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fHESiS SUMHTTEO FOR THE 0E6REE OF DOCTOR OF PHILOSOPHY (SCIENCE) OF T»^ UNIVERSITY OF NORTH BENGAL RAJARAMMOHUNPUR. DARJEELING. 1984

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CHANCHAL KUMAR SfNHA, M SC. Jhargram Raj College, Midnapur West Bens^l, India. 1,^

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ACKNOWLEDGEMENTS i SYNOPSIS ii -iv

PART' I^

'•STUDIES ON Trypanosoma IN SOME LOWER VERTEBRATES"

1. INTRODUCTION 1-2

2. REVIEW OP LITERATURE 2 - 17

3. !VIATERIA,LS 18 - 24

4. METHODS 24 - 25

5. OBSERVATIONS.

i* T^panosoma clariae Montel (1905) 26 - 30 ii. Tirypanosoma mukasai H€>are(1932) 31 - 36 iii. Trypanosoma n,sp.(a) 37 - 40 iv. Trypanosoma cancili Mandal (1978)41 - 45 V. Trypanosoma gachuii Misra,Chandra & Choudhury (197 3) 46 - 50 vi, Trypanosoma armeti Mandal(1975) 51 - 55 vii. Trypanosorna pancali Mandal (1975)56 - 59 viii. Trypanosoma rotatorium (Mayer,

184 3) 60 - 59 PAGE ix. Trypanosoma vittatae Robertson (1908) 70 •- 76

6. APPENDIX

i. Trypanosoma enhydris Sinha £ Mandal(1976) ... 76a

ii. Trypanosoma qangetlca Slnha(1978) 76b

7, CliECK-LIST OP Trypanosoma OF SOME LOWER VERTEBRATES

IN INDIA . .. 77-88

PART II

' STUDIES' ON Haemoqreqarina IN SOME CFIELONIANS AND OPHIDIANS "'

li. INTRODUCTION 89-91

2.. REVIEW OP LITERATURE 91-99

3. MATEROiLS 100 - 102

4. METHODS. ' 102 - 103

5. OBSERVATIONS i, Haemoqreqarin a lave rani Simond (1901) 104 - 109 •^i •• Haemogreqarina n . sp. (a)' 110 - 116 iii'. H^aemogreqarlna gangetiica (Misra,Nandi,Raut &

Choudhury, 1974) 117 -122

iv.. Haemogreqarin a n.sp. (Jb) ., 123 - 131

V. Haemogreqarin a n*sp. ic] .. 132 - 137b

6,. CHEa<:-LIST op Haemoqreqarina IN SOME CHELONIANS AND

OPHIDIAI^S: IN INDIA 138 - 139 PART III PAGE' '•' STU15IES ON ) IN SOME SNAKES'"^

1. . INTRODUCTION 140 - 141

2. REVIEW OF LITERATURE: 142 - 143

3. M.azrERIAL,S 144 - 146

4. METHODS 146 5. OBSERVATIONS ^* Hepatozoon n.sp.(a)...... 147 - 155 ii. Hepatozoon n.sp«(b)...... 155 - 162 iii. Hepatozoon n.sp. (£)...... 163 - 170

6. CPIEQC-LIST OF Hepatozoon FOUND IN SNAICES

OF INDIA 171

PART IV

"STUDIES ON SOME BLOOD PARASITES IN HIMALAYAN- PLYING SQUIRRELS"

1. INTRODUCTION 172 2. REVIEW OF LITBRATURS 173-174 3. MATERIALS 174 -176 4. METHODS ." 176 5. OBSERVATIONS

^» Trypanosoma (Herpet'osoma) indicim Luhe{1906) 177 -182 PAGE ii» HepatPzoon n.sp. (d) 183 - 189 iii. Anaplasma marqinale Theiler(1910).... 190 - 193

^^' Anaplasma centrale Theiler(1911),..,. 190 - 193 v.- Rickettsia sp, 193 - 194 vi. Microfilariae from Hylopetes alboniqer 195 - 196 vii. Microfilariae from Petaurista maqnificus 197 - 198

6. CI-IECK-LIST OF Trypanosoma^, Hepatozoon, Anaplasma^ Rickettsia AND MICROFIIARIA OF SOME HB-mT.AYAN PLYING' SQUIRRELS 199-201

REFERENCES' 202 - 236

APPENDIX (jVi) 237. A C K Cr O W L E D G E M E H" T S

I express my sincere gratitude to Professor (Dr.) B.Dasgupta,M.Sc,,Ph,D.(London)/Principal, Darjeeling Government College,Darjeeling,West Bengal,India for suggesting this problem of the thesis. I humbly acknowledge his untiring guidance,supervision and skilful direction through­ out the course of investigation.

I wish to tend my sincere thanks to Professor (Dr.), S.K.Dasgupta, former Head, POst-Gradu ate Zoology Department,Darjeeling Government College,Darjeeling,at present: Head of the Department of Zoology,Presidency College, Calcutta, VJest Bengal, India for his kind encouragement. Thanks are gratefully extended to my learned colleagues of Post-Graduate Zoology DoDartment v/here the major part of this v/ork is accomplished. Thanks are due to Dr.A.K.Mandal, Superintending Zoologist, zoological Survey of India,Calcutta, Dr. }3.C.Nandi, Lecturer, Zoology Department, HbOvghly Mohasin College, Dr.(Mrs.) D.Pradhan ,Lecturer,St.Joseph's College, Darjeeling, Dr. M.Banerjee,Lecturer,Zoology Department,Burdv/an University, Shri Sukdeb Sinha,Research Officer,National Institute of Communi­ cable diseases,Delhi, and Shri Sayantan Bandyopadhya,Research Scholar, Zoological Sujrvey of India,Calcutta, IX

SYNOPSIS

.The research project which has been undertaken in course of the present investigation is entitled " Observations on certain protozoan parasites in the blood of some vertebrates". The entire work has been divided into four parts. Part I is entitled "Studies on Trypanosoma in some lower vertebrates". It records eleven sioecies of Trypanosoma foimd in some fresh water fishes, Anurans, Ghelonians and Ophidians collected from some parts of West Bengal State and Manipur State. Out of these eleven soecies, one is considered new species from a fish host. Morphological features seen in perixoheral blood smears are described and compared with allied species of Tr^'-panosoma. Besides that,description of ten species ( including tvra species described elsewhere* ) are also included. A check-list containing knov.^n Trypanosoma in some lower vertebrates in India is also incorporated in this study. Part II is entitled "Studies on Haemoqreqarina in some Clielonians and Ophidians". It records the description of three, species along with two knov/n sneci^s

*Vide appendices 6 (i) and 6 (ii), Ill

from turtles and snakes collected from some parts of West Bengal State. The tissue phase of Haemogregarin a n.SD. (b)' in the sections of liver of snake host indicates the presence of megaloschizonts which has been detected in this study. Macrogamete and small immature oocysts of this parasite are also found in the section of leech. New haemogregarines are also compared v/ith the knovm species. The check-list provided includes the knov/n from Indian Chelonians and Ophidians. Part III is entitled"Studies on Hepatozoon in some snakes". It communicates the descr­ iption of three nev/ species of Hepatozoon from some snakes collected from some parts of West Bengal,India. Development of these parasites in liver and lung tissues of snakes are also studied which reveals macro and micro schizonts and their merozoites. Nev/ soecies of Hepatozoon are also compared v/ith the allied species. A check-list^ °f Hepatozoon from Indian snakes is also incorporated.

Part IV is entitled "Studies on some blood parasites in Himalayan flying squirrels". This chapter deals vrith the species of Trypa.nosoma/Hepatozoon XV

Anaplasma , Rickettsia along vrith microfilariae from some Himalayan flying squirrels,collected in Darjeeling (altitude 2000 -2010 m), West Bengal, India. Out of these parasites,, Hepatozoon is considered new to science and is compared v/ith knovm species of this genus. A check-list containing the blood parasites of Himalayan flying squirrels is also included in this study.

New names have not been provided for the parasites mentioned in the thesis. In lieu of this, small letters viz., (a) / (b^) / (c^) etc. have been used to denote new species. This procedure has been followed to satisfy the Criteria of Availability (Articles 9,10,11) of the International Code of Zoological Nomenclature. Names which are proposed to be assigned to these new para­ sites at the time of actual publication are listed in Appendix (iii). After publication the holotype and paratypes will be deposited with the National Zoological Collection of Zoological Survey of India, Calcutta, PAR T. I,

" STUDIES ON Trypanosoma IN SOME LO^ffiR VERTEBRATES' INTRODUCTION

Flagellates of the genus' Trypanosoma are all parasitic and may comprise of amastigote, promastigote, epimastigote,sphaeromastigote and trypomastigote forms at some stage of their life cycles, &nong these, tri'-pomastigotes are the typical forms occurring in blood of vertebrate hosts. ik^cording to Baker (1969) the trypomastigotes bear flagellum and are slender individuals with kinetoplast and basal body situated near the posterior end. Flagellum emerges through a short pocket,which is detectable only in electron micrographs. Flagellum proceeds through body surface and on account of its undulation a fin-like expansion of body is drawn out to form what is known as undulating membrane.

Most of the trypanosomes have been known only in peripheral blood of vertebrates and it is thought possible that an invertebrate host exists for every one of them with the possible exception of Trypanosoma equiperd\3m which is transferred directly from horse to horse during the sexual act. Trypanosomes have been recorded from almost every class of vertebrates and these have been accorded considerable importance because of the fact that some of these are knox^m to cause severe illness, as in the case of some mammalian hosts including man. The present v/ork was undertaken as it was felt that the study of trypanosomes in Xndia has so far been inadequate, and a survey is likely to yield interesting results.

REVIEW OF LITERATURE

A. Trypanosoma in Fishes of fresh water

Piscine haemoflagellates we^e first observed by Valentin (1841) in the blood of a trout,Salmo fario. It is uncerta to the genus whether they belonged . Trypanosoma or a Tryp an op1asm a. Remak (1542); detected a definite fish Trypanosoma in the- blood of pike,Esox lucius ( cited by Lorn,1979), According to Wenyon (1926),Mitrophanov in Europe described for the first time in 1883 two named species from fish of Trypanosoma,viz.,Trypanosoma carassii and Trypanosoma cobitis from Carassius carassius and Misgumus fossilis respectively. Since then many investigators encountered fish tr^^panosomes from fresh water in various parts of the world. Uanilev?sky (1885

Mfeiva Sc" Pinto, 1926 in the fish S^'-brjnchus rnarinoratus collected from Para State,NOrth Brazil. 7

B.. Trypanosoma in Anura.

Anuran trypanosomes were fiiist observed by Gluge(1842) in the blood of 'froq,Rana esculenta. Mayer(1843) described various forms of the same parasite from European frogs (Rana spvi.) under the names of Amoeba rotatorium, Parameci'um loricatum and costat\3m. Gruby(1843) also studied the above parasites and created the genus Trypanosoma (type species Tr^rpanosoma sanguinis Gruby,1843). Amoeba rotatorium was identical with Trypano­ soma sanguinis which now became a synon^mi of Tr^-'panosoma rotatorium and considered as type species of the genus, Lankester(1871) recorded Undulina ranarum from the blood of frog and this was nov; considered Trypanosoma ran arum:- (Lankester).. Button & Todd (1903) reported Trypanosoma sanguinis, Tr'/panosoma mega/ and Trypanosoma karyozeukton in the blood of African; frogs. Sergent & Sergent (1904)

described Trypanosoma inoplnatum from Rana esculenta of Algiersi Laveran S: Mesnil(1904) reviewed earlier work on. trypanosomes of ji^phibia and concluded that Trypanosoma rotatorium of frogs appeared to be a species of v.'-ide geographical distribution throughout the v/orld, J3utton .- et al_., (1907) also reported Trypanosoma kair'^^ozeukton from Bufo reqularis/ Rana mascarensis. Rana occipitalis•and Rana 8

oxyrhynchus from Africa. Franca & Athias (190'6) studied trypanosomes of Rana esculenta and observed Tr-xrpanosoma Ipricatim costatXTO^ Trypanosoma rotatori-um and Trypanosoma 'inopinatum Sergent & SGrgent,1904 along with tvro nev7 species viz., Trypanosoma undulans and Trypanosoma elegans. Same authors in 1907 recorded Tr^/panosoma rotatorium in I-^la arborea. Wenyon (1909) found several types of trypanosomes in Bufo regularis: in Sudan. Bouet (1909) reported trypanosomes in Bufo reqularis of the then French West Africa. Mathis & Leger(19119 described Trypanosoma chattoni from Bufo melanostictus of Viet Naxa. Stevenson (1911) observed thc.t trypanosomes rotated rapidly on its long axix and concluded that the name rotatorium was given for this characteristic movement. Macfie (1914) recorded Trypanosoma rotatorium^ Trypanosoma me_qa and a possibly new trypanosome from Bufo regularis in Nigeria. Trypanosomes v/ere also reported from Japan and adjacent countries by Koidsumi(191T),Ogawa (1913) and Oqawa & Uegaki (1927)ih anurana* Martin,et al.^(1909) found Tn/panosoma mega /Trypanosoma rotatorium and Trypanosoma elegans in the blood of Bufo regularis of Zaire. Kudo (1922) . described Trypanosoma parvum and Trypanosoma rotatori\jm from North American frogs, Tanabe(l931) described four morphological types of Trypanosoma rotatorium from Ran a niqrom.aculata in Korea. Fantham.et al*, (1942) described Trypanosoma lavalia ^Trypanosoma qaimiontis and Trypanosoma montrealis from Bufo americanus in Canada. These authors also reported the occurrence of Trypanosoma rotatorium and Trypanosoma inopinatum from frogs of Canada in the same year. Nigrelli (1945) also reported Trypanosoma

rotatorium from North limerican amphibians. Hbare(1932) found a Trypanosoma in a small frog,liyperolius- sp.caught on Buqala Island,Uganda and considered as Trypanosoma rotatorium. j'3iamond(1950) described Trypanosoma pipientis in the leopard frog, Rana pipiens. Diamond(1958,1965) listed 26 species

of anuran trypanosomes-as distinct species. Mohammed & Mansour:(1959a,b) described the polymorphic: forms of Trypanosoma rotatori\im in Egyptian toads. Bardsley(1969) found Trypanosoma rotatorivim in the Hull-frog, Rana catesbeiana. Woo (1969) described Trypanosoma canadensis.in amphibian of Southern Ontario. M- Bardsley & Harmsen (1973) reviewed the literature of anuran trypanosomes and listed 34 valid species except for" Trypanosoma aurorae Lehmann, 1959, which is: nomen nudum. MiYata(1976) described three morphological forms of Trypanosoma rotatoriijm collected from tadpoles and adult frogs in Mogi, near Nagasaki City. Werner & Walewski (1976) described Trypanosoma pseudopodium in Bufo americanus collected from Michig; Baker (1977) described some trypanosomes of African Anuran.

*Ayala(1970) described Tryp_ajip^s_cma bjLi_fop_hJ^e_b£torni in Bufo boreas halophilus collected from California. to

• , ., , ; . Miyata(1978) described 14 types of anuran trypanosome v/hich was detected from 10 species of frog captured in Kyushu? and Ryuikyu. islands. This author described Tr^npanosoma rotatorixm,Trypanosoma loricatum and Trypanosoma chattoni as ]\no\-m species and six new species viz., Trypanosoma n aq as al-c ien se / Trypanosoma ishigakiense^ Trypanosoma miyagii^ Tr^^^panosoma tsukamotol/ Txypanosoma ruqosae and Trypanosoma tsunezomiyatai and other five types which were not identified as distinct species or knovm species. He stated that the number of known anuran trypanosomes became 40 species by the addtiion of 6 new species /however,much more species might be described in near future in various parts of the vrorld,because some more trypanosomes figured in various literature under the name Trypanosoma rotatorium or Trypanosoma sp,,xv'ere not identical with one of those known species morphologically,.

C. Trypanosoma in Chelonia and Ophidia

Chelonian Trypanosoma was first encountered by Kunstler(1883) in the blood of mud-tortoise. Laveran & Mesnil (1902) described Trypanosoma damoniae from a tortoise/Damonia reevesii of Par East. Dutton S; Todd(1903) and Dutton,et al./(1907) noted the presence of trypanosomes- in different tortoises of Gambia. Robertson(1908) described XI

Trypanosoma vittatae from Emyda vittata collected from Sri Lanlca. Trs^panosoma vittatus Robertson (cited by Klinke & Elkan/,1965) v/as also reported from Lissemys punctata granosa. Bouet (1909): reported Trypanosoma pontyi from a tortoise of Africa. Joyeux(1913) described Trypanosoma sp. from Pelusios sinuatus of Guinea. Commes(1919) described described Trypanosoma lerovi in ICinixys home an a in Mali. Wenyon(1926) reviev/ed the reptilian trypanosomes of Order Chelonia.i :; :- : , .; Roudabush & Coatney (1937) described Trypanosoma chr^^semydis from Chrysemys belli margin at a. in United States of Anerica, Ploch & Abonnec (1942) described Trypanosoma testudinis and Trypanosoma platemysi from Testudo esculenta and Platemys platicephala respectively collected from South .America. Dias(1951) described Trypanosoma sheppardi and Trypanosoma neitzi from Pelusios sinuatus zuluensis in the then Portuguese Africa, Wallil^er (1965) revievred the literature of reptilian trypanosomes and listed 12 chelonian trypanosome of X'/hich tv/o were reported as Tiypanosoma sp.

Ophidian Trypanosoma v/as first detected by Button.-et al^., (1907) in puff-adder snake,Bitis arietans of Gambia. V?enyon (1908, 1909) described 12 described Trypanosoma erythrolampri and Trypanosoma najae from Erythrolamprus aesculapii and Naja niqricollis of South iimerica and Sudan respectively. 3ouet(1909) reported

Trypanosoma clozeli from Tropidonotus ferox of Western Africa,

Mathis 8c Leger (1909); described Trypanosoma primeti from

Tropidonotus piscator of Indo China. B3rumpt(1914) described

Trypanosoma brazili from Helicops modestus of Hrazil. Macfie

(1919) described Trypanosoma voltariae from Naja niqricollis of Ghana. Pess6a (1928) described Trypanosoma phylodriasi f^o'^ Philodri nattereri of Brazil. Arantes S.i Ponseca

(1931) described Trypanosoma butantanense and Trypanosoma merremii from Ophis merremii of Brazil. Fonseca(1935)

again reported Trypanosoma mattaqros sen se from Cycloqras gigas of Brazil. Schwetz (1944) found Trv'-panosoma sp. in Gray a omata. Fantham & Portec (1950) described Trypanosoma psejnmophis and Trypanosoma sebae in Psammophis sibilans and

Python sebae of Soyth Africa. Wallilcer (1965) reviewed

the reptilian tr^^panosomes and listed fifteen snake trypanosomes

of which three v/ere reported as Trypanosoma sp.

WORK DONE IN JKDlA,

h.,. Tii^panosoma in:; £resh water fishes

Lingard (1904) recorded Trypanosoma

sp, from fishes viz./Puntius (=Barbus) camaticuS/ Channa 13

(=Qphlocephalus) striatus and Ryncobdella aculeata collected from river at Pune,Maharastra. He(1904) also described Trypanosoma sp. from Channa striatus/ Mystus seenghala, Mystus tengara and Trichogaster fasciatus collected from Jamuna river,Uttar Pradesh, de Mello S: Valles. (1936) described Trypanosoma clarlae batrachi from Clarias batrachus•obtained from Goa, Qadri(1951) reported Trypanosoma sp. in Channa striatus' collected from Hyderabad, Andhra Pradesh. Qadri(1955) described Trypanosoma striati from Channa striatus caught in Hyderabad,i^jidhra Pradesh. The same author again (Qadri,1952) repoi~ted Trypanosoma batrachi and Trypanosoma danilewskyt saccobranchi in Claries batrachus and Heterop~ neustes (— Saccobranchus) fossilis; caught in Hyderabad, Andhra Pradesh. Hasan & Qasim(1962) described Trypanosoma punctati in Channa punctatus also caught from Hyderabad,Andhra Pradesh. Ray Chaudhuri & Misra (1973) described Tr^/panosoma elongatus and Trypanosoma mukundi from Channa punctatus and Heteropneustes fossilis- obtained from the local markets and from the lakes end ponds in and around Calcutta,West Bengal. Misra,et ad., (1973) described Trypanosoma qachuii in: Channa gachua collected from ponds in and around Calcutta,West Bengal. Tandon &•Joshi(1973) described Trypanosoma vittati andd Trypanosoma maguri from Mystus vittatus and Clarias batrachus obtained in 14

Calcutta markets,West Bengal. Pandey & Pandey (1974) described Tr^^/panosoma balqulensis from two different fishes' viz./Cirrhina reba and Qsteobrama cotio collected at Fainital, Uttar Pradesh. Tandon & Joshi (1974) observed Trypanosoma sp. in the blood of Channa punc t atus, Cl arias batrachtis, Mys tus seencfhala and Heteropneustes. fossil is collected from Luclcnov/, Uttar Pradesh. Mandal ( 1975,1977,1978,1979,1980) descr­ ibed seven species; of Trypanosoma lfiz.,1rypanosoma armeti Trypanosoma pancali, Trypanosoma choudhuryi, Trypanoaoma cancili ,Tr\'-panosoma anabasi, Tr;:^'-panosoma bengalensis and Trypanosoma tandoni from Mastocembelus armatus, Mastocembelus pancalus, Telapia mossambica, Xenentodon cancila, Anabus testudineus, Mystus bleakeri amd Mai 1ago attu respectively collected from Champahati ,,3agmari,Canning and Raidigi, 24- Parganas,VJest Bengal. Joshi (1976,1978) described Trypano­ soma mrigali, Trypanosoma seenghali, Trypanosoma batai and Trypanosoma stigmai from Cirrhina mriqala, Mystus seenghala, Labeo bata and Parbus stigma collected from Gomati river and Chinhat Lake,I,ucknov/,Uttar Pradesh. Tandon & Chandra (1977) found Trypanosoma sp, in the blood of Clarias batrachus I-lacrones seenghala, Mastocembelus armatus, Mys tus seenghala ^"^- ^'fallago attu collected from Lucknow,Uttar Pradesh. Joshi (1979) described the presence of Tr^?-panosoma sp. in the blood of Notopterus notopterus, Puntius stigma, Labeo 15

bata, Mystus aor/ Channa qachua , Heteropneustes fossilis Wallago attu, Mystus seenghala, Mastocembelus armatus^ CIarias batrachus and Channa punctatus collGcted from Lucknow,Uttar Pradesh. Joshi S: Dabrol (1979) found Tr^/panosoma sp. in Heteropneustes fossilis collected at Luc] enow/Uttar Pradesh, Mukherjee & Kalder(1979) found Trv'-panosma sp. in the blood of Handus nandus collected from Kalyani, Nadia, West Bengal. Narasimhamurti & Saratchandra (1980) described Trypanosoma channai and Trypanosoma qadrii in Channa punctatus and Clarias batra­ chus collected from ponds in and around Vlsakhapatnam and SrikakulxiTi/itedhra Pradesh. .Sinha (1980^ reported Trypanosoma sp. in the blood of Lepidocephalus guntea collected from ponds of Bongaon,24-Parganas,West Bengal, Gupta & Jairajpuri (1981) described Trypanosoma tricho­ gaster i from Trichogaster fasciata collected from Aligarh, Uttar Pradesh. Joshi (1982,1983) described Trypanosoma aori and Trypanosoma rupicoli in Mystus aor and Nemacheilus- rupicola from Gomoti river, Lucknov; and Kosi river near Kosi station, 11 km^. Noipth of A.lmora,Uttar Pradesh. Nandi. et. a_l •, (1983) listed 20 named species of Trimamosoma and 32 unnamed species of Trypanosoma from Indian fresh water fishes.

_ t. -^ll' "'' e '"•^"i \i . • 87542 -••'•ilt "2 JAN 1985 : -Cv Oh'' ' • 16

B» Trypanosoma in #ipura.

According to Wenyon(1926) anuran trypanosomes v;ere first recorded by Donovan in Bufo melano- stictus, Rana hexadactyla and Rana cyanophlyctis» Berestneff (1903) described Trypanosoma sp. from Rana tigrina and Rana limnocharis collected from Bombay. Pattoa}(1908) found Trypanosoma rotatorf-um in Rana tiqrina and Trypanosoma inopinatum (—bendersoni) in Rana tigrina as well as Rang hexadactyla from South India. Scott (1926 ,1927) reported Tr:^/pan03oma rotatorium in the blood of Rana tigrina . Pujati'. ,(1953) recorded Trypanosoma rotatoriiim from Rana cyanophlyctis and Rana tigrina from South India. Damayanthi & Rao(1979) reported Trypanosoma ranarum in the heart muscle of some frogs of VJarangal, Z^andhra Pradesh, The occurrence of this parasite v^as also obser^/ed in tadpoles by these authors. Ray & Nandi (1978) recorded Trypanosoma rotatorium in Rana limno­ charis from VJest Bengal. Ray ( 1979a,b, 1980) reported Trypanosoma rotatorium, Trypanosoma chattoni , Trypanosoma kar^^ozeukton, Trypanosoma loricatum, Trsnoanosoma malabarica and Tr^^panosoma systoma in Bufo melanostictus, Bufo stomati- cus, Microhyla ornata, Rana cyanoph|yctis, Rana hexadactyla, Rana limnocharis, Rana malabarica, Rana tigrina, Rhacophorus maculatus, Rhacophorus:malabaricus and Uperodon systoma collected from different pa-rts of India. Sinha (19814 reported Trypanosoma rotatorium from Manipur toads(Bufo 17

melanostictus). Ray & Choudhury (1980, 1981) observed Trypanosoma rotatorlum in Rana tiqrlna collected from West Bengal. Ray & Choudhur;/ (1983), described nine species of Trypanosoma viz./ Trypanosoma rotatorinm/ Trypanosoma loricatum, Trypanosoma karyozeukton^Trypanosoma chattoni ,Trypanosoma inopinatum, Trypanosoma ranarum, TryTDanosoma taprobanica, Trypanosoma malabarica and

II "^i in I I .III 111 1 111 I I-*- I . I mt iii»wiidnrr.^.M,. i • , m , „ • i, Trypanosoma systoma in Indian anurans collected from ** -^ I II »M»Wii—IIi.ii»Mi^i.i^-i- miinn different districts of West Bengal,Orissa,Bihar,Assam, Andaman Islands, Tripura ,Andhra Pradesh,Kerala and FTdva Goa. C. Trypanosoma in Chelonia and Ophidia. Sinha(1978) described Trypanosoma gangetica from a fresh water turtle,Trionyx gangeticus collected from Bonqaon,24-Parganas,Vfest Bengal. Haq & Mohiuddin (1956) described an unnamed species of Tr^.^'panosoma from Xenchrophis (=• Matrix ) piscator. Sinha S Mandal (1976) described Trypanosoma enhydris from a fresh water snake,Snhydris enhydris collected from Chakdah,Nadia, I'Jest Bengal, 18

MATERIALS

The following hosts were examined for Trypanosoma infection in their peripheral blood,

^* Clarias batrachus (Linnaeus)

It is popularly known as 'Magur' in Bengal. It belongs to Family Clariidae under Order Cypriniformes. It is found in fresh and brackish waters of plains of India and can live in mud. It possesses accessory respiratory organs and is able to breathe even when it is out of water.. Body is elongate, greyish-black with a laterally compressed head. Dorsal fin is more than two thirds of the length of body. This fish is recommended by dieticians as food for patients during the period of convalescence.

Thirty specimens were collected from ponds and ditches of Chakdah,Nadia,West Bengal and examined. Five of these fishes were found to harbour Trypanosoma in their peripheral blood,

2. Tilapia mossambica. (Peters) This fish popularly known as 'Jimerican Koi" belongs to Family Cichlidae under Order Perciformes. It is an exotic species introduced in India 19

in 1952 by the Central Marine Research Station ,Mandapam. Body is compressed and dull brownish-olive or blackish in colour. Dorsal and caudal fins are with yellow edges.

Twenty five specimens were collected from ponds of Bongaon,24-Parganas,West Bengal and examined. Two of these were found positive for Trypanosoma infection.

3« Eepidocephalus quntea (Hamilton)

It is popularly kno\Nni- as 'Gtile'' belonging to Family Cobitiidae under Order Cyprini- formes. It is found all over Xndia except for Malabar Coast and South of Krishna, Body is compressed and dirty yellow in colour. A light band extends from centre of snout-to a black ocellus. Numerous rows of dark spots are present on dorsal and caudal parts of body and often there are two rows of such spots on anal fin. Lateral line is absent.

Twenty of these specimens were collected from ponds of Bongaon,24-Parganas,West Bengal for studying Trypanosoma and two of these were found positive for the parasite. 20

^' Xienentodon cancila (Hamilton)

This fish is commonly known as 'Kekle' in Bengal. It belongs to Family Belonidae under Order Heloniformes. rt: is a fresh water species found throughout India. Body is stout almost cylindrical and upper part is green while lower part is whitish. It has silvery streak with dark margin from orbit to middle of caudal base.

Twenty specimens were captured from ponds of Bongaon/24-Parganas/West Bengal and examined Three of these were found too harbour Trypanosoma in their peripheral blood.

5. Channa gachua (Hamilton)

This fish is known as 'Cheng' in Bengal belonging to Family Ophiocephalidae under Order Ophiocephaliformes. It is found in fresh waters throu­ ghout greater parts of India. Body is hrovm V7ith dark bands running forwards from dorsal ridge to Just below lateral line.

Twelve specimens were collected from Baruipur^24-Parganas, West Bengal and examined. Two specimens were found infected with 21

Trypanosoma in its blood.

6. Mastocembelus armatus (Lacep^de)

This fish is popularly known as *Baam' in Bengal. It belongs to Family Mastocembelidae under Order Mastocembeliformes. It is fovmd in fresh and brackish waters in plains and hills of India. Upper part of body is brownish in colour::and the lower part is pale. Black or dark-brown irregular zigzag pattern is present in between lateral line and dorsal ridge. Fifteen specimens were qollected from Chakdah^Nadia^West Bengal and examined for Trypanosoma. Only two of these specimens were positiite for the parasite.

7* Mastocembelus pancalus (Hamilton)

It is popularly known as •Pankal• in Bengal. It belongs to Family Mastocem­ belidae under Order Mastocembeliformes, It is found in large rivers and estuaries. It is greenish-olive along black and yellowish colour below with yellowish spots along sides. 22

Fifteen specimens were collected from ponds of Bairuipur, 24-Parganas,West Bengal and examined. Tvro of these were found positive for Trypanosoma in their peripheral blood.

®* Bufo melanostictus Schneider

This toad is popularly known as "Kuno'in Bengal. It belongs to Family Bufonidae under Order ilnu.ra. It is found in damp places all over the country. Adult one has uniform brov/nish or greyish-olive above. Crown of head is deeply concave. Parotid glands: are elongate and longer than head. Upper parts are densely covered with tubercles and warts. Tympan-um is v/ell developed. Legs are short. Length of hind limb is not much more than that of the body.

Pour specimens were collected and blood smears V7ere drawn by Mr. Ekendar Singh of Manipur. Tvio of these specimens were found to harbour Trypanosoma.

9. Lissemys punctatus (Bonnaterr^)

It is popularly known as mud- turtle. It belongs to Family Trionychidae under Order Chelonia. The animal is timid and lives in ponds. Bbdy is olive-brown above with large v/ell-defined spots. 23

First marginal bone is much longer than the second. Entoplastral callosity is usually small in adult. Head and carapace bearr yellov/ spots. ' It is distri­ buted in the Ganges, and the ':'., Indus and in their tribu­ taries. Twelve mud turtles V7ere collected at different times from Bongaon,24-Parganas,West Bengal and examined. Five of these animals were found to harbour Trypanosoma.

^^* Trionyx gangeticus Cuvier

It is a common turtle found in the Gangetic river system. It belongs to Family Trionychidae under Order Chelonia. Its carapace and plastron are covered v;ith soft skin. Snout ends in a proboscis. Jax^s are concealed under fleshy lips. Ears are hidden and tail is short. Head is provided v/ith a black longitudinal streak between ^he eyes extend­ ing to the nape. Dorsal part is olivaceous,v/hile ventral part is yellowish. Fifteen specimens were collected from the Ichamati river of iBongaon, 24-P'arganas,West Bengal and examined. Txvo of these specimeBS were found positive ^°^ Trypanosoma. 24

11. Bnhydris enhydris (Schneider) This snake is popularly known as 'metuli' in Bengal. It belongs to Family Colubridae under Order si^uamata. It is an aquatic and non-poisonous snake but is often found on land in the vicinity of water. It is brovjn or grey or olivaceous in colour above and bounded on either side by a pale stripe which is most distinct on the hinder part of the body. Head is indistinctly variegated with grey or with an indis­ tinct dark stripe on each side of the eye. It is distri­ buted in North Eastern India and is very common in Bengal. It chiefly feeds on fishes. Fifty snakes were collected at different times from the ponds and ditches of Chakdah^Nadia/ West Bengal and examined. Thirty of these v^ere found to harbour Trypanosoma in their peripheral blood.

METHODS

The follov/ing methods were adopted for the preparation of blood smears from different specimens.

In fishes/blood was collected from branchial blood vessels or caudal veins v;ith' the help of a sterilised needle. 25

In amphibians/blood smears were draxvn by puncturing the facial vein at the angle of mouth. In turtles/blood was collected by pricking the limbs and sometimes by puncturing the heart directly when the animals are Icilled. In snakes/blood was collected by clipping the tail region or from the facial vein. Blood smears were stained with Leishman"s and Giemsa's stains. For staining v/ith Giemsa's stain the air dried slides were fixed in acetone-free methyl alcohol for five minutes and allowed to dry. For Leishman's stain sepatare fixation was not necessary /since the solution itself acted as fixatives^. Measurements of'trypanosomes were made from fixed and stained materials and Camera lucida drav/ings were made.

Nuclear index (Nl) and kinetoplastic index

(KI).' were calculated to determine the position'of nucleus and the kinetoplast after the methods advocated by Dias & Freitas (1943). and Keymer (1967). respectively for the parasites. 0: B S E R V A T I 0 N S 26

Trypanosoma in Fishes Parasite No.l. Trypanosoma clariae Monte1 ( Pigs. 1 - 12 )

1905. Trypanosoma clariae Montel, C.r.Soc.Biol.^58 :1016; Mathis & Leger, 1910, C.r.Soc.Biol.,69 : 349; Mathis & Leg6r,1911,C.r.Soc.Biol., 71: 185.

Occurrence The parasite was encountered in peripheral blood plasma of Clarias batrachus(Linnaeus) collected from Chakdah, Nadia, West Bengal, India.

Morphology Trypomastigotes were only found in blood plasma. These showed a single morphological type. These (Figs.1-12) were slender,elongate with pointed extremities. Narrow cell body was widest at its middle part. Cytoplasm stained light blue and contaiend 3-4 vacuoles. Volutin granules were found towards anterior part of cell body. Nucleus was oval, sub-terminal and stained light purple. A karyosome was not observed. Kinetoplast was terminal,conical and stained deep purple. A flagellurn emerged from base of kinetoplast forming an undulating membrane with 3-4 folds. Free part of flagellurn was short. 5 Aim

10 11 12

FIGURES.. 1 - 12. Camera luclda drawings of Trypanosoma clariae ^fOntel (1905)^ showing trypomastigote forms. 27

Dimensions Measurements of forty trypanosomes were given in the

following table 1. y TABIxE r

Measurements (in micrometeiS/ /um ) of Trypanosoma clariae Montel^

1905.

Range Average

Total length of body (including free flagellum). 27.5 - 37.5 30.7 Length of cell body. 20 ~ 24.5 23 Width of cell body. 1.1- 2 1.4

Posterior extremity to posterior kinetoplast. 0.01 - .03 .02 itoterior kinetoplast to posteriori nucleusi 6.3 - 8.9 7.7 Anterior nucleus to anterior end. 7.7 - 9.9 8.6

Length of free flagellum. 7.5 - 10 8 Length of nucleus. 1I-.38 -1.99 1.77 Width of nucleus .45 -1.2 .95 Length of kinetoplast. .^7 - 1.2 1

Width of kinetoplast. .33 - .4 .38

Nuclear index(posterior end to centre of nucleus / nucleus to anteriorfend). .91' - 1.04 1.02

Kinetoplastic index( posterior end to centre of nucleus / centre of kinetoplast to centre of nucleus). 1,04 - ,1'06 »r»t>5' 28 Discussions Different species of Clarias have been repo­ rted to harbour Trypanosoma in their peripheral blood plasma, Montel(1905) reported for the first time the occurrence of a trypanosome in Clarias macrocephalus of North Viet Nam and named the parasite Trypanosoma clariae. * Dutton e_t al_., (1906 ,1907) described trypanosomes in Clarias angolensis collected from Kinshasa in the Zaire which existed in three varities of differ­ ent size and assigned no name to them. Wenyon(1908) reported Trypanosoma sp. in the blood of Clarias anguillaris which abounded in the Nile river and in Laike itoibodi of Sudan. He did not give any description and measurements of the parasite. Bouet{1909) found a trypanosome in the same fish and suggested that it might be the sssss. same parasite as that from Clarias angolensis and therefore, no detailed account was given. He also described Trypanosoma toddi a parasite from Clarias anquillari in the then French West Africa which Baker(1960) considered a valid species. Zupitza(1909) also described an unnamed species of Trypanosoma i" Clarias sp. from Cameroon. Mathis & Leger (1910, 1911) described two forms of Trypanosoma clariae Montel,1905 and named Trypanosoma clariae var parva and Trypanosoma clariae var magma in the blood of Clarias macrocephalus from Tonkin, Trypanosoma clariae parva was small,narrow with pointed ends. Cytoplasm was granulated. Nucleus was large and lay at middle. Kinetoplast was rounded and near the posterior part of cell body. These form measured 39/am in total length and 2.75 Aim in breadth.

*• Hfe did not give measurements and adequate description of the Darasite. 3QJ authors gave any adequate description of the parasite. The : . parasite under report resembles the small form of trypanosomes described by Mathis & Leger (1910, 1911) and is identified as Trypanosoma clariae Montel(19051. The only difference is that the large forms ( Trypanosoma clariae var magma) were not found in the present study,as described by these authors.

Diagnosis Family Trypanosomatidae Doflein,1901; emend Grobben^l905. Single nucleus and a kinetoplast ; flagelliom arising from base of kineto­ plast. Genus. Trypanosoma Gruby^l843. Kinetoplast posterior to nucleus; flagell\3m forming outer margin of undulating membrane along anterior end. Specific characters parasite in the present study measuring 27 .5/urn - 37.5/um X 1.1/urn - 2pm incl­ uding free flagellxjm. Cytoplasm contai­ ning volutin granules; kinetoplast ter­ minal ; nucleus oval, sub-central. The present parasite is referred to as Trypanosoma clariae Montel(1905)

Host. CIarias batrachus(Linnaeus) Site of infection . Blood plasma. Locality: Chakdah,Nadia,West Bengal, India. Trypanosoma in Pishes Parasite No.2. Trypanosoma mukasai Hoare (Figs. 1' - 12)

1932, Trypanosoma mukasai Hoare, parasitology, 24 : il 210 ; Baker, 196©, Parasitology,. 50 r 515. 1997. Trypanosoma choudhuryi Mandal, Acta Protozool., 15 : 1.

Occurrence The parasite was found in peripheral blood smears of Tilapia mossambica (PetersX collected from Bongaon, 24- Parganas,.West Bengal, India. Morphology Trypomastigotes showed a single morphological type in blood plasma. These (Figs.1-12) were small and slender with bluntly pointed extremities. Cytoplasm was light purple and contained a fev/ volutin granules and vacuoles. Nucleus was oval, sub-central and stained purple in colour:-. It lay towards the anterior part. A karyosome was not found. Kinetoplast was small,sub-terminal,dot-like and deep purple in colour. Flagelltim arose from base of kinetoplast forming an undulating membrane with 2-3 folds. Free part of flagellvim was short at the anterior tip. Dividing forms of these trypomastigotes were not: seen except for one individual (Fig. 12) where the cell body contained nuclei but the kinetoplast remained undivided. 10 11 12

PXGURES* 1-12. Camera lucida drawings of Trypanosoma mukasai Hbare(1932),showing trypomastigote forms. FIG. 12. Dividing form of trypcMnastigote showing two nuclei. 32

Dimensions Morphometric measurements ( in micrometers, /am) of; thirty trypomastigote focms of Trypanosoma mukasai Hbare, 1932 were given in table 1,

TABLE 1

Range Average Total length including free flagellxim. 18.9 - 24.1' 21.4 Length of cell body. 14.3 - 18.8 16.2 Width of cell body. 1.2 - 2.2 1.5 Posterior end to posterior kinetoplast. ,01 - .5 .2 Anterior kinetoplast to postterior nucleus. 5.5-7.9 7 Anterior nucleus to anterior end. 4.4 - 8,8 5,8 Length of free flagellum. 4.4-5.8 5.1 Length of nucleus. 1.7- 2.4 1.9

Width of nucleus. ,6-1.2 1 Length of kinetoplast. ,7-1.1 .9 Width of kinetoplast. ,2 - ,6 .3 Nuclear index ( posterior end to centre of nucleus / centre of nucleus to anterior end. 1.1- 1.35 1.3 Kinetoplastic index (posterior end to centre of nucleus * / centre of kinetoplast to centre ofnucleus). 1.02 - 1.12 1.04 33

Discussion Trypanosomes have been recorded in differ­ ent species of Tilapia from Africa and India. Wenyon (190^) described an unnamed species of Trypanosoma in Tilapia zilli of the Nile river in Sudan. Leg^r &" Ldger (1914) found trypanosomes in Tilapia lata from the river Niger in the then French West Africa but assigned no specific name to the parasite, These authors believed that the trypanosomes were identical with those described by Wenyon(1909) from Tilapia zilli. After a lapse of many years,Dias (1955) described three species of trypanosomes viz./ Trypanosoma napolesi/ Trypanosoma rebeloi ( in which were included the trypanosomes described by Wenyon, 1908 in Tilapia zilli) and Trypanosoma serranoi (in which were included the trypanosomes which Neave,1906 reported from Synodontis schall) from Tilapia mossambica in Mozambiique.

Baker(1960) reviewed the trypanosomes- of African fresh water fishes and considered Dias's three species as variants of a single species and regarded them as synonyms of Trypanosoma mukasai Hoare(1932). Baker (1960) also reported the occurrence of Trypanosoma mukasai Hoare(1932) from Tilapia nilotica, Tilapia esculenta, Tilapia variabilis and Tilapia leucosticta of George and '/ictoria lakes of East Africa. Mandal(1977) described Trypanosoma choudhuryi in Tilapia mossambica collected from Champhahati, 24-Parganas, West Bengal/India. 34

The present parasite under discussion is identified as Trypanosoma mukasai Hoare(1932) as redescribed by Baker(1960) from different species of Tilapia on the basis of the morphological characters described above, Trypanosoma choudhuryi Mandal(1977) resembles very much the small forms of Trypanosoma mukasai Hoare(1932) in its morphology and dimensions. Therefore,Trypanosoma choudhuryi Mandal(1977) and the present parasite are both, identified as Trypanosoma mukasai Hbare(1932). The only difference is that the large forms were not found in the present study nor were these reported in Trypanosoma choudhuryi Mandal(1977). A comparative study on the measurements of these trypanosomes;is given in table 2: which reveals that differences in dimensions, which almost overlap each other,are minor in nature and not sufficient for considering Trypanosoma choudhuryi Mandal (1977) and the present parasite as valid species distinct from Trypanosoma mukasai Hoare(1932) 35

TABLE 2 Comparative measurements( in micrometers, /um) of Trypanosoma mukasai Hbare (1932)'/Trypanosoma choudhuryi Mandal(1977) and the present parasite.

Trypanosoma Trypanosoma The present mukasai Ho are choudhuryi parasite. (1932) as des- Mandal(1977) cribed by Baker (1960) Range Range Range Range Small forms Large forms TL. 22 - 35 45-62 23 - 37.8 18.9 -24.1

BL. 16 - 27 33 - 54 16.5-25.3 14.3-18.8 BW. 1-2.7 2-6 1.5-1.8 1.2 -2.2 FP. 5-12 7-17 6.5 - 12.5 4.4- 8.2 PK. 0-2 .5-2 .8 - 2.5 .01-.5 NL. 1.7-3.5 3 - 5.5 3.5 -5.2 1.7-2.4 NW, .75-1.7 1.5 - 5.7 1 - 2.5 . .6-1.2 KN.. 5-17 14 - 29 5.5-9.5 5.5-7.9 NA., 4.5-11.5 13 - 19 4.5-8.5 4.4-8.2 KL. .25-1.5 .5 - 1.5 1 - 1.5 .7 - 1.1 KW. .25-1 .5-1 1 - 1.05 .2 - .6

Abbreviations: TL= total length of the body including free flagellum; BL==-length of cell body excluding free flagellxm; BW =• width of the cell body; FP —length of free flagellxom; PK =- the distance from posterior: end to kinetoplast; NL==length of nucleus; Hlf =width of nucleus; KN ^anterior kinetoplast to nucleus; NA =^nterior nucleus to anterior end; KL —length of kinetoplast; KW=width of kinetoplast. 36

Diagnosis Family Trypanosomatidae Doflein,1901; emend C3robben,1905. Single nucleus and a kinetoplast; flagellum arising from base of kineto­ plast. Genus Trypanosoma Gruby,1843. Kinetoplast posterior to nucleus; flagellijm forming outer margin of undu­ lating membrane extending along anterior end. Specific characters Measuring 18.9/am - 24.1 yum X 1.2/um - 2.2/um in total length including free flagellum; kinetoplast sub-tecminal; nucleus towards anterior part of body;. The present parasite is referred to ^s Trypanosoma mukasai Hoare(1932).

Host. Tilapia mossambica (Peters) Site of infection. Blood plasma. Locality: Bongaon,24-parganas,West Bengal,India. 37 Tr;7,P^"oso"^a ^'^ Fishes Parasite No.3. Trypanosoma n . sp. (aj'

( Pigs, 1 - 5 )

Occurrence The parasite was encountered in peripheral blood smears of Eepldocephalus guntea (Hamilton) collected from Bongaon, 24 -Parganas,West Bengal,India.

Morphology Trypomastigotes were only found in blood plasma which showed a single morphological type. These v/ere elongate, slender (Figs. 1 ~ 4) or broad ( Fig.5), The posterior end is narrowly pointed while the anterior tip is bluntly pointed. Cytoplasm was homogeneous,non-granular and contained a few vacuoles. Xt stained light blue in colour. Nucleus was either oval (Figs. 1 - 4) or sub-spherical and pink in colour. A karyosome was not observed, Kinetoplast lay almost at the posterior end (Figs.l - 4). or away from the posterior part of the cell body{Fig. 5). * Flagellum arose near kinetoplast and trailed anteriorly along undulating membrane forming 4-8 folds. Free part of flagellum was present at the anterior tip.

•Kinetoplast was conical (Figs. 1 - 4) or dot-like (Fig.5) and stained deep purple in colour. 5 Aim I 1 FIGURES. 1-5. Camera lucida drawings of Trypanosoma n.sp, (a). Pigs, 1 -4. Slender trypomastigote forms. Pig. 5. Broad trypomastigote form. 38

Dimensions Measurements ( in micrometers//um) of twenty trypomas-

tigotes of Trypanosoma n.sp. {a) were given in table 1.

TABLE 1

Range Average Total length including free flagelltun. 38 - 50 43.5 Length of cell body. 28 - 41 38 Width of cell body. 2.2 - 3.4 2.7 posterior end to posterior kinetoplast. ,8-3 1.5 interior kinetoplast to pHiterior nucleus. 10.5-15.4 13 Anterior nucleus to anterior end. 12.8 -19 15 Length of free .flagellum. 7 - 10 8.5 Length of nucleus. 1.5 - 3.3 2.2

Width of nucleus. -J- • ^ •" -^ • -D 1.6 Length of kinetoplast. .5 - 1.1 .8 Width of kinetoplast. .4 - .6 .4 Nuclear index ( posterior end to centre of nucleus / nucleus to anterior end). .93 -i.©Z i.Oi Kinetoplastic index ( posterior end to centre of nucleus / centre of kinetoplast to centre of nucleus). J.(1B-1.21 1.14 39

Discussion Parasite under discussion possesses an undulating membrane and a poateriorr: kinetoplast.. So it has been placed under genus Trypanosoma. Sinha (1980) reported Trypanosoma sp, from Lepidocephalus gxintea collected at Bongaon, 24-Pairaganas, West Bengal, India, The same parasite is;- described here from the same host fish and considered a new species^ on the"basis of the morphological characters described above. The problem of new speciation remains complicated for the trypanosomes from fishes as also pointed out earlier by various investigators ( Baker, 1960; Becker, 1967,1970,1977; Joshi,1978,1982; Mandal,1979), Recently Frees et al.,(1978,1979) and Grogl et al.,(1980) considered species specificity and varying morphometric characteristics:to create new speciation. 25 species of Trypanosoma have been recor­ ded so far from Indian fresh water fishes. Among the knov/n mono- fOrphic forms,Trypanosoma n.sp, (^) resembles to some extent Trypano­ soma ophicephali Pearse (1933);, Trypanosoma punctati Hasan &" Qasim, (1962),Trypanosoma elongatus Raychaudhuri & Misra(197 3) and Trypano­ soma qadril Narasimhamurti &:Saratchandra(1980) due to: body confi­ guration but differs in the following morphometric measunssnents and in host range given belov/ (measurements in micrometers, /am).

Trypanosoma Trypanosoma Trypanosoma Trypanosoma Trypanosoma : ophicephali pxonctati elongatus qadrii n. sp (_a) 1. 33.6 34 4 3 - 44,5 20 - 32 28 - 41 2. 7.7 14.6 17 - 18.5 5-14 7-10 3. 5.2 2.7 6-6.5 2.5 - 4 1.5 - 3.3 Host Channa Channa Channa Clarias Iiepidocenhalus striatus pxinctatus punctatus batrachus guntea Locality (si ami Hyderabad Calcutta yisakhapath- Bongaon Thailand. India. India, nam, India. India. Thus, Tr^pianosom a n.sp. (a.) does not fit in other known st:)ecies,,and can not be considered as identical. i\bbreviations. 1.=- Body length; 2. length of free flagellum 3. Length of nucleus. 40

Diagnosis Family Trypanosomatidae Doflein/19D1; emend Grobben,1905 Single nucleus and a kinetoplast; flagellum arising from base of kinetopl­ ast. Genus, Trypanosoma Gruby,1843 Kinetoplast posterior to nucleus; flagellijm forming outer margin of undulating membrane extending along anterior end. Specific characters

Trypanastigotes measuring 38 Am - 50 /um X 2,2 Ami - 3,4 /am in total length including free flaqell^um* Cytoplasm non-granular; nucleus oval or spherical; kinetoplast terminal or subterminal; undulating membrane forming 4-8 folds. For the present the parasite is referred to as Trypanosoma n,sp,(a)

Host, Lepidocephalus guntea (Hamilton) Site of infection. Blood plasma. Locality:Bbngaon,24-Parganas, West Bengal,India, 41

Trypanosoma in Pishes Parasite No.4. Trypanosoma cancili Mandal ( Figs, r - 4 )

1978. Trypanosoma cancili Mandal, itogew. Parasitol,, 19 :: 158,

Occurrence The parasite was found in peripheral blood smears of Xenentodon cancila (Hamilton) collected from Bongaon,24- Parganas,West Bengal/ India.

Morphology Trypomastigotes were only present in blood plasma and showed a single morphological type. These (Pigs.1-4) were long, slender, elongate and both the extremities were pointed. Cytoplasm was basophilic and contained volutin granules. Nucleus was oval,sub-central and light pink in colour. No karyosome was observed. Kinetoplast was sub- terminal, small and stained deep purple, Flagellum emerged from the base of kinetoplast forming an undulating membrane with 1-2 folds. Free part of flagellum was present at the anterior tip. FIGURES. 1-4. Camera lucida drawings of Trypanosoma cancili Mandal(1978) showing trypomastigote forms. 42

DTMENSICNS Measurements (in micrometers/ /am) of thirty trypomastigotes;of Trypanosoma cancili Mandal/1978 were given in the following table 1.

TABLE r

Range Average

Total length including free flagellum. 30 - 37 33.3

Lengthh of cell body. 26 - 33 28.4

Width of cell body. 1.8-2.7 2.2 Posterior: end to posterior kinetoplast. .4 - .7 .5

Anterior kinetoplast to pwterior nucleus. 10 - 14 11.8

Anterior nucleus to anterior end. 9.5 - 14.5 12.4

Length of free flagellum. 4.4 - 6.8 4.8

Length of nucleus. 1.6 - 3.2 2.6

Width of nucleus. r.2 - 1.7 1.5

Length of kinetoplast. .8 - l-.l .9

Width of kinetoplast. .4 - ;6 .5 Nuclear: index (posterior end to centre of nucleus / centre of nucleus to anterior end>>l.or -1.08 1.04 Kinetoplastic index ( posterior end to centre of nucleus,/ centre of kinetoplast to centre of nucleus). 1.04 --rlfog ,1'07 43

Discussion The : - parasite is placed under the genus Trypanosoma on account of an undulating membrane and a posterior kinetoplast, Mandal(1978) described Trypanosoma cancili from Xenentodon cancila collected at Raidighi,24-Parganas/West Bengal^ India. Parasite under discussion is also reported from the same host and is identified as Trypanosoma cancili Mandal (1978)' on the basis of the following characters. Parasite is monomorphic,slender and atten­ uated at both ends. Cytoplasm is strongly basophilic and con­ tained vacuoles. Nucleus is ovoid and almost central, kineto- plast is oval and stains deep purple. Free part of flagellum is present.

However, the following differences are also noted which are given in the following table 2 (measurements in micrometers, /um). TAI3LE 2 Trypanosoma cancili Present parasite, Mandal (1978) Range Range Total length. 27-40.3 30-37 Length of cell body. 18.5-28.5 26-33 Width of cell body. _- 1.8-2.7 Length of free flagellum. 8.5 -11,8 4.4 - 6.8 Length of nucleus. 2.5-3.5 1.6-3.2 Width of nucleus. 1.3-2.8 1.2-1.7 44

Posterior end to kinetoplast. 1 - 2.5: 1.2-1.8 Kinetoplast to posterior nucleus. 8.5 - 10.5 10 - 14 Anterior nucleus to anterior end. 7.5 - 10.5 9.5 -14.5

Host locality. Raidighi,24-Parganas, Bongaon,24-Parga- West Bengal. nas,West Bengal,

Above differences are minor in nature and not sufficient for considering the present parasite as new one. However, the range of dimensions of Trypanosoma cancili is amended in this study and shown in table 3. Therefore, the parasite under report is identified and described as Trypanosoma cancili Mandal,1978 svibject to the above amendment.

TABLE 3 Range Total length including free flagellum, 27 - 40.3 Length of cell body. 18.5-33 Width of cell body. 1.8-2.7 Length of free flagellum. 4.4-11.8 Length of nucleus. 1.6-3.5 Width of nucleus. 1.2 - 2.8 Posterior end to kinetoplast. 1 - 2.5 Kinetoplast to posterior nucleus. 8.5 - 14 Nucleus to anterior end. 7,5 - 14.5 Nuclear index. 1.01- 1.08 Kinetoplastic index, 1,06- 1,(15.', 45

Diagnosis

Family Trypanosomatidae Doflein,190I; emend Grobben,1905. Single nucleus and a kinetoplast; flagellum arising from base of kineto­ plast. Genus Trypanosoma Gruby^1843. Kinetoplast posterior to nucleus; flagellum forming outer margin of undu­ lating membrane extending along anterior end. Specific characters Trypomastigotes exhibiting a single morp­ hological type ; 30 - 37/um X 1.8 -2.7/urn in total length; kinetoplast sub-terminal; nucleus sub-central; cytoplasm basophilic; The present parasite is referred to as Trypanosoma cancili Mandal (1978).

Host. Xenentodon cancila (Hamilton) Site of infection. Blood plasma. Locality : Bongaon, 24-Parganas,West Bengal,India. 46

Trypanosoma in Pishes Parasite No.5, Trypanosoma gachuli Misra,C3iandra Si Choudhury (Pigs. 1 - 4 )

1973. Trypanosoma gachuii Misra,Chandra & Choudhury, Arch. Protistenk./ 115 i 18.

Occurrence The parasite was found in peripheral blood smears of Channa gachua (Hamilton) collected from Baruipur, 24- Parganas, West Bengal, India.

Morphology Trypomastigotes were only found in peripheral blood plasma and showed a single morphological type. These (Pigs. 1 - 4). were slender with an anterior blunt end. Cytoplasm was homogeneous,non-granular and light blue in colour. Nucleus was elongate or oval and pink in colour. Nb kary- osome was observed. Kinetoplast was terminal,small,oval and stained deep purple. Flagellum emerged near kinetoplast and trailed anteriorly along the border of undulating membrane forming 1 - 2 folds. Free part of flagellum was present at the anterior tip. 47 Dimensions Measurements ( in micrometers. Am ) of thirty trypomastigote forms of Trypanosoma gachuii Misra,Chandra & Choudhury, 1973 were given in table 1,

TABLE 1

Range Average Total length including free flagelliom. 24.5 - 29 26.5 Length of cell body. 16,5 - 18 16.5 Width of cell body. 1-1.4 1.2 posterior end to posterior Icinetoplast. ,1 - .5 .2 Anterior kinetoplast to posterior nucleus. 6-8 7 Anterior nucleus to anterior end. 5-7,5 6 Length of free flagellxom. 7 - 9.5 8.4 Length of nucleus, 1.6 - 2 1.8 Width of nucleus. .5 - .8 .6 Length of kinetoplast, .6 - .9 .7 Width of kinetoplast. .4 - .6 .5 Nuclear index (posterior end to centre of nucleus / centre of nucleus to anterior end). 1^.2- 1.3 1.2 Kinetoplastic index ( posterior end to centre of nucleus • / centre of kinetoplast to centre of nucleusX. 1.06 -1.1 1.07 48

Discussion Several species of trypanosomes have been described from the Family Ophiocephalidae. Lingard(1904) reported for the first time the occurrence of a Trypanospma sp, from Chann.a (= Ophiocephalus) striatus of Pune. He did not give any measurements and description of the parasite. Wenyon (1909);found a trypanosome in Channa obscurus of the river File from Sudan. Mathis & L^g6r(1911) also reported Trypanosoma sp» in Channa maculatus of Tonkin. * After a lapse of many years, Qadri(1951) found a trypanosome in Channa striatus collected from Hyderabad, todhra Pradesh,India and later named as Trypanosoma striati in 1955. Hasan & Qasim(1962), Raychau- dhury & Misra(1973) and Narasimhamurthi & Sartchandra(1980) described Tiypanosoma punctati, Trypanosoma elongatus and Trypanosoma channai from Channa punctatus while Misra.. ejt al., (1973), described Trypanosoma gachuii from Channa gachua respectively,. Trypanosoma sp. was also reported from India by Tandon & Joshi(1974) and Joshi(1979) from Channa pvmctatus and Joshi(1979) from Channa gachua. None of these authors gave any description of the parasite. The parasite under report resembles short slender form of Trypanosoma qachuii Misra,Chandra & Choudhury (197 3) and accordingly the trypanosome in this study is identified as Trypanosoma qachuii Misra/Chandra& Choudhury(1973

* Pearse(1933) described Trypanosoma ophicephali in Channa striatus from THotfct"«wict. 49

Short slender form is elongated with pointed extremities.

Cytoplasm contains a few vacuoles and granules. Nucleus

is oval and s\ib-central. Kinetoplast is terminal or sub-

terminal. The long slender form of Trypanosoma gachuii

is not found in the present study. the following differences

are also noted which are given in table 2 (measurements in micrometers, /om),

TABLE 2 Trypanosoma gachuii Present parasite Misra,Chandra& Choudhury(1973)

short slender Long slender

TL. 34.4 - 38.9 51.1 - 54.4 24.5 - 29

BL. 23.3 - 26.4 38.4 - 44.4 16.5-18

3W. 2.2 - 2.7 2.5 - 5.6 1 - 1.4

FP. It) - 12.5 10 - 12.2 7 - 9.5

NL. 2 - 2.8 2.2 - 3.3 1.6 -2

HB. 2 - 2.7 2.2 - 3.3 .5 - .8

H^st Locality. Local markets of Calcutta, Baruipur,24- West Bengal,India. Pargan as, VJe s t Bengal,India.

Above differences are not sufficient

for considering the present parasite as new one. Therfore,

the parasite under report is identified and described as

Trypanosoma gachuii Misra-, Chandra & Choudhury (1973) 50

Diagnosis Family Trypanosomatiaae Doflein,1901;emend Grobben,1905. Single nucleus and a kinetoplast; flagelluin arising from base of j<:inetoplast. Genus Trypanosoma Gruby,1843, Kinetoplast posterior to nucleus ; flagell\im forming outer margin of undul- , ating membrane extending along anterior end. Specific characters In this study,the trypomastigotes measuring 24/um - 29/um X. 1/um - 1.4/urn in total length including free flagelltim; kinetoplas-t sub-terminal or teinninal; nucleus elongate and sub-central. The present parasite is referred to as Trypanosoma gachuii Misra,Chandra& Choudhury (1973).

Hess. Channa gachua (Ttemilton) Site of infection. Blood plasma. liOcality : Baruipur, 24-Paraganas,West Bengal, India. 51

Trypanosoma in Fishes Parasite No.6 Trypanosoma armeti Mandal ( Figs. 1 - 3)

1975. Trypanosoma armeti Mandal, Angew. Parasitol., 16 I 87.

Occurrence The parasite was encountered in peripheral blood smears of Mastocembelus armatus(Eacepede) collected from Chakdah/Kadia ,West Bengal,India.

Morphology Trypomastigobes were only found in blood plasma. These exhibited a single morphological type. These(Figs. 1 - 3) were long,slender and both the extremities were bluntly end. Cytoplasm was light blue in colour and contained volutin granules. A few vacuoles were also present. Nucleus was oval and stained light pink in colour. A k=>ryo-- some was not observed. Kinetoplast v/as terminal,dot-like or conical and stained deep purple. Flagellijm emerged near kinetoplast and formed an undulatiinj membrane with 3 - 5 folds. Free part of flagellum was present at the anterior tip. 5 Ajm I 1

FIGURES. 1-3. Camera lucida drawings of Trypanosoma armeti Mandal(1975),showing trypomastigote forms. 52

Dimensions Morphoraetric measurements (in micrometers^/um) of twenty trypomastigote forms of Trypanosoma armeti Mandal/ 1975 were given in table 1. TAP.LE t

Range Average Total length including free flagellum. 32 - 40 35 Length of cell body. 24 - 31 27.2 Width of cell body. 1.8 - 2.3 2 Posterior end to posterior: kinetoplast. .o£ - .2 .1 Anterior kinetoplast to posterior nucleus, 9.5 - 10., 8 10.5 Anterior nucleus to anterior end. 13 - 14.6 13.8 Length of free flagellum. 6.5 - 8.8 7.3 Length of nucleus. 1.4 - 2 1.6

Wideh of nucleus. .9 - 1.1 1 Length of kinetoplast. .7 - 1.1 .8 Width of kinetoplast. .3 - .5 .4 Nuclear index (posterior end to centre of nucleus,/ centre of nucleus to anterior end). .79 - .84 .82 Kinetoplastic index ( posterior end to centre of nucleus / centre of kinetoplast to centre of nucleus);, 1,03 - .1-Ob 1.04 5 3 Discussion

Mandal(1975) recorded the occurrence of

two species of Trypanosoma viz. / Trypanosoma armeti and Tr^nsanosoma

pancali from Mastocembelus a^rmatus and Mastocembelus pancalus

c^ollected at Champahati, 24-Parganas,West Bengal, India. -Tandon & Chandra (1977) and Joshi(1979) reported Trypanosoma sp. in Mastocembelus armatus collected from Lucknow,Uttar Pradesh. These authors did not give any adequate description of the parasite. The parasite is reported from Mas toe embelus armatus and is identified as Trypanosoma armeti Mandal(1975) on the basis of the follox^ring characters. Parasite is long, slender v;ith blunt extremi­ ties . Cytoplasm -.iis granular and contained vacuoles. Nucleus is ovoid and does not cover the vfhole v/idth of the body. Kineto- plastis terminal or sub-terminal. The only difference is that the stumpy forros vrere not found in the present study. Differences in dimensi** ons which almost overlap each other and in host locality noted in the following table 2 may be minor in nature and can not be consi­ dered as separate species. Therfore,the parasite under report is identified as Trypanosoma armeti Mandal(1975) ( measurements''in micrometers. Am). TABLE 2 Trypanosoma armeti Present parasite Mandal(l975) Stumpy Slender Total length including free flagellum. 48 - 50 53 - 57 32 - 40 54

Length of the cell body. 38 - 40 42 - 45 24-31

Width of the cell body. 4 - 5.5 5:- i.5 1.8 -2.3 Ijength of free flagellum. 10 - 11 11-12 6.5-8.8 Length of nucleus, 4-5 5-5.5 1.4-2

Width of nucleus. 3-3.5 2T2.5 .9-1.1 Length of kinetoplast, 1-1.5 .79 - t; .7-1.1 55

Diagnosis Family TrypaEiosomatidae Doflein,1901; emend Grobben,r905. Single nucleus and a kinetoplast; flagellian arising from base of kinetoplast. Genus- Trypanosoma Gruby,1843. Kinetoplast posterior to nucleus; flagellum forming outer margin of undulating membrane extending along anterior end. Specific characters Trypomastigotes measuring 32;Lim - 40 /uiTi X 1.8 /um - 2.3 yum in total length including free flagellum. Cytoplasm containing volutin granules, kinetoplast terminal, nucleus sub-central. The present parasite is referred to as Trypanosoma armeti Mandal,1975.

Host. Mastocembelus armatus (Lacepede) Site of infection. Blood plasma. Locality:; " Chakdah, iradia,West Bengal,India. 56^ Trypanosoma in Pishes

Parasite No.7

Trypanosoma pancali Mandal ( Figs. 1 - 3 )

1975. Trypanosoma pancali Mandal/ Anqew. Parasitol.,

16 :: 87.

Occurrence

The parasite was found in peripheral blood smears

of Mastocembelus pancalus (H&milton) collected from Baruipur,

24-Parganas,West Bengal,India.

Morphology

Trypomastigotes were only present in blood plasma

and exhibited a single morphological type. These(Figs.1

- 3) were slender ,elogate and attenuated at both ends.

Cytoplasm was alveolar, blue in colour: and contained volu­

tin granules. Nucleus was oval,sub-central and light pink

in colour. A karyosome was not found. Kinetoplast was small,round or conical,terminal and stained deep Durple.

Flagellum origiriated near kinetoplast forming an undulating

membrane with 3 - 7 folds. Free part of flagellum was

present at the anterior tip. .5 Aim

FIGURES, 1 - 3 . Camera lucida drawings of Trypanosoma pancali Mandal(1975),showing trypomastlgote forms. 57

nimensions Morphometric measurements ( in micrometers/ Aun ) of thirty trypomastigote forms of Trypanosoma pancali Mandal/1975 were given in table 1.

TiffiLE 1

Range Average Total length including free flagellum. 24,5 - 32,5 27,2 Length of cell body. 19,5 - 25.5 21.7 Width of cell body, 1.2 - 2.3 1.6 posterior end to posterior: kinetoplast. .01 - -^Sf :'.D3 itoterior kinetoplast to posterior nucleus. 7.5 - 10.5 8.8 -anterior: nucleus to anterior end. 8 - It,5 9.5 Iiength of free flagellum, 4.5-7.8 5.7 Length of nucleus. 1.2-1.6 1' Width of nucleus, ,6-1,1 ,7 Iiength of kinetoplast. ,4 - ,7 ,5 Width of kinetoplast. .3 - .5 ,3 Nuclear index ( posterior end to centre of nucleus / centre of nucleus to anterior end), .97 - V.l 1 Kinetoplastic index ( posterior end to centre of } nucleusf / centre of kinetoplast to centre of nucleus). i.04 -1,08 /.OS 58 Discussion

•.. Parasite under report is placed in the genus Trypanosoma on account of an undulating membrane and a posterior kinetoplast,

Mandal(1975) described a dimorphic (

Trypanosoma armeti) and a raonomorphic(Trypanosoma pancali) trypanosome from Mastocembelus armatus and Mastocembelus pancalus collected at Charapahati, 24-Parganas,West Bengal/India^jrespectively.

The present parasite is also reported from Mastocembelus pancalus

and is identified as Trypanosoma pancali Mandal(1975) on the basis of the follov7ing characters.

Parasite is slender,elongate and attenuated

at both ends. Cytoplasm contains volutin granules. Nucleus is either oval or sausage-shaped and occupies almost the entire width of the body. Kinetoplast is terminal or siib-terminal. undulating membrane forms 3-7 folds.

However,the following differences are also noted which are given in table 2. (measurements in micrometers//UTn) TABLE 2

Trypanosoma Present parasite cancili Mandal (1975) Total length including free flagellum, 35 - 40 24.5 -32.5 Length of body. 22-25 19.5-25.3 Length of free flagellum. 13-14 4.5-7.8 Host locality. Champahati,24-Parganas. Baruipur,24- Parganas. Above differences are minor in nature and not sufficient for considering the present parasite as new one. Therefore, the parasite under report is identified and described ^s Trypanosoma pancali Mandal (1975) . 5SF<

Diagnosis Family Trypanosomatidae Doflein,1901; emend Grobben, 1905. Single nucleus and a kinetoplast; flagellum arising from base of kinetoplast. Genus Trypanosoma Grtiby,1843. Kinetoplast posterior to nucleus; flagellxjm forming outer margin of undulating membrane extending along anterior end. Specific characters Monomorphic; measuring 24.5 Aim - S^'S/om X 1.2 /om - 2 .B /om with a free flagelltmi ( 4.5/um - 7^'8/um); cytoplasm alveolar containing volutin granules and few vacuoles. Nucleus oval, sub-central, kinetoplast terminal. The present parasite is referred to as Trypanosoma pancali Mandal,1975.

Host. Mastoceiabelus pancalus (Hamilton) Site of infection. Blood plasma.

Locality: Baruipur,24-Parganas,West Bengal,India. 60' Trypanosoma in Anura Parasite No,8. Trypanosoma rotatoriiom (Mayer) (FIGS. 1 - 7)

184 3, aiaoeba rotatoria Mayer, 'SDicilegitom observationiim anatomicarum de organo electrico in RalJ._s anelictricis et de Haematozois'/Bonnae; Chaussat^1850,Pes hematozo- aires (Ph.D. Thesis,Paris), 1885. Trypanosoma ranarum Danilewslvy,Biol .Zbl., 5 : 529; 1889, La parasitologic comparee du Sang 1. Nouvelles recherch- es Sur les parasites du sang des Oiseaux,Kharkoff,Partim, 1885, Trypanosoma rananim Danilev7s]cy,Ibid,; 1889, Ibid. 1885, Trypanosoma sanguinis Danilewsky,Ibid. ; 1889, Ibid, ^^^^* Tryoanosoma snaguinis ranarum Shalashnikov,Arhh. Vet, Hauk.S. Peterburg.,1 : 65, 1889, Trypanomonodes ranarum De.nilw,';s]^r>, 1-^63, Z£«!. Si^. ^[2^>^^.^^^^r^ 8, p. I. 1907. Tjrypanosoma loricatum Dutton,Todd & Tobey, Ann .trop.Med.

Paras it. ,1^ : 287, Part im.

1908. Trypanosoma sp. Patton, A Rep.King.Inst.Prev.Med.Guindy

(1907), 1_ : 3; Berestnef f, 190'3, Arch.Protistenk., 2: 34; Scott,

1926,Proc.Zool.Soc.London,1 : 231; 1927, Proc. zool.Soc.

London,2. : 173; Wenyon,1926,protozoology,Vol.l,p.5e8.

1908. Tr^^panosoma hylae Franca,Archs R. Inst.Bact., 2; 271,Partiin.

1911fei. Tri^^panosoma borreli' Mathis S, Leger, C. r. Se anc. Soc. Biol.,

70 : 956,Partim.

Occurrence

The parasite was encountered in peripheral blood smears of Bufo melanostictus Schneider a common toad,collected from Manipur, India.

Morphology

Trypomastigote forms of parasite were found in blood plasma. These exhibited four morphological t^rpes. Type. I.

These (Pigs. 1-2) may be described as thin slender with a long free flagellum. These were thin,narrow, slender,and both the extremities were pointed . 62

Posterior part of cell body stained deep blue and anterior: part took light blue. Cytoplasm was homogeneous and less vacuolated. Nucleus was circular and situated towards posterior part of cell body and stained pink in colour.* A karyosome v/as not observed. Kinetoplast was small, round ,deep purple in colour and adhered behind the nucleus, Flagellum rose from the base of kinetoplast and formed well-developed undulating membrane with 4-6 folds. Free part of flagellum was long.

Type. II.

This may be regarded as broad slender form (Fig. 3),. It was long,broader^ slender and both the extremities were pointed. Cytoplasm was homogeneous/ basophilic and non-granular. Nucleus I'ras round, light purole in colour, and was ver^'- close to kinetoplast. A karyosome was not found. Kinetoplast was dot-like deep pink in colour and situated away from posterior part. FlagelluTi rose from base of kinetoplast and formed an undulating membrane v/ith 5-7 folds. Free part of flagellurn was present. 63

Type. III.

These were regarded as leaf-like forms (Figs. 4-6). These v/ere v/ith a broad posterior end and a narrov/ pointed anterior end. Cytoplasm was basophilic and • in some individuals (Figs.5 - 6) several pellicular striations of myonemes v/ere present. Nucleus was round/light purple in colour and adhered to kinetoplast. A karyosome v/as not present. Kinetoplast v/as dot-like, deep punole in colour: and situated away from posterior part of cell body. Flagellum emerged from base of kinetoplast and formed undulating membrane v/ith 7-8 folds. Free part of flagellum was present.

Type. TV.

This, was described as round form (Fig.7), It :. ;, was round or oval in shape. Cytoplasm xiras homogeneous, non-granular,non-vacuolated and stained light bl\xe in colour. Nucleus was oval,sub-central and light pink in colour. A Karyosome was not found. Kinetoplast was small,conical, deep purple and lying av/ay from posterior tip. Flagellxim rose from base of Icinetoplast forming irregular undulating membrane v/ith one or tvro folds. Free part of flagellum v/as present. 6 Aom

FIGURES. 1 - 7, Camera lucida drawings of Trypanosoma II I 1^ II I Pii I' in rotatorium Otayer,1843). Figs. 1-2. Thin slender trypomastigote forms. Fig". 3. Bi-oad slender trypcwnastigote form. Pigs. 4-6. Leaf-like trypomastigote forms. Figs, 5 - 6. showing myonemes. Fig. 7. Round form of trypomastigote form. 64

Measurements (in micrometers/jm). of Trypanosoma rotatopium (Mayer^ 1843) . Tyoe, I. Thin slender forms.

Range. Average. Total length of body including free flaqell\jm. 45 - 58 51.8 Ijongth of cell body. 23 - 36 34 Width of cell body. 2.5- 5 4 Length from posterior extremity to posterior border of kinetoplast. 5-9.8 7.7 Length.from anterior border of kinetoplast to posterior nucleus. .1 - .4 .2 Length from anterior border of nucleus to anterior extremity. 16 - 25 20.7

Length;-, of free flagellimi. 16.S - 22.8 20

Length of nucleus. 1 - 1.6 1.2

Width of nucleus. .8 - 1.4 .9

Length of kinetoplast. .4 - .9 .6

Width of kinetoplast. .4 - .8 .5 Nuclear Index ( posterior end to centre of nucleus/centre of nucleus to anterior: . .AA extremity).

Kinetoplastic Index C posterior end to centre of nucleus j?feentre of kinetoplast to centre of nucleus) ^ - 7.^ 7' 5 — v'-^ 65 Measurements ( in micrometers/ ,/um) of Trypanosoma rotatorium

(Mayer, 184 3) Type. II. Broad slender form.

Total length of body including free flagellum, 62.4 Length of cell body. 49.2 Width of cell body. 6.6 Posterior extremity to posterior kinetoplast. 11.4 Anterior kinetoplast to posterior nucleus. .2 itotsrior nucleus to anterior extremity. 36 Length of free flagellum. 12.2 Length of nucleus. 1.2 Width of nucleus. 1.2 Length of kinetoplast, ,4 Width of kinetODlast. .4 Nuclear Index ( posterior end to centre of nucleus / centre of nucleus to anterior .-^A extremity. ). Kinetoplastic Index ( posterior end to centre of nucleus > / centre of kinetoplast to centre of 12-8 nucleus. 66

Measurements (in micrometers,Xim) of Trypanosoma rotatoriiam (Mayer, 1843). Type. Ill, Leaf-like forms.

^___™- Average, Total length of body including free flagellum. 45 t 49.5 47 Length of cell body. 37 - 40 38 Width of cell body. 14 - 17 15.5 Posterior extremity to posterior kinetoplast. 6.8-7.8 6.9 Anterior kinetoplast to posterior nucleus, 1.2-1.6 1.3 Anterior nucleus to anterior extremity. 29-33 31 Length of free flagellum, 7-9.5 8 Length of nucleus. .9-1.4 1,1 Width of nucleus. .8-1.3 I Length:' of kinetoplast.. .2 - .4 .3 Width of kinetoplast. ,1 - .3 .2 Nuclear Index ( posterior end to centre of 29 nucleus / centre of nucleus to anterior "27 - '3i •extremity. Kinetoplastic Index ( posterior end too centre of nucleus ^/ centre of kinetoplast to centre of nucleus). 4*2 ~ 5'05^ ^•5 67

Hfeasurements ( in micrometers, Ann) of Trypanosoma rotatorium (Mayer, 1843) . Type, IV, Round form.

Total length of body including free flagellum. 32.4 Length of cell body. 22.8 Width of cell body. 15.6 Posterior extremity to posterior kinetoplast. 6 Anterior 1-cinetoplast to posterior nucleus. .36 Anterior nucleus to anterior extremity. 11.6^ Length of free flagelliam, 9,6 Length of nucleus.. 2.5 Width of nucleus. 1.6 Length of Icinetoplast. .4 Width of kinetoplast. ,2 Nu3:lear Index ( posterior end to centre of nucleus / centre of nucleus to anterior extremity). '62

Kinetoplastic Index ( posterior end to centre of nucleus^ / centre of kinetoplast to centre of ^'A nucleus). 68

Dlsonssion

The present parasite is placed under genus Trypanosoma on account of undulating membrane and a posterior- kinetoplast. Parasite has been described as Tr'].T3anosoma rotatorium (Mayer/1843) because of the following characters. Parasite is polymorphic/showing four morpho­ logical types viz./ thin slender /broad slender,leaf-like and round forms. Poor stainabilty of nucleus is a most characteristic feature of Trypanosoma rotatorium is also noticed v/hich was reported earlier by many vrorkers ( Dutton/ et al./1907 7 \ : Balfour/1908; Stevenson/1911; Macfie/1914 ; Mohammed & Mansour /1959i). Morphological variation due to polymorphism in rotatorium from different species of anuran hosts is also pointed out by Fantham,- et al., 1942 and Mohammed & Mansour/1959*^ Therefore/the parasite under report is identified as Trypanosoma rotatorium (Mayer/1843).

Diagnosis Family, Trypanosomatidae Doflein/1901;emend Grobben/1905, Single nucleus and a kinetoplast; flaqellvim arising from base of kinetoplast. 69

Genus. Trypanosoma Gruby,1843. Polymorphic; kinetoiolast posterior to nucleus; flagellima forming undulating .membrane, Specific characters. Trypomastigotes exhibiting four morpholo­ gical types. Type, I. Thin slender forms measuring 45 Aim - 58 /um X 2.5 /urn - 5 /um. Type. II, Broad slender form measuring 62.4 /um X 6.6 /umia Type. III. Leaf-like forms measuring 45 Aim - 49.5 yum X 14 /um - 17 /um . T^'-pe. IV. Round form measuring 32.4/um X 15 .6 /um. In all trypomastiqote forms nucleus stains poorly. The present parasite is referred to as Trypanosoma rotatorium (Mayer,1843)

Host. Bufo melanostictus Schneider Site of infection. Peripheral blood. Loc ality : Man ipur ,In di a. TO Trypanosoma in CheIonia Parasite No.9. Trypanosoma vittatae Robertson (FIGS. 1 - 8 )

1'908. Trypanosoma vittatae Robertson, Spolia. Zeylan.,5 : 178 ; 1909, Q.Jj.microse.3ci.^53 :665,

Occurrence The parasite v;as found in peripheral blood smears of Lissemys punctatus (Bonnaterre) a mud-turtle,collected from Bongaon,24-Parganas,West Bengal,India.

Morphology In blood plasma,trypomastigotes exhibited three morphological types. Type. I. These may be regarded as thin slender forms (Pigs. 1-3). These were slender,elongate and curved. interior tip was pointed while posterior tip was either pointed or blunt. Cytoplasm was basophilic and contained a few vacuoles. Nucleus was sub-central,oval or. ': 1. circular,light pink in colour and lying towards posterior part of body. A kairyosome was sometimes present (Fig.2). Kinetoplast was small,dot-like,deep purple and located terminally or sub-terminally. Flagellum rose from base of kinetoplast and formed an undulatinq membrane with 3 -5 folds. Free part of flagellum was present at the anterior 71' tip. In one case, a trypomastigote (Fig.3) was fo\ind to contain two nuclei and a single kinetoplast.

Type. II. These were described as intermediate forms (Figs. 4-5). These were larger than the thin forms^^ having a slender elongate body. Both the extremities were pointed. Cytoplasm was homogeneous,basophilic and contained a few vacuoles. Nucleus was oval and light purple in colour:'and lay posteriorly. Karyosomes- were sometimes present (Fig.5). Kinetoplast was small, dot-like,sub-terminal and deep purple in colour. Plagellum emerged from near the kinetoplast forming an undulating membrane with 6 - 10 folds. Free part of flagellum was present at the anterior tip.

Type. III. These should be regarded as broad slender form (Figs.5-8). These were-large,slender, elongate and v/ith pointed extremities. Cytoplasm was homogeneous, basophilic:, and contained a few vacuoles. Nucleus was sub-spherical,sub-central,light purple in colour and lay posterior part of cell body. A karyosome was sometimes present (Fig.5). Kinetoplast v/as small, j^aiiikK dot-3il

C /urn- 5 Aim

FIGURES. 1 - 8'. Camera lucicl.a drawings of Trypanosoma yittatae Robertson ('1958) . Figs. 1-3. Thin slender trypomastigote forms. Fig, 3. Slender trypomastigote showing two nuclei, Figs. 4-5. Intermediate forms of trypomastigotes. Figs. 6 - 8. Broad slender trypomastigote forms. 72

Measurements ( in micrometers, Aun) of Tn/panosoma vittatae Robertson,1908. Type. I, Thin slender forms.

Total length of body including free Range. Average. fl age Hum. 23.9 - 30.6 27.4

Length of cell body. 17.4 - 23 19

Width of cell body. 1.4 - 6.2 3.1 Posterior extremity to posterior kinetoplast. .05 - .8 .4 interior Icinetoplast to posterior nucleus. 1.4-2.5 2.1 Anterior nucleus to anterior extremity. 11.5 - 20 14.5 Length of free flagellum. 6-12.6 8.4 Length of nucleus. 1.6-3 2.2 Width of nucleus. 1.2-2.1 1.6 Length of kinetoplast, ,4-1,1 .7 Width of kinetoplast, ,2 - ,5 .3 Nuclear Index ( posterior end to centre of nucleus / centre of nucleus to anterior .OT . ^ '21 - '2-8 ^' extremity). *" Kinetoplastic Index ( posterior end to centre of ?! nucleus - / centre of kinetoplast to centre of nucleus). i'D^)- ^'^

Movement of Parasite In citrate solution,parasites shovfed wriggling movement. 73

Measurements ( in micrometersv™\) of Trypanosoma vittatae Robertson,1908. Type. II. Intermediate forms.

Range. Averagg:, Total length of body including free flagellum. 35-42 38.9 Length of cell body. 23-33 28.1 Width of cell body. 2.4-3,5 2.6 Posterior extremity tooposterior kinetoplast, .01-3.4 1.8 Anterior kinetoplast to posterior nucleus. 1.2 --2.4 1.4 Anterior nucleus to anterior extremity. 18 - 23.5 21 Length of free flagellum. 8-12 10.4 Length of nucleus. 1.4-2.9 1.6 Width of nucleus. 1.4-2 1.5 Length of kinetoplast. .4 -.7 .5 Width of kinetODlast. ,3 - .5 .4 Nuclear Indeix (posterior end to centre of nucleus / centre of nucleus to anterior '2 extremity. •12- -32 Kinetoplastic Index ( posterior end. to centre of nucleus / centre of kinetoplast to centre of nucleus). 1-09- 1'9 ^'^

Movement of parasite

In citrate solution,parasites showed wriggling movement. T4

Measurements (in micrometers. Aim) of Trypanosoma vittatae Robertson,1908. Type. III.. Broad slender forms.

Range. Average. Total length of body including free flagellum. 54 - 70.8 60.3 Length of cell body. 37 - 44.5 41.1 Width of cell body. 2.2-6 3.3 Posterior extremity to posterior kinetoplast. 3.8-6.6 5.2 Anterior kinetoplast to posterior nucleus. 2.6-4.8 4 Anterior nucleus, to anterior extremity. 24.9 -31.5 27.8 Length of free flagellum. 12.4 - 23.4 18 Length of nucleus. 1.1-3.5 2.4 Width of'nucleus. 1-2.1 1.7 Length of kinetoplast. .7-1.3 1 Width of kinetoplast. .2 - .5 .3 Nuclear Index ( posterior end to centre of nucleus / centre of nucleus to anterior extremity). '2 9-- '-43 '^^ Kinetoplastic Index ( posterior end to centre of nucleus / centre of kinetoplast to centre of nucleus.). Z- ol - I'l ^

Movement of parasite In citrate solution, parasites shov/ed v/riggling movement. 75

Discussion

Parasite under discussion belongs to genus Tr^/panosoma on account of an undulating membrane and a posterior kinetoplast and has been identified as Trinoanosoma vittatae Robertson,1908 on the basis of the following characters. Parasite is polymorphic, shov/ing three distinct morphological types viz., small,intermediate and large forms. These also come close to the m.easurements as described by Robertson. However,the following differences are also noted with the present parasite. For example,myonemes of large forms are not present and divisional staqes are only encountered in small forms as in the present case. Differences noted above are minor in nature and not sufficient for consid-sring the present parasite as new one. Hence,parasite under report is identified as Trypanosoma vittatae. This is the first record of Tr^npanosoma vittatae from a host other than Emyda vittata .

Diagnosis Family. Trypanosomatidaee Doflein,1901;emend Grobben,1905. Single nucleus and a kinetoplast; flagellum arising from base of kinetoplast 76

Genus- Trypanosoma Gruby^1843. Pol;^miorphic ; kinetoplast posterior to nucleus; flagellum forming undula­ ting membrane. Specific Characters. Trypomastigotes exhibiting three morph­ ological types. Type. I. Thin slender foriTis measuring 23.9/um - 30.6/um X 1.4 Aim - 6.2/um including free flagellum. Type. II. Intermediate forms measuring 35 /um - 42 /um X 2.4 /ura - 3.5 /um inclu­ ding free flagellum. T\-p.e. III. Broad slender forms measuring 54/Jim - 70.8/um X 2.2/um - 6/um including free flagellum. The present parasite is referred to as Trypanosoma vittatae Robertson,1908.

Hdst. Lissemys punctatus (Bonnaterre) Site of infection. Peripheral blood plasma. Locality : Bongaon,24-Parganas,West Bengal,India. APPEITOIX (6 - i). ACTA PROTOZOOLOGICA VOL. 15 (No. 4) WARSZAWA, 31 XII 1976 pp. 393-398

Zoological Survey of India, 8, Lindsay Street, Calcutta-700016, India

C. K. SINHA and A. K. MANDAL

Trypanosoma enhydris sp. n. from a Fresh Water Snake Enhydris enhydris (Schneider)

Synopsis. The description of a new species of Trypanosome, Trypano- somo enhydris sp. n. (TrypanosomaUdae) from a fresh water snake, En­ hydris enhydris collected from West Bengal, India is incorporated in the paper. Its affinities with the known species of the genus and differen­ ces to consider it as new species are also incorporated.

This is the first instalment of the series deals with the haemoflagel- lates of some Indian reptiles. It includes the description of a new species f>i trypanosome from a fresh water snake Enhydris enhydris (Schneider). The snakes were captured from Chakdah, Nadia, West Bengal, India du­ ring the month of March to Jxme 1975, and brought to the laboratory for examination. Out of the 50 snakes examined 30 were found positive for trjrpanosome. Blood films were obtained by clipping the tail and stained with Giemsa's, Leishman's stain and Wright stain. Tissues fixed in Bouins Duboscq brasil's fluid for studying endogenous stage. No tisstie phase is seen in the preparation. However, observation to note down the tissue phase, intermediate vector along with some cross transmission experiments are being carried out in th^ laboratory. The results of which will be pub­ lished in due course. Drawings were made with the help of a camera lucida with the uniform magnification 1600 X. Measurements were made from camera lucida drawing along a line drawn from the anterior to the posterior end through the middle of the parasite. Estimated parasitemia 2-5 trypanosomes per cubic mm of blood films. The slides will be deposited to the National collection of the Zoological Survey of India.

Description Trypanosoma enhydris sp. n.

Holotype: Z. S. I. Registration No 1835 Type Host: Enhydris enhydris (Schneider) I Type Locality: Chakdah, Nadia, West Bengal, India. 394 C. K. SINHA and A. K. MANDAL

The trypanosoma is poljonorphic. Three distinct forms viz. small with free flageUum of considerable length, intermediate with a lor^ free fla- geUum and a large form with a short flageUum are seen in the peripher­ al blood. Some undergoing division along with the epimastigote forms have also been encountered. In the citrate solution, they were found sluggish and sometimes form a knot. Measurements of the organism are given in Table 1.

Table 1 Measurements of Trypanosoma enhydris sp. n. (in microns)

Fonn Small Intermediate Large

Length from posterior end to the kincto- plast 8.5 (7.5-9)* 16 (15.5-17.5) 39 (38-49) Length from kinetoplast to the nucleus 2.15 (2-3.5) 2.5 (2-3) 1.5 (1-2) Length of the nucleus 1.75(1.50-2.50) 2 (2-2.5) 6(5-7) Length from nucleus to anterior end of body 15 (14-16.5) 18.5 (17-19.5) 60 (55-62) Length of the free fiagellum 13 (12-14) 15 (14.5-17.5) 10 (9-11) Length of the kinetoplast 0.86 (0.5-1) 0.85 (55-1.5) 1.5 (1-2) Width of the kinetoplast 0.45(0.40-0.86) 0.65 (55-0.86) 0.40(0.40-0.42) Width of cell body 2 (1.5-2.4) 5 (4-5.5) 27 (24-30)

* Mean and range (in parantheses).

Small form

Slender, elongated and sharply pointed at both ends (Fig. 1). "nieir niHnber varies from 5 to l" in the films. Cytoplasm homogenous with scattered small vacuoles. It is deeply stained with Giemsa and more to­ wards the border opposite to undulating membrane. Cytoplasm towards periphery stains faintly and become almost without any stain at iiie post­ erior extremity. No myonemes or granular arrangements are found on the cytoplasm. Nucleus exhibits a round or oval shape situated almost at the middle. The chromatin granules are arranged towards the distinct nuclear mem­ brane sometimes leaving a decided gap at the centre. Kinetoplast almost subspherical situated slightly posterior to the nu­ cleus and appeared as a black dot with a halo around, hardly conical form with deep stain is noticed in the preparation. FlageUum originates from the base of the kinetoplast, trails anteriorly bordering the undulating membrane and extends beyond the body as free flageUum. TRYPANOSOMA ENHYDRIS SP. Ni 395

Figc. 1-6. Trypanosoma enhydris sp. n. 1 — Small form, 2 — Intermediate form, 3 — Large form of T. enhydris in peripheral blood, 4 — Epimastigote form of T. enhydtis in the peripheral blood> 5 — Trypanosome (divisional stage) with two nuclei and two kinetoplast, 6 — Two daughter individual after division. (Figs. 1, 2, 4, 5 are in one, and Figs 3, 6 in other magnification) 396 C. K. SINHA and A. K. MANDAL

The undulating membrane is conspicuous with three or four folds and stains light red with Giemsa. The body margin bordering the undulating membrane is evenly curved and does not follow the undulations of the membrane. y

Intermediate form

This form (Fig. 2) resembles the small one morphologically but varies in size (Table 1) and the number of folds ui the undulating membrane. The folds touch the body five or six times. They are in great abundance in the peripheral blood approximately 15-20 in each film.

Large form

They are few in number sometimes about 3 to 5 in one film; heavily stained body with a narrow undiilating membrane and a short free fla- gellum (Fig. 3). Cytoplasm coarse, vacuolated and takes a deep stain. Nucleus oval, stains very light with faintly granular chromatin materials. Kinetoplast adheres to the nucleus and stains deeply. The folds of the imdulating membrane waved 12 to 14 times bordered by the thick fla- gellum with a short free portion at the anterior end. In addition to these, some epimastigote forms (Fig. 4) are also encount­ ered in the peripheral blood where a dark blue kinetoplast is located at the anterior end very close to the nucleus. This form measures about 23j (inii to 27 fim (mean 25 (xm) by 3.5 ^nx to 4.5 |.im (mean 4 iim) with a free flagellimi 9 (i to 10 jim in length. Moreover, some divisional stages with two kinetoplasts and two nuclei almost divided with their cytoplasm (Fig. 5) have been found in the peripheral blood along with some daiight- er trypanosomes (Fig. 6).

Diagnosis of Trypanosoma enhydris sp. n.

The described haemoflageUate is polymorphic; small, intermediate and large; measuring 40 [xm by 2 [im, 54 fim by 5 (xm, and 116 um by 27 [xm respectively in total length including the free flagellum. Cytoplasm homogeneous with few vacuoles and without any volutin granules. Kinetoplast always away from posterior end, nucleus sub-cen­ tral and situated at the middle of the cytoplasm; undulating membrane distinct, prominently bordered by the thick flagelliim with considerable free end. The body cytoplasm does not participate in forming the folds of undulations. TRYPANOSOMA ENHYDRIS SP. N. 397

Discussion

Walliker (1965) reviewed the r<8ptilian trypanosomes and listed fifteen snake trypanosomes of which three were reported as Trypanoso­ ma sp. The species under report resembles Trypanosoma primetti Mathis et Legger, 1909 (Host: Torpidonotus=Natrix piscator) due to body configurat­ ion and shape, but the former is polymorphic and the latter is dimorphic. Moreover, the total length of the polymorphic forms including the free flagellum, measure about 40 ]xm by 2 ^m, 54 jim by 5 ycm, and 116 ixm by 27 um, of the species encountered; whereas in Trypanosoma primetti the dimorphic forms measure 57 ^m by 7 um and 105 i^m by 14 [xm. Another species of trypanosome from Natrix piscator was reported in Pakistani Science congress by Haq and Mohiuddin (1956), without giving any detail account. So the specimen dealt herewith could not be possible to compare with that species. However, efforts were made to inoculate the present trjrpanosome to Natrix piscator in the laboratory but were unsuccessful. Simultaneously the present trypanosome was ino^ culated to infection free Enhydris enhi/dris, the natural host and found * to be positive after 3 to 5 days. These experiments led us to conclude that the present trypanosome is , quite different from those of the species occurring in Natrix piscator. In addition, the present species does not approaches any other known try­ panosomes hence it is described as new and designated as Trypanosoma enhydris sp. n. The specific name of the parasite was given after the specific name of the host.

ACKNOWLEDGEMENTS

We are very indebted to Dr. S. Khera, Deputy Director-in-Charge, Zoological Survey of India for providing us laboratory facilites. Thank are also due to the Dept. of Science and Technology, Govt, of India for kindly grant the financial assistance to one of us (C. K. Sinha) and to Dr. T. D. Soota, for constant encoura­ gement.

RfiSUMfi

L'article s'agit de la description d'une nouvelle espfece, Trypanosoma enhydris (Trypanosomatidae), recueillie d'un serpent, Enhydris enhydris (Schneider), qui vit dan I'eau douce du Bengale occidental de I'lnde. Son affinite avec les especes con- nues du genre et ses caracteres tr^s distingu6s sent aussi mentiones. 398 C. K. SINHA and A. K. MANDAL

REFERENCES

M a t h i s C. and L e g e r M. 1909: Sur un nouveau trypanc«ome des serpents du Tonkin. C. r. Seanc. Soc. Biol., 67, 572-574. Haq S. M. and MohiuddinA. 1956: On a new trypanosome species from fresh water snake Natrix piscator. Proc. 8th Pakistan Sci. Conf. Biol., p. 48. Walliker D. 1965: Trypanoscmia superciliosae sp. nov. from the lizard XJranos- codon superciliosa. Parasitology, 55, 601-606. Received on 10th April 1976 APPENDIX (6 — ii) ACTA PROTOZOOLOGICA VOL. 17 (No. 1) WARSZAWA, 31 III 1978 pp. 9-12

Post-Qraduate Zoology Depaitment, Darje^ng Govemaaoent College, Daileeilng. mdia

C. K. SINHA

Trypanosoma gangetica sp. n. from a Fresh Water Turtle Trionyx gangetieus Cuvier Synopsis. The paper deals with a new species of monomorphic Trypa­ nosoma, Trypanosoma gangetica sp. n. (Trypanosomatidae) from a soft leathered turtle Trionyx gangetieus Cuvier collected from West Bengal, India. The morphology of the haemoflagellate has been described and it is compared with other known chelonian trypanosomes to consider it as new species.

Trypanosomes have been reported from the peripheral blood of different species of chelonians. L a v e r a n and M e s n 11 (1902) describ­ ed Trypanosoma domoniae from the tortoise Damonia reevesii. Button and Todd (1903) and Dutton et al. (1907) noted the presence of trypanosomes in different tortoises of Gambia. Bouet (1909) reported Trypanosoma pontyi from a tortoise Stemotherus derhianus of Africa. Trypanosoma chelodina was recorded from Chelodina longicollis by Johnson (1907) from Australia. Walliker (1965) reviewed the occurrence of reptilian trypanosomes and enlisted twelve chelonian try- panosome of which two were reported as Trypanosoma sp. In India, studies on chelonian trypanosomes have not so far been recorded. The turtles Trionyx gangetieus Cuvier, were collected from local markets of Bongaon, West Bengal, India In March 1975 with a view to study haematozoa. Fifteen tiutles were examined and five were positive for a haemoflagellate of the genus Trypanosoma in the circulating blood. No ecto-parasitic infestation was noticed. Thin blood films were drawn and stained with Giemsa and Leishman stain. Drawings and measure­ ments were made with the aid of camera lucida, along a line drawn from posterior to anterior end of the flagellate at the imiform magni­ fication of 1500 X.

Results

Trypanosoma gangetica sp. n. Type Host: Trionyx gangetieus Cuvier Type Locality: Bongaon, 24 Parganas, West Bengal, India. 10 C K. SmHA

Trypanosoma gangeUca sp. n. showed monomorphic form and were abxmdant in the blood film. AH the forms were long and slender. This form (Fig. 1 1-4) of flagellate is elongated with sharply pointed ends, Cytoplasm is homogenous and stains light pink with Giemsa. It contains 7-10 vacuoles. Nucleus is round or oval in shape. It always lies at the nxiddle of the body with a distinct nuclear membrane. Kinetoplast is a spherical dot like structure situated away from the pcsterior part of the nucleus and takes deep stain with Giemsa and Leishman. Flagellum is found to emerge from the kinetoplast and has 6 to 8 attachments with the undulating membrane proper and leaves it as a distinct free flagellum. A well stretched form exhibits the maximum width of the

Fig. 1 1-4. Trypanosoma gangetica sp. n. Camera lucida drawmgs. ,^-3—Jf°f slender form, note the distinct free flagellum and the vacuoles. 4 —WeU stretched condition of the long slender form TRYPANOSOMA GANGETICA SP. N. 11

Table 1 Measurements of Trypanosoma gangetica sp. n. (in \aa)

Characters Range Mean Total length of the parasite (including free flagellum) 53.4-58.0 55.7 Length of the cell body 33.7-36.7 34.7 Breadth of the cell body 1.8- 4.8 2.45 Length of the free flagellum 19.2-21.9 20.9 Length of the nucleus 2.2- 2.4 2.3 Breadth of the nucleus 1.3- 1.8 1.5 Length of the kinetoplast 0.6- 0.9 0.8 Breadth of the kinetoplast 0.7- 1.0 0.8 Post-kinetoplast distance 4.1- 4.6 4.38 Anterior edge of kinetoplast to posterior edge of nucleus 12.1-14.50 13.55 Anterior ed^ of nudeiis to anterior end of cell body 12.8-14.80 13.7 cell body- (Fig. 14). No, divisional phase has been observed so far (Table 1).

Diagnosis of Trypanosoma gangetica sp. n. The described haemoflageUate is monomorphic measuring 55.7 |im by 2.45 (un in total length including the free flagellum. Cytoplasm is honaogenous having 7-10 vacuoles and without any volutin grantjles. Kinetoplast is always away from the posterior part of the nucleus. Nucleus is always centrally situated in the cdl body. A prominent vmdulating membrane with 7-8 folds. The body cytoplasm does not enter inside the fold of it. A long free flagellum measuring 19.2 (im is also the characteristic f eatvire of this parasite.

Discussion

Trypanosomes appeared to be non-pathogenic to their host. The peculiarity of the present species of trypanosome described in the paper is that it has a long free flagellum measuring 19.2-21.9 jun and mean length being 20.9 |xm. Generally vertebrate trypanosomes reveal more than one morphological feature in the peripheral blood. Robertson(ije«^ 1909) discovered Trypanosoma vittatae in the soft tortoise Emyda rnt- tata which was polymorphic. The parasite under report lacks this feature. t/vt«i. by Trypanosoma vittatxis was also recorded by Robertson (JUi'nKt*^^'^*^^*''*^) from a tortoise Lissemys punctata granosa from Ceylon. The parasite 12 C. K. SINHA was 70 nm long and is different from the present form. The species under report draws a close affinity with Trypanosoma chrysemydis Roudabush and Coatney (1937) from Chrysemys belli marginata but it differs in many respect. T. chrysemydis measures 46.8-50.0 (im by 3.15-4.05 [xm. Moreover, it has got a short free flagellum measuring 13.05 urn. However, this flagellate differs from all other known chelonian trypanosomes. Considering all these aspects the trypanosome from Trio- nyx gangeticus Cuvier, has been assigned a new species status and named Trypanosoma gangetica sp. n. This turtle is recorded for the first time as a host for trypanosome. The holotype and paratypes of Trypanosoma gangetica sp. n. will be deposited in the national zoological collection of Zoological Survey of India, Calcutta in due course.

ACKNOWLEDGEMENTS

The author is indebted to Dr. B. Dasgupta, Principal, Darjeeling Government College for his valuable guidance and constant encouragement during the course of observations. Thanks are due to Shri Nilachal Chattaraj and Shri Sukdeb Sinha for their manifold help.

ZUSAMMENFASSUNG Trypanosoma gangetica sp. n. wird von einer Siisswasser Schildkrote Trionyx gangeticus Cuvier beschrieben. Die Morphologie des Parasiten und seine Bezie- hungen zu anderen bekannten Arten Wird diskutiert

REFERENCES

Bouet G. 1909: Sur quelques trypanosomes des vert^brfes k sang froid de I'Afrique occidentale frangaise. C. r. Seanc. Soc. Biol., 66, 609. Dutton J. E. and Todd J. L. 1903: First report of the Trypanosomiasis Expe­ dition to Senegamtoia (1902). Liverp. Sch. Trop. Med., Mem. 11, 1. Dutton J. E., Todd J. L. and To bey E. N. 1907: Concerning certain parasitic protozoa observed in Africa. Ann. Trop. Med. Parasitol., 1, 287. Johnson E. A. 1907: A trypanosome found in the River Murray turtle (Chelo- dina longicMis). Australas. Med. Gaz., 26, 2fi. Laveran A. and Mesnil F. 1902: Sur quelques Protozoaires parasites d'une tortue d'Asie (Damonia reevesil). C. r. Acad. Sci., 135, 609-414. Robertson M. 1908: A preliminary note on Haematozoa from some Ceylon reptiles. Spolia Zeylan., 5, 178-185. Robertson M. 1909: Studies on Ceylon Hematozoa. No. I. The life cycle of Trypanosoma vittatae. Q. J. microsc. Sci., 53, 665. Roudabush R. L. and Coatney G. R. 1937: On some blood protozoa of reptiles and amphibians. Trans. Am. Microsc. Soc, 56, 291-297. WaHiker D. 1965: Trypanosoma superciliosae sp. nov. from the lizard UT(inos- codon superciliosa. Parasitology, 55, 601-606. Received on 13 May 1977 77

CPiEa<-LIST OF Trypanosoma FOUND IN SGMJE LO^^JER

WmTEBRATSS IN INDIA.

( * indicates new species described in the thesis) ( ** indicates Icnoi-m species reported here)

Family. Trypanosomatidae Doflein,1901;emend Grobben^l905. Genus, Trypanosoma Gruby,1843. Trypanosoma in Fresh vrater fishes. r. Trypanosoma sp.. Lingard,1904 In the blood of Channa striatusy collected from Jamuna river,> Uttar Pradesh and also from Pune,Maharastra, Qadri,1951,

In the blood of same host fish,, coSilected from Hyderabad, Andhra Pradesh, 2. Trin^anoson^a sp. Linqard, 1904. In the blood of Puntius camaticus,collected from Pune, Maharastra. 78

3* Trypanosoma sp. Lingard,1904. Xn the blood o£ Ryncobdella aculeata , coilected from Ptme/M'aharastra. 4. Trypanosoma sp. Lingard,1904. In the blood of Trichogaster fasciatus , collected from Jamuna river,Uttar Pradesh.

5 .Trypanosoma sp.. Lingard, 1904. In the blood of Mystus seenghala , collected from Jamuna river,Uttar Pradesh. Tandon &-Joshi, 1974; Tandon & Chandra, 1977; Joshi,1979. In the blood of the same host fish ,collected from Lucknov/,Uttar Pradesh,

6. Trypanosoma sp. Lingard,1904. In the blood of Mystus tengara, collected from Jamuna river,Uttar Pradesh. 7. TrA;^panospma sp. Tandon & Joshi,1974 ; Joshi', 1979. In the blood of Channa punctatus', collected from Lucknow,Uttar Pradesh. 8. Trypanosoma sp. Tandon & Chandra,1977; Jbshi,1979. In the blood ;'• of Mastocembelus , arTTiatus, collected from Luckhow,Uttar Pradesh. 19?

5» Trypanosoma sp. Tandon & Joshi,1974/ Tandon & Chandra,1977; Joshi,1979. In the blood of Clarias batrachus, collected from Luckno\'>7,Uttar Pradesh.

10. Trypanosoma sp. Tandon & Chandra,1977; Joshi,1979. In the blood of Cirrhina mrigala , collected from Luc>Enovj,Utt*&r: Pradesh. 11., Trypanosoma sp. Tandon & Chandra, 1977 ; Joshi, 1979. In the blood of Wal1ago attu,collected from Lucknov/,Uttar Pradesh. r2. Trypanosoma sp. Joshi, 1979, In the blood of NOtopterus notopterus, collected from Lucknow,Uttar Pradesh. 13. Trypanosoma sp. Joshi, 1979. In the blood of Puntius stigma,collected from Lucknow,Uttar Pradesh. 14. Trypanosoma sp. Joshi,1979. In the blood of Labeo bata,collected from Lucknovr,Uttar Pradesh. 15. Trypanosoma sp. Joshi,1979. In the blood of Mystus aor,collected fHOM Lucknow, Uttar Pradesh, 16. Trypanosoma sp. Joshi,1979. In the blood of Channa gachua,collected from Lucknov/,Uttar Pradesh. 80

^"^-^ Trypanosoma sp. Mukherjee & Haldar, 1979. In the blood of Nandus; nandus,collected from I<:alyani,Nadia,West Bengal. 18. Trypanosoma sp. Sinha,1980. In the blood of Lepidocephalus g\xntea^ collected from Bongaon,24~Parganas,West Bengal. ^5» Trypanosoma clarlae batrachi de Mello S: Valles,1936. In the blood of Clarias batrachus_, collec­ ted from Goa. 20. Trypanosoma striati Qadri,1955. In the blood of Channa striatus,collected from Hyderabad,Andhra Pradesh, 21. Trypanosoma batrachi Qadri,1962. ,, In the blood of Clarias batrachus^collec- ted from FIyderabad,Andhra Pradesh. 22. Trypanosoma danilewski var saccobranchi Qadri,1962. In the blood of Hetegopneustes fossilis, collected from IiEyderabadjAndhra Pradesh. 23.. Trypanosoma punctati Hasan & Qasim/1962. In the blood of Channa punctafcus/collected from Hyderabad,Andhra Pradesh. 24. Tr-^/panosoma elongatus Raychaudhuri &, Misra, 1973, In the blood of Channa punctatus,collected from local market of Calcutta,West Bengal. 81

** 25. Trypanosoma qachuii Mlshra^Chandra & ChoudhurY,1973. In the blood of. Channa gachua,collected from local markets of Calcutta,West Bengal. Reported here from the same host fish/ collected from Baruipur/24-Parganas,West Bengal. 26. Trypanosoma mukundi Raychaudhuri & Misra,1973. In the blood of Heteropneustes fossilis, collected from local markets of Calcutta, West Bengal. 27. Trypanosoma vittati Tandon & Joshi,197 3. In the blood of Mystus vittatus,collected

from Calcutta,West Bengal.

28.Trypanosoma maguri' Tandon £ Joshi,1973. In the blood of CIarias batrachus,collec- ted from Calcutta,West Bengal. 29.. Trypanosoma baiqulensis Pandey & Pandey, 1974'.

In the blood of Cirrhina reba and Osteobrama cotio^collected from Nainital, Uttar Pradesh. **30. Trypanosoma armeti Mandal,1975. In the blood of Mastocembelus armatus:^ collected from Champahati,24-Parganas, West Bengal. Reported here from the same host fish, collected from Chakdah,Kfadia,West Bengal. 82

**31, Trypanosoma pancali Mandal/1975 In the blood of Mastocembelus pancalus^ collected from Champahati,24-Parqanas, West Bengal.. Reported from the same host fish,collected from Baruipur,24-Parqanas,West Bengal. 32. Tr^njanosoma mrigali Joshi, 1976 In the blood of Cirrhina mriqale,collected from Gomati river ,IJUcknow,Uttar Pradesh, 33.:. Tr^n^anosoma seenghala Joshi, 1976. In the blood of Mystus seenghala,collected from Gomoti river,Lucknov/^Uttar Pradesh.

** ^^•' Trypanosoma cancili' Mandal,1978. In the blood of Xenentodon cancila,collected from Raidighi,24-Parganas,West Bengal. Reported from the same host fish,collected from Bongaon,24-Parganas,West Bengal, 35. Trypanosoma anatoasi Mandal,1978. In the blood of Anabas testudineus,collected from Canning/.24-Parganas,West Bengal, 36. Trypanosoma batai Joshi,1978. In the blood of Labeo bata,collected from Gomati river and Chinhat Lake,Lucknow, 1 Tlttar Pradesh,. 83

37. Trypanosoma stlqmai Joshi,1978. In the blood of Barbus stigma/ collected from Gomati river and Chinhat Lalce,Lucknow, Uttar Pradesh,

38. Trypanosoma benqalensis Mandal,1980 In the blood of Mystus bleekeriycollected from Canning,24-Parganas,West Bengal, 39.. Trypanosoma tandoni f4andal, 1980. In: the blood of Wallaqo attu,collected from Champahati,24-Parganas>West Bengal,

40. Trypanosoma channai Naraslmhamurti Si Saratchandra, 1980, In the blood of Channa punctatus,collected from Visakhapathnam and Srikakulum, Andhra,Pradesh,

41.Trypanosoma qadrli Naraslmhamurti Si Saratchandra, 1980, In the blood of Clarias batrachus, collected from Visakhapathnam and Srika- kulum,Andhra Pradesh. 42. Trypanosoma trichoqasteri Gupta & Jairajpuri,1981. In the blood of Trichogaster fasciata, collected from Aligarh,Uttar Pradesh. 43.Trypanosoma aori Joshi,1982. in the blood of Mystus aor,collected from Gomoti river,Lucknow,Uttar Pradesh. 84

44. Trypanosoma rupicoli Joshi,1983. In the blood of Hemacheilus rupicola collected from Kosi river near Kosi station,Uttar Pradesh. **45. Trypanosoma clariae Montel,1905. In the blood of Glarias batrachus/colle- cted from Chakdah,Nadia,West Bengal. **46. Trs^panosoma mul< as ai Ho are ,1932. In the blood of Tilapia mossambica,colle-. cted from Bongaon,24-ParganaS/West Bengal, *47. Trypanosoma n.sp.(a) In the blood of Lepidocephalus guntea, collected from Bongaon,24-Parganas, West Bengal,

Trypanosoma in Anura

1. Tjrypanosoma sp. Donovan (cited by Wenyon, 1926). In the blood of Bufo melanostictus> Locality not stated. 2. Trypanosoma sp. Donovan (cited by Vfenyon, 1926) In the blood of Rana hexadactyla. Locality not stated. 85

3. Trypanosoma sp. Berestneff,1903. In the blood of Rana tigrina,collected from Boinbay^Maharastra.

'^* Trypanosoma sp, Berestneff, 1903. In the blood of Rana limnocharis:,collected from Bombay,Maharastra. 5, Trypanosoma sp. Patton,1908 (Cited by Wenyon,1926) In the blood of Rana hexadactyla. Locality not. stated. 6, Trypanosoma sp.^ Scott, 1926 &,: 1927. In the blood of Rana tigrina. Locality not stated. T. Trypanosoma rotatorium (Mayer,1843) Berestneff,1903; Efetton,19e8, ; Scott,1926,1927;Ray,1979a,b; Ray &,. Handi,1978,In the blood of Rana Ilmnocharis,collected from different parts of India. -' • Trypanosoma rotaterium (Mayer, 184 3) Wenyon,1925; Ray,1979 a,b,1980; Sinha,1981. In the blood of Bufo melanostictus,collected from some parts of India. Berestneff,1903;Patton,1908; Scott,1925,1927;Pujati,1953; Ray,1979 a,b,1980 ; Ray & Choudhury,1980,1981,1983. In the blood of Rana tigrina, collected from different parts of India. Pujati,1953; Ray,1979 a ,b,1980. In the blood of Rana cyanophlyctis, collected from some parts of India. 86

Ray, 1979 a,b,1980.. In the blood of Bufo stomaticus,Rhacophorus maculatus and Rhacophorus malabaricus ,collected from different parts of West Bengal and other states.

8... Trypanosoma loricattjm (Mayer, 1843) Ray, 1979b, 1980. In the blood of Rana limnocharls,collected from Balitha and Bishnupur,Bankura,West Benqal. 9. Trypanosoma ranarum (Lanlcester,1871) Damayanthi S.Rao,1979. In the blood of Rana sp, and Tadpoles;collected from Warangal,Andhra Pradesh.

Ray Si: Choudhury ,1983, In the blood of Rana tiqrina,collected from Bankura, Vfest Benqal. 10. Trypanosoma kar^^^ozeukton Dutton & Todd, 1903.

Ray,.1979b, 1980; Ray S-Choudhury, 1983. In the blood of Rana hexadactyla,collected from Salikona,,Hooghly,West Bengal. ir. Trypanosoma Inopinatiau Serqent & Sergent, 1904.

Patton,1908;Ray & Choudhury/1983. In the blood of Rana hexadactyla,collected from South India^and Bankura,West Bengal.

Patton,1908; Ray & Choudhury,1983. In the blood of Rana tigrina,collected from South India and Bankura,Vfest Bengal.. 8T

12. Trypanosoma chattonl Mathis &Leger,1911. Ray,1979a,b,1980; Ray & Choudhury,1983, In the blood of Bufo melanostictus^ Bufo stomatlcus ,Rana limnochards:- , Ran a tiqrlna, Rhacophorus:: maculatus, Rhacophorus malabaricus, Microhyla orntLta,collected from Volpoi',Mollem and Bondla,Goa. 13.. Trypanosoma taprobanlea Ray & Choudhury,1983. In the blood of Kaloula pulchra taprobanica ,collected from Santaldi, Purulia,West ^ngal. 14. Trypanosoma malabarica Ray & Choudhury,1983. In the blood of Rana malabarica, collected from Volpoi', Goa. 15.Trypanosoma systoma Ray & Choudhury,1983. In the blood of Uperodon systoma Midnapore, West Bengal.

Trypanpsoma in CheIonia

1. Trypanosoma gangetica Sinha,1978. In the blood of Tirionyx gangeticus collected from Bongaon,24-Parganas, West Bengal, 88

**2. Trypanosoma vlttatae Robertson,1908 In the blood of Llssemys punctatus collected from Bongaon,24-Parganas, West Bencfal.

Trypanosoma in Ophidia

1. Trypanosoma sp. Haq S: Mohiuddin, 1956. In the blood of Xenchrophis piscator;-. Locality not stated, 2. Trypanosoma enhydris Sinha & Mandal,1976, In the blood of Enhydris enhydris; collected from Chakdah, Nadia, West Bengal. ^^a

"^*^ 0» SSgmoc [?ec S£^a i^ SOMS ^^Loux^ ^S mu: OPBIDlAJrs« i 89

USTRODirCTIOff

Haemogregarines were termed " haemo-cocciTlia?'^ by Reichenow (1910),. OHiese parasites are found to adapt themselves to: the circulating; cells of the blood of different vertebrate animals. according to Wen^n (1926) haemogre­ garines fall into two groups^viz.^ type and type, depending on the nature of their processes of fertilization;. In the former type,the male gametocyte produces' a large number

. of microgametes which develop not in association"'with macrogamete. The sporozoites? are the only forms in red blood corpuscles. Adelea type is characterized by male gametocyte developing in association with female gametocyte,the so called syzygy,and.:. producing two or four microgametes. The forms; in blood corpuscles are gametocytes,and occasionally,sehizonts.

According to Grasse (1953) Family Haemogregarinidae Leger ,1911, contains•four genera viz,, :^emogre g arin a Danilewsky,1885, Hepatozoon Miller,1908, Labbd',1894 and Shiith & Johnson, 1902. According to Levine,et al., (1980) the suborder Adeleina consisted of three families viz., Haemogregarinidae , Hepatozoidae and under the Class Sporozoea. 90

The Family Haemogregarinidae contained a single genus H&emogregarinct which was known to occur: in cold­ blooded vertebrates. Recently/Lainson(1981) created another genus under the Family Haemogregarinidae to include two fish haemogregarines-which showed more than eight sporozoites in a mat\are oocyst. He stated " it is significant that both of the species of Cyrilia described are parasites;of fish. It may well be that the genus is limited to these animals and Haemogregarina to terrestrial vertebrates?'.. Accordingly,in this chapter the haemogregarines of vertebrates who are primarily terrestrial,are placed under the genus H&emogregarina till more knowledge about their life cycles both in vertebrate and invertebrate hosts are known. Haemogregarina are on record from a large number of cold-blooded vertebrates. According to Sinton (1925)/ and Wenyon (1926) Haemogregarina recorded from man,, monkeys and ox are simply vegetable organisms. In blood films,Haemogregarina are found in the form of gametocytes in various stages of developmentvas schizonts and as merozoites. Asexual reproduction also takes place in the internal organs of vertebrate -.hosts. 91

A good number of reptilian haemogregarines have been named and described by various investigators' from time to time under genus Haemogre garin a depending on the morphological characteristics of gametocytes seen in peripheral blood smears. There are only a few records of schizogonic or sporogonic stages. Morphological features in erythrocytic forms do not always provide a clear basis for identifying the parasite. In India, there has been little work on Haemogreqarina . In the present chapter, the results^ of a survey of Chelonian and Ophidian Haemogregarina encountered in some parts of world West Bengal are described. The geographical and hostal differences relating to the haemo­ gregarines described in this work appear to be enogh to A distinguish them from previously named species given the present very imperfect state of our knowledge of this group. REVIEW OF LITERATURE

First haemogregarine known to science was Haemogregarina minima, a parasite of the edible ^^^9 ( Rana esculenta),described and figured by Chaussat in 1850, He considered it to be a minute nematode and named it" Anguiirula minima . Lankester (1871) also noticed and figured the free sporonts of Haemogregarina minima. Gaule (1880) described the endo-corpuscular forms of the frog haemogregarine,which he termed "BlutwUrmchen"' and stagd that 92 these vrere simply the degenarating product of blood corpuscles and tissue cells. Lanlcester(1882) disagreed v/ith Gaule and upheld the parasitic nature of Haernogreqarina minima, which he proposed to call Drepanidium ran arum..

Haernogreqarina in Chelonia

CheIonian Haemogreqarina was first detected by a Russian worker,Danilev/sky (1885) in the blood of an European water tortoise,Bmys orbicularis which he named Haemogregarina steioanov/i and its develop­ mental staqes v/ere found in the leech Flacobdella cateni- gera. Bomer (1901) described Haemogregarina labbei -^" Chrysemys scripta elegans • and Platemys su. from North Jimerica. Simond (1901) described Haemogregarina billet! in a turtle (. Trionyx cartilaqineus) of Tonkin. Laveran & Mesnil (1902) described Haemogregarina stepanowiana snd Haemogregarin a rara in tortoises (Damonia reevesii) of Sri Lanka. Doucloux (1904) described Haemogreqarin a bagensis ih'Clemmys (=^mys) leprosa from Algiers. Castellani & Willey (1904) described Haemogregarina nicoriae from the blood of Geoemyda trijuga and described its life cycle in leech,Oizbranchus shipleyi collected from Sri Lanka. Robertson(1908) described Haemogregarina vittatae in ISmyda vittata of Sri Lanka. Hahn (1909 ) reported Haemogregarina stepanowi in the blood of turtles. Johnston (1909) described Haemogreqarina clelandi in Che 1 Odin a oblong a from Australia, Lave ran :&' Pettit (1909, 1910) described Haemogreqarina chelodinae and Haemo g re q ar in a pelleqrini in Chelodina lonqicollis and Damonia subtrijuga of Algiers and. Asia, Reichenow (1910) worked on the biology of Haemogreqarina stepanowi and gave a detailed account of its structure and life cycle,including an account of its development in leech. Robertson (1910) also studied the life cycle of Haemog­ reqarina nicoriae and noted that certain features of this parasite were similar to those described by Reiche­ now for Haemogre g ar in a stepanowi, Prowazelc (1910) described Haemogregarina clemi'nydis and Haemogregarina emydae in Clemmys japonic a and Srnyda japonic a from Japan, Franca (191Qa) described Haemogregarin a sternothaeri in Stemothaerus der-bianus' and Cinixys belliana from Portugu­ ese Guiena, Johnston S Cleland (1910,1912) again- reported Haemogregarin a clelandi from Australian turtles ^. Thiroux(1911) described Haemogregarin a trionyxis in turtles (Trionyx triunguis and Trionvx SD.) of Sene"qal(tUii-''> ,„.,> Wenyon(1926) reviewed chelonian Haemogregarin a and listed 20 named sqecies and 28 unnaned species of Haemogregarina , Hoare(l932) described a haemogregarine in oternothaerus sinuatus obtained from Lake Victoria,near : Ehtebee,Uganda 94 and considered it Haemogregarina stemothaeri. Roudabush & Coatney (1937) observed a haemogregarine in Chelydra serpentina and considered it to be Kaemogregarina stepanowi. Mackerras (1951) reported Haemoqregarina infection" in the blood of three tortoises.collected from Australia. Wang & HOpkins (1965) described Haemogrecf arin a sp. in the blood of Texas fresh V7ater turtles (Pseudemys ' scripta elegans). Marquardt(1966) observed Haemogregarin a stepanowi in five turtles from Southern Illinois. Desser(1973) described a Haemoqregarina sp, in the snapping turtle/Chelydra serpen- tin a. Acholonu (1974) described Haemogregarin a pseudemydis- in turtles from Louisiana. Paterson & Desser(l976) described Haemogregarin a balli from a cornmon snapping turtle, Che lydr a serpentina serpentina along with its developmental stages found in gastric caeca of leeches ( Placobdella parasitica and Placobdella omata) collected from Lake Sasajewan,Algonquin Park/ Ontario,Canada.

Haemogregarina in Ophidia.

Pfeiffer(1890) mentioned the occurrence of haemoprotozoa in blood of snakes. Snake haemogregarines were first described by Billet (1895) from three s-oecies of snakes at Cao-Bang in North Viet Nam and named two of these parasites as Dan i lev/sky a python is and SB

Laverania bungari. These parasites are now knovm as Haemoqreg arin a pythonis:and Haemogregarina bungari. Hagenmuller (1898$ described a haemogregarina from a snake (Macroprotodon cucullatus) of Mgeria and ; '.: Spain. Borner (1901) described Haemogregarina colubri in Coluber longissimus from North America. Lutz (1901) reported about ten unnamed species of Haemogregarina and one named species ( Haemogregarina serpentium ) from different snakes of South America. Laveran (1902) described four species of Haemogregarina viz«,Haemogregarina n aj ae/ Haemogregarin a zamensis from India and Algeria respectively and Haemogregarina moreassini and Haemogregarina crotali from North America. Billet (1904) described Haemogregarin a viperini in Tropidonotus viperinus from AJ-geria. Castellani & Willey (1904) reported Haemogregarina mirabilis from a snake-in Sri Lanka. Robertson(1906) found a Haemogre garin a sp. in Python sp. from Sri Lanka. Sambon &• Seligmanni 1907^ 1909 ) recorded some observations on haemogregarines in various snakes collected from different countries in AJirica, J'lmerica and India^and described about fifteen species viz., Haemogregarin a terzii Haemogregarina rarefaciens, Haemogregarin a brumpti^ Haemogre­ garina luhej., Haemogregarina wardi^ Haemogregarin a brendae^ Haemogregarina pococki^ Haemogregarina mansoni/ Haemoqregarina refrinqens^ Haemogregarin a shattocki, Haemogregarin a cantliei, Haemogregarina robertsoni, Haemogregarina bradfordi, Haemoqre- 96: gar in a plirameri and Haemogregarina seliginanni. Dobell (1908) described three unnamed species, of Haemogregarina from snakes of Australia and Brazil. Cuenod(1909) desc- cribed Haemogreq arin a qiganllnensis from a snake of Tunis, Laveran & Pettit (1909,. 1911') described Haemogregarina pituophiS/ Haemogregarina sebai and Haemogre q arin a seurati from snakes of Algeria, Mexico and Senegal. Carini (1910) described Haemogregarina philodr^^'asi in Philodryas.' schotti from iBrazil. Gilruth,et_ al_., (1910) described Haemogregarina megalocystis from a snake of Australia, Franca(1910^ desc­ ribed Haemo g re g ar in a manceauxi, Haemo g re g ar in a luisieri and Haemogregarina coronellae in snakes of Tunis and Portugal. Seidelin (1911) described Haemogregarina imperatoris in Boa imperator from Mexico. Johnston (1909, 1910) described Haemogregarina pseudechis, Haemogregarina amethystina and Haemogregarina moreliae from snalces of Australia. Johnston & Cleland (1910,1912) described Haemogregarin a dendrophidis^ and Ijaemogregarina bancrofti from snakes of Australia. Conor (1912) described Haemogjregarina vreissi in Haja tripudians from Africa, Phisalix & Laveran(1913) recorded Haemogregarin a roulei in Lachesis alternatus from Brazil. Phi3alix(1914) described Haemogregarina boodoni in a snake,,Boodon fuliginosus of Africa. Hoare (1918,1920) described Haemogregarina tigrinae Haemogregarina dogieli' and. Haemoqreqaein a sp. from snakes of Japan and Uganda. Pantham(1925) described Haemoareoarina 97

arietans in a snake of South Africa. Wenyon(1926) reviewed thfe literature of snake haemogregarines and listed about 52 species of Haeniogreqarina and 103 unnamed species of Haem.ogregarina from the various

Darts of the v/orld. Iioare(193 2) described Haemogrega- rina vubirizi, Haemogregarina mustoae/ Haemogregarina crotaphopeltis and Haemogregarina enswerae from snakes collected near Entebbe and Kampala in Uganda. Roudabush

& Coatney (1937) redescribed three knov/n snecies and a nevj species of Haemogregarina from snakes, Mohiuddin, et al., (1967) described Haemogregarina echisi in a snake,

Echis carinatus of the Sind region of West Pakistani

Pessoa.et. al_./ (1971) described haemogregarines of Brazili­ an snaj'ces. Toshioka (1970) described 7 types of haemo­ gregarines in the blood of Japanese snaJ^ies. Miyata{1974) detected Haemogregarina tigrinae and two unnamed species;

of Haemoqregarin a in Elaphe quadrivirgata. El aphe dim a corphora, Rhabdophis tigrinus tigrinus and Agkistrodon halys from Japan. According to this author,about 7o

named haemogregarines v/ere kno\7n from ophidian hosts but most of these were vsjcy difficult to be identified because

the descriptions and figures given by early worlcers v/ere

not distinct. Ball(1967) Qescribed haemogregarines

from snakes of East Africa. Zaharyan &• Vashetko(1978)

reoorted Haemogregarina lebetna, Haemogregarina echisi 98 and Haemoqreqarina sp. from snakes of Central Asia and Kazahstan. Zaharyan & Ermatova (1981) described haemoqregarines:from poisonous snakes in Tashkent.

WORIC DONS IK Il'JDIA.

Chelonian Haemoqreqarina vras first repo­ rted by 3imond(1901) who described Haemoqreqarina laverani and Haemoqreqarina mesnili from two turtles ( Emyda qranosa and Kachuqa tectum ) in Agra, The samE author described another Haemoqreqarin a which: vjas similar tc Haemogregarin a stepanov;i along with an infection of ' (cited by Gamharn,1966) from. Chitra in die a a soft leathered turtle, collected from river Jamuna in Agra. Patton (1908) repor­ ted the occurrence of Haemogregarina nicoriae.: from Erayda qranosa collected in Madras. taveran S Hattan-Larrier(19I2) described Haemogregarin a testudinis from a tortoise,Testudo emys but the exact locality was not stated by these authors. Bhatia (1936) listed Haemogregarina vittatae Robertson/1908, Haemogregarina malabarica de Mello,1932, Haemogregarin a xaveri de Mello,1932 and Haemogregarina nicoriae Castellani' SWilley, 1904 in turtle. Lis semys' punctata qranosa collected in.Goa and Madras. Misra,^ al./(1974) and Misra(1976, 1981) described Haemogregarin a gangetica (=simondi) from a turtle,Trionyx gangeticus collected in Calcutta market. 99 Narasimhainurti S Saratchandra(1979) recorded Haemogregarina sp. from turtles- ( Lissemys punctata) of Waltair:.

Otihidian Haemogreqarina was first seen by Siraond(r901) who reported three unnamed species from snakes ^ Coluber sp.,Maja tripudians • and Kaja sp.) . The author: did not state the exact locality. Laveran (1902) described FTaemogregarina naj ae from Waja tripudians from Pondicherry v/hich was subsequently reported from Southh India by Patton(1909),Plimmer(1912,1913,1914, 1916)and Scott(l926). . "' " . Haemogregarina , , in Eryx conicus was ' named Haemogregarina cantliei by Sambon Sf Seligmann (1907) , Haemogregarina pococlci was described by Sambon S Seligmann in Python molurus and the same parasite was rep­ orted by Patton (1909), Phisalix(1913), Plimmer (1912,1913:, 1914:, 1916,1917 and Scott(1926) ., Patton (1908,1909) recorded Haemogregarina sp. in snakes(Tropidonotus stolatus,Dendrophis pictus,Dryophis mycteri- zans,Bungarus caeruleus,Vipera ru5sellii,Eryx johnii,Zaocys mucosus and Tropidonotus piscator collected in Madras, Out of these the last five snakes v/ere also reported for Haemogregariilg sp, by Plimmer(1912,1913,1914> 1915),, Scott (1926)found Haemogregarina sp. ^^"^"^ Sryx johnii and Tropidonotus piscator. Eal 1(1958)described Haemogregarina mirabilis from Hatrix piscator collected in Bombay. Svahn(1955) described Fl&emogregarina bipileata in Naj a tripudians^. Sinhaffi Dasqupt a (1977)described Haemogregarina sp, in a fresh water snake,Snhydris enhydris of West Bengal. Choudhury,et al., (1978) reported a haemogregarine in P/thon molurus. Sinha(igSlfc) found a water* haemogregarine in v a/Vesifi^ snake eiiicij t^^-dLev^&p'imtTat^tkfc'j^ Dasgupta,. et al_*,.(1982) found a haemogregarine in Python molurus:. roo

Mi>»TERIJ\LS

The following hosts: were examined fore Haemogreqarina infection in their peripheral blood.

^' Lissemys punctatus (Bdnnaterre)

Twenty of these mud-turtles' were collected from time to time at Bongaon,24-Parganas/West Bengal and examined. Five of these animals were found to harbour Haemogregarina in their peripheral blood,

*2»- Trionyx gangeticus Cuvier

Twenty of these specimens were collected from in and around Bbngaon, 24-P&rganas;»West Bengal and examined for Haemogregarina . Four of these were found to harbour the parasite in their peripheral blood.

*2* Enhydris enhydris (Schneider)

Fifty specimens were collected from the ponds and ditches of Chakdah,Nadia, West Bengal/and examined for Haemogregarina. Twenty of these animals were found to harbour Haemogregarina in their peripheral blood.

* indicates that the description of the host has been given in page no. z% - 'T.A ^ 101

4. Xenchrophis piscator (Schneider)

This snake,popularly known as "checkered keel back" , belongs to Family Colubridae in Order Squamata. Body is stout,yellowish or olivaceous above and bears black spots. Scales are strongly keeled. Hfead is olive brown in colour above,with two oblique black streaks. Of these streaks,one is below and the other is behind the eye. Dorsal spots are arranged in five series forming a chess-board pattern. Nostrils are directed upwardsi The animal feeds on frogs, toads and lizards. The snake is distributed throughout India and Pakistan,

Six specimens were collected from different ponds of Baruipur,24-Parganas,West Bengal and examined. Three of these were found positive for H^emogreqarina in their peripheral blood.

The following ecto-parasites were examined in order to trace the developmental stages of Haemogreqarina, 1. Placobdella emydae Harding,1920.

It is commonly known as fresh water leech. It belongs to Family Glossiphoniidae 102

under Order Rhynchobdellida. It is greenish in colour with longitudinal stripes and v/ith dorsal warts. Body is dorso-ventrally flattened. Each segment bears three annuli. Anterior sucker is pedunculate bearing mouth and one pair of eyes. It: is ecto-parasitic on tortoises/ turtles/ frogs and fishes.

2. Haemadipsa zeylanica montivindicis Moore,1924. It is a common land leech belonging to Family H^emadipsidae in Order Ghathobdellida. It is small in size and olive-green in colour with spots. Integument is hard. Buccal ring is bordered by a thin membranous fold. Naphridiopores are directed upwards; The leech abounds in damp ravines and dripping forests in moderately high altitudes of Darjeeling district , Sikkim and in Assam.

METHODS

The following methods were adopted for the preparation of blood smears and tissue sections for the study of the developmental stages of 103

parasites in the vertebrate and in the invertebrate hosts. Blood was collected from turtles by pricking tlie limbs and sometimes by puncturing the heart directly when the animals were killed. Blood of snakes were obtained either by clipping the tail region or from the facial vein. Hfeavily infected turtles and snakes were- anaesthesised and dissected. Small pieces of tissue from liver, lung, heart ,spleen and kidney were fixed in Bouin's and Carney's fixatives. Leeches which fed on infected turtles and snakes weree killed after several days and fixed in Bouin's- and Camoy "s f ixatives ^ for further processing. Blood smears were stained in Leishman'"iS and Giemsa''s stains. Tissue sections were stained in Heiden- hains iron-haemato?cylin and haematoxylin-eosin stains:^ Measurements of parasites were made from fixed and stained materials and Camera lucida drawings were made. Photomicrographs were also taken. Q B:.S E_ R-¥ A T X Q N S 104 Haemoqreqarina in CheIonia Parasite No. 1, Haemogreqarina layer an i' Simond ( FIGS. 1 - 11.)

1901. Haemogreqarina laverani Simond, iton^. Inst. Pasteur:, 15 : 319.

Occurrence

The parasite was found in peripheral blood smears of mud-turtles,Lissemys punctatus (Bonnaterre) collected from Bongaon, 24-Parganas,West Bengal, India.

Morphology The parasite occurred in the erythrocytes of the host and a few mature gametocytes were seen in extra- corpuscular condition. In the circulating blood, parasites occurred in the form of trophozoites,schizonts and gametocytes.

Trophozoites (Figs. 1 & 4 ) These were broad and oval in shape, measuring 7.7/um - 10.3/Jm X 4/um - 5.9/um ( average 9.1/oia X 4.8/jra ). Cytoplasm was homogeneous, non*- granular and non-vacuolated and took up very faint stain. Nucleus was large,terminal or central and oval with compact 105

chromatin granules^which stained pink. Average measure­ ment of nucleus was 4.4/um X 4/jim.

SChizonts (Figs, 2 & 3 ) These were intra-erythrocytic and multinucleate form showing 2-3 nuclei and measured 8.5/ram - 12/um X 5.5/um - 10/um.( average 10.4 Aim X 7.7 Aim ), Cytoplasm was unstained, Miclei were deep pinlc and compact lying scattered in cytoplasm. Limiting membrane of schizo- nts was found intact and distinct. Average measurement of nucleus was 3,5/um X 2.5 Aim.

Merozoites Fully formed merozoites were not found.

Gametocytes (Figs. 4 - 9 )

These were abundant in blood smears and most of them were intra-erythrocytic, A few broad reniform gametocytes were seen lying in plasma. Two distinct morphological types were observed with reference to size, shape and staining properties, The two types might represent two different sexes which could not, however,be conclusively distinguished. 106

Type,. I.

This may be described as broad,bean- shaped and reniform. These ( Figs. 4-6 & 9: ) measured r2.3/um - 16,2/am X 4.6;)/um - 7.8/urn ( average 14.3/Um X Syum) . Cytoplasm was homogeneous,non-granular and strongly basophilia- containing few. granules. Two roiond bodies;stained light pinK' xvere found on one of the poles of these forms and these bodies- remained at a distance from each other. Sometime one such body was found near nucleus and other towards; one pole (Figs. 4 - 6 ) , Such bodies never occurred in both the poles. Sometimes one such body was found (Fig. 9), Nucleus was sub- central, non-compact or irregularr in outline,pink in colour and the average measurement of nucleus was 4.6/um X 3.5/am. Parasites infecting anucleated cells was also noticed (Fig.6). Extra-cellular gametocyte (Fig.9) measured 15,9/urn:, :,XS .8/um.

Type. ir.

This was regarded as oval form. These (Figs. 7-8) were more or less oval with a broader end measuring 12.3/um - 14.8/um X 4/um - 6/tim (average 13/um X 5.4/um ),. Cytoplasm was homogeneous,non-granular,non-vacu- olated and unstained. Nucleus was sub-central,large,oval compact and light pink in colour. Average measurement of the nucleus was 5,5/um X 4.8Am^. 10 11

FIGURES. 1 ~ 9. Camera lucicla drawings of Haemogregarlna laverani Simond(1901). Figs. 1 - 4. Trophozoites. Figs. 2 &' 3. Schizonts. Figs. 4-6/9. Gametocytes Type.I. Figs. 7 & 8. G&metocytes Type. II. Figs. 10 - 11, Normal erythrocytes. 107

Effect of parasites on host cells;

Invasion of this parasite caused slight linear hypertrophy of infected erythrocytes as shox^m in the following table ( measurements in micrometers,/um).

Range. Average, Length of normal erythrocyte. 15 - 20 17,8 Width of normal erythrocyte. 9,9-12,3 11 Length of normal erythrocyte nucleus, 5.4 - 6,8 6 Width of normal erythrocyte nucleus, 3,2-3,7 3,3 Length of infected erythrocyte. 18 - 21,6 20.1 Width of infected erythrocyte, 7.5-13.3 11.1 Length of infected erythrocyte nucleus. 5.4-6.9 5.9 Width of infected erythrocyte nucleus. 2.9 - 4.3 3,3

Nucleus of erythrocyte was usually^ markedly displaced to one side. Shape of hypertrophied infected cells depended on the position of parasite and on displaced host cell nucleus. Host cell nucleus was not; seen in fragmented condition.

Movement of parasite. In citrate solution,parasite moved slowly v/ithin host cells. 3108

Discussion

Parasite under report belongs to genus

Haemogregarina on account of intra-erythrocytic schizonts in

the blood of vertebrate host and identified as Haemogregarina

laverani Simond,1901 for the following characters.

Gametocyte is broad, reniform and possesses two spherical bodies in the cytoplasm which is the distinguishing

character of Haemogregarina laverani and not found in any other

species of Chelonian Haemogregarina*

However, the material available to Simond

did not show any erythrocytic schizont. The following differ­

ences are also noted. In Simond"s material young stages were

amoeboid and 3yum in diameter with a small nucleus. Some

young stages were in the form of a minute vermicule with limbs.

These vermicular forms did not extend beyond half the length of

the corpuscle and possessed a bulging end and a short drawn-out

tail.

Differences noted above are not sufficient

for considering the present parasite as new one. The parasite

is, therefore, identified as Haemogregarina laverani Simond,1901.

This is the first record of Haemogregarina Ihverani from a host other than Emyda granosa. 109

Diagnosis

Family, Haemogregarinidae Leger,1911, Schizogony intra-erythrocytic in vertebrate host ; gametocytes and merozoites in erythrocytes. Genus. Haemogregarina Danilewsky,1885. Intra-erythrocytic ; non-pigmented; erythrocytic schizogony; sexual dimorp­ hism of gametocytes not prominent. Specific characters Gametocytes exhibiting two morphological types. Type. I, Broad reniform. 12.3/um - 16.2AimX4.6Ajm- 7.8/um, T^Tpe. 11. Oval form, 12,3/um -

13,4A™ X 4/um - 5/wm, Trophozoite. 7.7/um - 10.3 Aim X 4/Jm -5,9/am, Schizont, 8,5/um- 12/um X 5,5/um - 10/jm-*

The parasite is referred to as Haemogrega­ rina laverani Simond,19011

Host, Lissemys punctatus (Bonnaterre) Site of infection. Peripheral blood. Locality ; Bongaon/ 24-Parganas,West Benggl,India. 110

Haemogreqarina in Chelonia Parasite No. 2. Haemogreqarin a n.sp. (3)

( FIGS. 1-15 )

Occurrence The parasite was encountered in peripheral blood smears of Kissemys gunctatus (Bonnaterrel a mud-turtle, collected from Bongaon,24-Parganas,West Bengal,India.

Morphology Parasite was mostly intra-erythrocytic and only a few merozoites were seen extra-corpuscular condition. In the circu­ lating blood , parasites occurred in the form of trophozoites, schizonts, merozoites and gametocytes. Leeches of genus Placobdella were noticed on outer surface of body of turtles. These were collected and secti­ oned for obtaining developmental stages of parasite, but; result was negative.

Trophozoites (Figs. 1-3 )

Young forms (Figs. 1 & 2) were small,round or oval broad at one end and slightly tapering at other measuring 3.5 Aim - 4 /Um X 2.8 /am - 3.2 /um ( average 3.7 /um X 2 /urn ), A mature trophozoite (Pig. 3 ) attained 5 /um X 2/um in size. Ill

Cytoplasm was homogeneous,non-granular,non~vacuolated. Nucleus was large, central or sx±>-central,oval and deep pink in colour. Average measurement of nucleus was 1.8 /um X 1.2/um,

SChizonts ( Fig#ii 4 - 7 )

These were intra-erythrocytic and multinucleate forms showing 3,, 4, 5 or 8 nuclei and meas­ ured 5 Aim - 10/um X 3.2 Am - 5.5 Aim ( average 7.4/Um X 4.3/um ), Cytoplasm was vacuolated and stained light blue in colour. Nuclei were deep pink and compact. Average measurement of nucleus;was 2Aini X 1.1/um, Limiting membran© of schizonts was intact and distinct.

Merozoite (Pig. 8)

One individual was found to invade a normal erythrocyte (Fig. 8);. It: was slightly ellipsoi­ dal in shape with tapering ends ; and measured 7.4.Aim X 2.5 /um. Cytoplasm was reticulated and light blue in colour. « Nucleus was sub-central, large,oval non-compact and deep pink in colour. Average measurement of nucleus was 2.4 ./um X 1.2 /jm.. Gametocytes ( Pigs. 9 - 13 >

These were abiondant in blood smears and were intra-erythrocytic. Two distinct morpjiological 112

types were found depending on their size,shape and staining properties; The two types might represent two different sexes which colu could not ,however,be conclusively distinguished.

Type. I. These were teiroed as vermicular elongate form (Figs. 9 - 10 )• These were with or without tapering ends and measured 9.6 vm - 14 um X 2.4/um - 3.3/um (average 12/um X 3/um ). Cytoplasm was homogeneous, non-granular and stained faintly.

Nucleus was central,non-^compact and deep pink in COIOP* ur. Average measurement of nucleus was 3 Aim X 1.4/im.

Type,. II. These were regarded as broad form ( Figs, 11 - 13)' measuring 6.8/um - 9.4/um X 2.6/um - 3.3 um ( average 8/um X 3/um }, Cytoplasm was reticular and light blue in colour . Nucleus was sub-central, non -compact and deep pink in colour. Average measure­ ment of nucleus was 2.8 Ami X 2.3 AJ™* Double infection of erythrocytes i^?as also common (Fig. 12 ) , FIGURES. 1 -13. Camera lucida drav/ings of Haemogregarina n.sp. (a) Pigs. 1 - 3. Trophozoites. Figs. 4 - 7. Schizonts. , T r ' Pigs, 8, Merozoite inV^.ading a normal erythrocyte. Figs. 9-10. G&metocytes Type. I. Figs. 11 - 13. Gametocytes Type . II. Pigs. 14-15. Normal erythrocyte. US-

Effect of parasites on host cells:

Invasion of this parasite caused slight linear hypertrophy of infected erythrocytes as sho\^m in the followinq table ( measurements in micrometers,^jm),

Ran ge. Ave rage, Length of normal erythrocyte. 13-17.6 15.8 Width of' normal erythrocyte. 7.5-9.5 8.5 Length of normal erythrocyte nucleus. 4.2-5.5 4.5 Width of normal erythrocyte nucleus. 2-3 2.7 Length of infected erythrocyte, 15 - 19 17.6 Width of infected erythrocyte. 8-11.6 9.4 Length of infected erythrocyte nucleus. 4.5-6.5 5 Width of infected erythrocyte nucleusi 2.8-4.5 3.3

Kri2Gleus of erytbrocyte was usually markedly displaced to one side. Shape of hypertrophied infected cells depended on the:position of parasite and on displaced host cell nucleus. Host cell nucleus was not seen in fragmented condition.

Movement of parasite. In citrate solution,parsite moved slowly within host cells. 114

nisGUssion

Haemogregarines have been classified by various- taxonomists ( Wenyon, 1926 ; Grasse, 1953; Honigberg et al^., 1964; Irvine, 1970; Levine et_ a]^«/1980 ). According to Levine et al. ^ (1980): the genus Haemogregarina is placed under the Family H&emogregarinidae of the sub-order Meleina in the Class Sporo- zoea. i^ecies of Haemoqreqarina are kno\^m to occur in a turtle of the genus Lissemys. Bhatia(1936) mentioned the occurrence of Hlmogregarin a nicoriae Castellani & Willey, 1904 in Lissemys punctata qranosa. Hfe also cited three more haemogregarincparasites viz./ H&emogregarina vittatae Robertson (1908) , Haemogregarina malabarica de Mello (.1932) •. . and Haemogregarina xaveri de Mello(1932) in Ixissemys punctata qranosa from Portuguese India,Nova Goa. Narasimhamurti & Saratchandra (1979) reported Haemog re g arin a sp. in Lissemys pxmctatus collected from South mdia. These authors did not give adequate description and measurements of the parasite. Parasite under study resembles Haemogregarina stepanowi Danilewsky, 1885, H.priogregarina nicoriae Castellani &: Willey,1904, Haemogregarina pseudemydis Acholonu,1974, Haemogregarina gangetica ( Misra,Nandi,Raut & Choudhury,1974) and Haemogregarina balli Patterson &' Desser, 1970: in the possession of erythrocyiric schizonts but differs from them on the basis of the following characters,

Haemogregarina stepanowi Danilewsky,1885 is sexually dimorphic, the microgametocyte having denser 115

cytoplasm and a smaller nucleus than in microgametocyte. Moreover, Reichenow (1910) recorded schizonts • v/ith 12-24 merozoites in erythrocytes in the bone-marrow. Xn Haemoqre«^ gagjna nicoriae Castellani & Willey,1904, the gametocytes are elongate,gregarine-like with one end granular and the other; end is clear. In Haemogregarina pseudemydis Acholonu,1974 the erythrocytic schizonts xvith 6-8 nuclei are absent rather the schizonts contain 35 - 140 merozoites in erythrocytes and leucocytes. In Haemogregarina gangetica ( Misra, Handi,Raut & Ghoudhur^^, 1974), gametocytes are sexually dimorphic. Microgame­ tocyte is characterized by tvra band-shaped structures of nucleus while macrogametocyte has ribbon-like nucleus. In Haemogrega­ rina balli Paterson & Dfesser, 1976 the sex of gametocytes is apparent where macrogeimetocyte has a basophilic cytoplasm and micro with a short recurved tail. Thus, the parasite under report does not resemble any known species;of H&emogregarina from turtlesi Therefore,it is concluded that the parasite should be regarded as a new species.

Diagnosis

Family Haemogregarinidae I.eger,1911. schizogony intra-erythrocytic in vertebrate host; gametocytes and merozoites in erythrocytes. 116

Genus Haemogregarina Danilewsky^1885. Intra-erythrocytic schizonts; gametocytes intra-erythrocytic ; Sexual dimorphism of gametocytes not prominent. Specific characters Gametocytes : exhibiting two morph­ ological types; non-capsulated. Type r. measuring 9,6/um - 14/um X

2.4 /um - 3.3 /um.

T\''pe 11. measuring 6.8/um - 9.4/um X

2.6 Affii - 3.3 /um. Trophozoites measuring 3.5/um - 5/um X 2 /um - 3.2 /um. Schizonts;measuring 5/um - 10/um X 3.2/ur 5.5 /um. Merozoite measuring 7.4/um X 2.5/um. For the present the parasite is referred • to as Haemogregarina n.sp.(a)

Host, Lissemys punctatus (B'onnaterre ) Site of infection, peripheral blood. Locality;:- Bongaon, 24-Parganas,West Bengal, India. 117/

Haemogregarina in'Che Ionia Parasite No. 3. Haemogregarina gangetica (Misra,Nandi,Raut and Choudhury): C FIGS. 1 - 17 )

1974, Haemogregarina simondl Misrs,Nandi,Raut and Choudhury, Acta. Protozool.,12 : 345, 1976,. Haemogregarina qanqetica Misra, ^ta. Protozool., 15; 21; Misca, 1981, Proc. Zool, Soc. ^Calcutta^, 32 : 141,

Occurrence The parasite was encountered in /the peripheral blood smears of Trionyx gangeticus Cuvier a common •-.turtle,collected from the Ichamati river of Bongaon,24-Parganas,West Bengal, India.

Morphology Parasite was mostly found in erythrocytes: and a few extra-corpuscularr merozoites were present. In erythrocytes, parasites were in the form of trophozoites,schizonts, merozoi­ tes and gametocytes. Leeches of genus Placobdella were also noticed attached to body surface of turtle. These were collected forr the study of developmental stages but the result was negative. 1X8

Trophozoites ( Figs. 1 - 4' )

Young forms ( Figs.1-3) were small, oval or irregular in shape measuring 4.5 Am - 8 A^im X 2.8 Aim - 3.5 /um ( average 6.1 Aim X 3.1 /om), Cytoplasm non-granular, non-vacuolated and unstained. Nucleus was large,central or terminal,round or oval and deep pink in colour. Mature trophozoites (Figs, 3 - 4) were broad and oval in shape measuring 11/am - 13.3 yum X 4-.8Aim - 7 ,8/Um ( average 11.8 /Um X 6/um ). Average measurement of nucleus was 5.7/um X 4.8AHn, Jkn erythrocyte was found to harbour both immature and mature parasites lying side by side (Fig. 3). Parasites infecting enucleated cells were also noticed ( Fig. 2 ),

Schizonts ( Figs. 5 - 9 ) These were intra-erythrocytic and multi-nu nucleate forms showing 2, 3, 4, 6 or 8 nuclei (Figs. 5 - 9 ) and measured 7/um - 14.5/um X 5,5/um - 9.8/um ( ave#Qge 11,2 /um X 7.5/um ),. Cytoplasm was vacuolar and basophilic. Nuclei were compact,oval or irregular in outline and stained deep pink. Average measurement of nucleus was 5.6 Am X 2.3 /um. Limiting membrane of the schizonts was intact and distinct, Merozoite (Fig. 10) This was found to onvade a normal erythrocyte (Fig. 10). It was oval with tapering end at one side and 119 globular at the other. Cytoplasm was reticular and light blue in colour. Nucleus was sub-c^entral,large,oval, non-compact and deep pink in colour. Nucleus measured 2.2/Um in diameter. Gametocytes (Pigs. 11" - 15 ) These were intra-erythrocytic and abundant in blood smears. Depending on their size, shape and staining properties;two distinct morphological types were noted. Thee two t3fpes might represent tWo different sexes which could not, however,be conclusively distinguished. Type. I, (Figs, 11' - 13)

Young forms were sausage-shaped (Fig. 11) measuring 6.8/jm - 1,3/am X 1,3 Ami - 3.3 Aim ( average 6,9yum X 2 yum ), These were enclosed within a capsule. Cytoplasm was homogeneous, non-granular and faintly stained. Nucleus was oval,5:entral and deep pink in colour. As development proceeded,body became bent giving rise to a hook-like shape,with a pointed end ( Fig. 12 ). Mature form vras seen to contain two unequal limbs with a sub-central and elongate nucleus. This form measured 13.6 vm X 2.2 um and nucleus measured 2,2/um X 1,1 yum. 120

Type. ir. (Figs. 14 - 15)

Young; form (Fig. 14> was oval in shape measuring 6.5/lira X 2.8/um.. Cytoplasm was homogeneous, faintly stained aad non-granular. Nucleus was large, terminal,non-compact and stained light pink in colour. Mature form (Pig. 15) measured 10.5/um X 4.4/um. Cytoplasm was alveolar and light blue in colour. Nucleus was sub- central, oval or elongate with irregular outline,deep pink in colour and measured 3.5yum X 1.2 Aim. Parasites were also enclosed in a capsule.

Effect of parasites on host cells

Invasion of this parasite caused slight linear hypertrophy of infected erythrocytes as shown in the following table ( measurements in micrometers, /urn).

Range. Ave rage, Length of normal erythrocyte, 14-17.8 16.1 Width of erythrocyte (normal). 7.4-12 10 Length of normal erythrocyte nucleus. 3.8-5.6 4.8 Width of normal erythrocyte nucleus. 2.9-4 3.6 Length of infected erythrocyte,. 16.5-19,5 17.8 Width of infected erythrocyte. 8.2 - 12.8 10,6 Length of infected erythrocyte • nucleus. 4.4-6,5 5,4 Width of infected erythrocyte nucleus. 3.5-4.8 3.9 FIGDRES. 1 - 15. camera luclda drawlnas 6 /um. Of Haemoqreq^n-na aanaetloa t^asra,^T&ncli,' • Raut £FfehouahurY,1974) • Pigs;i6-17.Homal erythrocytes, r.g. 10 Mercolte invading a normal- erythrocyte. ^-^s. 11 . 13. Ga.etocJ*e..Type.I.- Plg3. 14-15.Gametocytes ' "^ 121 Nucleus of erythrocyte was usually markedly displaced to one side. Shape of hypertrophied infected cells depended ont the position of parasite and on displaced host cell nucleus. Host cell nucleus was not seen in fragmented condi­ tion.

Movement of parasite In citrate solution,parasite moved slowly v/ithin host cells.

. Discussion Haemogregarina are kno\'m to occur in different species:: of the host turtle of this genus Trionyx. Simond(1901) described Haemogregarina billeti in Trionyx cartila- gineus3collected from Tonkin. Thiroux (1911) described Haemogrega­ rina trlonyxis in Trionyx' triunguis : and Trionyx sp. from Senegal(tiCot ^ iteholonu (1974) reported the occurrence of Haemogregarina pseud»» : r . emydis in Trionyx spinifer collected from South Eastern Louisiana. Misra et al., (1974,1976/1981) described Hiaemogregarina gangetica in a soft-leathered turtle (Trionyx gangeticus) inhabiting the river Ganges-and its estuary. Parasite under discussion is placed under genus Haemogregarina because of erythrocytic schizonts in the blood of turtle. It resembles Haemogregarin a gangetica(Misra,Nandi,Raut & Choudhury,1974) on the basis of the following characters. Parasite shows erythrocytic schizonts: with 8 developing merozoites. Gametocytes are of tv/o types. The infection occurs in same species of turtle host. However, the follov/ing differences are also noted. Gametocytes of Haemogregarina gangetica (Misra,KrQndi,Raut :. & Choudhury,1974) are described as micro and macrogametocyte»: Microgametocyte is characterized by two band-shaped structures of nucleus whie the macrogametocyte has ribbon-like nucleus. Micro­ gametocyte measures 9.16/um X 3.2 Aim and macrogametocyte attains 9.6 Aim X 4.8 Aim. Tisse stages are reported from the endothelial cells of the lung. 122

The sexes of the gametocytes in this study can not be contsonclusively distinguished.. Gametocytes of type l measures 6.8 Aim - 13 Aim X 1.3 Aim - 3.3 Aim^ and type 2 measures. 6.5/um - 10.5 Aim X 2.8 Am - 4.4 Aim. Tissue stages are not: found. Differences noted above are not sufficient for considering the parasite under report as new one. The parasite is ,therefore,identified and described as Haemogre g arin a gangetica ( Misra,Nandi,Raut & Choudhury,1974).

Diagnosis

Family Haemogregarinidae Leger,1911, Schizogony intra-erythrocytic in verte­ brate host; gametocytes and merozoites-: in erythrocyte S3. Genus Haemogregarina Danilewsky,1885. Intra-erythrocytic schizonts; sexual dimorphism not prominent. Specific characters : In the present work/the gametocytes; exhibiting two morphological types; type 1. 6.8 Aim - 13.6 /am X 1.3 Aim - 3.3 Aim; type 2 measuring 6.5 Aim - 10 Ajm X 2.8/um - 4.4 Aim; trophozoites 4.5 Aim - 13.3 Aim X 2.8 Aim - 7.8 /am: schizont 7 AMI - 14.5 Am X 5.5'^ 9.8 /um; merozoite 6.5 /un X 3 Aom. The present parasite is referred to as Haemogregagina gangetica ( Misra,Nandi Raut &' Choudhury,1974)

Host. Trionyx qangeticus Cuvier Site of infection. Peripheral blood Locality :: B©Eigaon, 24-ParganaS/West Bengal, India*. 123:

Haemogregarina in Ophidia

Parasite No. 4.

Haemogregarin a n.sp. ( b)

{ PIGS. 1 ~ 24 I

Occurrence

The parasite was encountered in the peripheral blood smears of Enhydris enhydris (Schneider),a common fresh water snake collected from Chakdah ,Nadia ,West

Bengal/India.

Morphology

The parasite was found mostly in the erythrocytes although free extra-corpuscular merozoites were also present.

The parasite occurred in the blood in the form of troohozoites, merozoites, schizonts and gametocytes.

Developmental stages of this parasite were found in the sections of liver where meqaloschizonts were present showing distinct cytomeres.

No ecto-parasite was noticed on the body of snakes.

Experimental transmission of this Haemogregarina to inverte­ brate host was attempted with leeches which are believed to be a most potential vector. Leeches ( Haemadipsa zeylanica montivindicis Moore,1924) were collected at Darjeeling 124

(altitude 2000 m ) and allowed to feed on the infected snakes. One of the leeches exhibited a few developm­ ental stages of this parasite.

Trophozoites (Pigs. 1 &' 2 )

Young forms (Figs.l & 2) were small,oval and sometimes broad at one end and tapering at other. These measured 6,4/am - 9 A^im X 2.8 Aim - 4.4 Aim ( average 7,5 A^nn X 3.4 Aun ). Cytoplasm was homobreneous,non-granular and stained very faintly. Nucleus was elongate or oval measuring: 3.5 /am - 6.6/um X 5 Aim - 3.3Aira and stained deep pink. Doxible infection by these parasites was also observed (Fig. 2) where the host cell nucleus was slightly pushed to upper side.

Schizontsi (Figs. 3 - 10)

Ijumaturar schizonts (Figs. 3 -4) appeared as serpentine forms in the peripheral blood circulation with two broad arms,one recurved upon the other. Cytoplasm was flocculent and basophilic. Nucleus was large,oval, compact,sub-central and deep pink in colour. These forms measured-ij2Aro - 19 Aim X 3.2/um - 4.6 \OT( average 17.5 Aim X 3.6 Aim), Average measurement of nucleus was 6/urn X 3.4 Aim, As the 12 F

development advanced, the two arms fused together (Fig.5) and nuclear division began. Intra-erythrocytic schizonts became mature (Pigs. 6 - 10). These were multinucleate forms shox"7ing 4, 6, 8, 9 and 11 nuclei and measured 10/um

- -i9,5/om X 5/am - li/um (average l4.lAim X 8/um ).

Cytoplasm was vacuolated and light blue in colour, Nuclei were deep pink ,compact and were found scattered throughout cytoplasm. Average measurement of nucleus was 4 Aim X 2 /Um,

Limiting membrane of the- schizonts was found intact and distinct. to extreme case of hypertrophied erythrocyte

(Pig, 10) showing doiible infection by the schizonts with 11 nuclei in each case was encountered. These two schizonts measured 24/Um - 25,2/im X 10,5 - 11.4/um ( average 24,6/um

X 11 /jm ) and their nuclei were 3,8/um - 7,2/um X 1,4/um

- 2.8/um ( average 5.4/um X 2,2/jm). Nucleus of the host cell was deformed and shifted to one side, Hypertrophied host cell measured 28/um X 24/um and the nucleus was 7,2/um

X 2.4 /um.

Merozoites:( Pigs, IT - 12 )

These were found in blood plasma

( Pigs. 11 - 12 ), One of these was found invading a normal erythrocyte (Pig.11', ). These were more or less V26n conical in shape and measured 8.7 Aim - 12/ara X 3,5/uin - 4 Aim ( average 10/um X 3.6/um ). Anterior part was tapering while the posterior part is rounded. Cytoplasm was vacuolated and stained faintly. Nucleus was central, large,compact ,oval and deeply stained, Average measurement of the nucleus was 2.8/umX 2.4/um.

Gametocytes (Figs. 13 - 17 ) These occurred in large nimber in blood smears. Two distinct morphological types were present depending on their size, shape and stain­ ing properties. Sexes could not be identified with certainty. Type. I. ( Figs. 13 - 15) Young form was 10.5 yUm X4:,2/um, Cytoplasm was alveolar,non-granular and faintly stained. Nucleus was central,non-compact and took up pink colour. Mature forms (Figs. 14 - 15 ) were encapsulated measuring 16/urn - 18.2 Aim X 3.2Aini - 3.8 Aim ( average 16,5 Aim X 3.2Aini )• Inside the capsule,parasite remained in the form of a vermicule whose one end was bent and sharply pointed. The other end was rotmded. Cytoplasm was alveolar,unstained and non-granular. Nucleus was non- compact, central or sub-central and stained dark purple. t27

Average measurement of the nucleus was 4.5/Um'X 3yuni.

Type. II. (Figs.16 - 17 ) These were slender,elongatee and terminal ends were slightly pointed. These measured 22.5/urn - 24.6 Aim X 2.4/urn - 3.2/Jin (average 23.7 Aim X 2.7/um)). Cytoplasm was homogeneous, faintly stained and devoid of any inclusions. Nucleus was oval,compact and stained dark purple. Average measurement of nucleus was 5/um X 2.5/um. Invasion of these forms caused a marked hypertrophy of infected corpuscles.

Tissue phase (Figs. 21 - 24 ) Schizogony of this parasite also took place in the internal organs of the snake host. It was in the form of megaloschizonts which were remarkable structure detected only in the serial sections of liver. These were either sub-spherical or oval (Figs.21 & 22) There were spaces or vacuoles with small nuclei scattered throughout the cytoplasm v/hich stained light pink with eosin. Cytomeres formed at a later stage which increased in size and produced more nuclei. Several cytomeres were formed and these ranged in size from 10/Um - 25 Aim X 6 A:im - l5Aini. Mature megaloschizonts were 155/um - 10 11

FIGURES. 1-10. Camera lucida drawings of H&emogre garin a n.gp. (h) • Figs. 1-2. Trophozoites. Figs. 3-4. Immature er^'-throcyti schizonts. Pigs. 5 - 10, Erythrocytic schizonts. Figs. 6' - 10. Erythpocytic sclaizonts v/ith 4 -11 nuclei . Fig, 10, Double infection by schizonts. Fig, 11. Normal erythrocyte. PIGtRES 21 -24:. 128

250yum X lldyum - 160 yumi average 195 Am X 125 Aim) and contained al large niimber of merozoites; (Pigs. 23 & 24 ): measuring 1.5/urn - 2.6/ura X 1.2/am - 2/urn ( average 2/urn X l.Tvum ).

Sporogony stages ( Figs.18 - 20 )

Sporogony of this parasite was found in serial sections of leech ( Haemadipsa zeylanica montiv- dicis Moore,1924) seven days after it had fed on an infec­ ted snake xirith HStemogreqarina in the blood. A macroga- mete was detected in the crop. It was oval with an irregular outline (Fig. 18) measuring 7/um X 4.4 Aun* Cyi:oplasm v/as homogeneous,non-granular,non-vacuolated and stained light' pink with eosin stain. Nucleus was central, compact,oval and stained blue with haematoxylin. Oocysts (Figs.19 & 20) were found in the lumen of intestine along with haemolysed blood,(Fig These were immature, oval or sub-spherical in shape measuring 6/urn - 10.5 yum X 3.5 Aim - 5.5/um ( average 9 A^m X 4.3/urn ). Cytoplasm was vacuolated and stained light pink in eosin. Fertilization of macro and microgametes was not obseirved. Oocysts were foiond to develop also in the inner lining of intestine. Immature oocysts were found to contain 3-6 nuclei which stained blue colour in haematoxylin. FIGURES, ir - 20. Camera lucida drav/lngs of Plaemogreqarlna n.sp.(b). Pigs. 11 - 12. Morozoites. Pig.11. Merozoite invading a normal erytbcrocyte. sPlns.l3 - 15. Gnmetocytes Type. I. Figs. 16 - 17. Oametocytes TJipe.ir. .Fig. 18. Macrogamete in the section of Crop of leech. Figs. 19 - 20. Immature oocysts in the intestine of leech. i2gF

Effect of parasites on host cells.

Invasion of this parasite caused marked hypertrophy of infected erythrocytes as shown in thiB following table ( measurements in micrometers, yum,);.

Range. Average. Length of normal erythrocyte, 13-20,5 17.6 Width of normal erythrocyte. 8.5-11.2 9.3 Length of normal erythrocyte nucleus4.2.**-8.8 6.5 Width of normal erythrocyte nucleus. 2.6- 5.6 4.2 Length of infected erythrocyte nucleus. 6.5-10 9 Width of infected erythrocyte nucleus. 4-5.8 4 Length of infected erythrocyte. 14.5 - 26 20,5 Width of infected erythrocyte. 9-13 11

Nucleus of erythrocyte was usually markedly displaced to one side. Shape of hypertrophied infected cells depended on the position of parasite and on displaced host cell nucleus. Host cell nucleus was not seen in fragmented condition.

Movement of parasite. In citrate solution,parasite moved slov/ly within host cells. 130

Discussion The parasite under discuHsion:shows erythrocytic schizogony in peripheral blood and tissue schizonts in the serial sections of liver of vertebrate host. This character has beenn quoted for the Family Haemogregarinidae and hence the parasite has been placed under the above family. Bhatia{192'6) listed four named and seventeen unnamed species of Haemoqreqarina from different Ophidian hosts, but their systematic position remain uncertain due to lack of detailed characters. , The parasite,however,does not resemble any known species from snakes under this genus for its own charac­ teristic features. For example,it shows erythrocytic schi­ zonts containing 4-11 nuclei with developing merozoites. Tv70 types of gametocytes ware found and their sexes could not be ascertained. Megaloschizonts develop in the liver cells and consists of cytomeres; Experimentally the present ppar asite, is seen to develop in an xinnatural host leech. In the gutt of this leech the gametocytes becomes macrogamete which aftfer fertilization forms ultimately oocysts VJhich lie in the intestinal lumen along with haemolysed blood and also in the inner lining of intestine. Considering all aspects,it is concluded that the parasite under report should be regarded as a new species. 1'31

Diagnosis Family. Haemogregarinidae Leger/1911. Schizogony intra-erythrocytic in verte­ brate host; gametocytes and merozoites in erythrocytes. Genus. Haemogregarina Danilev7sky/1885. Intra-erythrocytic; erythrocytic and tissue schizonts; small oocysts without sporocysts in leech. Specific characters. Gametocytes exhibiting two morpholo­ gical types . Type. I. 16/um - 18.2/am X 3.2 Aim - 3.8AmT Type. II. 22.5/um- 24.6/im X 2.4 - 3.2/am. Trophozoites, 6.4/iam - 9/um X 2.8 Aim - 4.4/um. Immature schizonts. 12 vm - 19/um X 3.2 Aim - 4.6/um. Mature schizonts. 10/um-25 .2/um X 5/Um- 11.4/um, Merozoites. 8.7/um - 12/um X 3.5/um-4/um, Megaloschizonts, 155/um - 250/um X 110/Um - 160 yxm. Oocysts. 6/orn - 10.5 Aim X 3.5 /um -5.5 Aim. For the present the parasite is referred to as Haemogregarina n.sp. (b^) . Host. Enhydris enhydris (Schneider) Site of infection. Peripheral blood , Liver Locality: Chakdah,Nadia/West Bsngal,India. 132

Haemogreqarina in CDphidia

Parasite Ho.5. Haemogreqarina n. sp, ic) ( FIGS. 1-13 >

Occurrence The parasite was encountered in the peripheral blood smears of Xenchrophis:piscator (Schneider) a checkered keel back snake,collected from Baruipur,24-Parganas,West Bengal, India.

Morphology Parasite was intra-erythrocytic, although a few extra-corpuscular mature vermicular gametocytes were present in blood plasma. In the circulating blood,these were in the form of trophozoites, schizonts and gametocytes.

Trophozoite ( Pig. 1 )

It was broad and oval measuring 7.1 /um X 5,9 Aom. Cytoplasm was homog^ous; and appeared almost stainless. Nucleus was large,bean-shaped and filled up most of the cell body. It consisted of beaded mass of chromatin lying on one side of the limiting membrane and stained deep pink in colours Nucleus measured 6.6/um X 3.3/um. JJ33

Schizonts ( Figs. 2 - 3)

This stage was intra-erythrocytic and was detected in binucleated condition (Figs. 2 S3 ).

These stages measured 16A3m - 17.8 Aim X 5 Aim -5.4 Aim (average

17/um X 5.2 Aim), Cytoplasm was homogeneous, non-granular and faintly stained. Nuclei were deep pink,compact,oval or irregular in outline. Limiting membrane was found intact.

Gametocytes (Figs. 4 - 11 )

These were abundant in blood smears

and all of them looked alike. Most of these were intra-

erythrocytic but a few mature vermicular forms were extra-

corpuscular. Considering size, shape and staining

properties only one morphological type could be recognised.

This type was described as slender

vermicular form (Figs. 4-11 )'. Young forms were slender

or oval (Figs, 4 - 5) or with a broad end and a slightly

curved end. These measured 15 Aim - 17.7 Aim X 3/um - 5 Aim

(average 16.4/am X 3.9 Aim ). Cytoplasm was homogeneous

and stained light blue. Nucleus was oval or band-like,

sub-central,deep pink in colour and v;ith compact chromatin

lying inside nuclear membrane.. It occupied almost entire breadth of parasite. Average measurement of nucleus was

5 Aim X 3/jm . As development advanced, these became (Figs^, FIGURES. 1-11. Camera lucida drawings of Haemogregarina n.sp. (c) . Pig.l, Trophozoite. Figs. 2-3, Schizonts. Figs. 4-11, Gametocytes. Figs. 12 - 13. Normal erythrocytes. 134

6 — 7): broad and slender measuring 18.8 Aim - 21.3 Aim X

3.5 Aim - 5.5/am ( average 19.7 Aim X 4.6AUTV^. Cytoplasm stained light blue. Nucleus v/as sub-central ,large, compact and deep pink in colour. Averager measurement of the- nucleus was 6 A^ X 3.8 Aim. Free gametocytes ( Figs. 10 - 111 slender and elongate and had two unequal limbs narrow at one end with a sub-central and oval nucleusm which also stained deep pink colour. These exceeded the length of corpuscle and measured 26.4 Aim - 33 Aim X 3.8Aim - 4.8/um ( average 29.6/um X 4.3 Aim ). Average measure­ ment of the nucleus was 6.7/um X 2.8 Aim. Extra-corpuscular gametocytes (Figs. 10 - 11 ) were less in nvimber and resembled those v;ho are about to leave the erythrocytes. These measured 27.4/um - 30/um X 4/um - 5.5 Aim ( average 28.5 Ami!X 4.8 Aim ),and the average measurement of the nucleus was 6.2/um X 3.3 /um.

Effect of parasite on host cells

Invasion of this parasite caused slight linear hypertrophy of infected erythrocytes as shovm in the following table (measurements in micrometers, /um). 135

Range, Average. Length of normal erythrocyte, 11.8 - 14.7 13 Width of normal erythrocyte. li.e - 8.9 8.1 Length of normal erythrocyte nucleus. 5-5.6 5.3 Width of normal erythrocyte nucleus. 3.2 - 4 3.4 Length of infected erythrocyte. 15,4 - 21' 18.6 Width of infected erythrocyte, 8.8-13.5 11.3 Length of infected erythrocyte nucleus. 6.5-12.2 9.2 Width of infected erythrocyte nucleus. 1.5-3.3 2.3

Nucleus of erythrocyte was usually markedly displaced to one side. Shape of hypertrophied infected cells depended on the position of parasite and on displaced host cell nucleus. Host cell nucleus was not seen in fragmented condition.

Movement of parasite

In citrate solution^parasite moved slowly xirithin host cells. 136

Discussion! The parasite is placed under genus Haemogreqarina because of schizogony in erythrocytes. It resembles Haemogreqarina mirabilis Castellani S: Willey (1904) in the fact that infection occurs in same species of snake ( 3fenchrophis =- Tropidonotus piscator ). However,the present parasite differs from Haemogreqarina mirabilis in morphological characters. Gametocytes of Haemogregarina mirabilis were reniforro/ thick and smaller measuring 12/um in length and wass enclosed in a capsule. Erythrocytic schizonts were also H. not described inTmirabilis.. Ball (1958) redescribed the same parasite and concluded that the parasite exliibited several features related to genus Hepatogoon espeeially in tissue schizonts. Gametocytes as described by Ball were enclosed ina a clear capsule with a definite limiting membrane . In contrast, the present parasite does not have a capsule and exhibits erythrocytic schizonts in the peripheral blood. Parasite under study also resembles Haemogregarina n.sp.(b^) because of erythrocytic schizonts but differ from it due to many other morphological features: ( sees table 1. page no.i37a,b.). Morphological differences noted here are sufficient to justify the conclusion that the present parasite does not resemble^ with other known genus of the species and therefore, it has been considered a new additiom 137

to genus H&emogregarina.

Diagnosis Family. Haemogregarinidae L^ger,1911. Schizogony intra-erythrocytic7 gametocytes in erythrocytes. Genus. H&emogregarina Danilewsky,1885, Intra-erythrocytic ; non-pigmented; erythcocytio schizont; gametocytes in erythrocytes. Specific characters. Gametocytes exhibiting single morphological type. Type. I. 15 Attn - 33 Aim X 3 Aim - 5.5/um. Trophozoite broad with a large nucleus; trophozoite, 7.1 /uivSg: 5.9 /jm. Schizonts. 16/um - 17.8 /um X 5 /um - 5.4 /um . For the present the parasite is referred to as Haemogregarina n.sp. (c). Host. Xfenchrophis piscator (Schneider ) Site of infection. Peripheral blood. Locality : Bariiipur, 24-parganas,West Bengal, India. I37CU Comparative s:^dy of Haemogreqarina it»isp» (a)':, (b); & (£> in chelonian and ophidian hosts collected from some parts of West Bengal, Ijndia ( measurements in micrometers. Aim)

Haemogregarina n.sp. Haemogre garin a n.sp. Haemogregarin a (a) (b) n.sp.(£) Blood forms Trophozoite 3.5 - 5 X 2 - 3.2 6.4 - 1Q: X 2.3 - 5.5 7.1 X 5.9' SChizODt intra- Intra-erythrocytic Intra-erythrocytic erythrocytic with 3,4,5 &•. schizonts with 6,8,9 & schizonts with 8 nuclei. 11 nuclei. 2 nuclei. Merozoite Merozoite in blood Merozoite not in blood plasma(7.4 X2.5). plasma(10 X 3.6). found. Gametocytes Tv;o types Tv;o types Ohe type Type I. 9.6 - 14 X 2.4 - 3, 3 Type 1.10.5 - 18.2 15 - 33 X 3 -5.5 X a.2 - 4.2 Type II.6.8-9.4 X2.6-3.3 Type II. 22.5-24.6 X 2.4 - 3.2 Non-capsulated, Capsulated. Non-c apsulated.

Sexual dimorphism. Dimorphic hut sexes Dimorphic but sexes Monomorphic, could not be conclusively could not be conclusively distinguished. distinguished.

Tissue phase unknovm. In cells of the liver tissue Unknown in the form of megaloschizonts.

Invertebrate host Placobdella occurs as Partial development of Unkno^-zn ecto-parasites, but sporogony sporogony stage in a leech stages not found. (Haemadipsa zeylanica montivindicis) 13Tb

Haemogre g ar in a Haemogregarina Haemogregarina n. sp. (a) n . sp. (h) n . sp. (£)

Site of infection Peripheral blood, Peripheral blood Peripheral and in the liver tissue. blood.

Host Lissemys punctatus Enhydris enhydris Xenchrophis (Bonnaterre) (Schneider) piscator (Schnei

Reptilia : Chelonia Reptilia :; Ophidia der) Reptilia :Ophidia

Locality: Bongaon, 24-Parganas, Chakdah/Nadia Baruipur/ 24- West Bengal/ India. West Bengal/ India. ParganaS/West Bengal/ India. T38

CHECK-LIST OF H&emoqreqarina IN SOME CHELONIANS

AND OPHIDIi?^S IN INDIA. (*For the present,erythrocytic schizonts or small oocysts without sporocysts will be considered generic feature)

(** indicates new species described in the thesis)

(*** indicates knox^m species ) reported here.

Family, Haemogregarinidae Leger,191T, * Genus. Haemoqreqarina Danilewsky,1885.

Haemogregarina in CheIonia

* ** 1, Haemogregarina laverani Simond,1901. In the blood of Lissemys punctatus,collected from Bongaon, 24-Parganas,West Bengal, 2, Haemogregarina nicoriae Caste Hani S: Willey, 1904; Patton,1908. In the blood °f Emyda granosa ,collected from Madras, Tamil Nadu . r39

*** 3. H&emogreqarina qanqetica (Misra ,Nandi, Raut & Choudhury,1974); Misra,1976, 1981. In the blood of Trionyx qanqeticus,collected from Bongaon, 24-Parganas,West Bengal.

4. Haemogreqarina n.sp. ( a ) In the blood of Lissemys punctatus, collected from Bongaon,

24-ParganaS/ West Bengal.

Haemogregarin a in Ophidia.

!• Haemogreqarlna mirabilis Castellani & Wiliey,19047 Ball,1958. In the blood of Xenchrophis piscator collected from Bombay. ** 2* Haemogreqarlna n.sp. (b) In the blood of Enhydris enhydris collected from Chakdah,Nadia,West Bengal.

** 3. Haemogregarina n.sp. (c) In the blood of Xenchrophis piscator, collected from Baruipur, 24-Parganas, West Bengal. PART. III.

STUDIES ON HepatOZOOn IN SOME SNAICES 140

INTRODUCTION

Hepatozoon is an intra-corpuscular non-pigmented parasite of vertebrates. Levine,et al»,(1980) placed Haemogregarina Danilewsky, 1885, Hfepatozoon Miller, 1908 and Karyolysus Labb^,1894 in three distinct: families viz,,

Haemogregarinidae Leger,1911,„ Hepatozoidae WenYon,1926 and Karyolysidae Wenyon,1926 of the suborder Adeleina under the Class Sporozoea, The characteristic features of the genus Haemogregarina Danilewsky,1885 has • been given in Part II, page nimber. 151, Genus Hepatozoon Miller,1908 is characterised by the presence of schizogony cycle in cells- of the internal organs ( liver, spleen, kidney, lung and bone- marrow etc. ) of vertebrate hosts. At an interval of several generations, some merozoites enter: erythrocytes or: leucocytes as in the case may be, and develop into gametocytes. Sporogony stages are knovm to occur in arthropod hosts. Zygote develops into oocyst stage. These increase in size and become ^quite large. Oocysts contain num.erous sporoblasts and ultjjnately to sporozoites.

Genus Karyolysus Labbe'', 1894 is distinguished from Haemogregarina and Hepatozoon' on the basis of the following characters. 141

Schizogony occurs in the endothelial cells of the blood vessels and the gametocytes enter the red blood corpuscles of vertebrate hosts., Sporogony occurs in invertebrate host (mite) v/here large oocyst deveops containing sporoblasts. These escape from oocyst as motile vermicules and enter the egg in v/hich these become sporocysts within v/hich sporozoites are-: developed.

The above three genera have been reported to occur in the peripheral blood of cold-blooded vertebrates, Gametocytes-of these genera do not always provide a reliable clue to their generic differentiation. As a result, most of the snake haemogregarines have been placed under genus Haemogreqarina although in many cases erythrocytic schizogony - and sporogony of these parasites are unknown.

Many species of Hepatozoon have been described from v/arm-blooded vertebrates but there are very- few reports about their pccurrence in cold-blooded animals.

In India/ there has-been little work on Hepatozoon. A syrvey of Hepatozoon in ophidians from some parts of West Bengal was undertaken. Results of this survey are given in this chapter.

The geographical and hostal differences relating to the haemogregarines described in this work appear to be enough to distinguish them from previously named species given the present ver^^ imperfect state of our knowledge of this group. 1'42

REVIEVI OE LITERATURE

Hepatozoon was first seen by

Bentley (1905) in leucocytes of dog from rndia,and it was named Leucooytozoon canis by James(1905). Balfour

Sudan and named it.Leucooytozoon muris in 1906, Miller

(1908) founded genus Hepatozoon for a parasite of leuco­ cytes of rat and named it Hepatozoon perniciosijm,

PTepatozoon in Ophidia.

An ophidian Hepatozoon was discovered by Garnham (1950) from an African sv/amp snake,

Crotaphopeltis deqeni' which he named Hepatozoon minchini.

Hull Sc Camin (1960) described some haem.ogregarines in snalces and stated that these parasites should be placed under genus

Hepatozoon,all belonging to a single species, Marguardt

(1966) described Hepatozoon sp. in some snaEes in Southern

Illinois. Mkrtchyan (1967) described Hepatozoon" ophidia from the blood of Russian snake,Elaphe hohenackeri. Ball, e^t al., (1967) transferred Haemogreqarina rarefaciens to genus Hepatozoon on the basis of thee study of its life cycle,the material being collected from Drymarchon corasis' in Coliraa and Te^ic,Mexico, Clark & Bradford(1969) desc­ ribed five types of a haemoqregaine of the genus Hepatozoon in a single blood smear of a snake,Pituophis melanoleucus mj

catenifer collected from Pacific North West. Ball^et al.^ (1969) described Ifepafeozoon fusifex in Boa constrictor collected in Colima ,Mexico. Pessoa S:; Cavalheiro (1969a/ b) and Pessoa.et. CLI ., (1970) described the sporogonic stages of three species of Hepatozoon in water snakes of Brazil. Booden/et al.,(1970) described Hepatozoon sp. in Boa constrictor and its transmission to a lizard by mosquito vector. Oda.et^; al., (1971) observed Hepatozoon in a snake and its transmission. Pessoa Sc 3iasi(1973) described several species of Hepatozoon parasites of Br^silian snakes. Ball S Chao(1973) observed the sporogo- nous stages of Hepatozoon rarefaciens cultured in a Culex pipiens

WORIC DCN'fi IN INDIA.

Sinha (1980^ described Hepatozoon sp.

in a snake/itophiesma stolata collected from West Bengal.

Hfe (1981^ also recorded Hepatozoon'sp, Ih a rat snake,

Ptyas mucosus collected from Bongaon,24-Parganas,West

Bengal. 144

RSrERIALS

The following hosts were examined

for Hepatozoon Infection in their peripheral blood.

-'-• Pt.yQ.s mucosus (Linnaeus) This is commonly known as Dhaman* in Bengal. It belongs to Eanily Colubridae under Order Squamata. It inhabits plains,often in the vicinity of human habitations. It is diurnal,non-poisonous,timid and is capable of climbing trees. Itr is olive-green,brown,yellowish in colour- above with', irregular cross-bars black in colour on posterior half of boSy. Ventral side is yellowish in colour . Posterior ventrals and sub-caudals are edged black. Head is disiilnct from neck. Eyes are large with a round pupil. It is known as rat- eater althbugh it has no particular choice of food. rt is distributed throughout India, Indo-China ,Sri'Lanka and adjoining countries.

Five specimens were collected from Bongaon,24-Parganas,West Bengal and two of these were found to harbour Hepatozoon in their peripheral blood.

2» Anphiesma stolata (Linnaeus)

This snake is known as striped keel-back. It belongs to Family Colubridae an under Order Squamata. Body is olive-green or brown in colour above t45

with black spots or reticulated cross-bars intersected by- two dorso-lateral yellow stripes. These stripes were well marked on the^hinder part of body. Ventral parts are white sometimes with a black spots on the side of each ventral shield. Head is olive-green in colour and the shields are edged with black. It . : is diurnal^non- poisonous and can be handled easily. It feeds chiefly on frogs and toads. I-tj:. is distributed throughout India, Sri Lanka and adjoining countries.

Six speciemens were collected from Chakdah, Nadia/West Bengal and examined. Two of these were positive for Hepatozoon in their peripheral blood,

3. Naja naja (Linnaeus)

This poisonous snake is popularly known as'Gdkhura'in Bengal. It belongs to Family Slapidae under Order Sguamata. It is olivaceous or brovTnish to black in colour. Head is not very dis­ tinct from neck. Neck region is dilatable which forms hood. Nostrils are large. Scales are placed obliquely, Eyes are round. It; feeds on rats,mice,toads and frogs. It occurs in Bengal,Eastern Himalaya and in Nepal. 146"

Pour specimens were collected from

Baruipur,24-Parganas,West Bengal, India,for examination and

one was foxind to harbour Hfepatozoon in its peripheral blood,

METHODS

The following methods were adopted for the prepararation of blopd smears and tissue sections.

Blood of snakes were obtained either by clipping the tail region or from the facial vein.

Infected snakes were killed and small pieces of tissue from liver and lung were fixed in Bouin's•and

Camoy's fixatives.

Blood smears were stained in Eeishman's and Giemsa's stains.

Tissue sections were stained in Heidenhain

"^ iron-haematoxylin and haematoxylin-eosin stains.

Measurements of parasites were made from

fixed and stained materials and Camera lucida drawings were made,

Photomicrographs were also taken in some cases. OB; •^S-iLAT I On 147/

Hepatozoon in Ophidia.

parasite No. 1.

Hepatozoon n. sp. (a)

(FIGS. 1 - 2fe )

OcKurrenoe

The parasite was encountered in the peripheral blood smears of rat snakes ( Ptyas mucosus Linnaeua ) collected from Bongaon,24-Parganas/West Bengal,Tndia.

Morphology

Parasite in peripheral blood were intra-erythro- cytic gametoocytes which were at various stages of develop­ ment.

Gametocytes (Pigs.l - 10.)

These were abundant in the blood smears.

Two distinct morphological types were found depending on their size,shape and staining properties. Sexes of these gametocytes could not however,be conclusively distinguiished.

Type. I. ( Figs. 1-4.)

Young individuals (Pigs.l - 2 ) were oval or bean-shaped measuring 5.5/um - 9.2/Jm X 1.67um - 3.8/um

(average 7 Aim X 3.2/um ). Cytoplasm was homogeneous, non­ granular and stained very faintly . Nucleus was central, non-compact and irregular in shape. It stained deep pink in colour. Average measurement of the nucleus was 3.2/um

X 2. 3 Aim • 148

Type. II.. As the development advanced, parasites were enclosed in a polar cap at each end which stained deep blue and fitted over parasite as a cup. Polar caps were very prominent xirith a maximiam length of 2.6 /am. Mature gametocyt^s ( Pigs. 3-4) measured 11/um - 13.2/um X 2.7 AMI - 3.3 Aim ( average 12/um X 3/am ), Cytoplasm was homogeneous, basophilic: and non-granjalar . Nucleus was compact, sub- spherical or oval,central or sub-central v;ith deeply stained chromatins. Average measurement of the nucleus was 3.3 /am X 2.6 /um.

Type. rr..

Young individuals (Figs. 5 -6j., were thin and narrow,one end lying broader than other. These measured 9.7 Aim - 11.2 Aim X 1.4/um - 1.6/um ( average 10.5/um X 1.5/um X. Cytoplftsm was homogeneous,non-granu­ lar and faintly stained. Nucleus was elongate or oval sub-central and occupied entire v/idth of cell body. As the development advanced, a capsule developed. Inside the capsule,parasite gradually assumed the shape of a charact­ eristic vermicular form. One end of parasite was sharply bent forming a hook like structure. Other end was broad or tapered slightly. Mature forms ( Pigs. 7 - 10) 4

5 nrr\

T 8 10 11 12

FIGS.I - 10, Camera lucida drawings of Hfepatozoon *H.Sf. (ou). FIGS.l - 4. Intra-erythrocytic gametocytes,T^pe I, FIGS.5 - 10. Intra-erythrocytic gametocytes. Type IX. PIGS.11 - 12. Normal erythrocytes* 149

measured 12/um - 16 Am X 2 Aim - 2.5 Aim ( average 14.2 Aim X 2.2 >um ). Cytoplasm showed similar features as in young forms. Nucleus was sub-terminal and deep pink in colour. Average measurement of the nucleus was 4.3 Aim X 2.4 AJtia,

Tissue phase. NO divisional stages were ever encountered in peripheral blood smears. Schizogony occurred in liver and lung of snake and these could be studied in sections:*

Trophozoites (Pigs. 13 - 14) Early forms as noted in the sections of liver were ovoid to sub-spherical measuring 4.4 Aim - 7.7 Aim X 4.8 Aim - 6.5 Aim ( average 6 .8 Aim X 5 .5 Aim ) . Cytoplasm was homogeneous,non-granular,non-vacuolated and light pink in colour with eosin stain. Nucleus was large, oval and deep blue with haematoxylin stain. Average measurements of nucleus was 3.3 Aim X 1.6 Aim. In sections,trophozoites were found surrounded by a clear empty space. Witl further development the parasite became sub-spherical tto oval and the nucleus divided into two daughter nuclei ( Fig. 15), 150

schizontsi ( Pigs.16 - 26)

Schizonts with two nuclei (Fig,15 ) measured 6.8 Am X 5.6/um. Cytoplasm was homogeneous,non-granular and took up eosin stain. Huclei were deep blue in haemat- oxylin. Average measurement of the nucleus was 2.4 Aim X 1.6yum. As the development proceeded,two types of schizo­ nts formed. Mature macroschizonts ( Pigs. 21 - 25 ) measured 10 /um - 13/um X 8/um - 11/um (' average 11.5 Aim X 9 /um) and produced two (Fig.27 );,four (Figs.21 - 24, 28 - 29 ) and eight (Figs. 25, 29) macromerozoites. Microschizonts (Figs. 16 - 20 ) were sxib-spherical to oval measuring 7,7/um - 11 /jm X 6 .5 /um - 8.8 /tim. ( average 9,5 /um X 7 .2 Aim ) with thirteen (Fig. 18);,sixteen ( Pig. 17), twenty four (Fig. 19 ) and thirty tv7o nuclei (Pig. 20). Ci^toplasm contained a reticular net-work of fibres with dark blue nuclei scattered throughout the cytoplasm. Nuclei were arranged around the peripheral margins of the cytoplasm as dense spherical masses about 1.2/um in diameter.

Merozoites (Figs. 21 - 26 ) Mature macroschizonts (Figs. 21 - 2ifc ) were found to contain tv;o,four and eight macromerozoites. These had a pointed anterior end and a broadly rounded posterior end. Cytoplasm resembled a loose net work with a terminal 151

spherical nucleus. Macromerozoites measured 3.5 Aim - 12 yumXl.6Ami-4/im ( average 5.7/urn X 2.4 Am). Average measurement of the nucleus was 1.2 Aim in diameter. Mature micromerozoites were not found. In one individual ( Fig.23) a mature macroschizont was found to contain four elongate merozoites with a large amount of residual body by their side. Cytoplasm was reticular and light pink in colour while nuclei were deep blue. Macromerozoites ( Pig. 26 ) were also found in lung smear. Two of these lay on two sides of the periphery of macroschizont and measured 10 Aim - 12 Aim X 3.3 Aim - 4 Aim ( average 11.5 Aim X 3.3 Aim ).

Effect of parasites on host cells

Invasion of thiss parasite caused slight linear hypertrophy of infected erythrocytes as shown in the following table.( measurementss in micrometers, Aun )

Ran ge. Ave rage. Length of normal erythrocyte* 12 - 16.5 15 Width of normal erythrocyte. 7,5-11.5 9.8 Length of normal erythrocyte nucleus. 3.3-6.6 5,3 Width of normal erythrocyte nucleus. 2.2-3.8 2.9 Length of infected erythrocyte. 16,3-20.9 18.2 Width of infected erythrocyte. 7-12.2 9.5 FIGS. 13 - 26 . Camera j^cida drawings of the tissue stages of Mepatozoon yusp. (a.). FIGS.13 - 14. Trophozoites in tissue sections of liver. FIG. 15. Blnucleate schizont in tissue sections of liver. FIGS.16 - 20. Immature and mature microschizonts in tissue secticn s of lung. FIGS. 21 - 24. Diimature and mature mncromerozoites in tissue sections of lung. FIG,25. Macromerozoltes in tissue sections of liver. FIG. 26. Macromerozoltes in lung smear. 3. X 1300

EIGURE|; r - 3. Photomicrographs of macromer©zoites of ^' Ifepatozpon n.sp. (a) ,in the sections of 1%'er and limg tissues. 1'. Two macromerozoites in liver section, 2. : I 3. Four macrometozoites;in lung section Eiqht raacrom^rozoites:; in liver section. , k 152

Length of infected erythrocyte nucleus. 5.2-10 7.1 Width of infected erythrocyte nucleus 1.6-3.3 2.2

Nucleus of erythrocyte v/as usually markedly displaced to one side. Sape of hypertrophied infected cells depended on the position of parasite and on displaced host cell nucleus. Host cell nucleus was not seen in fragmented condition.

Movement of parasite

In citrate solution,parasites moved slowly within host cells.

Discussion

Occurrence of schizogony in cells of the liver and lung of the snake host identifies the parasite as a member of the genus Hepatozoon (Miller,1908; Garnham,1950; Mohammed & Man sour, 195%; Btay,l964). Levine et al., (1980) placed the genus Hepatozoon in the Family Hepatozoidae of the sub order Adeleina under the Class Sporozoea. Landau(1973a, b) and Landau ejt al., (1"972)described a Haemoqreqarina sp. in an arthropod and a Hepatozoon sp. in a leech. They 153

reported tissue cysts in the Hepatozoon which are capable of infecting predators. Tissue cysts were not observed in the parasite described here. The parasite under discussion resembles Heoatozoon minchini Garnham (1950) , Heptntozoon rarefaciens Ball et al,, (1967) and Hfepatozoon fusifex Ball et^ &!_•» (1969) in the general pattern of schizogony in tissues of the snake but differs in other morjphological features as v;ell as in the types of tissue schizonts and merozoites. In Hepatozoon minchini^ gametocytes in peripheral blood and schizonts and merozoites in lung sections exhibit only a single morphological type. In Hepatozoon rarefaciens and in Hepatozoon fusifex gametocytes are of one type and three types( depending on the nature of infected ery­ throcytes) respectively. Ball et al./ (1969) considered that more than one species of haemogregarine occurred in Boa constri­ ctor. A comparative study of these species of Hepatozoon with the present parasite is given in table 1 which reveals that the present parasite does not completely resemble any of or any other known species of the genus, and it is therefore considered as a new soecies. 15 4' TABLE 1

Comparative study of Hepatozoon minchinl Gamham,1950; Hepatozoon

rarefaciens Ball ejt ai., (1967); Hepatozoon fusifex Ball et. al^., (1969)

and the present parasite,Hepatozoon n.sp.(a) ( measurements CRongeJ in

micrometers. Aim) .

H. minchinl H.rarefaciens H.fusifex H.n.sp, (a) Gametocytes( Intra-erythrocytic)

One type. One type Three types Two types as per description of gametocytes.

13 - 14 X 3 - 4. 11-18 X 2.5 -9 9,2-17.3 X 2.3-8.1 5.5-13.2 X 15-23 X 4.6-11.5 3.3-9.2 15-18.4 X 4.6-9.2 9.7-16 X 1.4-2.5 Sexual dimorphism Monomorphic. Monomorphic, Trimorphic, Dimorphic; sexes not promi­ sexes not nent. stated.

Development in vertebrate host: In liver In lung tissue. In liver,lung^spleen. In lung,liver, pancreas &. heart tissues.spleen,brain,heart •, " SEkir^ney tissues. ^

Types of tissue schizonts:; One type with Two typess Two types vjith Tv/o types raerozoites with mero2;oites. merozoites. with merozoite

Vector Culex tarsalls. Culex tarsalls Unknown. Unknown. Anopheles albimanus Aedes togoi, . I II .1.1 .1 . II . -^des sirrensis itoblyomma dissimile Site of infection: Peripheral blood. Peripheral Peripheral Peripheral blood. blood blood Vertebrate host. Crotaphopeltis Drymarchon Boa constrictor Ptyas mucosus degeni corals Constrictor constrictor Locality: KavironKavirondd o Gulf of: Colma & Tepic Colima, Mexico Bongaon, Lake Victoria,East Africa. Mexico North jsmerica. West bengax, India. r55

Diagnosis

Family. Hepatozoidae Wenyon,1926. Schizogony in cells of internal organs of verteberate host. Genus. Hepatozoon Miller,1908 Intra-corpuscular ; non-pigmented parasite; schizogony in cells of liver and lung tissues of snake host; gametocytes intra-erythrocytic; sexual dimorphism of gametocytes not prominent. Specific characters. Gametocytes exhibiting two morphologiffial types; Type. I. 5.5/um - 11 Aim X 1.6'"- 3.S/um Type. II. 9,7/um -16 Aim X 1.4^- 2.5 Am Development in vertebrate host(Tissue phase). Trophozoites. 6.4 Aim - 7.7/um X 4.8/um -6.5Afflaf Schizonts aree of two types: Type.I. Macroschizont, (10/um - 13/um X 8/um - 11 Aim) with macromerozoites (3.5/um - 12/um X 1.6 /um - 4 /um) Type. II. Microschizont ( 7.7/um - 11 ;um X 6.5 /Um - 8.8 /um ) . For tthe present the parasite is referred to ^^ Hepatozoon n. sp, (a^), Host. Ptyas mucosus (Linnaeus) Site of infection. Peripheral blood. Liver and lung tissues, Locality : Bongaon,24-Parganas,West Bengal,India. 156

Hepatozoon in Ophidia

Parasite No.2* Hepatozoon" n. sp. (B) ( PIGS. 1 - 16)

Occurrence The parasite was observed in peripheral blood smears of sttriped keel -back snalces ( Amphiesma stol^ta [Linnaeus] ) collected from Chakdah,Hadia/West Bengal,India.

Morphology Parasites in the circulating blood were either intra- or extra-corpuscular. These were in the form of gametocytes which are at various stages of development.

Gametocytes (Figs. 4-14) These were abundant in blood smears and only one morphological type is recognised depending on their size, shape and staining properties. Young individuals (Figs. 4-5) were small, oval or bean-shaped measuring 8.8/um - 9.4/am X 2.2 Aim - 2.4 Aim (average 9/um X 2.3 Aun ). Cytoplasm was homogeneous,non-gra­ nular, non-vacuolated and stained light blue. Nucleus was elonqate or oval ,terminal or sub-teeminal, deep pink in colour,occupying the greater part of cell body. Average measurement of the nucleus was 4.5/um X 2.5 Aim . Cases of double infection of 157

erythrocytes were noticed. Ohe young parasite and a mature parasite could be seen occurring in both sides of host cell nucleus (Fig.5 ). As development proceeded, mature young forms (Pigs. 6 - 7 ) were enclosed in a capsule with a limiting membrane. These became slender and sausages--shaped measuring 11 Ann - 13 Aim X 2.2/um - 4.4 /om (average 12/um X 3.3/um ). Cytoplasm was homogeneous and faintly stained. Nucleus was sub-spherical to oval, sub-central and deep pink in colour. Mature forms(Figs. 8 - 11 ) were long slender and vermicular. These were

capsulated forms measuring 16,5 Aim - 19.8 Aim X 2,5/UIW*3AMI ( average 18/um X 2.7 Aim)* Cytoplasm and nucleus showed similar features as in mature young forms. Average measure­ ment of the nucleus was 4/um X 1,7/um. Double and triple infection of erythrocytes by mature gametocytes were also found. Two to three gametocytes were seen crowding inside host cell which was much distorted (Figs, 9 - 10). Parasites; infecting anucleated cells were also seen and double infection of such case was also observed (Fig.11 ), Ex-capsulation of these gametocytes were encountered in extra-cellular form in blood smears, where parasites were seen to leave capsule. In one case (Fig. 12),the parasite was about to escape,and only a part of its body remained inside capsule. Capsule was colourless,transparent,permiable measuring 17,6 Aim X 5.5 rss

Ex-capsulated extra-cellular gametocytes (Pigs.13 - 14) were narrow longi'.a'*! elongate with tapering ends of which one end was more pointed than the other. These forms measured 17.8 Am - 19.3 Aim X 1.5 Aim - 2.2 Aim { average 18. 4/uni X 1.9 Aim ), Cytoplasm and nucleus showed similar features as in the case of late young forraau Averaqe measurement of the nucleus was 4.5/um X 1.8Aim,

Tissue phase.

No erythrocytic schizogony was seen in peripheral blood smears. Parasite showing various stages of development were detected in sections of liver and lung tissues. Trophozoites (Fig.l ) Early forms were spherical to ovoid measuring 6 Aim - 9 ^im X 4/um - 6.5 Aim ( average 7.2 Aim X 5 /um ), Cytoplasm was homogeneous/non-granular,non-vacu- olated and stained light pink with eosin stain. Nucleus was large, oval and composed of dark-staining chromatin granules which took up blue colour with haematoxylin stain. Average measurement of the nucleus was 2.5/jmK; 2 Aim* Trophozoite (Fig.l) was surrounded by a clear vacuole. It was amoeboid with four cytoplasmic processes and a centr­ ally placed large nucleus which measured 2.2/um in diameter. 159

Sehizont (Fig.2)

Early form contained four nuclei measuring 7.5/Um X 6/umia Cytoplasm was homogeneous> non-granular and stained light pink with eosin. Nucleus was spherical /deep blue with haematoxylin and measured 2 /um in diameter. As the development advanced/two kinds of schizonts were formed. Mature macroschizonts measured 11 Aim X 7.7 Aim^and produced four macromerozoites. Microschizonts (Figs. 15 - 16) were sub-spherical to oval measuring 7.5/um - 16/am X 7.Aim - 10.5 Aini ( average 11 Aim X 8/um )) with sixteen ,thirtysix and sixty or more nuclei. Cytoplasm was reticular,pink in colour and contained dark blue nuclei. Xn some the nuclei were also arranged arotind peripheral margins of schizocyst as dense spherical masses, measuring 1.8 Am in diameter.

Merozoites (Fig.3)

Mature macrosclzonts were found to contain four macromerozoites (Fig. 3). These were with a blunt or somewhat pointed anterior end and a broad poster­ ior end. Macromerozoites measured 5.5 Aim - 8 Aim X 3 Aim - 4.5 Aim (. .average 6.5/um X 3.2 Aim ). Cytoplasm was reticular/non-granular and light pink in colour with eosin. Nucleus was spherical/sub-terminal,compact and stained deep blue with haematoxylin. Average measurement of the nucleus 10 11: 12 13 14 5*193. 13-14. Free gametocytes, FIGURES 1-14. Camera lucida drax-zings of Hepatozoon n.sp.(b) 1. Trophozoite in liver section. 2. Quadrinucleate schizont in liver section. 3. Four macromerozoites in liver section. 4-14, Gametocytes•in erythrocytes. 4 - 6. Developing gametocytes in erythrocytes. 5. Double infection by immature and mature gametocytes 9 - 11. Double infection,triple infection and gameto­ cytes infecting anilcieated erythrocytes. 12. Mature gametocyte escaping from capsule. i&. photc»nic IS of microschizonts of: Hfepatozooi n. at). (b) in the^ sections' of^ lunl reo was 2.5 Aim in diameter. No residual mass was present with macromerozoites. Microschizonts were not fotmd to contain mature micromerozoites.

Effect of parasite on host cells. Invasion of this parasite caused slight linear hypertrophy of infected erythrocytes as shown in the following table ( measurements in micrometers,/mi)

Range. Average, Length of normal erythrocyte. 13.5-17,8 15.7 Width of normal erythrocyte. 8.5-11.5 9,9 Length of normal erythrocyte nucleus. 5.5-6.6 5.7 Width, of normal erythrocyte nucleus, 2.2-4.4 3,2 Length of infected erythrocyte, 16-22.5 19,4 Width of infected erythrocyte.nucleus. 8.8-13.2 11 Length of infected erythrocyte nucleus. 5.6-9 7.2 Width of infected erythrocyte nucleus. 2.2-3.3 2.7

Nucleus of erythrocyte was usually markedly displaced to one side. Shape of hypertrophied infected cells depend on the position of parasite and on displaced host cell nucleus. Host cell nucleus was not seen in fragmented condition. Movement of parasite In citrate solution/ parasites moved slowly within host cells. 1'61

Discussion Developmental stages in lung and liver tissues of the snake host indicates that the parasite belongs to Adelea type of genus Hepatozoon. Parasite under discussion resembles Hepato zoon minchinxGamham (1950), Hepatozoon rarefaciens Ball e^ B.1. , (1967) ,Hepatozoon fusifex Ball et al.,(1969) and Hepatozoon n.sp.(a) in the general pattern of schizogony in tissue of verte­ brate host but differs in other morphological features, as well as in the type of tissue schizonts and merozoites. Erythrocytic forms are smaller in Hepatozoon minchini and tissue schizonts and merozoites are of one kind, Gametocytes of Hepatozoon rarefaciens and Hepatozoon fusifex are different in structure and measurements. Moreover, a capsule is not known in their mature forms. In Hepatozoon n.sp.(a) two morphological types are knov/n. The parasite described in the present study differs from all other known species from snake host on the basis of its morphological characters described above and therefore it appears necessary to consider it as a new species. 162

Diagnosis:

Family. Hepatozoidae Wenyon,1926. Schizogony in cells of internal organs of vertebrate host. Genus. Hepatozoon . Miller,1908. Intra-corpuscular; non-pigmented parasite; gametocytes intra-erythrocytic ; sexual schizogony in cells of liver and lung tissues of snake. Specific characters. Garnetocytes exhilfiiting single morpholo­

gical type; 16.5 Aim S.SAUT^ - 19.8/um X 2.2/um - Si/um* Development in vertebrate host ( Tissue phase) Trophozoites measuring 6 Aim - 9 Aim X 4:/am - 6.5 Aim; Schizonts exhibiting two types; Macroschizont ( 11 Aim X 7.7 ^Aim) with 4 macromerozoites; Microschizont C7,5/urn - 16:/im X 11 Aim - 8 Aim ). For the present parasite is referred to ^'^ Hepatoooon n. sp. (h) •

Host. Amphiesma stolata. (Einnaeus) Site of infection. Peripheral blood.^-iver and lung tissue Locality:: Chakdah,Hadia,West Bengal, India. m3

Hepatozoon in Ophidia. Parasite No.3 Hepatozoon n.sp. (c) (FXGS.. 1-11)

Occurrence The parasite was found in peripheral blood smears of a cobra,Naja naja (Linnaeus)_ collected from Bamipur, 24-Parganas,West Bengal,India.

Morphology Parasite in blood films was in the form of intra- erythrocytic gametocytes which were at various stages of development. No extra-corpuscular form of parasite was observed. Gametocytes (Figs. 1-5) These were abundant in blood smears. Two distinct morphological types were detected depending on their size,shape and staining properties. These two types might represent t\^o different sexes,which could not however, be conclusively distinguished. Type. I, Early forms of these gametocytes were not seen. Young forms (Figs. 1 -2) were encapsulated,elongate, oval or slightly broad with two globular ends. These measu­ red 12/um - 13.8/um X 2,7 yum - 3.3/urn ( average 12.8/um X 2.9 im

/um),. Cytoplasm was homogeneous,non-granular and stamhed light blue in colour. Nucleus was compact,sub-spherical deep pink and with an average measurement of 3.2/um X 2/jm. Mature forms were slender,encapsulated and with a blunt tapering end at one pole with a broad globular end at the other. These measured 14.8/um - 16.5/um X 2.6/um -3.4/um Cytoplasm shov/ed similar features like late young forms. Nucleus was central,compact,oval,deep pink in colour and the average measurement was 3.6/um X 2.4/um.

Type. ir.

These (Figs.4-5) were oval,capsulated measuring 12.2/um - 14/um X 2.2/um - 2.5/um( average 13.2/um X 2.3/um). Inside capsule,parasites were found to contain polar caps at both ends which took up a deep blue colour and fitted over parasite as a cup. Polar caps were 2.2/um in length and remained in oblique or slanting fashion. Cytoplasm was homogeneous,non-granular and basophilic. Nucleus was light pink,non-compact,elongate or stretched with dispersed chromatin granules occupying maximum part of cell body. Average measurement of the nucleus was 7.5/um X 1.3/am. Ii65

Tissue phase.

N5 intra-erythrocytic schizonts were ever encountered in peripheral blood smears. Parasites showing various stages of asexual reproduction in sections of liver were found. Mature schizonts were also observed in blood

smears. Trophozoites (Pigs. 7-8)

Early trophozoite (Eig.8 ) was ovoid and measured 5/um X 3.8/jm» Cytoplasm was homogeneous,non­

granular, non-vacuolated and took up light eosin stain.

Nucleus was round or oval and composed of dark blue chromatin

granules in haematoxylin stain. Nucleus was round or oval.

Trophozoites were surrounded by a vacuole. In one indivi­

dual (Fig. 7 )) the parasite was amoeboid or irregular in shape

and measured 9/um X 4.6/um. Nucleus was large,central,

spherical and measured 3.2/um in diameter.

Schizonts. (Figs. 9 - 12)

Development of parasite inside liver

indicated two types of schizonts. Macroschizont (Fig.9)

measured 13.2/um X 12/um containing two macromerozoites.

Microschizonts (Figs. 10 - 12). were sub-spherical measuring

11.5/im - 16.5/um X 10 ^,um - 14.5/um C average 14/um X 12/um )

with 11 - 12 nuclei. Cytoplasm was reticular and took up

light eosin stain containing scattered nuclei within it. FIGURES.1 -12. Camera lucida drawings of Hepatozoon n.sp. (c). Figs. 1 - 3. Gametocytes Type. I. Figs. 4-5. Gametocytes Type.ir. Fig.6.Normal erythrocyte. Pigs. 7-8, Trophozoites in liver sections. Fig. 9. Macromerozoites. Figs. 10 - 12. Microschizonts. 1. X 135w

FIGURE 1." Photomicrograph of microschizont off Hfepatozoon n.sp. ( c ); in liver smears. rea

Nuclei were arranged around peripheral margins of mature' schizocyst as dense spherical masses which stained dark blue with haematoxylin stain. Microschizont;; (Pig. 16) was also found in peripheral blood smears and measured 25/um X 17 /um. It contained 21 nuclei which took up dark pink colour in Leishraan's stain.

Merozoite (Fig. 9)

Mature macromerozoites ( Fig. 9) were two in number lying side by side. These were 8.5 Aim -9/um X 3/lim - 3.5/um (average 8.7/um X 3.2 Aim ). These were with a blunt or somewhat pointed anterior end and a broad posterior end. Cytoplasm was homogeneous^non-granular: and stained light pink with eosin stain. Nucleus v/as spherical,compact and sub-terminal. No residual body was detected. Micromerozoites were not observed.

Effect of parasites on host cells. Invasion of this parasite caused slight linear hypertrophy of infected erythrocytes as shown in the follov/ing table ( measurements in micrometers, /um) .

Range. Average. Length of normal erythrocyte. 14.3-17.7 16 Width of normal erythrocyte. 8.8- 11 9.9 167

Length of normal erythrocyte nucleus. 5.5-7.77 6.4 Width of normal erythrocyte nucleus. 2.7-4.4 3.5 Length of infected ;'. erythrocyte. 15.5-22 19.6 Width of infected erythrocyte. 7.7 - 13 9.6 Length of infe-cted erythrocytic nucleus 5.2-8.2 7.3 Width of infected erythrocyte nucleus?. 1.9-2.3 2.1

Nucleus of erythrocyte was usually markedly displaced to one side. Shape of hypertrophied infected cells depended on the position of parasite and on displaced host cell nucleus. Htist cell nucleus was not seen in fragmented condition.

Movement of Parasite. In citrate solution, parasite moved slowly within host cells. 168 Discussion Schizogony in the liver cells of cobra has placed the present parasite under genus Hepatozoon« The parasite resembles Hepatozoon minchini Garnham (1950), Hepatozoon rarefaciens Ballet a,l., (1967) , Hepatozoon fusifex Ball et_ al., (1969), Hepatozoon n.sp.(a^) and Hepatozoon n.sp.(b) in general pattern of schizogony in tissue of the snake host, but differs in other morphological features>as v;ell as in the type of tissue schizonts and merozoites. In Hepatozoon minchini , gametocytes in peripheral blood and schizonts and merozoites in the lunig sections exhibit single morphological type. In Hepatozoon rarefaciens and Hepatozoon fusifex the gametocytes are very distinct and different from the parasite described here. In Hepatozoon n.sp. (_a) and ih) the gametocytes in peripheral blood and the tissue schizonts and merozoi­ tes are quite distinct and different from the present case. A comparative study of the present three new species is given ( page no. 170 ) which indicates that these species are different frsm each other in their erythrocY^ic forms as well aaifltissue forms. Thus,the parasite described here is considered a new addition to science a^^d named here as Hepatozoon n.sp,(£)

Diagnosis Family Hepatozoidae Vtenyon,1926, Schizogony in cells of internal organs of vertebrate host. 169

Genus Hepatozoon Miller, 1908. Intra-corpuscular; tissue schizonts in the cells of liver of snake host} Se:-cual dimorphism not prominent . Specific characters In the present study, gametocytes exhibiting tv/o morphological types. Type I. 12/um - 16 .5 Aim X 2.6/um -3.4 A^n* Type II. 12.2 Aim - 14Aiin X 2.2/m - 2.5/urn. Development in vertebrate host i Trophozoite. 5/um - 9/um X 3.8/um - 4.6/jm Macroschizont. 13.2/um X 12 /um:/; with mnrozo- ites 8.5 Aim - 9/om X 3/wn - 3.5 Aom. Microschizonf* 11.5 ATO -25 Aim X 10 Aim - 17/am.

For the present,the parasite is referred to as Hepatozoon n.sp. (c)

Host. Naja naja (Linnaeus)' Site of infection. Peripheral blood S;-liver Locality: Baruipur, 24-Parganas, West Bengal,India. 170 Comparative study of Hepatozoon n. sp • (a),, (b). & ic) in snakes; collected in some parts of West Bengal (measurements in micrometers) Hepatozoon n.sp. (_a) Hepatozoon n.sp. (b) Hepatozoon n .sp. {c)

Blood forms

Gametocytes Tv/o types One type Two types Type I 5.5 - 13.2 X l,e - 3.8 16.5 - 19.8 X 2.5-3 12-16.5 X2.6-3.4 Type II 9.7 - 16 X 1.4 - 2.5 12.2 -14 X 2.2 -2.

Sexual dimorphism Dimorphic; saxes could Monomorphic, Dimorphic;sexes not be conclusively distinguished. could not be conclusive ly distinguished. Development in vertebrate host In the liver & lung tissues. In the liver & In the liver tissue. lunq tissues.

Trophozoite Trophozoite; Macroschizont Trophozoite; macro- macroschizont with with macromerozoites; schizont with macromerozoites; macromerozoites; Microschizont. microschizont. microschizont .

Invertebrate host. Unknown, Unknoim. Unkno;vn, Site of infection Peripheral blood,liver Peripheral blood. Peripheral blood, &': lung tissues. liver & lung tissues. Si liver tissue. Vertebrate host Ptyas mucosus(Linnaeus) itophiesma stolata(Linnaeus) Naja naja (Linnaeus) Loc al ity: Bongaon,24-Parganas Chakdah,Nadia,WestBengal. Baruipur,24-Par- West Bengal,India. India. ganas,West Bengal, India. 171:

CI-iECK-LIST OF Hepatozoon IN INDIMI SNSCES (REPTILIA)

( * indicates new species described in this thesis)

Family. Hepatozoidae Wenyon,1926. Genus.. Hepatozoon Miller^ 1908.

!• Hepatozoon sp. Sinha/1980. In the blood of amphiesma stolata#collected from Chakdah, lsradia,West Bengal. * 2. Hepatozoon n.sp.(a) In the blood of Ptyas mucosus. collected from Bongaon,24-Parganas~;, West Bengal, * 3. Hepatozoon n.sp. (b^) In the blood of Anphiesma stolata ,collected from Cha3cdah^ Nadia, West Bengal. * 4. Hepatozoon: n.sp. (c) In the blood of Naja naja , collected from Baruipur,24- Parganas, West Bengal. PAR T. rv. ^

" STUDIES ON SOME BLOOD PARASITES IN: HIMALAYAN FLYING SQUIRRELS. "= 172

INTRODUCTION

Flying squirrels are a group of interesting semi-arboreal mammals which have attracted the attention of many v/orkers from time to time for theirraultiplicity o f infection in blood. A good deal of work has been done on malaria-like parasi­ tes belonging to genera and Rayella ( Chatterjee,et al^. ,1970; Dasgupta, 1965,1967; Dasgupta & Chatterjee, 1969; Dasgupta & Pal, 1974; Dasgupta,.et al.,.1970,1971; Pal S Dasgupta, 1974,1976; Ray,1949,1960.).

Besides above, Trypanosoma^ Hepatozoon and microfilariae have also been observed in the blood of flying squirrels ( Chatterjee,fefc. al.,1970; Dasgupta,1965; Dasgupta,et al., 1967 ; Sinhaffi Dasgupta,1978) . m the present work observation; on some parasites from the blood of flying squirrels collected in Darjeeling C altitude 200Q - 2010 m-),West Bengal, India,are described. 173 REVIEW OF LITERATURE

A. Trypanosoma Trypanosomes belonging to" sub-genus Herpetosoma Dbflein,1901 was recorded from peripheral blood of a nxmber of ground scfuirrels but there was hardly any report of their occurr­ ence in flying squirrels. WORK DONE IK INDIA. Chatterjee,et al,„(1970) reported trypanosomes in the blood of a Himalayan flying squirrel (Petaurista magniflcus). Sinha & Dasgupta (1978) recorded Trypanosoma sp. from peripheral blood of Petaurista nobilis nobilis.

B:. Hepatozoon Hepatozoon was described from a number of mammals including the ground squirrels. However,no named snecies of HepatOEOon were described from flying sqxiirrels so far.

WORK DONE IN INDIA. ^ Hepatozoon from the flying squirrel (Pteromys petaurista). was first recorded by Donovan (cited by Wenyon, 1926) .. Dasgupta (1965) recorded Hepatozoon in the leucocytes of Petaurista magniflcus collected in Darjeeling (altitude 2000 m),West Bengal.

C,:.- Anaolasma

Anaplasma was known to occur in various mammals but its occurrence in flying squirrels was unknown. r74

D.. Rickettsia Rickettsia was also reported from various animals but nothing was knovm from flying squirrels.

E. Microfilaria Microfilaria from the blood of mammals inclu­ ding ground squirrels was reported by diffe3:~ent workers but there was seldom any report of their presence in flying squirrels.

WORK DONE IN INDIA.

Dasgupta,et al./(1967). reported microfilaria in the blood andd internal organs of Petaurista maqnificus. Chatterjee.et al.,(1970) also recorded filarial infection in Petaurista maqnificus. Dasgupta/et al.,(197S) described an adult filarial worm/Pipetalonema laemmleri corresponding to above microfilariae noted in the above flying squirrel.

MATBRIALS The following hosts were examined for blood parasites in their peripheral blood circulation.

^' Petauriata nobilis nrobills (Gray)

This is popularly knovm as "Gray's flying squirrel" belonging to family Sciuridae under Order Rodentia. It has a slender body with a long bushy tail. Body is deep maroon in colour with a bright yellow line which runs down the spine. 175

parachute is orange-yellow in colour. Proximities of limbs and tail tip are black. Under parts are rufous. It has an arboreal habit. It is distributed throughout Nepal and Darjeeling district of West Bengal. Pour specimens were collected from Darjeeling (altitude 2010 m ),West Bengal and examined. TWO of these were found to harbour Trypanosoma, "and Hepato-

zoon. 2• Petaurista maqnificus(Hodgson) This is commonly known as "Hodgson's flying squirrel". It belongs to Family Sciuridae^i' under Order Rodentia. Body and head are chestnut in colour. Upper part is always darker than the parachute. Shoulders and both sides of body are very much yellow and pale. Dorsal hairs are darkish in colour at the base. Feet are chestnut or black in colour. Tail is rufous having a well defined black tip while lower parts are pale rufous in colour. Xt is distributed in ETepal ano Sikkim.

Chatterjee et al.,(1970) examined nine 3. adults and six young animals collected in and around Darjee­ ling (altitude 2010 ml. Out of vrhich three adults and three youngs were found infected by Trypanosoma. These authors also noted microfilaria in the blood of flying squirrels; 3.. Hylopetes alboniger (Hodgson)

This is popularly known as "particoloured" flying squirrel. It belongs to Family Sciuridae under Order Rodentia. Dorsal surface is covered with grey hairs. Yentral surface is white. P^tagixim is blackish in colour. Tail is dorso-ventrally flattened. It is distributed throughout Eastern Himalaya. Two specimens were collected from Darjeeling (altitude 2000 m),West Bengal and examined. One of these was positive for Anaplasma^Rickettsia and micro­ filaria in its peripheral blood.

METHODS The following methods were adopted for the preparatidn of blood smears; and tissue sections for the study of the developmental stages of parasites in the vertebrate host. In flying squirrels,blood smears were drawn by puncturing the brachial vein of the fore-arm. Blood smears were stained v/ith Leish- man's and Giemsa's stains.. iSmall pieces of tissue from liver, liing and spleen were fixed in Bouin's and Carnoy's fixatives. Tissue sections were stained in Heidenhain''s iron-haematoxylin and haematoxylin-eosin stains. Measurements of parasites were made from fixed and stained materials and Camera lucida drawings were made. Photomicrographs were also taken in some cases. OBSERVATIONS 177

Trypanosoma in Elying Sqviirrel

Parasite ETo. 1.

Trypanosoma (Herpetosoma); indictBn (L\ihe) (FIGS. 11-12 )

1906:w Trypanosoma indicvim Liihe, Menses. Handb. Tropenkrankh, 3_. • ^^ ' TOdd> 1963,Arch. Protistenk., 107 :1 . 1964. Trypanosoma ( H^rpetosoma ) indicimi (Ltihe) Hoare, J. Protozool.,, 11 : 200.

Occurrence The haemoflagellate was found in peripheral blood smears' of Himalayan flying squirrels, Petaurista magnificus (Hodgson) and Petaurista nobilis nobilis (Gray) collected at Darjeeling ( altitude 2010 m ) West Bfengal,India.

Morphology The parasite exhibited only the trypomastigote forms in blood smears(. Figs. 1 - 12 ). These forms were monomorphic, small,elongate,slender and curved. Both the extremities were more or less pointed. Cytoplasm was homogeneous, light blue in colour and contained 3-5 vacuoles in some individuals (Figs. 6,10 ). Nucleus was sub-spherical to ovoid pink in colour and placed more towards posterior part of the cell body. In a few cases, chromatins were localized on two poles of nuclear membrane (Figs. 7 - 8). Kinetoplast was sub-terminal,dot-like and 178

stained deep pink in colour. A trypomastigote<| Fig. 11) was seen to contain two kinetoplasts showing without any sign of division of nucleus. Flagellura arose from the base of kinetoplast extending along the border of undul­ ating membrane and reached upto anterior end of body forming a short and free flagellum. Flagellum along withh undulating membrane formed 2-3 folds but in some individuals (Figs. 9-10) folds could not be detected. A single trypomastigote form (Fig. 12 )} was found in the process of binary fission ^with two kinetoplasts and two nuclei.

Dimensions Measurements of forty trypanosomes were given below. Measurements ( in micrometers, /tun ) of Trypanosoma (Herpetosoma);. indicum iMhe

Range. Average Total length of body (including free flagellum)'. 20.9 - 25.5 23.1

Length of cell body. 17.8 - 20.5 19

Width of cell body. 1.3 - 2.3 1.8 Eength. from posterior extremity to posterior border of kinetoplast . 1.1-2.8 1.8 FIGURES-1 Ttypanosoma ,1906. romatins on .ttwo

netoplasts. mastigote.

#ilfe' 179

Length from anterior border of kinetoplast to posterior border of nucleus. 4.7 - 7.4 5.9 Length from anterior border of nucleus• to anterior extremity* 6,4-10.4 8.9 Length of free flagellxrai. 2.9-5.5 4.1 Length, of nucleus. 1.3-2.3 1.8 Width of nucleus. 1-1.7 1.2 Length of kinetoplast. .7/- 1.2 .9 Width of nucleus. .3 - .7 .5 ffiiclear index (length from posterior end to the centre of the nucleus/ centre of nucleus to the anterior extremity). .96 - 1.08 .95 Kinetoplastic index (length from posterior end to the centre of '. nucleus / centre of kinetoplast to centre of nucleus). 4.23 -1.37 1.32

Movement of parasite In citrate solution, trypanosomes were found to move actively.

Discussion The parasite under report has been placed under genus Trypanosoma on account of a posterior kinetoplast and an undulating membrane. Blood smears of Petaurista magnificus previously prepared by Chatterjee.et al.,(1970) were also compared in this study and the haemoflagellates were found identical, and identified as Trypanosoma (Herpe- tosomaX indiciim (LUhe) on the basis of the follov/ing characters.

Parasites were small,elongate,slender and curved. Both the extremeties were more or less pointed. Cytoplasm was uniformly stained with vacuoles. Nucleus was spherical or oval and took a dark stain, Kinetoplast was dot-like or oval and stained darker than nucleus, parasite with.\ two kinetoplasts was present.

However,the parasite did not show tv/o morphological types, a slender and a broad type in the peripheral blood smears as described by Todd in 1963, In contrast,only single morphological type was seen in this study • • V7hich was identical to broad type of Tbdd's material.

Differences in morphometric measurements and in host range were also noted { Table.1) which are not sufficient for considering the . ' parasite as new one , -The parasite is,therefore,described as Trypanosoma (Herpetosoma) indicxmi (Luhe ,1906)-,,

This is the first record of Trypanosoma (Herpetosoma) indicum (LUhe) from a host other than Funambulus Palmaruin (Linnaeus) , 181-

TABLE. 1.

Tr,ypanosoma^ Present (Herpetosoma ) • J- f-r"^ \ parasite mdicum (Luhe) as described by Todd (.1963). (Measurements in micrometers/ /Um)

Range. Range. Total length of body (including- free fl age Hum ) 22.76 -41.46 20.9-25.5 Length of cell body. 19.74 - 33.84 17.8-20.5 Width of cell body. 1.41 - 4.70 1.3-2.3 Length of free flagellxim. 3.76- 8.46 2.9 - 5.5 Length of nucleus . 1.41 - 3.05 1.3 - 2.3 Width of nucleus. .89 - 2.35 1 - 1.7 Length of kinetoplast. .47 - 1.41 .7 - 1.2 Posterior extremity to posterior l-cinetoplast. 1.88 - 4.70 1.1 - 2.8 Anterior kinetoplast to posterior nucleus. 2.82 - 8.463 4.T - 7.4 jynterior nucleus to anterior extremity. 10.57 - 11.99 6.8- 10.4

Host. Punambulus Petaurista palmarum magnlficus

Pfetaurista nobiljs nobilis 182

Dlaqnosis

Family. Tiypanosoraatidae Doflein/1901' Single nucleus and a kinetoplast ; flagellum arising from base of kineto­ plast. Genus. Trypanosoma Gruby/1843. Kinetoplast posterior to nucleus; flagelliom forming outer margin of undulating membrane. Sub-genus, Herpetosoma Doflein^1901. Meditjm size; kinetoplast sub-terminal. Specific characters, T'rypomastigotes exhibiting single morphological type; measuring 20.9 Aunw 25,5/um X 1.3/um - 2.3/um ; free flage- IJyUm 2.9/um - 5.5 Aim ; nucleus sub-cent­ ral ; kinetoplast sxib-terminal; reprodu­ ction by binary fission. The present parasite is referred to as Trypanosoma (Herpetosoma) indicum (Luhe)

Hosts. Petaurista maqnificus (Hodgson) ^ Pgtaurista nobilis nobilis (Gray). Site of infection. Peripheral blood. Locality : Darjeeling ( altitude 2010 m) ,West Bengal, India, 183

Hepatozoon in Flying squirrels

Parasite No. 2.

HepatozooTt n.sp. (d^)

( PIGS. 1 - 15 )

Occurrence The parasite was encoiintered in peripheral blood smears of flying squirrels,Petaurista nobilis nobilis (Gray) =^^ Petaurista magnificus (Hodgson) collected from Darjeeling (altitude 2010 m ),West Bengal,India.

Morphology Parasites in peripheral blood were intra-leucocytic gametocytes which were at various stages of development. No extra-corpuscular form was seen.

Gametocytes (Figs. 1 - 10 ) These were few in blood smears. Depending on their size, shape and staining properties only one morph­ ological type was detected, Gametocytes were described as broad and oval form (Figs. 1 - 10 ). Young forms (Figs. ., 6/ , -10) were either oval or slightly bent at one pole and measured 11 /um - 13/uin X 4/um - 5.5/urn ( average 12/urn X 4.2/jm ). Cytoplasm was homogeneous, non-granular and stained light pink in colour. Average measurement of the nucleus was 6/um X 4/um. As development advanced, the parasites became encapsulated. V84

Mature forms (Figs. 1,'. - 5.. ) were broad and oval measuring 13.2/um - 14.5 Aim X 5.8/um - 7.5/um (average 13.5/um X 6.3 Aim ). Cytoplasm was homogeneous or reti­ culated/non-granular and stained very faintly. Nucleus was elongate or oval with irregular irf outline in some and stained deep pink. Average measurements of the nucleus was 7 /um X 4 /um.

Measurements of Hepatozoon n.sp. (d) from two species flying squirrels.

C Measurements in micrometers> Aim ). Range. Range. Length of parasite. 11-13.2 12-14.5

Width of parasite. 4-6 6-7.5 Length of parasite nucleus. 6 - 7.2 5 - 10.5 Width of parasite nucleus. 3.5 - 4.5 3-5

Host. Petaurista nobilis Petaurista nobilis (Gray), magnificus (Hodgson)

Tissue phase

No intra-erythrocytic schizont was encountered in peripheral blood smears. Parasites 185

showing early development in the spleen were however, fouad in serial sections of spleen.

Trophozoites (Figs. 11 - 15 )

Early developmental stages were trophozoites (Figs. II - 12 ) in the capillaries of spleen. These were either ovoid or spherical in shape and measured 5 yum - 9/um X 2.4/um - 7.8 Aim ( average 7/um X 4.4/jm ), Cytoplasm was homogeneous/non-granular,non-vacuolated and took up light eosin stain. Nucleus was round,terminal or sub-terminal and stained deep blue with haematoxylin. Sometimes an unstained area occurred around the nucleus (Fig. 3 ) . Average measurement of the nucleus was 3 /um in diam­ eter. A vacuole was also present around some trophozoites (Figs. 12 - 13 )). Late trophozoites (Figs. 14 - 15 ) were large and ovoid in shape measuring 10/um - 12.5/um X 5/um - 8/um ( average 11.2/um X 6.5/um )'. Cytoplasm was foamy in appearence and stained light pink with eosin. Nucleus was elongated and with dispersed chromatii, indicating the initial stage of division,terminal in position and stained deep blue with haematoxylin (Fig. 15 ) ..

Effect of parasite on host cells;

Invasion of this parasite caused linear hypertrophy of infected leucocytes as shovm in the following table (measurements in micrometers,/um ), FIGURES. 1-15. Camera lucida drawings of Hepatozoon n.sp.( sL )• Pigs. 1 - 10. Gametocytes. Pigs. 11 - 12. Trophozoites in spleen section. Figs. 13 - 15. Developing trophozoites in spleen section. Pig, 15. Showing initial stage of division in spleen section, 186

Range. Average. Length of normal leucocytes. 11 - 16.5 12.7 Width', of normal leucocytes. 7.8-12.8 10.7 Length of normal leucocyte nucleus. 8.5~13 11 V'fidth of normal leucocyte nucleus. 6.4-11 7.7 Length of infected leucocyte. 12.5-24 17 Width of infected leucocyte. 7.5 - 15 10 Length of infected host cell nucleus. 10 - 22 13 Width of infected host cell nucleus. 4.5- 12 7"

Usually nucleus of leucocyte was markedly displaced to one side. Shape of hypertrophied infected cells depended on the position of the parasite and on thee displaced host cell nucleus. In some,host cell nucleus was seen in fragmented condition.

Movement of parasite:: In citrate solution/parasites were found to move slowly within the host cells.

Discussion

Wenyon (1926) placed all the haemogreg- arines of mammals in genus Hepatozoon Miller (1908). Levine^_ 187

et _al_./(1980) placed the genus Hepatozoon in the Family e Hepatozoidae of the sub order Ade^Ji^ina under the class Sporozoea, Schizogony in the spleen of flying squirrels has placed the present parasite under the genus Hepatozoon. Parasites from blood smears of Petaurista maqnifitiuus previously prepared by Dasgupta (1965) were compared with those found in the blood smears of Petaurista nobilis nobilis. All the parasites thus studied were considered identical though a slight variation in size could be noted. The ' parasite resembles Hepato­ zoon petauri Welsh & Barling(1909), Hepatozoon sciuri (Coles, 1914)' and Hepatozoon qriseisciuri ClarK (1958) in general configuration of gametocyte but differs sufficiently in other morphological characters and in host range as shown in the following table ( measurements in micrometers,/um),

Hepatozoon Hepatozoon- Hepatozoon Hepatozoon 2 (=Haemogregarina) n. sp. (d)

Ijength of sciurj, griseisciuri Oval, Curved, gametocyte. 5-8 10 16 - 18 9.9-11.8 11-14,5 Width of gametocyte. 2.5 - 4 4.5 3.3-4 4-7.5 Wfcfth of gametocyte nucleus. 4.5 Length of gametocyte nucleus, - 8 4-3.3 5-10.5 188

Tissue staqe. unknoxvn liver, Ixing bone-marrow, spleen, & heart blood. liver & spleen.

Invertebrate host. unkno^/m. Ore hope as Euhaemogamasus unknown. wickMami ambulans & Echinolaelaps echidnus

Vfertebrate host, Petaurgs Sciurus Sciurus Petaurista norfolcensis vulgaris carolinensis magnificus &. Petaurista nobllis nobilis

It will be seen from the above table that the parasite under discussion can not be considered as identical with similar parasites described in other hosts. As such the parasite is considered new and is named Hepatozoon n.sp. (d) in this chapter. X89

Diagnosis: Family. Hepatozoidae WenyQnrl926.

Schizogony in cells of internal organs of vertebrate host ; gametocytes in peripheral blood smears. Genus. Hepatozoon^Miller,1908. Intra-leucocytic ; non-pigraented parasite; schizogony in capillaries of spleen. Specific characters, Gametocytes exhibiting single morphologi­ cal type ; broad oval foinn measuring 11 /am - 14.5 Aim X 4 Aim - 7.5 /am. Development in vertebrate host( Tissue phase) Trophozoites (5 Aim - 12.5 Aim X 2.4 Aim - 8 Aim),, in the spleen. For the present the parasite is referred to as Hepatozoon n.sp. (d) .

Hosts. Petaurista magnificus:(Hodgson) & Petaurista nobilis nobilig: (Gray) Site of infectionr:. Peripheral blood. Locality: Darjeeling ( altitude 2010 m). West Bengal, India. r 'I 000

FIGURBSr U - 6:w Camera lucida drawings of finaplasma margjnale Theiler^l910 and Anaplasma centrale Theiler,1911, in erythrocytes; 1-2. Anaplasma margin ale 3, Doiible infection by Anaplasma margin ale and'. Anaplasma cen-ferale ^ ~ ^' Anaplasma central^

fl!HWfllf!SSM;!SlSi«"^---igiS ." 190

jyiaplasma in Flying squirrel

Parasite Nos. 3 & 4 Anaplasma marginale Theiler, & Anaplasma centrale Theiler^ *, : ' ..' .

(FIGS. 1 - 6 ) 1910. $n§2l3^-iB^. Sa£.giEale Theiler, Bull, Soc. Path, .exot.,

1 : 135.; ' ' ' ^ '" 1911. Anaplasma centrale Theiler,Mrector Vet. Res.,Union of South Africa, p.7. Occurrence The parasite was found in peripheral blood smears of a flying scfuirrel,Hylopetes alboniger (HodgsonJ collected from Darjeeling ( altitude 2000 m) ,West Bengal,India,

Morphology Parasites were intra-erythrocytic and found in abundance in blood smears. Tv/o species of these parasites occurred as mixed infection in a single host.

^« Anaplasma marginale Theiler.1910.

These appeared as spherical chromatin granules which stained bright red with Leishman's stain-> measuring 0.2 - 0.40/um in diameter ( average 0.25 Aim ) (Figs. 1 - 3 ). These were found lying at the margin of red blood corpuscles and some individuals (Figs. 2 - 3 ) were present near to host cell limiting membrane. No cytoplasm was apparent. A parasitophorous vacuole was noticed (Fig.3), rg^ii

Double infection was also seen where two species ( Anaplasma marginale and Anaplasma centraleX were found present,

2. Anaplasma centrale Theiler^1911.

These parasites were morphologically very similar to Anaplasma marginale which was differentiated b^" its central position in erythrocytes (Figs. 3 - 6 ). These measured 0.2/am - 0,5/um in diameter ( average 0,28/um) No cytoplasm was apparent but a parasitophorous vacuole was present ( Fig. 3 ). Spherical chromatin granule stained deep red in colour.

Discussion Parasites under discussion belong to genus Anaplasma on account of the presence of spherical bodies in erythrocytes of flying squirrel and has been identi­ fied as Anaplasma marqinale Theiler/1910 and Anaplasma centrale Theiler,1911 for the following considerations. Diagnosis of Anaplasma marginale is made usually on the presence of the marginal bodies in eryth­ rocytes and no cytoplasm is apparent. Anaplasma centrale is characterized by its central position in erythrocytes and no definite cyto­ plasm is present. X92

Eaveran & Pranchini (1914) recorded above tv70 species of genus Anaplasma from a large number of animals including rodents but there is no report of occurrence of these parasites from flying squirrel so far.

This appears to be the first record of gjiaplasma marqinale and Anaplasma centrale from Hylopetes

It^D onijger;.

Diagnosis:

Genus, itoaplasma Theiler/1910.

Spherical chromatin body without

apparent cytoplasm in the corpus­

cles of vertebrates.

Specific characters.

Spherical marginal bodies in erythrocytes; staining bright red; spherical bodies at the centre of erythrocytes; staining deep red. The present parasites are identi­ fied as itoaplasma marginale Theiler^

1910 and Anaplasma centrale Theiler,

-•- • I Lii • J III I • •nil, ' 1911. 193

Host. Hylopetes alboniger (HodgsonJ Site of infection. Peripheral blood. Locality : Darjeeling ( altitude 2000 m), West Bengal, India.

Rickettsia in Plying squirrel

Parasite No. 5. Rickettsia sp.

( , :: . PIGS.1 - 2)

Occurrence The parasite was found in peripheral blood smears of a flying squirrel, Hylopetes alboniqer(Hodgson) collected in Darjeeling ( altitude 2000 m ),West Bengal,India.

Morphology Parasites were intra-leucocytic and detected in blood smears. These were rod-shaped,either straight or slightly cuarved,deep pink with Leishraan's stain,measuring 0.08/am - 1.5/am X 0.33/am - 0.66/um ( average 1.1/lom X 4/um) No cytoplasm was apparent. Parasites showed multiple infection in white blood corpuscles (Figs. 1 - 2 ). ^

I—-I

^

FIGURES' H. - 2. Camera lucida drawings of Rickettsia sp. 194

Discussion Parasite under discussion belongs to genus Rickettsia Rocha-Lima,1916 on account of the presence of minute rod-shaped structures in white blood corpuscles of flying squi- rrel. The relevant literature on this subject reveals that there is no report of occurrence of Rickettsia from flying squirrels so far. This appears to be the first record of Rickettsia £^om Hylopetes alboniger (Hodgsonj.

Diagnosis :

Genus. Rickettsia Rocha-Lima, 1916, Intracellular; minute rod-shaped structures in the leucocytes of vertebrate host. The present parasite is identified as Rickettsia sp.

Host. Hylopetes•alboniger CHodgson ) Site of infection . Peripheral blood. Locality : Darjeeling^ ( altitude 2000 m ), West Bengal, India. 195

Microfilaria in Flying squirrels

parasite iTo. 6.

(; . :.:: . FIGS.I — 2)

Occurrence This larval nematode v/orm was found in perip­ heral blood smears of a flying squirrel, Plylopetes alboniger (Hodgson_i collected in Darjeeling (altitude 2000 m). West Bengal/ India.

Morphology Larval nematodes were extra-cellular and found in abundance in blood smears (Figs, 1 - 2 ). These were unsheathed,long,slender and much coiled. Anterior: part was knob-like while the posterior:part was slightly pointed. Cephalic space was short and in some containing a few sparse nuclei. No stylet was present. Nuclei were crowded and extended upto the tip of tail. Nuclear columns were interrupted by considerable gap at the fixed points. Nuclei took up deep pink colour.

Dimensions The measurements of thiry microfilariae were given in the following table ( measurements in micrometers, /um ):. m

r. X 500 2. X 480

PIGURETS 11 - 2, Photomicrographs of microfilariae. 196

Range, Average. Total length of body. 57-88 70 Width of body. 3.2-5.5 3.6 Anterior part to excretory pore. 17 - 30 24 Excretory pore to anal pore, 31 - 40 33 Anal pore to tip of tail. 8-17 12

Discussion The parasite under report is identified as a larval nematode viz., microfilaria on account of the presence of nerve ring,excretory pore,excretory cells and anal pore. The specific determination can not be made in the absence of adult forms. This appears to be the first record of microfilaria from Hylopetes alboniqer ("Hodgsonj.

Host. Hylopetes alboniger (Hodgsonj Site of infection. Peripheral blood plasma. Adults. Unknown. Locality: Darjeeling ( altitude 2000 m ), West Bengal,India. 197

Microfilaria in Flying squirrel

Parasite NO*7.

( :: .. i, :: .. FIGS.I -2 )

Occurrence The parasite was encountered in blood smears of a filing squirrel^ Petaurista magnificus ( Hodgson ), collected from Darjeeling (altitude 2010 m),West Bengal, India.

Morphology Microfilariae (. Figs. 1 - 2 ) were extra-corpus­ cular and lying inn blood plasma. These were also unsh­ eathed, long, slender and coiled. One end of the worm was rounded and the other end is pointed. Body was filled up with nuclei which stained deep pinK in Leishman's stain. Cephalic space contained sparse nuclei. Nuclei were extended upto the tip of tail. Nuclear columns were interirupted by considerable gap at the fixed points; Mr unstainable part was seen at the posterior third of the bo body with one or more nuclei at lateral margins.

Dimensions The measurements (in micrometers, /am ) of twenty five microfilariae were given in the following table. V. X 428. 2. X 320.

FIGURES 1-2 Photomicrographs of microfilariae. 198

Range, Average, Total length of body. 68 - 95 76 Width of body, 3-5 4 interior part to excretory pore. 20 - 32 25 Excretory pore to anal pore. 33 - 45 39 Anal pore to tip of tail. 10 - 18 11

Discussion Parasite under discussion is identified as microfilaria because of the presence of nerve ring, excre­ tory pore and anal pore. For the present the microfilaria is tentatively identified as Dipetalonema laemmleri,described from Petaurista maqnificus (Hodgson ) by Dasgupta, e1: al,, (1978)

Host, petaurista maqnificus ( Hodgson) Site of infection. Peripheral blood plasma. Locality: Darjeeling ( altitude 2010 m), West Bengal, India, 199

CHECI^IST OP Trypanosoma^ Hepatozoon, Anaplasma^ Rickettsia and I4ICR0FILARIA OF FLYING SQUIRRELS IN INDIA.

( * indicates new speciAs described in the thesis, ) ( ** denotes new host record.)

Family. Trypanosomatidae Doflein,1901. ©fenusi Trypanosoma Gruby,1843. Sub-genus. Herpetosoma Doflein/1901.

1. Trypanosoma (Berpetosoma) sp. Chatterjee/ et al., (1970). In the blood of Petaurista magnificus(Hodgson)/ collected from DarjeelingCalti - tude 2010 m),West Bengal.

2. Trypanosoma (Eferpetosoma) sp. Sinha & Dasgupta, 1978. ^ In the blood of Petaurista nobilis- nobilis:-(Gray),collected from Darjeeling ( altitude 2010 m) West Bengal. , ** 3. Trypanosoma (Hferpetosoma) indicum Ltlhe, 1906. In the blood of Petaurista magnificus (Ebdgson) and Petaurista nobilis nobilis:(Gray),collected from Darjeeling (altitude 2010 m). West Bengal. 200

Family. Hepatozoidae Wenyon, 1926. Gfenus. Hepatozoon Miller/1908.

1. Hepatozoon sp« Donovan (cited by Wenyon,1926) In the blood of Pteromys petaurista. Exact locality not stated. 2* Hepatozoon sp. Dasgupta,1965, In the blood of Petaurista maqnificus (Hodgson)/Collected from Darjeelingr^ (altitude 2010 m ),West Bengal. * 3• Hepatozoon n.sp.(d) In the blood of Petaurista magnificus (Hodgson) and Petaurista nobilis nobilis (Gray),collected from Darjeeling (altitude 2010 m ), West Bengal.

Genus. Anaplasma Theiler,1910.

**l.ADaplasma marginale Theiler,1910. In the blood of Hylopetes alboniqer (Hodgson) ,collected from Darjeeling (altitude 2000 m ),West Bengal, 201

* *2. Anaplasmaa centrale Theiler,1911. In the blood of Hylooetes alboniger (HodgsonJ, collected from Darjeeling ( altitude 2000 m) West Bengal.

Genus. Rickettsia Rocha-Lima,1916.

**1.,Rickettsia sp. In the blood of Hylopetes: alboniger(Hodgsory,collected from Darjeeling (altitude 2000m), West Bengali

1. Larval form of a nematode viz., microfilaria ** In the blood of Hylopetes alboni­ ger (Hodgson) and Petaurista magni- ficus (Hodgson),collected from Darjeeling ( altitude 2000 ,2010 m) West Bengal. RE::FERENCES 202

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.-^fi^PSlTY LIBRAE? lAJA BAMMOaUNFOn APPENDIX (all? Z37

Names proposed at the time of actual publication to new parasites^ are listed below : —

PART. I,

iii.. Trypanosoma bongaonensis n.sp.

PART. IX.

ii. Haemogreqarina dasguptai n.sp. iii.. Haemogreqarina enhydris' n\ sp. V. Haemogregarin a bengalensis n.sp,

PART. IIX.

i, Hepatozoon mucosus n.sp. ii. Hepatozoon dasguptai n.sp « iii . Hepatozoon bengalensis n.sp.

PART. JV.

ii. Hepatozoon darjeelingensis n.sp.