für Protistenkunde, 53, Bhowmik, B., Mitra, A.K. & Bandyopadhyay, P.K. (2011) Studies on the biodiversity of aseptate gregarines from the oligochaetes of West Bengal, India. Proceedings of 22 nd National Congress of Parasitology, West Bengal, India, pp Boisson, C. (1957) Monocystidae parasites d'oligochates d' Indochine. Annales des Sciences Naturelles, 19, Boldt, M. (1910) Zweineue Gregarinenartenaus Octolasium complanatum ant. Dunes. Zoologischer Anzeiger, 36, Clopton, R.E. (2000) Order Eugregarinorida Léger, In: Lee, J.J., Leedale, G.F. & Bradbury, P. (Eds.), The illustrated guide to the Protozoa. 2nd Edition. Allen Press Inc., Lawrence, pp Ghosh, E. (1923) Monocystis from the earthworm of Calcutta. Journal of Royal Microscopical Society,1923, Hausmann, K., Hülsmann, N. & Radek. R. (2003) 3rd completely revised edition; E. Schweizerbart schebuchhandlung (Nägele u. Obermiller) Stuttgart, Protistology, 379 pp. Kar, A.B. (1946) Observations of Monocystis megascolexae from the seminal vesicles of the earthworm, Megascolex sp. Calcutta University Journal, Department of Science (New Series), 2, Levine, N.D. (1976) Revision and checklist of the species of the aseptate gregarine genus Lecudina. Transactions of the American Microscopical Society, 95, CHECKLIST OF MONOCYSTIS DESCRIBED FROM OLIGOCHAETE HOSTS Zootaxa 0000 (0) 2013 Magnolia Press 7

10 Levine, N.D. (1977) Revision and Check-list of the species of the aseptate gregarine family Monocystidae. Folia Parasitologica, (Praha), 24, Levine, N.D.(1988) The protozoan Phylum Apicomplexa. Vol. 1. CRC Press, Inc. Boca Ratan, Florida, 220 pp. Loubatieres, R. (1955) Contribution a 1'etude des gregarinomorphes Monocystidae parasites des oligochates du Languedoc - Roussillon. Annual Science of National Zoology, Pradhan, D. & Dasgupta, B. (1982) New gregarines in earthworms from Senchal and Goomti in Darjeeling district, West Bengal, India. Journal of Bengal Natural History Society (New Series), 1, Subbarao, B.V.S.S.R., Kalavati, C. & Narasimhamurti, C.C. (1979) A new aseptate gregarine, Monocystis pontrodrili sp. n. from the litoral oligocheate annelids Pontrodilus bermudensis Beddard. Acta Protozoologica, 18, Sarkar, S. & Bandyopadhyay, P.K. (2011) First record of a gregarine from an earthworm of Bangladesh. Proceedings of 22 nd National Congress of Parasitology, West Bengal, India, pp Zootaxa 0000 (0) 2013 Magnolia Press SARKAR & BANDYOPADHYAY

11 178 Original Investigation / Özgün Araştırma Description of Rhynchocystis silvae sp. nov (Apicomplexa: Eugregarinida) From Metaphire peguana Rosa (1890) of Dhaka, Bangladesh Dakka, Bangladeş den Bir Solucan Türü Metaphire peguana Rosa (1890) da Yeni Bir Gregarin Parazit Türü Rhynchocystis silvae sp. nov. (Apicomplexa: Eugregarinida) Sutapa Sarkar 1, Probir K. Bandyopadhyay 1, Bayram Göçmen 2 1 Department of Zoology, University of Kalyani, West Bengal, India 2 Department of Biology, Faculty of Science, Ege University, İzmir, Turkey ABSTRACT Objective: Biodiversity studies in search of endoparasitic acephaline gregarines of earthworms revealed a new species under the genus Rhynchocystis Hesse, Methods: The species has been obtained from the seminal vesicles of the earthworm, Metaphire peguana. The parasite was identiied using standard methodology. Results: Gamonts are solitary, elongated with a conical, enlarged head and blunt posterior end. The mature gamont measures (425.05±4.70) µm in length and the width at the widest portion is (47.88±2.85) µm and (16.50±1.14) µm in the posterior end. The nucleus is elongated and positioned at the posterior end. The nucleus measures (19.74±1.95) µm (13.40±1.95) µm. The measurement of the mucron is (15.47±1.86) µm (19.44±1.71) µm. Gametocysts are ovoid with two unequal sized gametocytes. The gametocyst measures (96.20±1.14) µm (79.56±1.86) µm. Large and small gametocytes measure (59.81±1.76) µm (72.48±1.71) µm and (34.47±2.74) µm (64.38±1.84) µm respectively. Oocysts are biconical, measuring (12.81±1.71) µm (7.70±1.20) µm. Conclusion: A new gregarine parasite species is described. (Turkiye Parazitol Derg 2012; 36: ) Key Words: Rhynchocystis silvae sp. nov, gregarine, earthworm, seminal vesicles, Bangladesh Received: Accepted: ÖZET Amaç: Toprak solucanlarının endoparazitik asefalin gregarinlerinin araştırılmasına yönelik biyoçeşitlilik çalışmaları Rhynchocystis, Hesse, 1909 cinsine dahil yeni bir türü ortaya koymuştur. Yöntemler: Yeni saptanan tür, Metaphire peguana nın seminal vesiküllerinden elde edilmiştir. Parazitin teşhisinde standart yöntemler izlenmiştir. Bulgular: Gamontlar soliter, konik şekilde uzamış bir baş ve küt bir posterior uca sahiptir. Ergin gamont uzunluğu (425.05±4.70) µm ve genişliği posterior uçta (16.50±1.14) µm iken en geniş kısımda (47.88±2.85) µm olarak ölçülmüştür. Nukleus uzamış olup, posteriorda yerleşir. Nukleus ölçüleri (19.74±1.95) µm (13.40±1.95) µm dir. Mukron ölçüleri (15.47±1.86) µm (19.44±1.71) µm dir. Gametosistler oval ve farklı büyüklükte iki gametosit içerir. Gametositler (96.20±1.14) µm (79.56±1.86) µm dir. Büyük ve küçük gametosit ölçüleri sırasıyla, (59.81±1.76) µm (72.48±1.71) µm ve (34.47±2.74) µm (64.38±1.84) µm dir. Ookistler bikonik ve (12.81±1.71) µm (7.70±1.20) µm boyutlarındadır. Sonuç: Yeni bir gregarin parazit türü tanımlanmıştır. (Turkiye Parazitol Derg 2012; 36: ) Anahtar Sözcükler: Rhynchocystis silvae sp. nov., gregarin, toprak solucanı, seminal vesikül, Bangladeş Geliş Tarihi: Kabul Tarihi: Address for Correspondence / Yazışma Adresi: Dr. Bayram Göçmen, Department of Biology, Faculty of Science, Ege University, İzmir, Turkey Phone: Fax: bayram.gocmen@ege.edu.tr doi: /tpd

12 Turkiye Parazitol Derg 2012; 36: Sarkar et al. A New Gregarine Parasite Species179 INTRODUCTION Gregarines are a diverse and successful group of protozoan parasites belonging to the phylum Apicomplexa. Gregarines are specific parasites of a wide range of invertebrates from Phyla Arthropoda (including crustaceans, insects, myriapods) and Annelida (including polychaetes, oligochaetes and leeches). Of the two major groups of gregarines, aseptate and septate, earthworms harbour the aseptate one. The aseptate or acephaline forms are characterized by a non-septate body and they are the common endoparasites of the seminal vesicles of earthworms. There are not many important studies on gregarine protozoan parasites infesting invertebrates throughout the world. The researches on aseptate gregarines have gained momentum since Ruston (1) described D. minuta from Lumbricus terrestris. Bandyopadhyay and Mitra (2) described one species, D. indica from Lampito mauritii collected from India. Later, a study of aseptate gregarines from earthworms revealed a new species of the genus Rhynchocystis Hesse 1909, which is described here. Levine (3) listed some species under the genus Rhynchocystis. These were Rhynchocystis awatii, R. cognetti, R. cuneiformis, R. pilosa, R. hessei, R. mamillata, R. oculata, R. ovata, R. pessoai, R. piriformes, R. porrecta. All of these species of Rhynchocystis were described from seminal vesicles of oligochaete hosts. During the present biodiversity studies of the aseptate gregarines from the earthworms of Bangladesh, a new species belonging to the genus Rhynchocystis previously defined by Hesse in 1909 was identified. This paper deals with a unique form of a Rhynchocystis species obtained from the seminal vesicles of the earthworm, Metaphire peguana, collected from North Badda of the Dhaka district, Bangladesh. This new species of aseptate gregarine is described for the first time in the present study. METHODS For the present study 30 host specimens were collected from the drainage soil of North Badda, Dhaka. The specimen collections were conducted between January 2011-June The collected earthworms were kept in soil in an earthen tub and brought to the laboratory alive. Some of the earthworms were dissected while alive and their seminal vesicles were carefully removed. The fluids of the seminal vesicle 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 and covered with a cover slip for examination of living protozoans under a light microscope. After initial study of living protozoans the content of the seminal vesicles were semidried and fixed in Schaudin s solution for 20 minutes. After fixation, the smears were stored in 70% ethanol for removal of mercuric chloride. The slides were then passed through a descending series of alcohol (5 minutes each) and placed in distilled water. These were transferred to a 3% iron alum solution (Over night) and stained with Heidenhain s hematoxylin solution for 20 minutes. Differentiation was done with 1% iron alum solution under the low power objective lens of the light microscope. The slides were then washed thoroughly, dehydrated in an ascending series of alcohol, cleared in xylin and mounted in D.P.X. All measurements are presented in micrometers (µm) as mean (±) SD followed in parentheses by the range. Photographs were taken with an Olympus Phase Contrast Microscope fitted with a digital camera. The methodology used to describe shapes of planes and solids of the gregarines is in accordance with Clopton (4). The following abbreviations are used for the gregarine parasite in this paper. GL=Gamont length, GWwa=Gamont width at the widest part (anterior end), GWp=Gamont width (posterior part), LN=Length of nucleus, WN=Width of nucleus, LG=Length of gametocyst, WG=Width of gametocyst, LGL=Large Gametocyte Length, LGW=Large Gametocyte Width, SGL=Small Gametocyte Length, SGW=Small Gametocyte Width, LO=Length of oocyst, WO=Width of oocyst. RESULTS Phylum-Apicomplexa Levine, 1988 Order - Eugregarinida Leger, 1900 Family- Monocystidae Biitschli, 1882 Subfamily - Rhynchocystinae Bhatia 1930 Genus- Rhynchocystis, Hesse, 1909 Rhynchocystis silvae sp. nov (Figure 1 a-c, Table 1, 2) Mature gamont measures (425.05±4.70) µm (16.50±1.14) µm. The widest part of it is (47.88±2.85) µm width. The nucleus measures (19.74±1.95) µm (13.40±1.95) µm. Mucorn measures (15.47±1.86) µm (19.44±1.71) µm. The gametocyst is (96.20±1.14) µm in length and (79.56±1.86) µm in width. Large and small gametocytes measure (59.81±1.76) µm (72.48±1.71) µm and (34.47±2.74) µm (64.38±1.84) µm respectively. Length of the oocyst is (12.81±1.71) µm and width is (7.70±1.20) µm. The genus Rhynchocystis was detected by Hesse in Bhatia, placed it in a new sub family Rhynchocystinae in 1930 and the subfamily was defined as, Gamont ovoid, spherical or elongated with a conical or cylindroconical trunk at the anterior end, solitary: oocyst biconical with similar non appendiculate ends, with eight sporozoites by Levine (1). The morphology of the present form obtained from the seminal vesicles of the earthworm, Metaphire peguana collected from Dhaka, the capital of Bangladesh is studied thoroughly. The trophozoite of the present form is usually long Figure 1. a-c) Photomicrographs of different stages of the life cycle of Rhynchocystis silvae sp. nov. obtained from the seminal vesicles of the earthworm, Metaphire peguana. a) A mature gamont; b) Gametocyst with two unequal sized gametocytes; c) Oocysts a b c

13 Sarkar et al. 180A New Gregarine Parasite Species Turkiye Parazitol Derg 2012; 36: Table 1. Summarized statistics of different body parts of the gamonts, gametocysts and oocysts. All measurements are in µm Characters Mean Range Standard Deviation Gamont length (GL) Gamont width at the widest part (anterior part) (GWwa) Gamont width (Posterior part) (GWp) Length of nucleus (LN) Width of nucleus (WN) Length of gametocyst (LG) Width of gametocyst (WG) Large gametocyte length (LGL) Large gametocyte width (LGW) Small gametocyte length (SGL) Small gametocyte width (SGW) Length of oocyst (LO) Width of Oocyst (WO) and slender. Its anterior end is broad and rounded but the rest of the body tapers towards the posterior extremity. It is cylindrical and blunt. The anterior end of the parasite is provided with a small conical mucorn which emerges from the base of a concavity formed by the infolding of the ectosarc. Endoplasm is dense with paraglycogen grains. Epicyte is thin, transparent and quite delicate. Ectoplasm is relatively thick. The nucleus is small, elongated and near the posterior end, typically contains a central karyosome surrounded by a layer of chromatic granules which lies half way between it and the thick nuclear membrane. Gametocysts have two unequal sized gametocytes. Oocysts are biconical. Detailed measurements are presented in Table 1. Taxonomic Summary Type Material: Rhynchocystis silvae sp. nov Type Host: Metaphire peguana. Type Locality: North Badda, ( N and E.) Dhaka district, Bangladesh Site of infestation: Seminal Vesicles. Prevalence: 21/30=(70%) Holotype: R1/301/2011, deposited at the Parasitology Laboratory of the Department of Zoology, University of Kalyani, Kalyani , West Bengal, India. Paratypes: R2/302/2011, deposited at the Parasitology Laboratory of the Department of Zoology, University of Kalyani. Kalyani , West Bengal, India. Symbiotype: MP/11/09, deposited at the Parasitology Laboratory of the Department of Zoology, University of Kalyani. Etymology: The species is named in honour of Dr. Joseph D Silva, former Professor of the Department of Zoology from the University of Dhaka, Bangladesh for his contribution in the field of Parasitology. DISCUSSION Being a parasite of seminal vesicles of oligochaetes and having a cylindrical anterior end, with little differentiation, the parasite justifies its inclusions under the taxa: family Monocystidae, Subfamily Rhynchocystinae and the genus Rhynchocystis Hesse, The genus Rhynchocystis is characterized by the presence of some sort of anterior epimerite like apparatus. Only ten Rhynchocystis species have been described so far from all over the world by Levine (1), and only few of them are described from India. The species presented here has been described from the earthworm of Bangladesh for the first time. Among the described species, R. pilosa and R. porrecta are more closely related with the new gregarine species presented in this study by Troisi (5). In R. pilosa the epimerite like apparatus is highly specialized for attaching the parasite with host cells, whereas the other species of the same genus rarely or never uses its anterior apparatus for the purpose of attachment and it may be reduced to a small, conical, protoplasmic structure called the mucorn by Troisi, (5). In R. pilosa the body is usually long and slender, anterior end broad and rounded, while the rest of the body tapers toward the posterior extremity, which is pointed. The average size of R. pilosa is 217.3µ 25.5µ. The present form differs from R. pilosa in having a more elongated and cylindrical body with a blunt posterior end and it measures (425.05±4.70) µm (47.88±2.85) µm. The anterior pole of R. porrecta and the present form possesses a very well developed mucorn which is made up of smooth hyaline protoplasm. But in R. pilosa this type of attaching apparatus surrounding the mucorn is lacking. The ectosarc of R. pilosa is thick, the cuticle thin with close longitudinal ridges. The cuticular hairs are also present in R. pilosa, but in R. porrecta the epicyte is thin, transparent and marked by 36 to 48 longitudinal ridges. Longitudinal myonems are also present in the myocyte of both R. pilosa and R. porrecta. Myonem is absent in the present form. The endoplasm of R. pilosa is viscous, packed with oval paraglycogen grains whereas the endoplasm of R. porrecta is composed of dense granular protoplasm within many ovoid paraglycogen grains which resemble the present species. Nucleus in the present form is small, elongated and found in the posterior portion of the gamont, measuring (19.74±1.95) µm (13.40±1.95) µm. However, in R. pilosa the nucleus is rounded and large while it is of a vesicular type in R. porrecta. The gametocysts of R. porrecta contain two approximately equal sized gametocytes. Spores are biconical, symmetrical and measures 27.7 µm µm 11.8 to 12.1 µm in R. porrecta. The cyst of R. pilosa is ovoid and measures 94.9 µm 4.2 µm. They are comparatively small and measure 13.3 µm to 5.0 µm. The gametocysts of the present form are also ovoid with two unequal sized gametocytes and the gametocyst measures (96.20±1.14) µm (79.56±1.86) µm. Tye oocyst of the present form is biconical. It is (12.81±1.71) µm in length and (7.70±1.20) µm in width. Besides these, the adult trophozoites of R. pilosa and R. porrecta undergo a process of asexual reproduction, plasmotomy. R. porrecta also undergo a process of degeneration in their life cycle. However it is not found in the present species. Moreover, the hosts are also different. R. pilosa was described from the seminal vesicles of Lumbricus castanues, L. terrestris, Helodrilus foetidus while the R. porrecta was obtained from the seminal vesicles of L.

14 Turkiye Parazitol Derg 2012; 36: Sarkar et al. A New Gregarine Parasite Species181 Table 2. A comparison between different characters of R. silvae sp. nov. with closely related species is presented. All measurements are in µm Species Characters R. pilosa Hesse (1909) R. porrecta Hesse (1909) R. silvae sp. nov Host(s) Lumbricus castaneus, Lumbricus rubellus and Metaphire peguana Lumbricus terrestris and Helodrilus foetidus Helodrilus foetidus Locality Europe (Philadelphia) Europe (Philadelphia) North Badda, Dhaka District, (Bangladesh) Gamonts Long and slender. Anterior Snake like with enlarged head. Elongated with a conical, end broad, rounded and Total length of the gamont is enlarged head and blunt posterior extremity is pointed. 21/2 mm. Anterior end swollen posterior ends. Mature gamont It measures 217.3µ in length by average 32µ to 36µ in diameter length µ in width and rest of the body tapers (425.05±4.70) µm and width at gradually to a point average the widest part is µ in diameter (47.88±2.85) µm. Width of the posterior end is (16.50±1.14) µm Mucorn Present. Cuticle hair Present. But Cuticle hair Present at the anterior part. is also present for attacking is absent It measures to the host (15.47±1.86) µm (19.44±1.71) µm Longitudinal Myonems Present Present Absent Nucleus Rounded, large with central Vesicular type Small, elongated and at the karyosome and at the anterior posterior end. It measures end (19.74±1.95) µm (13.40±1.95) µm Endoplasm Viscous, packed with oval Composed of dense granular Composed of dense granular paraglycogen grains protoplasm within many ovoid protoplasm with ovoid paraglycogen grains paraglycogen grains Gametocysts Ovoid, and measures Speherical, contains two equal Ovoid, with two unequal sized 94.9µ 84.2µ sized gametocytes. Each gametocytes. Gametocysts gametocyte produces measure (96.20±1.14) isogametes µm (79.56±1.86) µm Spores Small and measures 13.3µ to 5.0µ Biconical, symmetrical and Biconical and measures measures 27.7µ to (12.81±1.71) 28.0µ 11.8µ to 12.1µ µm (7.70±1.20) µm Plasmotomy Present Present Absent Degeneration Absent Present Absent References Troisi, 1933 Troisi, 1933 Present study rubellus and Helodrilus foetidus. On the contrary the present form has been described from the Metaphire peguana. A comparative table: Table 2 contains the comparison of the present species with other closely related species. It is seen from the table that the present form differs in morphology, morphometrics, host preference and distribution pattern from the other two previously described Rhynchocystis species. Moreover, it is evident that the present species obtained from Metaphire peguana is also totally different from the two closely related species R. porecta and R. polisa. After careful consideration, it can be concluded that the present form should not be accommodated into any known species under the genus Rhynchocystis. Therefore, a separate status has been proposed for the present form and it is also described for the first time from Metaphire peguana of Bangladesh. Hence the name Rhynchocystis silvae sp. nov. is being proposed here. Acknowledgments One of the authors (SS) is grateful to the authorities of the University of Kalyani, Kalyani, West Bengal, India for extending permission to work at the Parasitology Laboratory of the Department of Zoology, University of Kalyani, and Dr. C.K. Mandal of Zoological Survey of India, Calcutta for identification of the host specimen. Conflict of Interest No conflict of interest was declared by the authors. REFERENCES 1. Ruston J. Dirhynchocystis minuta n. sp. gregarine from the seminal vesicles of Lumbricus terrestris L. with a note on association of Rhynchocystis porrecta. Schmidt J Parasit 1959; 45: [CrossRef] 2. Bandyopadhyay PK, Mitra AK. Dirhynchocystis indica (Apicomplexa, Rhynchocystinae), a new gregarine species from the earthworm Lampito mauritii (Annelida, Oligochaeta) in India. Protistology 2006; 4: Levine ND. the protozoan phylum Apicomplexa. Vol.I.CRC Press Inc., Boca Raton, Florida USA, 1988; Clopton RE. Standard nomenclature and metrics of plane shapes for use in gregarine taxonomy. Comp Parasitol 2004; 71: [CrossRef] 5. Troisi RA. Studies on the acephaline gregarines (Protozoa, Sporozoa) of some Oligochaete Annelids. The American Microscopical Society 1933; 62: [CrossRef]

15 J Parasit Dis DOI /s ORIGINAL ARTICLE On the occurrence of a gregarine parasite (Apicomplexa: Eugregarinorida) from the seminal vesicles of earthworms (Annelida: Oligochaeta) of Bangladesh Sutapa Sarkar Probir K. Bandyopadhyay Amlan Kumar Mitra Received: 29 July 2011 / Accepted: 18 December 2011 Ó Indian Society for Parasitology 2012 Abstract In a survey of the endoparasitic acephaline gregarines in the Satkhira district of Bangladesh, seminal vesicles of earthworm, Metaphire posthuma were found to be infested with a new species of the genus Enterocystis Tsvetcov 1926, Enterocystis elongatum sp. nov. The new species is elongated and measures (67.7 ± 10.3) lm in length and (35.7 ± 1.6) lm in width. Nucleus ovoid and measures (6.6±2.2) lm in length and (5.3±1.1) lm in width. Gametocyst with two unequal sized gametocytes and measures lm (97.9±4.8) lm in length and (86.9 ± 7.7) lm in width. Oocysts ellipsoidal and measure (16.7±2.6) lm in length and (7.6± 1.1) lm in width. Prominent association or syzygy is present in life cycle. Keywords Protozoa Gregarine Enterocystis elongatum sp. nov. Earthworm Seminal vesicles Satkhira Bangladesh Introduction In India researches on gregarine protozoan parasites infesting invertebrates have not been carried out with great importance. Gregarines are a group of apicomplexan protozoan parasites with two forms, septate and aseptate. Only S. Sarkar P. K. Bandyopadhyay (&) Parasitology Laboratory, Department of Zoology, University of Kalyani, Kalyani , West Bengal, India prabir0432@hotmail.com A. K. Mitra Department of Zoology, Ranaghat College, Ranaghat, Nadia, West Bengal, India a few of over 350 species of Indian earthworms have been studied for the occurrence of gregarines, but this research has gained momentum since the 1980s. It has already resulted in the finding of representatives of the genera Apolocystis De Martis (1923); Monocystis Stein 1848 (Stein 1848) Nematocystis Hesse 1909; Stomatophora Drzewiecki 1907; Zygocystis Stein 1848; Enterocystis Tsvetcov (1926). Levine (1977) recognized eight species of Enterocystis of which E. ensis Tsvetcov 1926; E. ephemerae (Frantzius) Desportes (1963); E. palmata Codreanu 1940; E. racovitza Codreanu (1940); E. rhithrogenae Codreanu 1940; E. fungoides Codreanu 1940; E. grassei Desportes 1963 in Ephemeroptera and E. hydrophili (Foerster) Baudoin and Maillard 1972 in Coleoptera. After that E. bengalensis Sarkar 1983 was described from India. Since then no other species has been described from Indian sub continent. Noble (1938) described E. bullis from Urechis caupo, California. Besides these, E. yumushii Iitsuka 1933, E. greefi (Greeff) Noble 1938, were described from different hosts except earthworms. The biodiversity survey of gregarines from earthworms in the Satkhira district of Bangladesh revealed another species of the aseptate gregarine of the genus Enterocystis, Tsvetcov 1926 from this subcontinent which has been described here. This paper deals with the description of a new species of the genus Enterocystis. The morphometric comparison with closely related species and the discussion of its taxonomy and systematics are also presented. Materials and methods Samplings were carried out in search of earthworm hosts in the Satkhira district of Bangladesh. The host earthworms were collected during winter season (November 123

16 J Parasit Dis 2009 February 2010) and the collected earthworms were kept in soil in a tub and taken to the laboratory alive. Some of the earthworms 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 protozoan under a light microscope. After initial study of living protozoans the content of the seminal vesicles semidried and fixed in Schaudin s fluid for 20 min. The smears were stored in 70% ethanol for removal of mercuric chloride. The slides were then passed through a descending series of alcohol (5 min each) and placed in distilled water. These were transferred to a 3% Iron Alum solution (Over night) and stained with Heidenhain s Hematoxylin solution for 20 min. Differentiation was done with 1% iron alum solution under the low power objective lens of the light microscope. The slides were then washed thoroughly, dehydrated in an ascending series of alcohol, cleared in xylene and mounted in DPX. All measurements were made with a calibrated ocular micrometer (lm). In each case minimum and maximum values are given, followed in parentheses by arithmetic mean, standard deviation. For statistical analysis measurements of 20 specimens were taken into account. Photomicrographs were taken using an Olympus light microscope (model CH-2) and Olympus digital camera (model CX41). The methods of describing shapes of planes and solids have been done following Clopton karyosome absent. The nucleus measures lm. The gametocysts are unique shaped with two unequal sized gametocytes and measure lm. Oocysts ellipsoidal without any sporozoites. Oocyst measures lm. Taxonomic summary Species Enterocystis elongatum sp. nov Name of the host Metaphire posthuma Locality Satkhira, Bangladesh Habitat Seminal Vesicles No. of the host infested 12 No. of the specimens 20 Prevalence 12/20 (60%) Material examined Holotype: BD/15/09 deposited at the Parasitology laboratory of the Department of Zoology, University of Kalyani, Kalyani , West Bengal, India Syntype BD/EN/5/10 deposited at the Parasitology Laboratory of the Department of Zoology, University of Kalyani. Kalyani , West Bengal, India Etymology The name of the species is given after the shape of the parasite Results Enterocystis elongatum sp. nov. (Fig. 1, a f; Table 1). Phylum Apicomplexa Levine 1988 Order Eugregarinorida Léger 1900 Suborder Aseptatoria Chakravarty 1960 Family Enterocystidae Codreanu 1940 Genus Enterocystis Tsvetcov With the characters of the genus Enterocystis Tsvetcov 1926 as given by Levine 1988; gamonts are intracellular. Early syzygy, primite enlarging at its base and the satellite remaining more or less cylindrical that the two cells have sword or dagger shape, gametocysts without sporoducts, opening by simple rupture; oocysts ellipsoidal. The shape of the mature gamont is elongated, solitary, aseptate and cleaned gated with a tunnel. Both the ends of the gamonts are blunt and there is a clear distinction between ectoplasm and endoplasm. Ectoplasm smooth. Endoplasm brown and densed with the presence of paraglycogen granules. Mucorn absent. Gamont measures lm. Prominent association of mature gamonts are present. The nucleus is ovoidal with variable position within the gamont. Discussion The species under discussion shows great morphological variability while compared with other species of the genus Enterocystis described so far. The species resembles to some extent with E. bullis (Noble 1938) and E. bengalensis (Sarkar 1983). The gamonts of E. bullis are solitary, bulb like distention of the middle of the body, while the posterior end becomes pointed and dagger like. But here the mature gamonts are aseptate, elongated, solitary and clean gated with a tunnel. The size of the gamont is significantly larger in E. bullis than the species described here. Both the ends of the gamont of the present form is blunt and there is a clear distinction between ectoplasm and endoplasm. In E. bullis a cluster of fibrils was extended from the anterior end of the parasite which appeared to be a modification of epicyte for attachment. Nucleus of E. bullis was spherical with granular nucleoplasm, a large karyosome and additional chromatin bodies were also present. But here it is small, almost ovoid, contained with a group of granules and occupies variable positions within the gamont. Ectoplasm both in E. bullis and here is smooth. Endoplasm in both the cases are highly granular with large number of 123

17 J Parasit Dis Fig. 1 Photomicrographs of different stages of the life cycle of Enterocystis elongatum sp. nov. obtained from the seminal vesicles of the earthworm Metaphire posthuma. a. A mature gamont; b c. Gamonts in clusters; d. Gamonts in syzygy; e. Gametocyst; f. Oocysts. [Scale bars: 50 lm (a d), 100 lm (e), 20 lm (f)] paraglycogen granules. The gametocyst of E. bullis was pyriform, one end being sharply pointed. But in the species under discussion, the shape of the gametocyst is unique having two unequal gametocytes. In E. bullis the oocysts are spherical and without any gelatinous wall. The oocysts are ellipsoidal and without any sporozoites in the present form. In E. bengalensis, the primite is cylindrical with a bulb-head anterior end and a round or truncated posterior end with slightly concave sides. In the present form the bulb-like anterior end is absent without any distinct primite and the gamonts are elongated, solitary and with blunt ends. Late syzygy is observed in E. bengalensis whereas life history of present study encounters early syzygy. Nucleus in E. bengalensis is spherical but here it is ovoidal. 123

18 J Parasit Dis Table 1 Morphometric comparison of Enterocystis elongatum sp. nov. with E. bullis Noble, 1938 and E. benglalensis Sarkar 1983 Species? E. bullis Noble 1938 E. bengalensis Sarkar 1983 E. elongatum sp. nov. (n = 20) Characters; Host Urechis caupo Psocatropos sp. (Psocoptera) Metaphire posthuma Locality Drakes Bay, California Naihati, West Bengal, India Satkhira, Bangladesh Site of Infestation Gamonts Mucorn Posterior end of the midgut of Urechis caupo Solitary, characterized by a bulb-like distention of the middle part of the body while the posterior end become pointed. Adult gamont range 400 ( ) microns in length and 150 ( ) microns in width No mucorn but a cluster of fibrils is present which extended from anterior end of the parasite Midgut and hindgut The primite cylindrical with a bulb-head anterior end and a round or truncated posterior end with slightly concave sides. The satellite was cylindroconical or lanceolate slightly longer than the primite. Syzygy present in this stage No mucorn Seminal vesicles Elongated, solitary, aseptate and clean gated with a tunnel. Gamont with blunt ends and measures lm No mucorn Ectoplasm Smooth Smooth Smooth Endoplasm Nucleus Gametocyst Oocyst Highly granular and with a large number of paraglycogen granules Spherical with granular nucleoplasm and 10 l in diameter which was situated at about the center of the body. One large karyosome is characteristic, additional chromatic bodies were present Pyriform, one end being sharply pointed and measured 5/6 l Spherical and measured 185/200 l without the gelatinous wall. Spores unknown Highly granular and with a large number of paraglycogen granules Spherical with nucleoplasm which situated at near about the posterior end Granular with numerous paraglycogen granules Nucleus ovoidal, measures lm, which was not situated at about the center of the body Thin-walled gamatocysts, 63.5 in diameter. The gamatocysts are unique shaped with two unequal sized gametocytes, measuring lm Ellipsoidal with truncated and thickened end, measures 694lm and contained filiform sporozoites Oocysts ellipsoidal and measure lm, without any sporozoites Syzygy Lateral syzygy is present Caudo-frontal syzygy is present Syzygy of mature gamonts are present Reference Noble (1938) Sarkar (1983) Present study The gametocyst in E. bengalensis is rounded which is ovoid in the present form. Gametocyst of present species contains two unequal sized gametocytes which is lacking in E. bengalensis. Size of the gametocyst of E. bengalensis is smaller in comparison to the present form. Oocyst of E. bengalensis is ellipsoidal with truncated and thickened ends without any sporozoites. The size of the oocysts are significantly larger than E. bengalensis. Moreover, the hosts are also different. Both E. bullis and E. bengalensis were described from insect hosts whereas the present form has been described from oligochaete hosts. It is also quite different in morphology, host preference and distribution pattern from the other two Enterocystis species described earlier. So it is evident that the present species which is obtained from the seminal vesicles of the earthworm host Metaphire posthuma, is totally different from E. bullis (Noble 1938) and E. bengalensis (Sarkar 1983) and it is also found for the first time from the hosts of Bangladesh. Considering all these aspects the present form is designated as a new species and hence the name Enterocystis elongatum sp.nov is being proposed. Acknowledgments One of the authors (SS) is thankful to the authorities of the University of Kalyani, Kalyani for extending permission to work at the Parasitology Laboratory of the Department of Zoology, and Dr. C. K. Mandal of ZSI, Kolkata for identification of the host specimens. References Bhatia BL (1930) Synopsis of the genera and classification of haplocyte gregarines. Ibid 22:156 Clopton RE (2004) Standard nomenclature and metrics of plane shapes for use in gregarine taxonomy. Comp Parasitol 71: Codreanu M (1940) Sur quatre gregarines nouvelles du genre Enterocy-stis, parasites des Ephemeres torrenticoles. Arch Zool Exp Gen (Notes et Revue) 81: De Martis C (1923) Sul genera Monocystis. Monit Zool Ital Fire-nze 34:

19 J Parasit Dis Desportes I (1963) Quelques gregarines parasites d insectes aquatiques de France. Annales de Parasitologie Humaine et Comparée 38: Hesse E (1909) Contribution a l etude des monocystidees Oligochaetes. Arch Zool Exp Gen 5: Levine ND (1977) Revision and checklist of the species of the aseptate gregarine family Monocystidae. Folia Parasitol 24:1 24 Levine ND (1988) The protozoan phylum Apicomplexa, vol I. CRC Press Inc., Boca Raton, Florida Noble E (1938) A new gregarine from Urechis Caupo. Reprinted from tran-sactions of the Trans Am Microscopy Soc, 57(2): Sarkar NK (1983) Enterocystis bengalensis (Apicomplexa: Enterocystidae) from Psocatropos sp. (Psocoptera) of West Bengal, India. Curr Sci 52(8): Stein Fr (1848) Urber die Nature der Gregarinss. Miiller s Arch F Anat Phys U Wiss Med, pp Tsvetcov BN (1926) Eine neue Gregarinengattung Enterocystis ensis, ausden Larven einer Eintagsflicge Vorlanfige Mutteilung. Russk Arkh Protist

20 ISSN Rec. zool. Surv. India : 113(Part-3): 01-08, 2013 AN ANNOTATED LIST OF PROTOZOAN PARASITES BELONGING TO THE GENUS MONOCYSTIS VON STEIN, 1848 (APICOMPLEXA: CONOIDASIDA: EUGREGARINIDIDA: MONOCYSTIDAE) DESCRIBED FROM OLIGOCHAETE HOSTS SUTAPA SARKAR AND P. K. BANDYOPADHYAY Parasitology Laboratory, Department of Zoology, University of Kalyani, Kalyani , West Bengal, India. prabir0432@hotmail.com INTRODUCTION Apicomplexan are parasitic, with the gregarines exclusively infecting invertebrate hosts. Gregarines are a group with two forms, septate and aseptate. Aseptate gregarines contains 400 species while the septate gregarines contains about 900 species (Levine, 1977). In the aseptate gregarines (acephaline) the trophozoite has one compartment while in the septate forms (cephaline) there are several compartments. Gregarines are certainly significant from an evolutionary point of view because of their suspected early diverging position. The genus Monocystis was established by Von Stein in They are chiefly coelozoic or lumen dwelling protozoan of invertebrates, especially arthropods and annelids, considered as aseptate gregarines. Genus Monocystis Von Stein, 1848, are characterized by, without any mucorn, ovoid gamonts, short or elongated body, solitary, biconical oocysts, and symmetrical (Levine, 1988). Some species are especially important because they can cause diseases in invertebrates. Only few reports are available concerning biodiversity a m o n g M o n o c y s t i s s p e c i e s b a s e d o n morphological characterization. Levine, 1988 listed seventy four species of the genus Monocystis under the family Monocystidae, two of them have been described from non oligochaete hosts. Later on, many scientists worked on the aseptate gregarines. Many species of the genus Monocystis have been described from different oligochaete hosts of different geographical region. Till date eighty five species of Monocystis have so far been established throughout the world from the oligochaete hosts. But there is no systematic checklist of the species. In view of this it is considered useful to prepare a updated checklist of the species. The checklist is presented below in Table: 1. Tabulated are the type hosts, distribution, site of infection and the original reference to the data. REMARKS The paper contains the name of eighty six described species of the genus Monocystis under the family Monocystidae from oligochaete hosts. SUMMARY In this communication, eighty six species belonging to the genus Monocystis Von Stein, 1848 have been incorporated. The species have been described from different oligochaete hosts from different geographical distribution. The sites of infections have also been indicated. The paper will throw some light on the systematics of Monocystis species, since study of Monocystis is important from the evolutionary point of view. ACKNOWLEDGEMENTS One of the authors (SS) is thankful to the authorities of the University of Kalyani, Kalyani for extending their permission to work at the Parasitology Laboratory of the Department of Zoology, University of Kalyani, Kalyani , Nadia, West Bengal, India and the scientists of Zoological Survey of India, Calcutta for identification of the host specimens.

21 2 Rec. zool. Surv. India Table 1 : An annotated list of Monocystis species, type hosts, distribution, site of infection and original references are tabulated below. Sv=Seminal Vesicles, C=Coelom, Ints=Intestine, Ovr=Ovary, T= Testes Name of the Monocystis species Host (s) Distribution Site of infections M. agilis Von Stein, 1848 Lumbricus terrestris, L. rubellus England, France, Germany, SV L. castaneus, Allolobophora longa, Hungary, Poland, Sweden, Pheretima hypeiensis, Eisenia foetida USSR, Mexico M. perichaetae (Beddard, 1888) Labbé, 1899 Megascolex (Syn., Perichaeta) novaezealandiae, New Zealand, Mauritius C M. mauritii, Diporochaeta intermedia, M. armatus M. lumbrici (Henle, 1845) Cuénot, 1901 Lumbricus terrestris, L. rubellus, Poland, Sweden, England SV L. castaneus, Eisenia foetida France, Germany M. ciliata Drzhevetskii, 1907 Allolobophora longa USSR SV and C M. crenulata Hesse, 1909 Allolobophoro longa, A. caliginosa, France, Hungary SV A. caliginosa var. trapezoides M. hirsuta Hesse, 1909 Lumbricus casteneus France, Germany SV M. lememei Hesse, 1909 Allolobophora caliginosa, Octolasium complanatum France, Algeria SV M. duboscqi Hesse, 1909 Lumbricus variegates France SV M. bretscheri Hesse, 1909 Fridericia polycheta France C M. macrospora Hesse, 1909 Pheretima hawayana, P. rodericensis France C M. striata Hesse, 1909 Lumbricus terrestris, L. rubellus, L. castaneus, England, France, Germany SV M. turbo Hesse, 1909 Octolasium lacteum, Eisenia foetida France, Germany SV M. arcuata Boldt, 1910 Lumbricus castaneus, L. rubellus, Eisenia foetida Sweden, England, Germany SV M. piriformis Boldt, 1910 Octolasium complanatum, O. transpadanum, Germany, France, Hungary SV and C Fridericia galba, F. Hegemon M. catenata Mulsow, 1911 Lumbricus terrestris and L. rubellus Germany C M. thamnodrili Cognetti de martiis, 1911 Thamnodrilus incertus Equador C M. perforans Pinto, 1918 Glossoscolex wiengreeni Brazil T M. naidis von Voss, 1921 Nais elinguis or N. obtusa Germany C M. beddardi Ghosh, 1923 Eutyphaeus nicholsoni India SV M. bengalensis Ghosh, 1923 Pheretima posthuma India SV M. suecica Berlin, 1923 Eisenia foetida, L. terrestris, L. rubellus Sweden SV and C M. tubiformis Berlin, 1923 Lumbricus rubellus, L. castaneus Sweden SV M. acuta Berlin, 1924 Lumbricus rubellus, L. castaneus Poland, Sweden SV and C

22 SARKAR & BANDYOPADHYAY : AN ANNOTATED LIST OF PROTOZOAN PARASITES 3 M. carlgrenii Berlin, 1924 Lumbricus terrestris, L. rubellus Sweden SV M. caudata Berlin, 1924 Lumbricus rubellus, L. castaneus, Allolobophora longa Poland, Sweden SV M. densa Berlin, 1924 Lumbricus terrestris, L. rubellus, L. castaneus, Eisenia Poland, Sweden, England SV foetida, Allolobophora longa, A. caliginosa, and A. chlorotica M. hessei Berlin, 1924 Lumbricus terrestris, L. rubellus Sweden, England SV M. oblonga Berlin, 1924 Lumbricus terrestris, L. rubellus, L. castaneus, Sweden SV and C Allolobophora longa M. polymorpha Berlin, 1924 Lumbricus rubellus Poland, Sweden SV M. securiformis Berlin, 1924 Allolobophora caliginosa Sweden SV M. ventrosa Berlin, 1924 Lumbricus rubellus, L castaneus, Eisenia foetida Sweden, France, Germany Hungary SV and C M. wallengrenii Berlin, 1924 Allolobophora c. caliginosa, A. longa, England, Poland, Sweden, SV Lumbricus rubellus France, Germany M. pheretimae Bhatia and Chatterjee, 1925 Pheretima posthuma India SV and C M. mrazeki Hahn, 1928 Rhynchelmis limosella, R. komareki Czechoslovakia SV M. criodrilii Sciacchitano, 1931 Criodrilus lacuum Italy SV M. banyulensis Tuzet and Loubatiers, 1946 Octolasium complanatum and Dichrogaster baeri France SV M. octolasii Tuzet and Loubatiéres, 1946 Octolasium complanatum France SV M. setosa Tuzet and Loubatiéres, 1946 Allolobophora gigas France SV M. buccalis Tuzet and Loubatiéres, 1948 Allolobophora gigas France SV M. lloydi Ghosh, 1923 emend, Loubatiéres, 1955 Pheretima posthuma India SV M. dichogasteri Tuzet and Zuber-Vogeli, 1955 Dichogaster inermis France, West Africa SV M. hederacea Loubatiéres, 1955 Allolobophora rosea France SV M. lopadiformis Loubatiéres, 1955 Eisenia foetida France SV M. proteiformis Loubatiéres, 1955 Eisenia foetida France SV M. capillata Tuzet and Vogeli, 1956 Millsonia anomala France, West Africa SV M. eudrilii Tuzet and Vogeli, 1956 Eudrilus eugeniae Ivory Coast VS and C M. lumbricoides (Hesse, 1909), Meier, 1956 Allolobophora c. caliginosa, A. longa, England, France, Germany, India SV A. r. rosea, Dendrobaena tenuis, Eisenia foetida and Pheretima heterochaeta

23 4 Rec. zool. Surv. India Name of the Monocystis species Host (s) Distribution Site of infections M. lobosa Tuzet and Vogeli, 1956 Millsonia anomala France, West Africa SC M. omodeoi Tuzet and Vogeli, 1956 Dichogaster baeri France, West Africa SV M. longispora Boisson, 1957 Perionyx excavates Indo-China SV M. biacuminata Boisson, 1957 Glossoscolex corethrurus Indo-China SV M. minor Boisson, 1957 Pheretima peguana Indo-China SV M. nidata Boisson, 1957 Pontodrilus ephippiger Indo-China SV M. radiata Boisson, 1957 Glossoscolex corethrurus Indo-China - M. cambrensis Rees, 1961 Eisenia foetida Wales C M. lanceata Rees, 1961 Allolobophora caliginosa Wales SV M. mollis Bereczky, 1967 Dendrobaena platyura var. montana Hungary SV M. mammillae Segun, 1968 Dendrobaena mammalis England C M. rhabdota Giere, 1971 Lumbricillus lineatus Germany SV and C M. lumbricilli Giere, 1971 Lumbricillus lineatus Germany SV and C M. tupi Righi, 1974 Tupidrilus lacteus, Guaranidrilus oiepe Brazil SV M. loubatiersi Levine, 1977 Eisenia foetida France SV M. saigonensis Boisson, 1957, Pheretima saigonensis Indo-China - emend. Levine, 1977 M. tuzetae Levine, 1977 Octolasium complanatum France SV M. berlini Levine, 1977 Lumbricus rubellus Sweden SV M. boissoni Levine, 1977 Pheretima posthuma Indo-China SV M. eiseniae Levine, 1977 Eisenia foetida France SV and C M. abegbei Segun, 1978 Libyodrilus violaceus Nigeria Haemocoel M. libyodrili Segun, 1978 Libyodrilus violaceus Nigeria C and Body Muscle M. pontodrili Subbarao, Kalavati and Pontodrilus barmudensis Lalbag, Murshidabad, SV Narasimhamurti, 1979 West Bengal, India M. senchalensis Pradhan and Dasgupta, 1982 Apporectodae trapezoides Darjeeling, India SV M. lalbagensis Bandyopadhyay et al, 2001 Metaphire posthuma Lalbag, Murshidabad, West Bengal, India SV M. nadiensis Bandyopadhyay and Biswas, 2002 Metaphire posthuma Fulia, Nadia, India C

24 SARKAR & BANDYOPADHYAY : AN ANNOTATED LIST OF PROTOZOAN PARASITES 5 M. darjeelingensis Bandyopadhyay and Amynthus robusta Senchal, Darjeeling, India SV Mitra, 2005a M. ranaghatensis Bandyopadhyay and Eutyphoeus valtoni Ranaghat, West Bengal, India SV Mitra, 2005a M. levinei Bandyopadhyay et al., 2005b Eutyphoeus incommodus Kalyani, West Bengal, India SV M. clubae Bandyopadhyay, et al., 2006a. Lampito mauritii West Bengal, India SV M. apporectodae Bandyopadhyay et al., 2006b Apporectodae trapezoides Bankura, West Bengal, India SV M. metaphire Bandyopadhyay et al., 2006c Metaphire houlleti Madhyamgram, North 24 pgs, SV West Bengal, India M. amynthae Bandyopadhyay et al., 2006d Amynthas hawayanus Darjeeling SV M. arabindae Bandyopadhyay et al., 2007 Eutyphoeus incommodus West Midnapur, West Bengal, India SV M. elongatum Bandyopadhyay et al., 2008 Perionyx excavates Murshidabad, West Bengal, India SV M. septum Bandyopadhyay et al., 2009a Eutyphoeus orientalis Murshidabad, West Bengal, India SV M. constricta Bandyopadhyay et al., 2009b Eutyphoeus quaripallatus Calcutta, West Bengal, India SV M. lampitae Bhowmik et al., 2011a Lampito mauritii Bagdaha, West Bengal, India SV M. ayeshae Sarkar and Bandyopadhyay, 2011b Metaphire posthuma Satkhira, Bangladesh SV

25 6 Rec. zool. Surv. India REFERENCES Bandyopadhyay, P.K., Roychoudhuri, U.S. and Biswas, G Observation on an endoparasite (Apicomplexa: Sporozoa) from the earthworm of West Bengal, India. U.P.J. Zool. 21 (1) : Bandyopadhyay, P.K. and Biswas, G Studies on an endoparasite. Monocystis nadiensis sp. n. from the earthworm of Nadia, West Bengal, India, Trans. Zool. Soc. East. Ind. 6 (1) : Bandyopadhyay, P.K. and Mitra, A.K. 2005a. Observations on two new species of Monocystis Stein, 1848 (Protozoa: Apicomplexa: Monocystidae) Monocystis darjeelingensis sp. n. and M. ranaghatensis sp. n. from earthworms (Annelida: Oligochaeta) of West Bengal, India. Anim. Biol. 55: Bandyopadhyay, P.K., Bhowmik, B. and Mitra, A.K. 2005b. Observations on a new species of Monocystis Stein, 1848 (Protozoa: Apicomplexa: Monocystidae) Monocystis levinei sp. nov. from an Inidan earthworm (Annelida : Oligochaeta) Eutyphoeus incommodus. Zootaxa. 1016: Bandyopadhyay, P.K., Mitra, A.K. and Mallik, P. 2006a. Biology of Monocystis clubae sp. nov. (Apicomplexa: Eugregarinida) from an Indian earthworm Lampito mauritii (Annelida: Oligochaeta) of India. Zootaxa. 1120: Bandyopadhyay, P.K. Mallik, P., Göçmen, B. and Mitra, A.K. 2006b. Monocystis apporectodae sp. nov. (Protoza: Apicomplexa: Eugregarinida), from an Indian earthworm Apporectodea trapezoides Duges. Acta Parasitol. Turcica. 30(1): Bandyopadhyay, P.K. Mallik, P., Göçmen, B. and Mitra, A.K. 2006c. Monocystis metaphirae sp. nov. (Protista: Apicomplexa Monocystidae) from the earthworm Metaphire houlleti (Perrier). Acta Parasitol. Ture. 30 (1): Bandyopadhyay, P.K., Göçmen, B., Bhowmik, B., Mitra, A.K. 2006d. A new species of Monocystis Stein, 1848 (Protista: Apicomplexa: Eugregarinida) from the Indian earthworm, Amynthas hawayanaus Rosa, 1891 (Annelida: Oligochaeta). Acta Parasitologica Turcica. 30 (2): Bandyopadhyay P.K. Mallik P., Mitra A.K Observations on Monocystis arabindae n. sp. and Nematocystis majumdari n. sp. (Protozoa: Apicomplexa: Monocystidae) from Seminal Vesicles of an Earthworm Eutyphoeus incommodus (Beddard) from West Bengal, India. Acta Protozool. 46: Bandyopadhyay, P.K., Chakraborty, A, Mitra, A.K. Göçmen B., Monocystis elongatum n. sp. (Protoza: Apicomplexa: Monocystidae) from seminal vesicles of the earthworm Perionyx excavatus (Perrier) (Annelida: Oligochaeta) in West Bengal, India. North-Western J. Zool. 4 (1): Bandyopadhyay, P.K., Chakraborty, A, Mitra 2009a. Monocystis septum n. sp. (Protozoa: Apicomplexa: Monocystidae) from seminal vesicles of earthworms (Annelida: Oligochaeta) in West Bengal, India. Protistology. 6 (1): Bandyopadhyay, P.K., Mitra, A.K., Göçmen, B 2009b. Observation on Monocystis constricta (Protozoa; Apicomplexa; Monocystidae) from an Indian Earthworm, Eutyphoeus quaripapillatus Michelsen, Turkiye Parazitoloji Dergisi, 33 (3): , Bedard, F.E Note on a new gregarine. Proc. Zool. Soc. London Bereezky, M.C Txonomische untersuchungen iiber die parasitiernden Monocystideen (Protozoa) bei Regenwurmern (Lumbricidae). Opusc. Zool., Budapest. 7 (1): Berlin, H Uber drei neue Monocystiden. Ark. Zool. 15 (8): Berlin, H Untersuchngen uber Monocystideen in den vesiculae seminalies der schwedischen oligochaeten. Arch. Protistenk. 48:

26 SARKAR & BANDYOPADHYAY : AN ANNOTATED LIST OF PROTOZOAN PARASITES 7 Bhatia, B.L. and Chatterjee, C.B On some more gregarine parasites of Indian earthworm. Arch. Protistenk. 53: Bhowmik, B., Mitra, A. K., Bandyopadhyay, P.K. 2011a. Studies on the Biodiversity of Aseptate Gregarines from the Oligochaetes of West Bengal. Proc. Nat. Cong. Parasitol Boisson, C Monocystidae parasites d'oligochates d' Indochine. Ann. Sci. Nat. Zool. 19: Boldt, M Zwei neue Gregarinenarten aus Octolasium complanatum ant. Dunes. Zool. Anz. 36: Boldt, M In den samenblasen der ostprussichen Regenwurrner parasitierende Monocystedeen. Schr. Phys.-Okon. GeselIsch. Konigsberg. 51: Bosanquet. W. C Notes on a gregarine of the earthworm (Lumbricus herculeus). Q. J. Micros. Sci. 39: Cognetti De Martiis, L Contributa alla conoscenza delle Monocystidae e deil loro fenomeni reproducttivi. Arch. Protistenk. 23: Cuenot, L Recherches sur 1' evolution et la conjugaison des gregarines. Arch. Biol. 17: Drzhevetskii, V.F O resnichyatikh gregarinakh. C.R. St. Petersberg Trav. Soc. Nat. 38: Ghosh. E Monocystids from the earthworm of Calcutta. J. R. Microsco. Soc.1923: Giere, O Monocystis lumbricilli n. sp. (Gregarinida: Sporozoa) aus Lumbricillus linetaus, Neubeschreibung und Literaturkritik. Arch. Protistenk. 113: Hahn. J Monocystis mrazeki, une nouvile gregarine parasites de Rhynchelinis limosella et de Rh. komareki Hrabe. 1. Dissemination. Arch. Protitenk. 72: 1-12 Henle, J Ucher die Gaming Gregarine. Arch. Anat. Physiol. Wiss. Med. 1845: Hesse, E Contribution a 1'etude des monocystidees des oligochetes. Arch. Zook Exp. 3 Gen. 43: Kalavati, C Monocystis odontotermi n. sp. from the haemocoel of Odontotermes obesus Rambur. Indian J, Parasitol. 3 (1): Konsuloff, S Untersuchugen uber die Rotatorienparasiten. Arch.Protistenk. 36: Labbé, A Sporozoa. In Schulze, F.E. and Butschlii, O., eds. Das Tierreich Eine Zusammenst. Und Kennzeich. Der Rezen. Tierfor. 5: Levine, N.D Revision and check-list of the species of the aseptate gregarine family Monocystidae. Folia Parasitol. (Praha). 24: Levine, N.D Revision and checklist of the species (other than Lecudina) of the aseptate gregarine family Lecudinidae. J. Protozool. 24: Levine, N.D Check list of the species of the aseptate gregarine family Urosporidae. Int. J. Parasit. 7: Levine, N.D Check-list of the species of the aseptate gregarine families Aikinetocystidae, Diplocystidae, Allantocystidae, Schaudinnellidae, Ganymidae and Enterocystidae. J. Invert. Pathol. 29: Levine, N.D The Protozoan Phylum Apicomplexa, Vol. 1. CRC Press, Inc. Boca Roton, Florida, USA

27 8 Rec. zool. Surv. India Loubatiéres, R Contribution a 1'etude des gregarinomorphes Monocystidae parasites des oligochates du Languedoc -Roussillon. Ann. Sci. Nat. (Zool.) 17 (11) 17: Meier, M Die Monocystideent'auna der oligochawn von Earlangen und umgebung. Protistenk. 101: Arch. Mulsow, K Uber Fortpflanzungserscheinungen bei Monocystis rostrata n. sp. Arch. Protistenk. 22: Pinto, C.D.M Contribuicao ao estudo des gregarines. Mem. Inst. Oswaldo. Cruz. 15: Pradhan, D. and Dasgupta, B New gregarines in earthworms from Senchal and Gomti in Darjeeling district, West Bengal, India. J. Bengal. Nat. Hist. Soc.(N. S.). 1 (2): 4-16 Rees, B Studies. on monocystids gregarines. Three Monocystis species including two new specics, M. cambrencis and M. lanceata. Parasitol. 51: Righi. G Alguns Sporozoa (Gregarinida a haplosporida) de Oligochaeta (Naididae en Enchytracidae ) Brasilciros. Pap. Avul. ZooL Sao. Paulo. 28: Segun, A. O Studies of acephaline gregarines in British earthworms, their systematic, occurrance and possible mode of transmission. Ph.D. thesis. University of London. 318 pp. Segun, A. O Monocystid Gregarine Parasites of Nigerian Earthworms. J. ProtozooL 25: Subbarao, B.V. S. S. R., Kalavati, C. and Narasimhamurti C. C A new aseptate gregarine, Monocystis pontrodrili sp. n. from the litoral oligocheates Pontrodilus bermudensis Beddard. Acta Protozool. 18 (2): Sarkar, S., Bandyopadhyay, P.K. 2011b. First record of a Gregarine from an earthworm of Bangladesh. Proc. Nat. Cong. Parasitol Sciacchitano, I Su alcune gregarine parasite des Criodrihis lacuum Hoffm. Bol. Zool. Napoli. 2: Tuzet, O.and Loubattieres, R Notes sur les monocystidees. Archs. Zool. Exp. Gen. 84: Tuzet, O. and Zuber-Vogeli, M Gregarines et cities parasites des vesicules serninales de Dichogaster inermis Michaelson oligochete de Man (A.O.F.). Bull. Inst. France. Aft. N. 17A: Tuzet, O. and Zuber-Vogeli, M Deuxieme contribution a 1'etude des monocystidae parasites des oligochetes Africains Millsonia anomaly Omodeo, 1954 et Dichogaster baeri Sciacchitano. Oligochaetes de Gagrnoa (A.O.F.). BuIL Inst. France. Afr. '- 11 8A: Von Stein, Beitragc Zur kenntniss niederer thiere. Z. Wiss. Zool., 1: Von Voss, H Monocystis naidis n. sp., eine neue Colomgregarine der oligochaeten. Arch. Protistenk., 42: Manuscript received : ; Accepted :

28 Bangladesh J. Zool. 40(1): 51-58, 2012 STUDIES ON THE BIODIVERSITY OF ASEPTATE GREGARINE PARASITE FROM THE OLIGOCHAETE HOST OF BANGLADESH Sutapa Sarkar and Probir K. Bandyopadhyay* Parasitology Laboratory, Department of Zoology, University of Kalyani, Kalyani , West Bengal, India Abstract: Biodiversity studies in search of endoparasitic acephaline gregarines of the earthworms of Dhaka, Bangladesh, revealed a new species under the genus Nematocystis Hesse, The species was obtained from the seminal vesicles of the earthworm Metaphire peguana Rasa, It is a perfectly ribbon like organism, solitary, cylindrical, with several bulges and foldings in the middle portion of the body, especially in mature forms. Mature gamont measures ( ± ) µm in length and ( ± 2.135) µm in width. Nucleus small and rounded, measures ( ± 2.992) µm in length and ( ± 2.282) µm in width. Gametocysts spherical with two approximately equal sized gametocytes. It measures ( ± 4.908) µm in length and ( ± 5.823) µm in width. Oocysts biconical, measuring ( ± 3.870) µm (13.26 ± 2.08) µm. Keywords: Gregarine, parasite, Nematocystis bangladeshensis sp.nov, seminal vesicles, earthworm, Metaphire peguana, Bangladesh. INTRODUCTION Levine (1977) listed 27 species of Nematocystis Hesse, Segun (1978) describe a species under the genus Nematocystis. Later on Pradhan and Dasgupta (1980) and Bandyopadhyay et al. (2005, 2006, 2007), Mallik et al. (2009 and 2010) worked a lot on Nematocystis. A total 39 species of Nematocystis have been reported from oligochaete hosts, of which only15 species have been from India. The present paper deals with the description of a new species of Nematocystis obtained from the seminal vesicles of the earthworm Metaphire peguana Rasa, 1890 collected from Dhaka, Bangladesh for the first time. The present study is related with the biodiversity of the acephaline gregarine from the oligochaete host of Bangladesh. The objective of the present study is to describe a new species of aseptate gregarine from the oligochaete host, Bangladesh. MATERIAL AND METHODS The biotic survey of gregarine parasites in oligochaetes was performed in Dhaka, Bangladesh. Samplings were carried out in search of earthworm hosts in Dhaka, Bangladesh. The host earthworms were collected during the months of April -June 2011 from the drainage soil and the collected earthworms were kept *Corresponding author: prabir0432@hotmail.com

29 52 Sarkar and Bandyopadhyay in soil in a tub and taken to the laboratory alive. Some of the earthworms were dissected while alive and their seminal vesicles were carefully removed. These were placed on clean glass slide 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 the examination of living protozoan under a light microscope. After initial study of living protozoans, the content of the seminal vesicles was semidried and fixed in Schaudin s fluid for 20 minutes. The smears were stored in 70% ethanol for the removal of mercuric chloride. The slides were then passed through a descending series of alcohol (five minutes each) and placed in distilled water. These were transferred to a 3% Iron Alum solution (Over night) and stained with Heidenhain s hematoxylin for 20 minutes. Differentiation was done with 1% iron alum solution under the low power objective lens of the light microscope. The slides were then washed thoroughly, dehydrated in an ascending series of alcohol, cleared in xyline and mounted in DPX. All measurements were made with a calibrated ocular micrometer. In each case minimum and maximum values are given, followed in parentheses by arithmetic mean, standard deviation. For statistical analysis measurements of 20 specimens were taken into account. Photomicrographs were taken using an Olympus light microscope (model CH-2) and Olympus digital camera (model CX41). The methods of describing the shapes of planes and solids have been done following Clopton (2004). RESULTS AND DISCUSSION Phylum : Apicomplexa Levine, 1977 Class : Sporozoa Labbe, 1899 Order : Eugregarinida Lager, 1900 Family : Monocystidae Biitschli 1882 Sub family : Monocystinae Bhatia, 1930 Genus : Nematocystis Hesse, 1909 Nematocystis bangladeshensis sp. nov (Figs 1-4, Tables 1-2) Mature gamont length (GL) ( ± ) µm and width (GW) ( ± 2.135) µm. Gamont width at the middle part ( ± 4.825) µm and gamont width at the posterior end (28.73 ± 5.706) µm. Nucleus small and rounded. The length of nucleus (LN) ( ± 2.992) µm and width (WN) ( ± 2.282) µm. Gametocyst spherical with two approximately equal sized gametocytes. Length of gametocyst (LG) ( ± 4.908) µm and

30 Studies on the biodiversity of aseptate gregarine parasite 53 width (WG) ( ± 5.823) µm. Oocyst biconical. Length of oocyst, ( ± 3.870) µm and width (13.26 ± 2.08) µm. Figs 1-4: Photomicrographs of different stages of the life cycle of Nematocystis bangladeshensis sp.nov. obtained from the seminal vesicles of the earthworm Metaphire peguana. 1. Anterior part of mature gamont. 2. Posterior part of mature gamont. 3. Gametocyst. 4. Oocysts. Magnifications : 15x40(1-3), 15x100 (4). With the characters of the genus Nematocystis Hesse, 1909 as given by Levine (1977) Gamonts large, cylindrod, nematoid, often with mucorn at anterior end solitary, oocyst biconical. In the present form the gamonts are solitary and cylindrical, with an enlarged head. The anterior end is usually swollen and rounded, but the edges are not perfectly parallel, because of some foldings. The posterior end ended gradually to a point. The mucorn well developed, made up of smooth hyalin protoplasm. Ectoplasm and epicyte thin, transparent and quite delicate. Sarcocyte is composed of dense homogeneous protoplasm. Endoplasm is composed of dense protoplasm without any paraglycogen grain and in the head region it is more transparent in comparison to other portion of the body. Nucleus elongated and present at the anterior end.

31 54 Sarkar and Bandyopadhyay Gametocyst spherical with two approximately equal sized gametocytes, Oocyst biconical. The presence of large and long solitary nematode like gamont and biconical oocyst justifies the inclusion of the present form under genus Nematocystis Hesse Table 1. Summary of Measurements (µm) of different body parts of the gamonts, gametocysts and oocysts are presented below. Different body parts of the gamonts, gametocysts and oocyst. Mean Range Standard deviation Gamont length (GL) Gamont width (GWa) (anterior part) Gamont width (GWm) (Middle part) Gamont width (GWp) (Posterior part) Protruted anterior part length Length of nucleus (LN) Width of nucleus (WN) Length of gametocyst (LG) Width of gametocyst (WG) Length of oocyst (LO) Width of oocyst (WO) The species under discussion has some peculiar characters (cylindrical body with roughly parallel sides ending in a v shaped pointed posterior end, several folding along the middle part of the body, anterior end is lobe like and with mucorn). While comparing the species it has some similarities with Nematocystis levinei, N. gardenica and N. kalyaniensis, but dissimilarities are predominant with N. stephensoni (Bhatia and Setna 1926), N. bayrami, N. vinodae. The length of the gamont of present form is greater than that of N. bayrami, N. gardenica, N. kalyaniensis, N. vinodae, N. majumdari and also from N. stephensoni. But, it does not show much difference with the length of N. indica and N. Levinei. The width of the gamont of N. indica and N. levinei has some differences with the present form. The shape of the present form also varies with the earlier described species of Nematocystis. The size and position of the nucleus also differs with the previously described species. In N. indica the nucleus is elongated. Whereas, it is spherical in N. kalyaniensis, rounded and compact in N. majumdari and N. bayrami, oval in N. gardenica, ellipsoidal in N. stephensoni. The gametocyst of N. indica possess two almost equal gametocytes which resembles the present species. But in N. bayrami, the gametocyst bears two unequal sized gametocytes. The shape of the Oocyst in the present species differs from that of N. gardenica and N. kalyaniensis, but it shows some degree of similarities with the previously described species in regard to body dimensions. Moreover, the hosts are also different. N. gardenica, N. kalyaniensis

32 Studies on the biodiversity of aseptate gregarine parasite 55

33 56 Sarkar and Bandyopadhyay

34 Studies on the biodiversity of aseptate gregarine parasite 57 and N. indica were described from Amynthas diffriengens and Amynthas hawayanus whereas N. bayrami and N. vinodae from Eutyphoeus orientalis and Eutyphoeus nicholsoni, respectively of Indian subcontinent. On the other hand, the host of the present form is obtained from Metaphire peguana of Bangladesh. So it is evident, that the present form obtained from the seminal vesicles of Metaphire peguana is totally different from the other species under the genus Nematocystis described earlier. It differs in shape, size and distribution pattern also and is found for the first time from different geographical region, i.e. Bangladesh. Considering all these aspects the present form is a new species and hence the name Nematocystis bangladeshensis sp. nov. is being proposed. TAXONOMIC SUMMARY Species: Nematocystis bangladeshensis sp. nov. Name of the Host: Metaphire peguana Rasa, Locality: Dhaka (Lat, N, Long, E) Bangladesh. Habitat: Seminal Vesicles. No. of the specimens examined: 20 No. of the host infested: 10 Prevalence: 10/20 (50%) Material examined: Holotype no: 301/BD/ DH/11 deposited at the Parasitology laboratory of the Department of Zoology, University of Kalyani, Kalyani , West Bengal, India. Paratype no: 302/BD/DH/2/11 deposited at the Parasitology Laboratory of the Department of Zoology, University of Kalyani, Pin , West Bengal, India. Etymology: The name of the species is given after the name of the country from where the hosts have been collected. Concluding remarks: The biodiversity study of protozoan parasites from oligochaete hosts of Bangladesh revealed that the seminal vesicle of Mataphire peguana are infected with a new species of the genus Nematocystis and while compairing the other species described earlier, it seems to be new to science and a new name has been proposed in this communication. Acknowledgments: One of the authors (SS) is thankful to the authorities of the University of Kalyani, Kalyani, West Bengal for extending their permission to work at the Parasitology Laboratory of the Department of Zoology, and to Dr. C.K. Mandal of ZSI, Kolkata, India for identification of the host specimens.

35 58 Sarkar and Bandyopadhyay LITERATURE CITED BANDYOPADHYAY, P.K. and MITRA, A.K Observation on two new species of Nematocystis Hesse, 1909 (Protozoa; Monocystidae) from earthworms (Annelida: Oligochaeta) of West Bengal, India. Anim Biol. 55: BANDYOPADHYAY, P.K., MALLIK, P. and MITRA, A.K Nematocystis indica sp. n. (Protozoa: Apicomplexa: Eugregarinida). An endoparasitic monocystid gregarine from the seminal vesicles of an Indian earthworm Amynthas diffriengens Baird (Annelida: Oligochaeta) Protistol. 4: BANDYOPADHYAY, P.K., MALLIK, P. and MITRA, A.K Observations on Monocystis arabindae n. sp. and Nematocystis majumdari n. sp. (Protozoa: Apicompleaxa:Monocystidae) from Seminal Vesicles of an Earthworm Eutyphoeus incommodus (Beddard) from West Bengal, India. Acta Protozool. 46: BHATIA, B.L. and SETNA, S.B On some more gregarine parasites of Indian earthworm. Arch. Protistenk. 53: CLOPTON, R.E Standard nomenclature and metrics of plane shapes for use in gregarine taxonomy. Comp. Parasitol. 71: LEVINE, N.D Revision and checklist of the species of the aseptate gregarine family Monocystidae. Folia. Parasitol. 24 : MALLIK, P. and BANDYOPADHYAY, P.K Nematocystis bayrami sp. n. (Protozoa, Apicomlexa, Eugregarinida) monocystid gregarine from Eutyphoeus orientalis Beddard (Annelida, Oligochaeta) N-W. J. Zool. 5: MALLIK, P., BANDYOPADHYAY, P.K. and GÖÇMAN, B Nematocystis vinodae n.sp. (Protozoa, Apicomplexa, Eugregarinida), a monocystid gregarine from Eutyphoeus nicholsoni (Beddard) Türkiye. Parasitol. Derge 34: PRADHAN, D. and DASGUPTA, B Record of some new gregarines in earthworms from the hill areas of Darjeeling district-i. North Bengal Univ. Rev. (Sci. Tech.) 1: SEGUN, A.O Monocystid gregarine parasites of Nigerian earthworms. J. Protozool. 25: (Manuscript received on September 29, 2011; revised on February 6, 2012)

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45 A New species of Monocystis Von Stein, 1848 (Apicomplexa; Conoidacida; Eugregarinida) from an Earthworm Host of Bangladesh Abstract The gamont of the present study is solitary, elongated with rounded head. Total length of the gamont is (343.2±4.39) µm and head region measures, (46.8±4.39) (41.6±4.39) µm. Middle portion of the gamont is flattened, broader than the posterior end and measuring, (78±8.78) µm. Posterior end is cylindrical and tube like which measures, (192.43±4.39) (20.8±4.39)µm. Rounded nucleus is present at the anterior head like region measuring, (11.04±1.03) in diameter. Gametocysts are oval with two unequal gametocytes. Gametocyst measures, (103.93±4.31) (83.13±4.39) µm. Oocysts are biconical measuring, (36.4±4.39) (26±4.31) µm. Key words: Endoparasite, gregarine, earthworm, seminal vesicle, Bangladesh. Introduction Gregarines are a diverse group of apicomplexan parasites that inhabit the intestines, coeloms and reproductive vesicles of marine, fresh water and terrestrial invertebrates. Approximately 250 genera and 1650 species have been described so far (Clopton, 2000; Hausmann et al. 2003; Levine and 1976, 1977 and 1988). The majority of apicomplexan protozoa infect a wide range of animals from molluscs to mammals. Many of them cause diseases of medical and veterinary importance and represent a significant economic burden and global health care challenge. There are two major groups of gregarines that is aseptate and septate. In aseptate gregarines the trophozoite has a single compartment, but in the other group there are several compartments. Arthropods tend to harbour the septate but aseptate gregarines frequently found in seminal vesicles and coelomic cavity of earthworm. The majority of the gregarines reported so far are septate gregarines from insects. Monocystid gragarines are parasitic protozoa which are frequently found in seminal vesicles and coelomic cavities of terrestrial oligochaetes. Many papers have been published on these parasites. Almost all the studies are taxonomic in nature and lists of species with their hosts have been described (Ruston 1959; Marek 1967; Segun 1971 a, b; Pizl 1989 a, b). Rees (1961, 1962 and 1963) have described some phases of monocystid life cycle. During the ongoing survey of aseptate gregarines in Bangladesh only one species of Monocystis have been reported by Sarkar and Bandyopadhyay (2011). Till now there are many records of the species Monocystis Stein, 1848 found in earthworms of India (Ghosh, 1923; Bhatia and Chatterjee, 1925; Kar, 1946; Kalavati, 1979; Subbarao et.al., 1979; Pradhan and Dasgupta, 1982). Recently Bandyopadhyay et al. (2001); Bandyopadhyay, and Biswas, (2002); Bandyopadhyay, and Mitra, (2005a); Bandyopadhyay et al, (2005b); Bandyopadhyay et al. (2006a); Bandyopadhyay et al. (2006b); Bandyopadhyay et al. (2006c); Bandyopadhyay et al. (2006d); Bandyopadhyay et al. (2007); Bandyopadhyay et al. (2008); Bandyopadhyay et al.

46 (2009a); Bandyopadhyay et al. (2009b) described a number of Monocystis species. Members of the genus Monocystis are characterized by having indistinct unmarked mucorn, ovoid, gamonts, short, elongate, solitary trophozoite and biconical, symmetrical oocysts (Levine, 1988). This is the first report of gregarine parasite from the host Metaphire posthuma Vaillant, The aim of the present study is to introduce the new species and describe the a Monocystis sp. Material and Methods Host specimens were collected from the Narayanganj district of Bangladesh during February 2011-June Live specimen were kept in soil in a tub and taken to the laboratory alive. Some of the earthworms were dissected while alive and their seminal vesicles were carefully removed. These were placed on clean glass with a drop of 0.65% Nacl solution. A thin film of seminal fluid was drawn out on a slide covered with a cover slip for examination of living protozoan under a light microscope. After initial study of living protozoans the content of the seminal vesicles semidried and fixed in Schaudin s fluid for 20 minutes. The smears were stored in 70% ethanol for removal of mercuric chloride. The slides were then passed through a descending series of alcohol (5 minutes each) and placed in distilled water. These were transferred to a 3% iron alum solution (Over night) and stained with Heidenhain s hematoxylin solution for 20 minutes. Differentiation was done with 1% iron alum solution under the low power objective lens of the light microscope. The slides were then washed thoroughly, dehydrated in an ascending series of alcohol, cleared in xylin and mounted in DPX. Camera Lucida drawings of different stages of gregarines were made and photomicrographs were taken with an olympus phase contrast microscope (15 40 magnification, Model CH-2, ) fitted with digital camera. All measurements were taken with the aid of calibrated ocular micrometers (μm). In each case minimum and maximum values are given, followed in parentheses by arithmetic mean, standard deviation (SD). The methods of describing shapes of planes and solids have been done following Clopton (2004). Results Monocystis sahadatae n. sp., (Table 1, Figs 1-3) Phylum: Apicomplexa Levine, 1988; Order: Eugregarinorida Léger, 1900; Family: Monocystidae Bütschli, 1882; Subfamily: Monocystinae Bhatia, 1930; Genus: Monocystis Stein, 1848.

47 Description of Monocystis sahadatae n. sp. Body shape of the species is irregular. Anterior portion is almost rounded with rounded nucleus. Middle portion flat. Posterior portion cylindrical and blunt. Endosarc and ectosarc identified. Endosarc packed with granules. Total length of the gamont (343.2±4.39) µm. Head region measures, (46.8±4.39) (41.6±4.39) µm. Middle portion measuring (78±8.78) µm. Posterior tube like portion measures, (192.43±4.39) (20.8±4.39) µm and rounded nucleus measures, (11.04±1.03) diameter. Gametocyst measuring, (103.93±4.31) (83.13±4.39) µm. Oocyst biconical and measures, (36.4±4.39) (26±4.31) µm. The members of the genus Monocystis Stein, 1848 are characterized by having a distinct mucorn, ovoid and solitary gamonts and biconical, symmetrical oocysts (Levine, 1988). In case of the present forms of the gamonts are solitary, elongated and with rounded end at the anterior surface. The significant charactaristics feature of the gamont is a rounded head like appearance at the anterior end and tube like posterior end. Nucleus is present at the head region of the gamont. Ectoplasm smooth. Endoplasm dense with paraglycogen granules. Gametocyst consists of two unequal gametocytes. Oocysts bi conical. 1 2 Fig. 1. Trophozoite, 2. Gametocyst, 3. Oocysts. Scale bars: (1-2) 100 µm, (3) 10 µm. 3

48 Taxonomic Summary Type material: Monocystis sahadatae n. sp. Type host: Metaphire posthuma (Vaillant, 1869) Type of locality: Narayanganj, Bangladesh. (latitude: 23 37' 23" N, longitude: / 90 30' 4" E). Site of infection: Seminal vesicles Prevalence: 15/45 (33.3%) Holotype: NA/01/12 is deposited in the Parasitology Laboratory of the Department of Zoology, University of Kalyani, Kalyani , West Bengal, India. Paratype: NA/02/12 is deposited in the Parasitology Laboratory of the Department of Zoology, University of Kalyani, Kalyani , West Bengal, India. Etymology: The species has been named after the name of Professor Sahadat Ali, Former Vice-Chancellor, Dhaka University, for his outstanding contribution in the field of Parasitology. Discussion Gamonts cylindrical, solitary with mucorn and resembling late syzygy of the parasite in an earthworm justify its inclusion under the family Monocystidae, Sub family: Monocystinae, Genus: Monocystis. All other species of Monocystis have been reported from terrestrial earthworms. The present species shows great morphological variability when compared with other species of the genus Monocystis from India and resembles only with Monocystis elongatum Bandyopadhyay et al, The gamont of the M. elongatum are more elongated, curved and constricted at the anterior end with mucorn but trapper sharply to the posterior end. The present species is much smaller than M. elongatum in size, head region is very much rounded. Middle portion of the present species is totally different in structure and also the tube like posterior part. The shape and position of the nucleus of the both species is also different in the present form. The nucleus is rounded and presented in the head like region. But in M. elongatum the nucleus is rounded to ovoid and present at the middle part of the body. The gametocyst of M. elongatum consists of two equal sized gametocytes where the present species consists of two unequal sized gametocytes. Oocysts in both species are bi conical. Syzygy is absent in both species. The species under discussion is also quite different in morphology, host preference and distribution from other Monocystis species M. elongatum described earlier from India. The morphometric comparison has been presented here in Table 1. The present investigation demonstrates that the parasite should be designated as a new species and hence the name Monocystis sahadatae n. sp. is being proposed. Table 1: Morphometric comparison of Monocystis sahadatae n. sp. with M. elongatum.

49 Species M. elongatum Bandyopadhyay M. sahadatae n. sp. (n=15) Characters et al., Host Perionyx excavatus Perrier, Metaphire posthuma Vaillant, Locality Murshidabad, West Bengal. Narayanganj, Bangladesh. Gamonts Elongated and curved body tapers towards its posterior end with sharply pointed ends, Solitary, irregular in shape, measuring (343.2±4.39) µm. measuring (310.0) (52.0) µm. Distinct mucorn present at the anterior end of the body. Ectoplasm Smooth. Smooth. Endoplasm Vacuolated with paraglycogen Vacuolated with paraglycogen Nucleus Gametocyst granules Rounded to ovoid measuring (19.4) (16.3) µm. Ovoid with two unequal gametocytes, measuring (82.0) (72.5) µm. Oocyst Biconical measuring (12.3) (9.4) µm. granules Rounded, measuring (11.04±1.03) in diameter. Ovoidal, measuring (103.93±4.31) (83.13±4.39) µm. Bioconical, measuring (36.4±4.39) (26±4.31) µm. Site of Infection Seminal Vesicles. Seminal Vesicles References Bandyopadhyay et al., Present Study. Acknowledegement: Thanks are due to the DST (Govt. of India) PURSE ( ), for their partial financial support to carry out the research work. References Bandyopadhyay P K, Roychoudhuri U S and Biswas G 2001 Observation on an endoparasite (Apicomplexa: Sporozoa) from the earthworm of West Bengal, India; U.P.J. Zool. 21 (1) Bandyopadhyay P K and Biswas G 2002 Studies on an endoparasite. Monocystis nadiensis sp. n. from the earthworm of Nadia, West Bengal, India; Trans. Zool. Soc. East. Ind. 6(1) 12-15

50 Bandyopadhyay P K and Mitra A K 2005a Observations on two new species of Monocystis Stein, 1848 (Protozoa: Apicomplexa: Monocystidae)Monocystis darjeelingensis sp. n. and M. ranaghatensis sp. n. from earthworms (Annelida: Oligochaeta) of West Bengal, India; Anim. Biol Bandyopadhyay P K, Bhowmik B and Mitra A K 2005b Observations on a new species of Monocystis Stein, 1848 (Protozoa: Apicomplexa: Monocystidae) Monocystis levinei sp. nov. from an Inidan earthworm (Annelida : Oligochaeta) Eutyphoeus incommodus; Zootaxa Bandyopadhyay P K, Mitra A K and Mallik P 2006a Biology of Monocystis clubae sp. nov. (Apicomplexa: Eugregarinida) from an Indian earthworm Lampito mauritii (Annelida: Oligochaeta) of India; Zootaxa Bandyopadhyay P K, Mallik P, Göçmen B and Mitra A K 2006b Monocystis apporectodae sp. nov. (Protoza: Apicomplexa: Eugregarinida), from an Indian earthworm Apporectodea trapezoids Duges; Acta Parasitol. Turcica. 30(1) Bandyopadhyay P K, Mallik, P, Göçmen B and Mitra A K 2006c Monocystis metaphirae sp. nov. (Protista: Apicomplexa Monocystidae) from the earthworm Metaphire houlleti (Perrier). Acta Parasitol. Ture. 30 (1) Bandyopadhyay P K, Göçmen B, Bhowmik B and Mitra A K 2006d A new species of Monocystis Stein, 1848 (Protista: Apicomplexa: Eugregarinida) from the Indian earthworm, Amynthas hawyanaus Rosa, 1891 (Annelida: Oligochaeta); Acta Parasitologica Turcica. 30(2) Bandyopadhyay P K, Mallik P and Mitra A K 2007 Observations on Monocystis arabindae n. sp. and Nematocystis majumdari n. sp. (Protozoa: Apicomplexa: Monocystidae) from Seminal Vesicles of an Earthworm Eutyphoeus incommodus (Beddard) from West Bengal, India; Acta Protozool Bandyopadhyay P K, Chakraborty A, Mitra A K and Göçmen B, 2008 Monocystis elongatum n. sp. (Protoza: Apicomplexa: Monocystidae) from seminal vesicles of the earthworm Perionyx excavatus (Perrier) (Annelida: Oligochaeta) in West Bengal, India; North-Western J. Zool. 4(1)91-98 Bandyopadhyay P K, Chakraborty A and Mitra A K 2009a Monocystis septum n. sp. (Protozoa: Apicomplexa: Monocystidae) from seminal vesicles of earthworms (Annelida: Oligochaeta) in West Bengal, India; Protistology. 6 (1) Bandyopadhyay P K, Mitra A K and Göçmen B 2009b Observation on Monocystis constricta (Protozoa; Apicomplexa; Monocystidae) from an Indian Earthworm, Eutyphoeus quaripapillatus Michelsen, 1907; Turkiye Parazitoloji Dergisi, 33 (3) Bhatia B L and Chatterjee C B 1925 On some more gregarine parasites of Indian earthworm. Arch. Protistenk

51 Clopton R E 2000 Order Eugregarinorida Léger, 1900 Pp in J. J. Lee, G. F. Leedale, and P. Bradbury, eds. The illustrated guide to the Protozoa 2nd Edition Allen Press Inc., Lawrence Clopton R E 2004 Standard nomenclature and metrics of plane shapes for use in gregarine taxonomy; Comp Parasitol Ghosh. E 1923 Monocystis from the earthworm of Calcutta; J. R. Microsco. Soc Hausmann K, Hülsmann N, and R Radek 2003 Protistology. 3rd completely revised edition; E. Schweizerbart sche Buchhandlung (Nägele u. Obermiller) Stuttgart, 379 pp Kalavati C 1979 Monocystis odontotermitis n. sp. from the haemocoel of Odontotermes obesus Rambur; Indian J, Parasitol. 3(1) Kar A B 1946 Observations on Monocystis megascolexae n. sp. from the seminal vesicles of the earthworm Megaloscolex sp Col. Univ.; J. Dept. Sci. (N.S.) Levine N D 1976 Revision and checklist of the species of the aseptate gregarine genus Lecudina. Trans. Am. Micros. Soc Levine N D 1977 Revision and check-list of the species of the aseptate gregarine family Monocystidae. Folia Parasitol. (Praha) Levine N D 1988 The Protozoan Phylum Apicomplexa. Vol I. CRC Press, Florida, USA Marek J 1967 The gregarines found in the seminal vesicles of Lumbricus rubellus Hoffmeister and Lumbricus terrestris L. from Wroclaw and the neighbourhood; Zool. Pol Pizl V 1989a Monocystid gregarines (Protozoa, Apicomplexa) of some Czechoslovak earthworms. Vest. cs. Spolec. zool Pizl V 1989b An addition to the Czechoslovak records of monocystid gregarines. Vest. cs. Spolec. zool Pradhan D and Dasgupta B 1982 New gregarines in earthworms from Senchal and Gomti in Darjeeling district, West Bengal, India. J. Bengal. Nat. Hist. Soc. (V. S.). 1(2) 4-16 Rees B 1961 Studies on monocystid gregarines. Three Monocystis species including to new species M. cambrensis and M. lanceata; Parasitology Rees B 1962 Studies on monocystid gregarines. Two new monocystid genera Cephalocystis and Dendrocystis; Parasitology Rees B 1963 Studies on monocystid gregarines. Six Apolocystis species including four new species A. lavernensis, A. perfida, A. rotaria and A. Spinosa; Parasitology Ruston J 1959 Dirhynchocystis minuta n. sp., gregarine from the seminal vesicles of Lumbricus terrestris L., with a note on the association of Rhynchocystis porrecta Schmidt; J. Parasitol

52 Sarkar S and Bandyopadhyay P K 2011 First record of a Gregarine from an earthworm of Bangladesh; Proc. Nat. Cong. Parasitol Segun A O 1971a Acephaline gregarines of british earthworms- their possible host specificity; Parasitology Segun A O 1971b Further additions to the British records of acephaline gregarines of earthworms; Parasitology Subbarao B V S S R, Kalavati C and Narasimhamurti C C 1979 A new aseptate gregarine, Monocystis pontrodrili sp. n. from the litoral oligocheate annelids Pontrodilus bermudensis Beddard; Acta Protozool. 18(2)

53 First record of a gregarine parasite from an earthworm of Bangladesh *Sutapa Sarkar and Probir Kumar Bandyopadhyay *Department of Zoology, Satkhira Government College, Satkhira, Bangladesh Parasitology Laboratory, Department of Zoology, University of Kalyani, Kalyani, West Bengal, India Abstract : The present communication deals with the description of a new species of the genus Monocystis (Apicomplexa:Eugregarinorida). During the study of aseptate gregarines from the earthworm hosts of Bangladesh, one species of the genus Monocystis Von Stein, 1848 has been recorded which appears to be the first record from Bangladesh. Keeping it in mind extensive survay on aseptate gregarines of Oligochaetes have been done. In this paper the description, taxonomy and systematics of Monocystis ayeshae n. sp., its morphology and morphometric comparisons with closely related species are presented. Key words : Gregarine, earthworms, Bangladesh. Abbreviations SD = Standard Deviation SE = Standard Error CV(%) = Coefficient of Curvature TL = Trophozoite Length TW = Trophozoite Width DN = Diameter of nucleus LG = Length of Gametocyst WG = Width of Gametocyst OL = Oocyst Length WO = Oocyst Width Introduction Gregarines are a diverse and successful group of protozoan parasites belonging to the phylum Apicomplexa, order Eugregarinorida Lager, The Eugregarinida are all parasitic and are restricted to invertebrates (Clopton 2002). Investigation on the incidence of aseptate gregarines from the annelids have immense important because of severe pathogenecity of the parasites. Aseptate gregarine fauna has been reported from various parts of the world including India. Bhatia (1929) listed fifteen species of Monocystis and seven species of related genera from the seminal vesicles of a single species of common earthworm, Lumbricus terrestris. Twenty seven species have been recorded from L. rubellus as stated by Meier (1956). Levine (1977a) listed seventy species under the genus Monocystis. Later on, five more species were added: two by Segun (1978), one each by Kalavati (1979), Subbarao et al (1979) and Pradhan and Dasgupta (1982). Prior to that, Kar (1946) described M. megascolexae. India has gained momentum since 1980 (Bandyopadhyay et al, 2001, 2002, 2005a, 2005d, 2006c, 2007, 2008) Pradhan and Dasgupta 1983a, 1983b, Roy Choudhury and Haldar, In course of the ongoing survey of the aseptate gregarines in India, 15 species of Monocystis have been recorded. Out of them 13 species have been reported from Oligochaete hosts. However, especially in Bangladesh, the search is far from this type of study. The biodiversity survey of gregarines from the Satkhira district of Bangladesh revealed a new species of the genus Monocystis Stein, 1848 from the seminal vesicles of the earthworm, Metaphire posthuma. This paper presents the description of a new species of the genus Monocystis.The morphometric comparison with other related species and the discussion of its taxonomy and systematics have been incorporated. Out of twelve host specimens studied about nine hosts were infested with this new species. The infestation rate of Monocystis was very significant. * Corresponding Author : prabir 0432@hotmail.com 22nd National Congress of Parasitology, Oct. 30-Nov. 01, 2011 Dept. of Zoology, University of Kalyani, Kalyani, W.B. 287

54 Materials and Methods Oligochaetes, host specimens were collected from the Satkhira district of Bangladesh. The host earthworms were collected during winter season (November 09 February 10) and the collected earthworms were kept in soil in a tub and taken to the laboratory alive. Some of the earthworms were dissected while alive and their seminal vesicles were carefully removed. These were placed on clean glass with a drop of 06% Nacl solution. A thin film of seminal fluid was drawn out on a slide covered with a cover slip for examination of living protozoan under a light microscope. After initial study of living protozoans the content of the seminal vesicles semidried and fixed in Schaudin s fluid for 20 minutes. The smears were stored in 70% ethanol for removal of mercuric chloride. The slides were then passed through a descending series of alcohol (5 minutes each) and placed in distilled water. These were transferred to a 3% iron alum solution (Over night) and stained with Heidenhain s hematoxylin solution for 20 minutes. Differentiation was done with 1% iron alum solution under the low power objective lens of the light microscope. The slides were then washed thoroughly, dehydrated in an ascending series of alcohol, cleared in xylin and mounted in DPX. Camera Lucida drawings of different stages of gregarines were made and photomicrographs were taken with an olympus phase contrast microscope (15 40 magnification, Model CH - 2, ) fitted with digital camera. All measurements were taken with the aid of calibrated ocular micrometers (µm). In each case minimum and maximum values are given, followed in parentheses by arithmetic mean, standard deviation and sample size, n = 12. The methods of describing shapes of planes and solids have been done following clopton (2004). Results Monocystis ayeshae n.sp. (Fig: 1-4, Table-1, 2). Fig 1-4. Photomicrographs of different stages of the life cycle of Monocystis ayeshae n.sp. obtained from the seminal vesicles of the earthworm Metaphire posthuma. Fig 1. A mature trophozoite, Fig 2. Gametocyst, Fig 3-4. Oocysts. Trophozoites are oval in shape, middle portion is slightly wide. Mucorn not visible. It measures µm (95.21±1.98) µm (53.40±3.24), n=12 and has alveolated protoplasm without any inclusions. Ectosarc thin, endosarc is more granulated. Nucleus is large and also oval in shape. It is placed at the anterior part of the trophozoites. There is no chromatin bodies in the nucleus. Nucleus measures µm (16.02±1.91) in diameter. Gametocyst almost rounded and consists two unequal sized of gametocytes. Gametocyst measures µm (136.83±4.04) µm (99.26±1.98). The large gametocyte is bean shaped or kidney shaped whereas the small gametocyte is broadly elliptoid. Oocysts are with pointed end and navicular in shape. It measures µm (11.78±2.17) µm (6.81±1.75) nd National Congress of Parasitology, Oct. 30-Nov. 01, 2011 Dept. of Zoology, University of Kalyani, Kalyani, W.B.