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STUDIES ON THE PROTOZOAN PARASITES

OF THE

EARTHWORMS OF SOUTHEAST ASIA

ABSTRACT

THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF

PHILOSOPHY (SCIENCE) OF THE UNIVERSITY OF KALYANI

SUTAPA SARKAR, M. Sc

PARASITOLOGY LABORATORY, DEPARTMENT OF ZOOLOGY

UNIVERSITY OF KALYANI, KALYANI – 741235

WEST BENGAL, INDIA

FEBRUARY-2014

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INTRODUCTION

The word ‗protozoa‘ was once a phylum name. Today however, the term is being used almost

colloquially, as a common noun that refers to a number of phyla. ‗Protozoa‘ consist of a

single cell although many species contain more than one nucleus during all or portions of

their life cycles and certain stages, such as spores, may be built from more than one cell. By

the middle of the nineteenth century many genera of protozoa had been described and their

enormous structural diversity, complexity and even beauty were widely recognized. At least

forty five thousand species of protozoa have been described to date, many of which are

parasitic. Parasitic protozoa causes harm and affect more people in the world than any other

group of disease organisms. For this reason studies on protozoa occupy a prominent place in

the history of parasitology. The one of the largest phyla of protozoa is ―Apicomplexa‖. All

Apicomplexans are parasitic. The ―Gregarines‖ are a group of Apicomplexan protozoa,

classified as the Gregarinasina or Gregarinia. The large (roughly half a millimeter) parasites

inhabit the intestine of a large number of invertebrates. They are not found in humans.

However, Gregarinasina is closely related to both Toxoplasma and Plasmodium, which cause

toxoplasmosis and malaria respectively. Approximately two hundred and fifty genera and one

thousand six hundred and fifty species of gregarines have been described so far (Levine,

1976, 1977 and 1988; Clopton, 2000; Hausmann et al., 2003). Gregarines are of two types:

aseptate or asegmented (Only found in Oligochaete hosts) and septate or segmented (Only

found in Arthropods Group). The present study deals with only on aseptate gregarines of

earthworm host.

The thesis contains identification, taxonomy and pathobiology of protozoan parasites

(aseptate gregarines) and their morphometric studies and systematics. SEM study and DNA

sequence analysis have also been done.

Characteristics of the Gregarines

Gregarines are single-celled apicomplexan parasites of invertebrates. The group is

characterised by the following general features:

Apical complex in the sporozoite stage.

Attachment to host via a mucron (in aseptate gregarines) or an epimerite (in septate

gregarines).

Relatively large spindle-shaped cells.

Trophozoites are of very diverse shaped.

Monoxenous life-cycle, requiring only one host.

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Inhabit extracellular body cavities of invertebrates such as the intestine, coelom and

reproductive vesicle.

Mitochondria with tubular cristae, often distributed near the cell periphery.

Syzygy—the process in which two mature trophozoites pair up before the formation

of a gametocyst, is present in the life-cycle.

Trophozoite contains a large conspicuous nucleus.

Hosts of gregarines

Gregarines inhabit different body spaces within the invertebrate host, such as the intestine,

coelom and reproductive organs. Most of the species are known to be host specific.

Monocystis sp. infects the reproductive organs of earthworms. Septate gregarines infect the

intestines of marine and terrestrial arthropods.

Study area and period

The studies have been carried out during the period January 2010 to June 2013 at the

Parasitology Laboratory, University of Kalyani, Kalyani, West Bengal, India. Host specimens

were collected from different region of Southeast Asia particularly India, Bangladesh,

Myanmar, Bhutan and Nepal.

Aim and Objectives of the research works

The aim and objects of the present study are:

1. To identify the different types of aseptate gregarines of earthworms of South East

Asia particularly in India, Bangladesh, Myanmar, Bhutan and Nepal.

2. To isolate and characterize the aseptate gregarines.

3. To identify the different stages of life cycle of gregarine parasites.

4. To determine the systematic position of the parasites of earthworms.

5. To observe the different morphological parameters of trophozoites, gametocysts and

oocysts.

6. To make a comprehensive list and distribution of gregarine parasites of earthworms of

South East Asia based on earlier published information.

7. To conduct the SEM study of some aseptate gregarines.

8. To study the pathological changes of the affected organs of the host.

9. To study the DNA profile of some aseptate gregarines.

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LITERATURE REVIEW

Dujardin (1835) discovered a gregarine, Proteus tenax, from an earthworm, Lumbricus

terrestris. Later on, Stein (1848) transferred this parasite of the genus Monocystis and

renamed it Monocystis agilis. A new species of gregarine, Zygocystis cometa was described

by Stein (1848) under the newly erected genus Lumbricus terrestris. Henle (1845) described

Gregarina lumbrici from Lumbricus terrestris, later it was transferred to the genus

Monocystis by Cuenot (1901). Cognetti de Martiis in (1923) transferred Monocystis

lumbricoides to a new genus namely Apolocystis.

Cognetti de Martiis (1923) also described Monocystis magna from Lumbricus terrestris Hesse

(1909) transferred it to the genus Nematocystis. Monocystis porrecta was described by

Schmidt (1854) Hesse (1909) transferred to the genus Rhynchocystis.

Rhynchocystis a new Genus was erected by Hesse (1909). Gregarina perichaetae from

Magascolex novaezealandiae was described by Beddard (1888). Labbé (1899) corrected the

systematic position and placed it under the genus Monocystis.

In 1907 Drzhevetskiy and Drzewieeki established the genus Stomatophora and described

Stomatophora coronate. Hesse (1909) established some new genera namely Rhynchocystis,

Nematocystis Pleurocystis and Monocystis respectively. Two new genera namely

Aikinetocystis and Nellocystis were erected by Gates (1926 and 1933) from the earthworms of

Burma which have been included under a different family Aikinetocystidae

Troisi (1933, 1940) investigated three new species namely Zygocystis wenrichi, Apolocystis

giganitea and Apolocystis minuta. Several new species from France belonging to the genera

Monocystis, Nematocystis, Rhabdocystis, Apolocystis, Zygocystis, Rhynchocystis and

Dirhynchocystis were described by Tuzet and Loubatieres (1946 and 1948), Tuzet and Vogeli

(1956), Tuzet and Zuber-Vogeli (1955).

One more new species from England under the genus Dirhynchocystis from Lumbricus

terrestris was reported by Ruston (1959). Rees (1961, 1962 and 1963) and Rees and Howell

(1966) established two new genera Cephalocystis and Arborocystis along with many new

species under the genera Apolocystis and Monocystis. Knowledge on transmission, nutrition,

distribution life history and systematic of aseptate gregarines of United Kingdom were

enriched by Miles (1962, 1963, a b, c and 1964). Bereczky (1967) from Hungary described

some species under the genera Zygocystis and Apolocystis. Details account on the

morphology and life history of Zygocystis limnodrili from the seminal vesicle of Limnodrillus

hofmeisteri was forwarded by Janiszewska (1968) from Poland.

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Valuable contribution to the taxonomy of monocystid gregarines were made by Segun (1968,

1971 a, b, c and 1972) from England and from Nigeria, Bohatier (1970) from France and

Giere (1971) from Germany. Levine (1977a) revised the family Zygocystidae.

Rhynchocystidae, Stomatophoridae and Oligochactocystidae. Levine (1971b, 1976, 1977b,

1977c and 1977d) revised the family Selenidildae Lecudinidae, Urosporidae and another six

families (Aikinetocystidae, Diplocystidae, Allantocystidae, Schaudinnelidae and

Enterocystidae) were brought together in a separate checklist. Besides, a numerical record in

the progress of taxonomy of the Apicomplexan protozoa till 1987 was also prepared by

Levine (1988).

In India a lot of work has been done in aseptate gregarines during the period 1923 to

1938. Ghosh (1923) worked on the systematics of aseptate gregarines in the earthworms of

Calcutta. In British India investigation on aseptate gregarine fauna were made by Bhatia

(1924a, b, 1929, 1930 and 1938); Bhatia and Chatterjee (1925); Bhatia and Setna (1926 and

1938); Setna (1927, 1931a and b); Chakravarty and Chatterjee (1936). Gates (1926 and 1933)

established two genera Aikinetocystis and Nellocystis from the earthworms of Burma.

Pradhan and Dasgupta (1980a and b; 1982 and 1983a and b) added many new species under

the genera Monocystis, Apolocystis, Zygocystis and Nematocystis. Roy Chowdhury and

Haldar (1984) and Roy Chowdhury and Ghosh (1988) described two new species of

Nematocystis and one new species of Stomatophora. Monocystis lalbagensis from Metaphire

posthuma of Murshidabad district and Stomatophora majumdari from Metaphire posthuma of

Nadia district of West Bengal were described by Bandyopadhyay et al. (2001a and b).

Bandyopadhyay and Biswas (2002) described Monocystis nadiensis from Metaphire

posthuma. In 2004, Bandyopdhyay and Mitra described Zygocystis levinei from Amynthas

nicholsoni from West Bengal. In the same year they described Apolocystis chotonagpurensis

and Stomatophora janovy from India.

Bandyopdhyay and Mitra (2005a, 2005b, 2005c, 2005d and 2005e) described Monocystis

darjeelingensis from the host Amynthas robusta, M. ranaghatensis from Eutyphoeus valtani,

Nematocystis gardenica from Amynthas diffrinegens and Nematocystis kalyaniensis,

Stomatophora cloptoni from Eutyphoeus orientalis, Monocystis levinei from Eutypheous

incommodus and Zygocystis perionyx from the earthworm, Perionyx gravelleyi in West

Bengal, India. Bandyopadhyay et al. (2006a, 2006b, 2006c, 2006d, 2006e and 2006f)

described Monocystis clubae, M. apporectodae, Monocystis metaphirea and a new genus

Stomatocystis, under the family Stomatophorinae; Nematocystis indica, Dirhynchocystis

indica from different hosts of earthworm in West Bengal, India.

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In 2007, Bandyopadhyay et al. made observation on Monocystis arabindae and Nematocystis

majumdari from the seminal vesicles of an earthworm from West Bengal, India.

Bandyopadhyay et al. (2008, 2009a, and 2009b) established Monocystis elongatum,

Monocystis septum and Monocystis constricta respectively from West Bengal, India. Mallik

et al. (2011a) found Nematocystis vinodae from Eutyphoeus nicholsoni. Bhowmik, Mitra, and

Bandyopadhyay (2011b) studied on the biodiversity of aseptate gregarines from the

Oligochaetes of West Bengal. Sarkar et al. (2012a) described Enterocystis elongatum from an

earthworm of Bangladesh. Sarkar and Bandyopadhyay (2012b) also described Nematocystis

bangladeshensis from Metaphire peguana of Bangladesh. Sarkar described (2012c)

Rhynchocystis silvae from Metaphire peguana of Bangladesh. Recently, an annotated list and

a checklist of Monocystis sp. have been established by Sarkar and Bandyopadhyay (2013a,

2013c). Two new Monocystis sp. i.e. Monocystis sahadatae and Monocystis apareshae have

been described from Bangladesh by Sarkar and Bandyopadhyay (2013b and 2014).

Warner (1968) studied the fine structure of Rhynchocystis pilosa. Electron

microscopic studies on the cortical region of monocystid parasites were performed by

Vinckier and Viver (1968) and Vinekier (1969). Vavra and Small (1969) studied aseptate

gregarine movement under scanning electron microscope. Macmillan (1973a and b) studied

on the attachment organelles of the gregarines. Details ultrastructural organization of

trophozoite, gamont, gamet, zygote and oocyst of Apolocystis were done by Martinucci and

Crespi (1979). In 2003a and b Leander et al., have worked on phylogeny of gregarines.

Sarkar and Bandyopadhyay (2013) worked on the Scanning Electron Microscopic

studies on aseptate gregarines. In the same year they also worked on the molecular aspect of

Monocystis of the earthworm collected from West Bengal. The gene sequence of Monocystis

sp. 1 PKB-2013 (Accession no. KC189250) and Monocystis sp. 2 PKB-2013 (Accession no.

KC296788) have also been done and presented in the thesis.

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MATERIALS AND METHODS

Permanent Slide Preparation for Light Microscopy

Live specimen were kept in soil in a tub and taken to the laboratory alive. Standard

methodologies were followed to prepare the permanent slides.

Dissected in of 0.65% Nacl solution

Seminal fluid was drawn out on a slide covered with a cover slip for examination of living

protozoan under a light microscope

Semi dried and fixed in Schaudins fluid (20 minutes)

Smears were stored in 70% ethanol for removal of mercuric chloride

Staining as follows (30% & 50% for 5 minutes each)

Washed with distilled water and preserved them in 3% aqueous Iron alum

Stained in Hematoxylin for 20 minutes

Differentiation was done with 1% Iron alum for light microscopy

Smears are washed in distilled water and upgraded in alcohol (30% 50% 70% 90%

100% alcohol)

Cleaned in Xylene and mounted in DPX

Camera Lucida drawings of different stages of gregarines and photographs were taken with

an Olympus Phase Contrast microscope (15 × 40 magnification, Model CH-2, 396250).

Measurements were taken with the aid of Calibrated Ocular Micrometers (μm). In each case

minimum and maximum values are given, followed in parentheses by arithmetic mean,

standard deviation (SD). The methods of describing shapes of planes and solids have been

done following Clopton (2004).

Preservation and identification of hosts

Identification of host specimens were done by Zoological Survey of India, Kolkata, West

Bengal, India. The Holotype materials have been deposited in the ‗Parasitology laboratory‘,

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Department of Zoology, University of Kalyani, Kalyani – 741235, West Bengal, India and the

Paratypes will be deposited in the national collection of Zoological Survey of India, Calcutta.

Histopathological Slide Preparation

Internal organs namely seminal vesicle, spermatheca and cuticle were aseptically dissected

out from both normal and infected earthworm. The organs were fixed in Bouins fluid 10%

formalin buffer. The fixed organs are then processed in the series of alcohol gradation.

Organs were routinely processed at the Parasitology Laboratory of the University of Kalyani,

Kalyani in the following solutions (in the order they have been listed):

70% ethyl alcohol - two separate one hour baths.

80% ethyl alcohol - two separate one hour baths.

95% ethyl alcohol - two separate one hour baths.

100% ethyl alcohol - two separate one hour baths.

Clearing agent [xylene] - two separate one hour baths.

Paraffin -two separate one hour baths.

Embedding

Organs were then placed in embedding molds to form blocks ready to be sectioned. Cold

trays and melted paraffin were used. Embedding dish was kept in top of the fridge plate for

cooling and solidity properly.

Trimming and Sectioning

To section the solidified blocks, they were trimmed properly using sharp razor blade. The

section cutting were made in uniformed thickness and straight.

Staining and Mounting

The sections were stained by haematoxylin and eosin. Permanent slides were mounted by

D.P.X.

Sample preparation for SEM Study

i. Seminal fluid was taken and centrifuged.

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ii. The pellet obtained was dissolved and washed in phosphate buffer and then smeared

on round glass coverslip.

iii. The material was then fixed in 0.25% gluteraldehyde for not more than 30 minutes

and then dehydrated in alcohol gradations such as 30% 50% 70% 90% 100 %

iv. The dehydrated material was then washed or cleared using the clearing agent isoamyl

alchohol, and then dried.

v. The dried material was then sput coated with gold and observed under microscope.

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OBSERVATIONS

TAXONOMICAL STUDIES OF ASEPTATE GREGARINES FROM EARTHWORM

HOSTS

Name of the host and families of some aseptate gregarines

During the tenure of research work fifteen species of Oligochaete hosts belonging to

four families have been studied for aseptate gregarine parasites.

The name of the host species and their families are given below:

Name of the Hosts Families

(1) Metaphire posthuma Vaillant, 1868 Megascolecidae

(2) Metaphire houlleti Perrier, 1807 Megascolecidae

(3) Metaphire anomala Michaelsen, 1907 Megascolecidae

(4) Metaphire peguana Megascolecidae

(5) Perionyx excavatus Perrier, 1872 Megascolecidae

(6) Amynthas diffringens Baird, 1972 Megascolecidae

(7) Lampito mauritii Kingberg, 1866 Megascolecidae

(8) Drawida nepalensis Michaelsen, 1907 Moniligastridae

(9) Glyphidrillus tuberosus Stephenson, 1916 Almidae

(10) Eutyphoeus incommodus Beddard, 1901 Octochaetidae

(11) Eutyphoeus comillahnus Michaelsen, 1907 Octochaetidae

(12) Eutyphoeus waltoni Michaelsen, 1907 Octochaetidae

(13) Eutyphoeus orientalis Beddard, 1883 Octochaetidae

(14) Eutyphoeus nicholsoni Beddard, 1901 Octochaetidae

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(15) Eutyphoeus quadripapillatus Michaelsen, 1907 Octochaetidae

Total one hundred seventy aseptate gregarines of different groups from different hosts

were isolated during the study period. Among them sixteen new Monocystis species, four

new Nematocystis sp., one new Apolocystis sp., one new Zygocystis sp., one new

Rhynchocystis sp. (Published as Rhynchocystis silvae), three new Dirhynchocystis sp., two

new Stomatophora sp., one new Enterocystis sp. (Published as Enterocystis elongatum) and

one new Aikinetocystis sp. Beside these, Monocystis ayeshae, Monocystis apareshae and

Monocystis sahadatae of Monocystis sp. and Nematocystis bangladeshensis of Nematocystis

sp. have been published in the journals of national and international repute. The aseptate

gregarines, isolated from three families i.e. Monocystidae, Enterocystidae and

Aikinetocystidae with four subfamilies such as Monocystinae, Zygocystinae,

Rhynchocystinae, Stomatophorinae belonging to the following genera Monosystis Von Stein,

1848; Nematocystis Hesse, 1909; Apolocystis Cognetti de Martiis, 1923; Zygocystis Von

Stein, 1848; Rhynchocystis Hesse, 1909; Dirhynchocystis Cognetti de Martiis, 1921;

Stomatophora Drzhevetskii, 1907; Enterocystis Tsvetkov, 1926 and Aikinetocystis Bhatia,

1930 respectively. Besides these some re-described species from different genera have also

been incorporated.

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Genus: Monocystis von Stein, 1848

Mucron not marked; gamonts ovoid, short of elongate, solitary; oocysts biconical,

symmetrical. The genus Monocystis was established by Von Stein, 1848 with the type species

Monocystis agilis obtained from the earthworm Lumbricus terrestris.

The present study deals with sixteen new species of Monocystis observed from the

earthworms of southeast Asia. According to the ‗International Code of Zoological

Nomenclature‘, species that have been encountered for the first time are named as Genus sp.

I sp. nov; Genus sp. II sp. nov. and so on. The observed parasites are as follow:

Monocystis sp. I sp. nov.

Monocystis sp. II sp. nov.

Monocystis sp. III sp. nov.

Monocystis sp. IV sp. nov.

Monocystis sp. V sp. nov.

Monocystis sp. VI sp. nov.

Monocystis sp. VII sp. nov.

Monocystis sp. VIII sp. nov.

Monocystis sp. IX sp. nov.

Monocystis sp. X sp. nov.

Monocystis sp. XI sp. nov.

Monocystis sp. XII sp. nov.

Monocystis sp. XIII sp. nov.

Monocystis sp. XIV sp. nov.

Monocystis sp. XV sp. nov.

Monocystis ayeshae Sarkar and Bandyopadhyay, 2011b.

Monocystis sahadatae Sarkar and Bandyopadhyay, 2013b.

Monocystis aparaeshae Sarkar and Bandyopadhyay, 2014.

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Genus: Nematocystis Hesse, 1909

Gamonts large, cylindroids, nematoid, often with mucorn at anterior end, solitary Oocysts

biconical.

The present study deals with four new species including a re-described species of

Nematocystis observed from the earthworms of southeast Asia. According to the

‗International Code of Zoological Nomenclature‘, species that have been encountered for

the first time are named as Genus sp. I sp. nov; Genus sp. II sp. nov. and so on. The observed

parasites are as follow:

Nematocystis sp. I sp. nov.

Nematocystis sp. II sp. nov.

Nematocystis sp. III sp. nov.

Nematocystis sp. IV sp. nov.

Nematocystis bangladeshensis Sarkar and Bandyopadhyay, 2012b

Nematocystis kalyaniensis Bandyopadhyay and Mitra, 2005 (re-described)

Genus: Apolocystis Cognetti de Martiis, 1923

Gamonts spherical and solitary oocysts biconical, Cognetti de Martiis, 1923 defined

the genus as follows: trophozoite spherical without the principal axis marked by presence of

any spherical peripheral organ; solitary, spore biconical.

The present study deals with one new species of Apolocystis observed from the earthworms

of southeast Asia. According to the ‗International Code of Zoological Nomenclature‘,

species that have been encountered for the first time are named as Genus sp. I sp. nov; Genus

sp. II sp. nov. and so on. The observed parasites are as follow:

Apolocystis sp. I sp. nov.

Genus: Stomatophora Drzhevetskii, 1907

Gamont spherical or ovoid, sucker petaloid, with radiating sides, Oocysts biconical,

with a flattened button at each end, attached to each other end to end in long chains inside

gametocysts.

The present study deals with two new species including one re-described species of

Stomatophora observed from the earthworms of southeast Asia. According to the

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‗International Code of Zoological Nomenclature‘, species that have been encountered for

the first time are named as Genus sp. I sp. nov; Genus sp. II sp. nov. and so on. The observed

parasites are as follow:

Stomatophora sp. I sp. nov.

Stomatophora sp. II sp. nov.

Stomatophora diadema Hesse, 1909 (re-described).

Genus: Rhynchocystis Hesse, 1909

Rostrum of gamont metabolic most often elongated into a conical or cylindrical trunk.

The present study deals with one new species of Rhynchocystis observed from the

earthworms of southeast Asia. The observed parasite as follows:

Rhynchocystis silvae, Sarkar et al., 2012c

Genus: Dirhynchocystis Cognetti de Martiis, 1921

Rostrum of gamont metabolic, most often elongated into a conical or cylindroconical

trunk. Gamont with projections at both ends.

The present study deals with three new species of Dirhynchocystis observed from the

earthworms of southeast Asia. According to the ‗International Code of Zoological

Nomenclature‘, species that have been encountered for the first time are named as Genus sp.

I sp. nov; Genus sp. II sp. nov. and so on. The observed parasites are as follow:

Dirhynchocystis sp. I sp. nov.

Dirhynchocystis sp. II sp. nov.

Dirhynchocystis sp. III sp. nov.

Genus: Zygocystis Von Stein. 1848

Gamonts pyriform, always in frontal (head to head) Syzygy, two to three in syzygy;

Oocysts navicular; in seminal vesicles or coelom of digochaetes.

The present study deals with one new species of Zygocystis observed from the

earthworms of southeast Asia. According to the ‗International Code of Zoological

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Nomenclature‘, species that have been encountered for the first time are named as Genus sp.

I sp. nov; Genus sp. II sp. nov. and so on. The observed parasites are as follow:

Zygocystis sp. I sp. nov.

Genus: Enterocystis Tsvetkov, 1926

Development intra-cellular. Early syzygy, the primate enlarging at its base and the

satellite remaining more or less cyndrical, so that the two sells have a sword or dagger shape;

gametocysts without sporoducts, opening by simple rupture; oocysts ellipsoidal.

The present study deals with one new species of Enterocystis observed from the

earthworms of southeast Asia. The observed parasite as follows:

Enterocystis elongatum Sarkar et al., 2012a

Genus Aikinetocystis Gates, 1926

Syzygy in pairs, tail to tail by the non-ramified ends; in coelom of oligochaetes.

The present study deals with one new species of Aikinetocystis observed from the

earthworms of southeast Asia. According to the ‗International Code of Zoological

Nomenclature‘, species that have been encountered for the first time are named as Genus sp.

I sp. nov; Genus sp. II sp. nov. and so on. The observed parasites are as follow:

Aikinetocystis sp. I sp. nov.

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MOLECULAR CHARACTERIZATION OF Monocystis sp. 1 PKB-2013 AND

Monocystis sp. 2 PKB-2013 BY 28S rRNA GENE SEQUENCING

Molecular property of Monocystis sp. 1 PKB 2013

The A+T (Adenine + Thymine) and G+C (Guanine + Cytosine) contents of the nucleotide

sequence of Monocystis sp. 1 PKB 2013 (KC189250) were obtained as 61.42% and 38.6 %

respectively. The Mol % of the nucleotides obtained have been shown in Fig. 38. The 28s

ribosomal RNA gene sequence (547 base pairs long) of Monocystis sp. 1 PKB-2013

(KC189250) showed that nucleotide sequence contains 161 Adenine bases, 100 Cytosine

bases, 111 Guanine bases and 175 Thymine bases. The complete sequence is given below:

CCACCATTAA ATTTTTATAT TGACGTTACA GACGTGCCAC TTTACACTCG ACTTCTACAT

TTTATTTACT GTATTCTCTT CAATAAATCA CGTTGCGTTT GCTGCCAATG AAACATCTGG

TAGGTTGACG TGCAGATGGA GCATTCACAT TTATGTTTCT GGAATGTGAT AGGTGTGAAG

AATTTCGACA AGGTGAAATG TTACTTACAT CAAAAAGTGA TACCGTCAGG AACTTGTAAA

ACTTTCAGCG ATGGATGTCT CGGATCTCGC AACGATGAAA AACGCAGCTA GCTGCGATAC

GCAGTGTGAC TTGCAAACTT CTGAGAATCA TAAGATTTTT GAACGCAAAC GGTGCTTCTA

GGCTCTGCCT AGCAGCATGT TCATTTCAGT GCTTTAACTT TTTTGTTGCA TTGGTTGCTA

ACGCAACGTG ATTGATTGCT GCGAATGCAG TAACTAAAAT GTTGAAATTG ATGTTTGAAG

TGGCACATCT CTAACTTCAA TTATAAAATT TGTAGAGCCT GAAATTGAAC AAGTATACCC

GTAGAAA

Molecular property of Monocystis sp. 2 PKB 2013

The A+T (Adenine + Thymine) and G+C (Guanine + Cytosine) contents of the nucleotide

sequence of Monocystis sp. 2 PKB-2013 (KC296788) were obtained as 39.3% and 60.7%

respectively. The Mol % of the nucleotides obtained have been shown in Fig. 39. The 28s

ribosomal RNA gene sequence of Monocystis sp. 2 PKB-2013 (KC296788) contained 139

Adenine bases, 78 Cytosine bases, 101 Guanine bases and 137 Thymine bases. The complete

sequence has been presented below:

GAGAATGGTC AAGTCGTAAC ATGGTATCTG TAGGTGAACC TGCGGATGGA TCATTCACAT

TTAGTTTTTT CTTTGAGTAT GAAAGTAGTG TGTAAATCTC GATATGAAAG AATGTGATCT

TTATCAACTC GAAAGACAGT CAATAACTCG TGAACTTTCA GCGATGGATG TCTCGGGTCT

CGCAACGATG AAGAACGCAG CTAGCTGCGA TACGCAGTGT GACTTGCAAA CTTCTGAGAA

TCATAAGATT TTTGAACGCA AACGGCGCTT CTAGGCTCTG CTTAGTAGCA TGTTCATTTC

AGTGCTTTAA CTATTTTGTT GCTCTAATAA TTAAAGCAAC GTGACTGGTT GCTACGCAGC

AAGCAACTAA GGTTGTCAAG TTGAAGTATG AAGTAGCAAA TCTGTAACTT CAATTATAAA

ATTTGAAGAG CCTGAAATTG AACAAGTATA CCCGT

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HISTOPATHOLOGICAL STUDY OF INFESTED ORGANS

Histology of the ―Study of cells‖ is a very important branch of science which is essential for

the accurate detection of anomalies of tissues which cannot be revealed in general

examination. The pathological changes of the tissues of earthworm host caused due to the

presence of Monocystis sp., Stomatophora sp. and Nematocystis sp. were observed. In this

case the most prominent pathological changes were observed in seminal vesicle and cuticle of

the host. Very remarkable deformation of seminal vesicle characterized by distoration,

breakdown and degeneration of the seminiferous tubules. An external coat made of thick

hyaline material around the cyst ectoplasm was observed in the present study also.

Inflammatory response of host tissue after the maturity of trophozoite has also reported

hypertrophy and vacuolization of cytoplasm in host cells surrounding the mature trophozoite

which envelop the whole Seminal Vesicle tissue. In cuticle, muscle fibres were arranged in

longitudinal pattern in normal host tissues but the tissue architecture was found to be

destroyed in infected condition. Oocysts of parasites were also present in the infected cuticle.

In spermathca, spermathecal duct, tubules, spermathecal gland could not identified clearly.

There were many blood clots and lesions present.

SEM STUDIES OF ASEPTATE GREGARINES

Scanning electromicrographs of different Monocystis, Nematocystis and

Stomatophora; syzygy, gametocyst and oocyst have been done. SEM study of infected organs

of earthworms (seminal vesicle, spermatheca and cuticle) have also been done.

SEASONAL PREVALENCE OF THE ASEPTATES GREGARINES

010203040506070

Seas

on

al p

reva

len

ce (

%)

Different aseptate Gregarines

Seasonal prevalence of aseptate Gregarines

Rainy

Summer

18

SUMMARY

The present study deals with the studies of the description and lifecycle of thirty new

species and two re-described species of aseptate gregarines obtained from different

earthworm hosts, collected from the different regions of India and Indian subcontinent

such as India, Bangladesh, Nepal, Bhutan, Myanmar and Thailand.

For each species detail description of developmental stages such as trophozoite,

gametocyst, and oocyst have been studied and the systematic position of each species

have been elucidated accordingly. Detail account of the holotype and paratype have

been provided.

Information of host, site of infection and prevalence have been presented in a list.

Thorough comparisons with the closely related species under different genera have

been described for each newly established species.

28SrRNA gene sequences and SEM photographs of some selected aseptate gregarines

have been done.

Histopathological changes of the affected organs of the host due to aseptate gregarines

have been observed.

Seasonal prevalence of the different aseptate gregarines have been presented in

Appendix.

19

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