CHAPTER- 1
INTRODUCTION
CHAPTER-1
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. This evolution in the use
and meaning of protozoa has come about largely as a result of electron microscopy. ‗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. Soon
after second World War, researchers recognized that the group was a large heterogeneous
assemblage whose members did not all confirm to a single body plan. In 1980 a committee of the
society of protozoologists revised the classification, recognizing seven phyla and further
revisions were recommended by a similar group of experts in 1985.
Because of the small size of most of them protozoa were not detected until Antonym Van
Leeuwenhock developed his microscopes in the seventh century. By the middle of the eighteenth
century other parasitic protozoa were being reported at a rapid rate and such discoveries have
continued unable to the present. 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 Apicomplexa is a protozoan phylum of around five thousand species, the majority of
which are parasitic, infecting a wide range of animals from molluscs to mammals (CavalierSmith, 1993). The emergence of parasite strains resistant to the few efficacious treatments that
are available underscores the urgent need to identify new classes of drug targets and novel anti
parasitic therapeutics (Aspinall et al., 2002; Trouiller et al., 2002). Members of the phylum
include: Plasmodium, the etiological agent of malaria, accounting for one-in-five deaths among
children under the age of five in Africa (World Health Organization, 2003); Toxoplasma gondii,
the causative agent of toxoplasmosis, infects nearly one-in-three of the adult population with
severe implications (Belanger et al., 1999); Cryptosporidium, a waterborne pathogen also with
implications for immune-compromised individuals (Hunter and Nichols, 2002); the invertebrate
parasite Gregarina, a useful model for studying Apicomplexan motility; and the agricultural
parasites, Eimeria, Neospora, Sarcocystis, and Theileria, which cause disease across a range of
livestock (Graat et al., 1996; Dubey 1999).
The Apicomplexan life cycle is complex and may be divided into three broad stages:
sporozoite, merozoite, and gametocyte. While the general life cycle is common to the phylum,
1
CHAPTER-1
there
are
striking
differences
between
species.
Some
require
a
single
host
(e.g., Cryptosporidium), whereas others are more complex, requiring sexual reproduction in the
vector species for transmission (e.g., Theileria and Plasmodium). Apicomplexans are
characterized by a number of defining organelles involved in host cell attachment, invasion, and
the establishment of an intracellular parasitophorous vacuole within the host cell. Proteins stored
in these vesicles are released through the apical complex at the anterior of the cell (Sibley, 2004).
In addition, all Apicomplexans examined to date (with the exception of the Cryptosporidium
and Gregarina) possess an apicoplast organelle that is hypothesized to be an ancient secondary
endosymbiosis with an algal cell (Zhu et al., 2000b; Fast et al., 2001; Toso and Omoto, 2007).
The genome of this plastid has reduced to the point where the remaining genes are
predominantly involved in organelle replication (Wilson et al., 1996). The order Eugregarinorida
Léger, 1900 belonging to the phylum Apicomplexa represents an interesting group ‗Gregarines‘.
Gregarines are distinct from coccidians, haemosporidians and piroplasms and are thought to be
the earliest lineage of Apicomplexan. As the gregarine-host association is an old one are not
capable to develop significant pathology. They are obligatory endoparasites, incapable of active
life outside their hosts and their resistant spore or sporocysts containing the sporozoites in the
normal and primitive means of distribution. Sporocysts or oocysts are the infective stage of their
life-cycle (Mackinnon and Hawes, 1961).
One of the largest phyla of protozoa is ―Apicomplexa‖. The ―Gregarines” are a diverse
group of Apicomplexa, classified as the Gregarinasina or Gregarinia. The large (roughly half a
millimeter) gregarine parasites inhabit the intestine, coelom and reproductive organ of
invertebrates. Approximately two hundred and fifty genera and one thousand six hundred and
fifty species of aseptate have been described so far (Levine, 1976, 1977 and 1988; Clopton,
2000; Hausmann et al., 2003). They are haploid throughout their life-cycles except for the
zygote. This stage is deployed but the first division after its formation is meiotic, so that all other
stages are haploid. Gregarines are of two types: Aseptate (Only found in Oligochaete hosts) and
Septate (Only found in Arthropods Group). The present study deals with only on aseptate
gregarines of earthworm host.
Some ancestral characters found in gregarines (e.g. extracellular feeding stages and a
monoxenous life-cycle). It gives the group a reputation of being ―primitive‖. Although some
lineages of gregarines have retained characters inferred to be ancestral for the group, and perhaps
Apicomplexans as a whole, most gregarines represent highly derived parasites with novel
ultrastructural and behavioral adaptations (Leander, 2008).
2
CHAPTER-1
They are divided into three taxa based on habitat, host range and trophozoite morphology.
Archigregarines are found only in marine habitats. They possess intestinal trophozoites that are
similar in morphology to the infective sporozoites. Phylogenetic analysis suggests that this group
is paraphyletic and will need division. Eugregarines are found in marine, freshwater and
terrestrial habitats. These species possess large trophozoites that are significantly different in
morphology and behavior from the sporozoites. Intestinal eugregarines are separated into septate
and aseptate (which are mostly marine) gregarines depending on whether the trophozoite is
superficially divided by a transverse septum. This taxon contains most of the known gregarine
species. Urosporidians are aseptate eugregarines that infect the coelomic spaces of marine hosts.
Unusually they tend to lack attachment structures and form gamont pairs that pulsate freely
within the coelomic fluid. Monocystids are aseptate eugregarines that infect the reproductive
vesicles of terrestrial annelids. These latter species tend to branch closely with Neogregarines
and may need to be reclassified. Neogregarines are found only in terrestrial hosts. These species
have reduced trophozoites and tend to infect tissues other than the intestine. DNA studies suggest
that the archigregarines are ancestral to the others.
1.1
Characteristics of Gregarina
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.

Tophozoites are of very diverse.

Monoxenous life-cycle, requiring only one host.

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.
3
CHAPTER-1
1.2
General life cycle and mode of transmission of aseptate gregarines to hosts
The transmission of aseptate gregarines to new hosts usually takes place by oral ingestion of
oocysts in both aquatic and terrestrial environments. Some gregarine oocysts might be
transmitted with host gametes during copulation e.g. Monocystis. In either case, four or more
sporozoites (depending on the species) equipped with an apical complex eventually escape from
the oocysts (Fig. a, stages 8 and 1), find their way to the appropriate body cavity and penetrate
the host cells. The sporozoites emerge from a host cell, begin to feed and develop into larger
trophozoites (Fig. a, stage 2). Some gregarines have sporozoites and trophozoites that are
capable of asexual replication, a process called schizogony (or merogony); most gregarines
appear to lack schizogony in their life-cycles (or at least this process has yet to be observed).
Two mature trophozoites eventually pair up in a process called syzygy and develop into gamonts
(Fig. a, stage 3). The orientation of gamonts during syzygy differs depending on the species (e.g.
side-to-side and head-to-tail). A gametocyst wall forms around each pair of gamonts, which then
begin to divide into hundreds of gametes (Fig. a, stage 5). This process is called gametogony
(Fig. a; process B). Pairs of gametes fuse and form zygotes (Fig. a, stage 6), each of which
becomes surrounded by an oocyst wall (Fig. a, stage 7). Within the oocyst, meiosis occurs to
yield four or more spindle-shaped sporozoites (Fig. a, process A = sporogony) (Kuriyama et al.
2005). Hundreds of oocysts accumulate within each gametocyst, and are usually released via
host feces or via host death and decay.
Fig.-a: Illustration of the general life cycle of gregarina: A. Spindle-shaped sporozoites are
produced in the oocyst via meiosis (sporogony); B. Inside the gametocyst gamonts divide into
hundreds of gametes (gametogeny).
4
CHAPTER-1
Fig. b showing the life cycle of the Monocystis species. The infection starts with the
hermaphroditic earthworm host ingesting an oocyst (1, 2) that releases sporozoites (3, 4) within
the intestines. The sporozoite is motile and penetrates the intestinal wall (5) and enters the dorsal
blood vessel (6). The sporozoite enters the seminal vesicles via the dorsoventral hearts (7). The
sporozoites feed on the host's developing spermatocytes (8) in the wall of the seminal vesicle.
The gregarines then move into the lumen of the vesicle where they mature into trophozoites.
Each ball of sperm within the seminal vesicle contains a young trophozoite (9). These
trophozoites are covered by remnants of the sperm cells and often superficially take on the
appearance of a ciliated eukaryotic cell. After consuming the sperm ball, the new mature
trophozoites pair up in syzygy (10). The trophozoites develop into gamonts, and a gametocyst
wall forms around each pair (11). Each of the two gamonts undergo multiple nuclear division to
produce many nuclei developing into gametes. Two gametes, each fuse and form zygotes, which
are surrounded by oocyst walls (12). Within this oocyst, the diploid zygote undergoes meiosis
and then mitosis to produce eight haploid daughter cells by a process known as sporogony. The
gametocysts, or oocysts if they have been released, leave the earthworm through the male genital
pore and are liberated into the soil (13 and 14). Infection of a new host occurs by oral ingestion
of an oocyst (or perhaps through the process of mutual cross fertilization during host sexual
reproduction).
Fig. b: An illustration of the life cycle of Monocystis sp. The infection starts with an earthworm
ingesting an oocyst, and the parasites then develop within the host's seminal vesicle.
5
CHAPTER-1
1.3
Classification of gregarines (Aseptate Eugregarines)
The classification of gregarines differed in various respect for a long time. In present day the
gregarines have been included under the phylum Apicomplexa Levine, 1980 and the class
Sporozoea. The present system of classification shows the multiple organs of gregarines,
extensive survey of literature on aseptate gregarines clarified this fact. Kudo (1966) recognized
ten families under Superfamily Acephalinoidea of the Suborder Eugregarinida and the Order
Gregarinida. The families are Monocystidae, Rhynchocystidae, Zygocystidae, Aikinetocystidae,
Stomatophoridae,
Schaudinnellidae,
Diplocystidae,
Urosporidae,
Allantocystidae
and
Ganymedidae. Levine (1970) established the Apicomplexa as a group in the Sub phylum rank
having Apical complex and consequently the gregarines and most of the sporozoans were
included under it, except the groups; Microspora, Myxozoa, and Asceptospora; which are
without an apical complex. Thereafter, Levine (1971b, 1977a, 1977b, 1977c and 1977d) made
lot of revision of all the species, genera, and families of Acephaline gregarines. Amongst these,
Levine (1977a) made a major revision of the families namely, Zygocystidae, Rhynchocystidae,
Stomatophoridae and Oligochaetocystidae into Subfamily status and placed them under the
Family Monocystidae. He considered the navicular structure of the oocyst as a common
character for all the members of the Family Monocystidae.
The committee of systematic and evolution of the society of protozoologists under the
chairmanship of Prof. Norman D. Levine proposed a newly revised classification of the protozoa
in the year 1980, where seven phyla have been accepted namely, Sarcomastigophora,
Labyrhynthomorpha, Apicomplexa, Microspora, Asceptospra, Myxozoa and Ciliophora. The
phylum Apicomplexa was further subdivided into two classes, Perkinsea and Sporozoea. Three
subclasses were included under the class Sporozoea namely Gregarinia, Coccidia and
Piroplasmia. But certain nomenclatural taxonomic changes were made under the phylum
Apicomplexa in 1988. A new class Conoidasida was included for the structure of its conoid.
Levine (1988) included the order Eugregarinorida Léger, 1900 and suborder Aseptatorina
Chakraborty, 1960. Levine (1988) performed a thorough revision and presented a comprehensive
picture of the aseptate gregarines. He recognized ten families namely Selenidididae,
Lecudinidae, Urosporidae, Aikinetocystidae, Monocystidae, Diplocystidae, Allantocystidae,
Schaudinnellidae, Ganymedidae and Enterocystidae under the Suborder Aseptatorina of the
Order Eugregarinida. This Order is included under the Class Conoidasida, Sub-class
Gregarinasina, Phylum Apicomplexa. Classification of gregarines have been followed of Levine
(1988) in the present work.
6
CHAPTER-1
1.4
Systematics and taxonomy of parasites
In general the world‘s invertebrates of which parasites make up a sizeable fraction are not nearly
as well known as are the vertebrates. Thousands of new species of protozoa, helminthes, and
arthropods are described every year. Taxonomy or the science of classification, is a vibrant an
area of biology today as it was a hundred years ago. A good part of the activity is due to the new
techniques, especially, biochemical ones, that have been adopted by taxonomists. Parasitologists
are constantly evaluating the criteria they use to make taxonomic decisions, trying to decipher
the relationship between biochemical and morphological characteristics of species and reexamining the genus, family and order groupings of the animals, they studied. Taxomomy is a
basic sub discipline of biology. A scientific name carries with it a massive amount of
information, some implied, some explicit and all of value to ecologists, immunologists,
epidemiologists and evolutionary biologists. Taxonomic criteria vary from parasite group to
group. In case of arthropods skeletal morphology is still of primary importance. The
classification of platyhelminthes is based on reproductive organs-primarily their numbers, sizes
and relative positions in the body. Nematode taxonomists must focus on reproductive structures,
including those at the posterior ends of the male, but the arrangements of sensory papillae and
other cuticular features, especially those around the mouth, are considered also. Protozoan
taxonomic characters include cyst morphology,
number and arrangement of flagella, and
biochemical properties. Members of the genus ―Leishmania‖ for example are ―typed‖ using
isozyme patterns and DNA buoyant density measurements. It is not only easier but more
advisable for experimental biologists to obtain described and documented organisms from such a
collection that it is for them to do the taxonomy themselves. A parasitological ecologist however
must-be prepared to identify animals and to describe them if necessary. Thus the researcher
quickly becomes familiar with the massive body of literature. Some of it published in obscure
and foreign journals that has accumulated since Linnaeas first described.
1.5
Ecology, ethology and distribution of earthworms
Earthworm is therefore major invertebrate group that plays an important role in soil formation.
Nutrient recycling and transport. Taxonomically, earthworm belonging to the phylum Annelida
and class oligochaeta. Earthworms belonging to three different categories; these are epigeic,
endozoic and anesic, and the division is on the basis of their vertical distribution in the soil strate,
feeding and defection activity in the soils. The epigeic worms prefer to live in litter heaps or in
any other organic matter on the soil surface. On the other hand, epigeic worms require nitrogenrich resource for completion of their life cycle in a very short time. They are voracious feeders
7
CHAPTER-1
and produce cast exclusively on surface soil. Different depth of soil strate are the habitat of the
endogeic worms and their preferred food is humified matter which at different levels of
degradation. They release much of their caste in the subsurface soil, the surface casting being
negligible which mostly released only under unfavorable conditions. Majority of the endogeic
worms have the intrinsic factors that would trigger them to enter into the seasonal diapauses.
Deep burrower worms, such as the anesic worms forms the characteristic subterranean furrows.
Anesic worms pack the burrow walls with their excrements, thus forming fine tunnels in the soil.
They collect liter from the surface soil and store it in the burrows as food. These worms help in
regular mixing of surface matter to lower strate and also loosening the soil. Lumbricid worms
posses a good musculature in the posterior position of the gizzard and thus they are good burrow
formers in the soil. The mesohuntic group requires a slightly higher level of humus matter and
remains active at a depth of ten to thirteen centimeter. The oligohumic worms are very deep
burrowers and they survive on almost mineralized and very poor organic matter. The polyhumic
group depends on pickets of humus materials in soil and is highly selective feeder restricted to
top ten centimeter of the soil. The high temperature and short spells of heavy rains favour high
microbial degradation and even leaching out of the nutrient materials. So a large number of
tropical worms have adapted to survive on the poor quality of the feed. The worms are also
found to be highly resistant to many pesticides. This quality of worms helps in the distribution of
the chemicals applied to soil. Some of the chemicals also get degraded in their system. In that
they minimize the persistence of toxicity of chemical for a longer period in the soil. The adverse
effect from these characters of the worm is the form of other animals prey upon the worms from
highly contaminated soils, the toxins accumulated in the worms impaired the life of the
predators.
1.6
Host parasite relationship and host specificity
The association of the parasite and the host is an intimate one and the host parasite relationship
may show low degree of specificity i.e. can flourish in a range of host species. The relation of the
gregarine parasites with their host has been one of the much studied topics. Various ideas have
been suggested regarding the relation of the gregarine parasites with their hosts as a result of
extensive observations conducted in recent past on them. Some of these observations have been
summarized by Chatterjee (1971). The host parasite relationship may show low degree of
specificity that is can flourish in a range of host species. Hence the association of the parasite and
the host is an intimate one. The problem of host parasite relationship raises a number of disputes.
The parasites always face a biological hazard especially to find a new host. They encounter some
problems to escape from the old host and also to infect a new host. Besides there are also
8
CHAPTER-1
problem relating to the factors that determine the host parasite relationship, to the degree of
mutual adaptation between the two and the nature of benefit obtained by the parasite and the
extent to which these are harmful to their hosts. Sometime there is a very high degree of
specificity which means that the parasite can only live successfully in a single host species or
small range of related forms. Hence, the problems involved are not solely ecological as evidence
by the varying degrees of specificity found in the host parasite relationship. Such situation is
commonly held to show that the relationship is an ancient one and that the specificity is of a
phylogenetic significance. In such cases, a taxonomical analysis of the parasite group may prove
to understand the taxonomy of the host. Gregarines often failed to draw attention and they have
been given unimportance, since they are parasites of invertebrates (Levine, 1977). However,
Levine (1977) suggested the need for their study as they may be used for biological control of
disease vector. Studies on the impact of gregarines on their hosts have been performed in many
developed and developing countries, but in India limited care has been paid for such perspective.
1.7
Importance of the present study
Taxonomy as a formal science however started with Linnaeus‘s publication of the tenth edition
of Systema Naturae in 1758. The discovery and description of new parasite species continues to
day just as does the description of new species in almost every group of plants and animals.
Although biologists have a massive catalogue of the plants flora and fauna this list is far
from complete. Indeed, based on the rate of new published descriptions scientists estimate that
humans are destroying species faster than they are discovering them, especially in the tropics.
Today systematic rely on published species descriptions as well as on studies of DNA proteins
ecological niches and geographical distribution to develop phylogenies or evolutionary history of
parasites when people became aware that parasites were trouble some and even serious agents of
disease, they began an ongoing effort to heal the infected and eliminate the parasites. Curiosity
about routes of infection led to studies of parasite life cycle, thus it became generally understood
in the last part of the nineteenth century. As more and more life cycles became known,
parasitologists quickly realized the importance of understanding these seemingly complex series
of ecological and embryological events. The biology of parasite does not differ fundamentally
from the biology of free-living organisms, and parasite systems have provided outstanding
models in studies of basic biological phenomenon. Gregarines are species specific, parasites of a
wide range of invertebrates from phyla arthropoda including crustaceans, insects, myriapods,
annelida inlcluding polycheates, oligochaetes and leeches, nemertea, mollusca, echinodermata
and urochordata. The gregarine life cycle can be related to that of the more medically important
parasites such as malaria. Gregarines are not often studied because they do not parasitize
9
CHAPTER-1
vertebrates and do not even seem to cause serious damage to their invertebrate host. There are
approximately one thousand six hundred named species, but most have not been studied
extensively. It can imagine that there are many more species yet to be described. This study will
investigate the gregarine parasites from the seminal vesicle of earthworms.
In India since the work has been done only on morphotaxonomy but based on the
modern concept of gregarines taxonomy most of the previous reports are not accurate and fully
explained. Keeping this in mind, the present study has been conducted on various groups of
aseptate gregarine form different region of Southeast Asia particularly India, Bangladesh, Nepal,
Bhutan, Myanmar and Thailand with their morphotaxonomy, SEM study, molecular structure
analysis, histopthology, which will contribute to the better understanding the group that would
be helpful in feeling up the gap of knowledge about aseptate gregarine of Apicomplexa, the
diverse group of protozoan parasite.
The study of the aseptate gregarines fauna is widely available from various part of the
world. But till date a comprehensive study has not been conducted on aseptate gregarines in
Southeast Asia. Since Southeast Asia belongs a tropical region and covers a vast geographical
area, so the species diversity will be proportionally high in comparison to other continents of the
world.
1.8
Choice of the host and site of infestation
Gregarine parasites are found in almost all invertebrate groups. Earthworms are readily available
and are fairly dependably infected with gregarines. The seminal vesicles of earthworm are highly
infested throughout the year. Gregarines complete their life cycle within a single invertebrate
host. Highly infested hosts, such as earthworms are easily obtained for use in the laboratory.
They are safe to use and the various life-cycle stages are large so it is easier for students or
researchers to observe. The gregarine life-cycle includes a large vegetative stage often with
interesting motility that is visible under the dissecting microscope. Even with the few commonly
used host species, diversity in the protozoan parasite is evident. In addition to being easier to use
in a laboratory setting, gregarines have special features that make interesting to study in their
own right. This laboratory study is suitable for a number of different laboratory courses from
general surveys of biological diversity, protozoology to parasitology. It is interesting to study a
group of closely related parasites called gregarines that complete their life-cycle within a single
invertebrate host such as ―earthworm‖. Levine (1977) stated that almost all members of different
families of aseptate gregarines occur in earthworms. Seminal vesicles are the site where almost
all stages of life cycles of aseptate gregarines are found at a time, beside this, perivisceral cavity
10
CHAPTER-1
Fig. c: Internal anatomy of an earthworm
Fig. e: Position of Seminal vesicles
(enlarged)
11
CHAPTER-1
the space between the ectoderm and endoderm is the site where different developmental stages
also found. According to Edward and Lofty (1972) gregarine infection may occur in alimentary
canal, coelom, blood system, testes, spermathecae, and seminal vesicles. Whatever may be,
Mackinnon and Hawes (1961) pointed out that the seminal vesicle is the ―locus classicus‖ of
Monocystis species.
1.9
Countries of Southeast Asia and its climate
Southeast Asia or Southeastern Asia is a sub region of Asia, consisting of the countries that are
geographically south of China, east of India, west of New Guinea and north of Australia. The
region lies on the intersection of geological plates, with heavy seismic and volcanic activity.
Southeast Asia consists of two geographic regions: Mainland Southeast Asia, also known as
Indochina, comprises Cambodia, Laos, Burma(Myanmar), Thailand, Vietnam and Peninsular
Malaysia,
and Maritime
Southeast
Asia,
comprises Brunei, East
Malaysia, East
Timor, Indonesia, Philippines, Christmas Island, and Singapore. The climate in Southeast Asia is
mainly tropical–hot and humid throughout year round with plentiful rainfall. Southeast Asia has
a wet and dry season caused by seasonal shift in winds or monsoon. The tropical rain belt causes
additional rainfall during the monsoon season. The rain forest is the second largest on earth (with
the Amazon being the largest). An exception to this type of climate and vegetation is the
mountain areas in the northern region, where high altitudes lead to milder temperatures and drier
landscape. Other parts fall out of this climate because they are desert like.
1.10 Biodiversity of earthworms and their distribution of Southeast Asia: An Overview
In India due to continuous biodiversity surveys of earthworms, number of new species is
increasing day by day. Although in comparison to more than three thousand global species
(Stephenson, 1923) the number of Indian species is far less (Only three hundred ninety). Indian
earthworms were well studied by Stephenson (1923), Julka (1988), Haldar (1998). The
earthworm fauna of Western Himalaya studied by Julka (1995) includes only thirty six species
but the proper collection localities were not mentioned. Later on Paliwal and Julka in 2005
reported twenty nine species from the unnamed collection of Zoological Survey of India and
mentioned nine species from Uttar Pradesh and in 2009 they worked on the faunal diversity of
pong damn, wetland ecosystem series. A recent survey was under taken in different ecological
regions of Uttarakhand during 2006 to 2008 and a total twenty one species were collected of
which seven species have been recorded for the first time from the state. Total number of the
12
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species of earthworms of Uttarakhand is now forty five under twenty genera and eight families.
Reynolds et al. (1976, 1981, 1989 and 1993) and Reynolds and Wetzel (2004) account for eight
thousand three hundred and twenty described species of oligochaetes world wide. From India,
one thousand and ninety three annelid species are recorded, but from West Bengal only one
hundred seventy nine annelid species have been reported (Dof-WB, 2000). Approximately two
hundred and seventy five species of earth worms belonging to sixty four genera, under ten
families are the total strength of Indian earthworm population. According to Julka (1988), Indian
mainland comprises about forty three endemic genera and twenty one are peregrine. They have
been introduced to this region presumably in soil around roots of exotic plants. Out of forty three
genera, thirty are exclusively Indian and thirteen are extra Indian distribution. Peninsula and
north east region of India are the site of endemic fauna. The peregrine genera are distributed
among eight families namely, Lumbricidae (eight genera), Megascolecidae (four genera),
Ocnerodrilidae (three genera), Acanthodrilidae (two genera), Eudrilidae (one genus),
Glossoscolecidae (one genus), Criodrilidae (one genus) and Octochaetidae (one genus). Mainly
due to tolerance to a wide range of ecological conditions and parthenogenetic mode of
reproduction, the successful colonization of the peregrine species had developed. According to
Julka (1988), peregrine lumbricids dominate over the endemic species in the Western Himalayas
and Darjeeling-Sikkim area due to their extensive extent of colonization. In 2008,
Bandyopadhyay et al. worked on the earthworms of North 24 Parganas, West Bengal, India and
their distribution. In 2010, Mandal et al. made a brief check-list on earthworms and their
distribution. In 1999, Jahan et al. worked on abundance of earthworm of Bogra. Mannan et al.,
(1994) worked on bio ecology of the earthworm Metaphire (posthuma), Pramanick and Jahan,
(2003) worked on the earthworm of Northwestern regions of Bangladesh. Reynolds, 1994
worked on the earthworms (Oligochaete, Megascolecidae, Moniligastridae and Octochaetidae);
in 1995, he also recorded some additional earthworms: Glossscolecidae, Ocnerotrilidae;
Reynolds, 1976 made a catalogue of names, descriptions and type specimens of the oligochaeta,
Das and Reynolds, 2003 made a checklist and distribution and distribution of the fresh water and
terrestrial annelida (Oligochaeta, Hirudinea and Polychaeta) of Bangladesh.
During the tenure of the research work 2010-2013, fifteen species of earthworms from
India, Bangladesh, Nepal, Bhutan, Myanmar and Thailand belonging to four families have been
studied for aseptate gregarine parasites. The host earthworms examined were Metaphire
posthuma, M. houlleti, M. anomala, M. peguana, Amynthas diffringens, Perionyx exavatus,
Eutyphoeus waltoni, E. nicholsoni, E. orientalis, E. comillahnus, E. quadripapillatus, E.
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incommodus, Glyphidrilus tuberosus, Lampito mauritii, Drawida nepalensis belonging to the
families Megascoleidae, Octochaetidae, Moniligastridae and Almidae respectively.
Metaphire posthuma is widely distributed in India, Bangladesh, Myanmar, Thailand,
Vietnam, Indonesia. Metaphire houlleti is distributed in West Bengal of India, Nepal,
Bangladesh, Myanmar, Thailand, Malay, Java and Indonesia. Metaphire anomala is present in
India, Myanmar, Thailand, Nepal, Bhutan and Bangladesh. Perionyx excavatus is endemic in
India, Myanmar and Thailand. Lampito mauritii is widely distributed in India, Srilanka,
Bangladesh, Myanmar, Thailand, Malay and Indonesia. Amynthas diffringens is found in India,
China, Srilanka, Nepal, Bhutan, Myanmar, Bangladesh and Indonesia. Drawida nepalensis is
widely distributed in India, Nepal, Bangladesh, Myanmar and Indonesia. Eutyphoeus waltoni are
widely distributed in India. Eutyphoeus nicholsoni, Eutyphoeus incommodus and Eutyphoeus
orientalis are widely distributed in India, Bangladesh. Eutyphoeus comillahnus is widely
distributed in Bangladesh. Eutyphoeus quadripapillatus is widely distributed in India,
Bangladesh. Glyphidrilus tuberosus is widely distributed in India and Myanmar. Gates in 1972
worked on Megadrile oligochaetes of Burma (Myanmar) with special reference to Southeast
Asia.
For studying the taxonomy of the parasites, all the species of earthworms were collected
from different regions of Southeastern countries mainly India, Nepal, Bhutan, Bangladesh,
Myanmar and Thailand. Majority of the collected earthworms are endogenin, geophagus, either
polyhumic or mesohumic worms. Collection of host earthworms were executed from different
niches like forest floor, manure heaps, kitchen drainage, cultivated land, garden, road side,
embankment of pond and irrigation canal etc. Largely, distribution of the host species depends
on the climatic condition and nature of soil of that particular area and moreover temperature,
humidity, rainfall, etc. also play a key role over parasitic load. The study areas (Fig.-p, Fig-q,
Fig-r) are shown in the Appendix of the thesis.
1.11 Aim and Objectives of the research works
The aim and objectives of the of the research work is to study the biodiversity, morphotaxonomy
and systematic as well as the biology of the isolated aseptate gregarines belonging to the
following genera namely, Monocystis, Nematocystis, Stomatophora, Apolocystis, Zygocystis,
Rhynchocystis,
Dirhynchocystis,
Enterocystis
and
Aikinetocystis
under
the
families
Monocystidae, Stomatophoridae, Rhynchocystidae, Dirhynchocystidae, Enterocystidae and
Aikinetocystidae.
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Thorough observations of identified aseptate gregarines from different countries of
Southeast Asia have been made to determining the systematic position. The study have been
elucidated by considering the various stages of life-cycle of the parasites. Different
morphological parameters such as, measurement of trophozoites, gametocysts, oocysts and
syzygy (whether it is present) with closely related species have been presented here. Besides
these the type host, type locality, prevalence, molecular study, SEM study, histopathological
study have also been taken into consideration.
1.12 Diversity of aseptate gregarines and their taxonomic studies
As far as earthworm host is concerned severer genera of parasitic protozoa belonging to the
Phylum-Apicomplexa Levine, 1988, Order-Eugregarinorida Léger, 1900; Family-Monocystidae
Bütschli, 1882 have been reported by earlier workers. The parasites are not equally prevalent
among earthworms of different countries of Southeast Asia. The present study deals with sixteen
new Monocystis species, four new Nematocystis sp., one new Apolocystis sp., one new
Zygocystis sp., three new Dirhynchocystis sp., two new Stomatophora sp., and one new
Aikinetocystis sp. Beside these, Monocystis ayeshae, Monocystis apareshae, Monocystis
sahadatae of Monocystis sp.; Nematocystis bangladeshensis of Nematocystis sp.; Rhynchocystis
silvae of Rhynchocystis sp. and Enterocystis elongatum of Enterocystis sp. have been published
in the journals of national and international repute. The copies of all the published papers have
been incorporated in this thesis.
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