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1
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
2
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.
3
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.
4
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.
5
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.
6
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.
7
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‘,
8
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.
9
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.
10
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
11
(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.
12
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.
13
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
14
‗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
15
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.
16
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
17
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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