1

CHAPTER 1

INCIDENCE OF PARASITIC INFECTION

INTRODUCTION

The first thorough description of the relation between a digenetic

trematode and its snail host resulted from the investigations into the life

cycle of a liver fluke, Fasciola hepaticaL. and its development in Lymnaea

truncatula (Mull.) by Thomas (1883). Ever since numerous biologist have

been attracted by the simingly inexhaustible variety of life cycles in which

these parasites utilizes molluscan hosts. Though many life cycles remain

unknown but the importance of the sub-class Pulmonata of the class

Gastropoda in the evolution of the modern digenean is clearly evident. The

digeneans are characterized by a complex life cycle in which usually at

least one of the hosts is a mollusc. The work was initiated early in 20th

century to undertake or investigate larval trematode infection in the

freshwater snails. During the fall of 1913 the study of the larval trematode

infestation in the freshwater snails was undertaken by William (1914) at

the suggestion of Prof. Henry B. Word as an attempt to open up this

underdeveloped field. The snails studied, which were obtained from

several sources, yielded a surprisingly large number of species of cercaria,

belonging to a wide variety of trematode groups. Ancient period of study

the grouping of cercariae is done following classification of Luhe (1909).

Some of the digeneans that develop in pulmonates are well known for their

medical or veterinary importance, which is largely due to the outstanding

success of their snail hosts in a variety of freshwater habitats.

Schistosomiasis and Fascioliasisremains urgentproblems, partly because

the snail hosts continue to be abundant, despite attempts to control their

population by chemical and non-chemical (biological) means.

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Fortunately, much of the extensive and rapidly increasing literature

dealing with relation between pulmonates and the Digenea is gathered into

reviews. Recent contributions cover the intermediate host of

Schistosomiasis andFasciolasis (Jordan and Webbe, 1969; Berrei 1970;

Kendall, 1970) and wider fields (Ulmer, 1971; Brooks, 1969). While

Wright (1971) and Erasmus (1972) describe the biology of trematodes with

special reference to experimental studies and most of them involving

pulmonate snails.Trematode parasites are essentially a component of the

fauna associated with aquatic environment and are generally overlooked

until epidemic disease makes the association inescapable (Erasmus 1972).

Free living digeneans larvae may be exceedingly abundant in water, an

infected snail may discharge clouds of cercariae and in the words of Basch

(1975) snails in their natural habitats may be subjected to a barrage of

miracidia derived from numerous species of trematode parasitic in local

vertebrates.

In the life histories of Digenea employ either two or three hosts, a

few cases are known in which a single species of snail may serves both as

first and second intermediate host. The egg - laying generation lives in

vertebrate and their eggs must reach water or dump soil where the

miracidium already developed insidethe egg, before leaving the host may

remain alive. No miracidium can withstand desiccation.

In order to proceed further life cycle of the parasite, it is necessary

for the miracidium, with perhaps a single exception, to get into the body

of a mollusc, such as an aquatic or terrestrial snail or a bivalve. Entry is

passive, the egg is eaten by the mollusc if. This is the usual method of

entry for miracidia of these trematodes which produce small eggs.

Examples are the Heterophyidae, Opisthorchidae, Brychylaemidae and

Plagiorchidae.It is also well known fact that both terrestrial and aquatic

snails are avid eaters of feces and that they gather around the dung of

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vertebrates to feed. The entry into the molluscan host by most miracidia

of medium or large size is brought about by their active penetration

through soft mucous surface tentacle, head-foot organ, the mantle or

possibly of the lining of the respiratory chamber into which they have

been observed to disappear.

The act of penetration into the molluscan host tissue may require

only a few seconds or possibly as much as a minute. Penetration is

accomplished by the joint action of muscular movements, activity of cilia

located on the epidermal plates and the lysis of the host cells, or perhaps of

the mucoproteins constituting the intercellular cements by the secretion

discharged from the penetration glands. These secretions probably

containhyaluronidase which has been found by Levin et al.(1948) in

cercaria possessing penetration glands. The actual process of penetration is

probably the same whether the miracidia attack a soft external surface of

the host or the epithelial lining of the digestive tract by those emerging

from ingested eggs.

In the field of parasitology, in particular d igenean parasitic infection

to the snail hosts, starts from swallowing of trematode‘s eggs or

penetration and enterance of miracidial larval form into the snail body.

After the enterance of the miracidia there starts the incubation period and

this lasts till the emergence or the start of release or discharge of cercaria

from the snail body called as period of prepatency. The start of cercarial

release is known as begin of patency period and this lasts till it is free of

infection. This start of cercarial release till complete shedding of the larvae

is called as period of patency. These two periods i.e. prepatency and

patency are different from parasite to parasite and snail host. This may be

depending on the infection rate i.e. number of miracidial infestation to the

snail.

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Whether the small miracidia which are ingested while the egg comes

into contact with suitable molluscan hosts appears to be largely a matter of

chance. The hazard involved in making connections with suitable mollusc

are truly enormous, a risk in which is compensated bythe worm and large

output of cercariae released by the sporocysts or rediae which develop in

the molluscan host resulting from the successful establishment of a

miracidium.

Almost all known digeneans have early stages which are parasitic in

mollusc (Gastropods, Scaphopods or lamellibranches) and the adults are

internal parasites of vertebrates. A common life cycle comprises a phase of

asexual multiplication in a primary (first intermediate) molluscan host,

followed by a period encystment within a secondary intermediate host and

finally development to maturity with sexual reproduction within a

vertebrate (definitive) host. In most complex life cycles the parasites

require a third intermediate host. Usually the first and second intermediate

hosts are molluscs. Penetration into the first molluscan host is achieved

either within water by a free swimmingmiracidium larvae or parasites‘ egg

may ingested by the host and hatch in its gut. The established miracidium

commonly develops into a mother sporocyst which gives rise in one type

of life cycle to one or more generations of mother sporocysts or in other

type of life cycle to one or more generations of rediae.

Sporocysts are sac like organisms which absorb nutrient through

their surface, while rediae are equipped with a pharynx and gut and can

ingest pieces of host tissue. Daughter sporocyst and rediae produces

another stage, the cercaria, which in most life cycle has a brief free

swimming existence, terminated by encystment as a metacercaria.

Encystment may occur either on vegetation (as a Fasciola) or within the

second intermediate host, which may be a different individual of the

species which serves as a first intermediate host. In some life cycles the

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second intermediate host is ingested by a definitive host. The Schistosomes

are unusual in having cercaria that penetrates directly into the definitive

host. An extensive range of information about the life cycle may be

obtained from Wright (1971) Erasmus (1972) and Canning and Wright

(1972).

Cort (1914) wrote his preliminary report ―Larval trematodes from

North American Fresh Water Snails.‖ The snails examined by Cort were

from different localities throughout the United States and from various

ecological situations. During his survey he described fourteen new species

of cercaria. Cort et al.,(1937)carriedout an extensive survey, by

determining the incidence of 17 species trematodes in over 7000

specimens of Lymnaea emarginataangulata (Sow) from two lakes in North

America.O‘Roke (1917) published a short paper on ―Larval Trematodes

from Kansas Fresh water Snails.‖ His study was less pretentious than that

of Cort‘s but added several new species in the field of Helminthology.

Later on Faust (1919) presented his finding in ―The American Naturalist‖,

which deals with a biological survey of cecariae. His probe includes

examination of total number of host record was 72. The net outcome of the

survey was 61 species of larval trematodes and there were eleven species

of larval trematodes recorded from two or more hosts. He states that ―The

molluscs most heavily infected are the ubiquitous species.

Planorbistrivolvis and Physagyrinathe Western species Lymnaea proxima.

The effect of the exposure dosage of miracidia on the biology of the

snail host and the subsequent development of the larval stages of the

parasites is an interesting problem and in recent years number of

researchers got attracted towards this area of research.(Najarian,1961;Chu

et al.,1966; Pan, 1963; Pesigan et al., 1958; Schreiber and Schubert (1949a

and 1949b); Hanson (1975) recently found that

Schistosomamansonicercarial embryos freed from sporocysts and

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developed to swimming cercariae when cultivated in presence of

Biomphalariaglabrata embryos (Bge) tissue culture. Resently, Paul and

Joseph (1977)made invitro developmental studies onSchistosomamansoni

cercariae.

The proportion of the total body weight of heavily infected snails

that is contributed by larval trematodes was estimated to be about 20% by

Hurst (1927) and Wesenberg Lund (1931) in the case of

Echionostomarevolutum (Froelich) in Physaoccidentalis Tryonand

LeucochloridiumparadoxumCarus in Succinaeaputris(L). But the data

given by Cheng (1971) indicated that this proportion can be about 27% for

Physasayii (Tappan) infected with Echionostomarevolutum.

Numbers of experiments were made on the cercarial release from

snail hosts. Krull (1941) reported that the snail

Pseudosuccineacollumellawhen exposed to a single miracidium of

Fasciola hepatica the number of cercaria emerged ranged from 14 to 629

per snail host. Rothschild (1939, 1942)has indicated that as many as a

million might emerge from a single host. Elon and George (1954) made

observations on the number of daughter sporocysts and cercariae produced

in Physagyrina after exposure to single and multiple Ochetosomatides egg

exposures. They have demonstrated the pattern of cercarial shedding for

the larvae of the Ochetosomatidesand the number of daughter sporocysts

resulting from one or more egg exposures and the duration of the cercarial

shedding period.

While studying population dynamics of the vector snail in the field,

the question arise and can be answered by laboratory studies under

controlled conditions (Sturrock and Sturrock, 1970). He observed a

reduced growth rate in B. glabrata infected at the age of two weeks. In

other experiments on the influence of S. mansoni on the growth of B.

glabrata or B. pfeifferi a temporary accelerated growth rate was found

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(Chernin, 1960; Pan 1963, 1965; Sturrock 1966 and Sturrock and

Sturrock, 1970). In some cases infected snails become eventually stunted

(Pan, 1963 and 1965). On the other hand ‗gigantism‘ has been supposed

to occur in field-infected specimens of some other species of gastropods

(Linke, 1934; Wesenberg-Lund, 1934; Rothschield, 1941, Boettger, 1953

and James, 1965).

Generally, a higher mortality rate (Chernin, 1960, Pan 1963 and

1965; Chu et al. 1966; Sturrock 1966 and 1967 and Sturrock and Sturrock,

1970) and an increased susceptibility to bad conditions eg. Desiccation

(Brumpt, 1941; Oliver et al., 1954) and high temperatures (Etges and

Gresso, 1965) has been found in snails infected with

Schistosomaheamatobium. During the past quarter of the century there has

been much interest on the lines of parasitic affects the behavior, ecology

and evolution of their hosts (Price, 1980; Rollinson and Anderson, 1985;

Dobson, 1988; Barnard and Behnke, 1990; Anderson and May 1991 and

Toft et al. 1991).

The snail counts per unit of time method measures the density of the

snail population (Oliver and Scheidermans 1956) in the marked area only,

not the total population. These authers investigated infection rate of

parasites by shedding and crushing method. A detailed description of the

various types can be found in Cheng‘s report (1973). Some are classified

according to the position and number of body suckers. Some are

categorized according to the shape and relative size of their tails, while

some cercariae are categorized morphologically by specialized body

structure like the Xiphidiocercarie, the stylet bearing cercariae. The various

species have one characteristic in common; they all have an anterior

marginof the oral sucker.

Molluscs provide an environment that the parasite exploits to

achieve considerable growth and reproduction. Intimate contact between

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parasite and host is required and undoubtedly the interaction extend to a

molecular dialogue, which likely triggers development in the parasite and

may or may not the full force of the snail internal defence.

One of the most characteristic features of trematode-mollusc

interaction is their specificity. Despite the fact that aquatic environment are

usually inhibited by several snails species, each host species is normally

found infected with specific larval trematodes and never with others,

despite the array of trematode miracidia present in the environment. The

use of the restricted group of host by the parasite is a phenomenon known

as ―host specificity‖. Trematodes show high specificity in their use of

molluscan intermediate hosts more than in their definite vertebrate host.

Only certain species combinations are compatible (i.e. the parasite

recognizes, penetrates and develops within the snail). Most digenean

species can develop successfully in only a single snail family, genus or

even species (Adema and Loker, 1977).

The occurrence of multiple infections in nature may provide a

possible mechanism for intermediate host switching the process by which a

trematode transfers to a new host. The miracidial penetration of the

―wrong‖ host would normally result in elimination of the incompatible

parasite, but if the invading parasite survives by overcoming the snails

internal defence system, the infection may persist.

Joosse and Van Elk (1986); while working on experimentally

parasitized trematode host snailLymnaea stagnalis by the miracidia of

Trichobilharziaocellata, determined the period of prepatency and patency.

Similar type of observations were made by Pan (1963 and 1965) and

Sturrock and Sturrock (1970) studied the process of egg-laying in

experimentally infected and non-infected planorbid snail,

Biomphalariaglabrata, during early prepatency, prepatency and patency

period of trematode infection.

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Transmission of digenetic trematodes from the snail to the next

host in the life cycle depends largely on the proportion of snails that

release cercaria as well as the number of cercariae released from each

snail (Anderson and May 1991). These factors may be quantified

respectively, in terms of the prevalence and intensity of patent infection

(Margolis et al., 1982, Bush et al., 1997). In field population both these

factors tend to increase with increasing snail size. Among freshwater

snails, for example, a positive correlation between snail size and the

prevalence of infection was reported in 73% of the field studies reviewed

by Sorenson and Minchella (2001); size may also be positively correlated

with intensity of infection (Smith, 1984). For both mammalian and avian

schistosomes, the prevalence and intensity of infection tend to be highest

among the largest snails (Sturrock, 1973; Kulesa et al., 1982; Loker,

1983, Woolhouse 1989 and Niemann and Lewis, 1990).

Large snails are older on an average, than small snails, within a

given population (Minchella et al. 1985). This age variation translates into

differential exposure to and duration of infection among snails of differing

sizes because larger older snails may have been exposed to more miracidia

or may have been infected for a longer period and thus have patent highly

productive infections. Snails size itself (regardless of age) might in fact

affect trematode development; if for example, a relative large snail

provides more space, greater energetic resources or both for production of

cercariae.

In recent year‘s wealth of information gathered on larval stages of

digenetic trematodes from freshwater snails, described throughout the

world. As not by Robert and Janovy (2000), the global prevalence of

several animal parasites has not changed in 50 years. Of particular interest

is Schistosomiasis where although the worldwide distribution of people

infected may have changed due to eradication programs, as in Japan the

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number of people at risk are (200 million) infected and dying (20,000/year)

from Schistosome infection have not diminished (Oliveria et al., 2004).

Parasitic Platyhelminthes are important economically and socially and

deserve the attention they receive. They deserve the attention not because

of they are important economically or medically, but because they are

fascinating animals that provide challenges to understand associations of

animals and model systems in any field of biology.

According to Kerney (1999), the pond snail, L.stagnalis is a

Holaretic freshwater snail and a common host for many trematode parasite

species (Loy and Haas 2001). One or more species of cercariae, sometimes

more than ten, may be found in freshwater and terrestrial gastropod (Ito,

1980). The freshwater snail Paludomuspetrosus is one of the freshwater

snail found in the mountains of Southern Thailand. This species is

common in areas where, Paragonimuswestermanione of the animal lung

fluke that lives in carnivores. Duangduen et al., (2003) reported four types

of larval trematodes from the freshwater snailP.petrosus, Ashrafi et al.,

(2004) studied 4830 different snails from Quiland Province, Iran, from the

point of larval stages of Fasciola, in the snail Lymnaea gedrosiana. Only

seven (0.35%) specimens were found infected with larval stages of

Fasciola species. Hamann (2006) described specimens of

Glypthelminsvitellinophilum from the anuran Lysapsuslimellus,

Cipangopaludinalecythis an edible snail as a host for a new Furcocercous

cercaria from Manipur (Gambhir et al., 2008). Very recently a faunistic

survey has been made by Sharif et al., (2010) to isolate cercariae from

Lymnaeid snails in Central areas of Mazandaran, Iran. Sami and Ghaleb

(2011) investigated larval stages of digenetic trematodes in the

prosobranch gastropod snails from freshwater bodies in Palestine.Most

freshwater snails can become intermediate hosts for trematode cercariae

which may be transmitted to people and animals.

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The present freshwater pulmonate snail Lymnaea acuminata is

inhabitant of varieties of aquatic habitats VIZ freshwater ponds, pools,

ditches and rivers is proned to get exposed varieties of natural and artificial

stress conditions.Through rain water runoff the water at their habitat gets

contaminated by varieties of contaminants and there can be chances of

trematode infection in the water due to human and other animal‘s excreta

entering through rain water runoff. With this insight the present chapter

was framed to cover various aspects as under:

This first chapter deals with the incidences of larval trematode infection

to the freshwater pulmonate snail Lymnaea acuminata. The topic includes

following subtopics as under.

i. Frequency of larval trematode parasitic infection in the snail Lymnaea

acuminata during two years of study period.

ii.Determination of period of patency (cercarial release period) in

naturally infected snails and larval release.

iii. Different types of larval trematode parasites in L. acuminata.

iv. Number of cercariae released during period of patency.

v. Frequency of particular type of parasitic infection during two years

study period in L. acuminata.

vi.Effect of infection on Survival of snail during patency period

vii.Host size dependence larval trematode infection and cercarial release

during patency period

viii.Effect of larval trematode parasitic infection on shell size and weight

of the snail during patency period.

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MATERIAL AND METHODS

The freshwater snail species of Lymnaea acuminata were procured

from different water bodies in and around the city Aurangabad during two

years of study (from Jan. 2009 to Dec. 2010). Immediately snails were

washed with tap water in the laboratory in order to remove mud particles

and algal material present on the shell. Normal sized intact healthy snails

(22 ± 1mm shell length) were sorted out and maintained in 100 ml

dechlorinated tap water. Dechlorination of tap water was done at laboratory

conditions.Water in the trough getting exposed to open air at least 24 hours

prior to use. Next day visual observations were made for parasitic infection.

i. Frequency of larval trematode parasitic infection in the snail

Lymnaea acuminata

In order to find out frequency of parasitic infection during every month

for total two years study period, release of cercaria in the snail water in the

beaker, indicative of natural infection to the snail.

Fortnight collection of snail was made and randomly sorted out

normal sized, intact and healthy snails showing creeping movements (70-80

numbers) were maintained individually in separate 250 ml capacity beakers.

Observations were made for continuous subsequent three days to check

normal and trematode infection to the snails. Number of infected snails was

counted, and infection rate in percent was calculated for every month

during two years of study period (Jan.2009 to Dec. 2010).

ii. Determination of patency period in naturally infected Lymnaea

acuminata

After getting sorted out naturally infected snails, after completion of

prepatency or incubation period, there starts the release of cercaria, this is

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called as begin of patency period. Cercarial release is with slow rate at the

beginning of patency for two days is called initial phase of patency period.

Then starts peak phase of patency period, during which there is intense

release of cercariae for three days and once again there is reduced rate of

cercarial release, this lasts for two more days and called as post phase of

patency. In this way patency period of Lymnaea acuminata is of seven

days, which starts from beginning of larval release to complete stop of

cercarial emergence or released by the snail.

iii. Different type of larval trematode parasites found in snail L.

acuminata

Different larval trematodes emerged during patency were collected

separately and got centrifuged. The larvae settled at the bottom of the tube

were transferred to cavity block. First of all the larvae got preserved in 4%

formalin. These preserved larvae then got processed for morphological

study. After getting washed with distilled water, got stained with either

heamatoxylinor neutral redand passed through increasing grades of alcohol

for dehydration. Parasites cleared in xylol and mounted over the micro

slide under DPX medium. Observations were made under compound

microscope for their identification. The trematode cercarial larval

characters such as position and number of suckers, shape and size of the

tail and morphologically by specialized body structures were taken into

account for their identification. While identifying the cercaria a systematic

key reference by Frandsen and Christensen (1984) was followed. These

different parasitic pathogens were photographed.

iv. Number of cercariae released during period of patency

Naturally infected medium sized snails got sorted out and were maintained

individually in separate beaker for cercarial release. 4-5 infected snails

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continued for cercarial release separately. After every 24 hrs.snails were

separated and transferred in new beaker with freshwater. The cercariae in

infected snailwater were killed with addition of formalin (2-3 drops). One

ml cercarial water taken in cavity block. With the help of capillary tube

dropper, cercariae were sorted and counted. The same procedure was

continued for total 7 days period of patency i.e. initial phase of patency,

peak phase of patency and post phase of patency. An average with

standard deviation of cercariae released during every day was calculated

for seven day of patency period.

v. Frequency of particular type of larval trematode infection in the

snail Lymnaea acuminata

Naturally infected snails got sorted out and maintained individually

in separate beakers of 250 ml capacity. 30- 40 infected snails were

identified and kept under observation for different type of larval trematode

pathogen identification. The cercariae released in the snail water was

collected and after killing the cercaria with addition of 2-3 drops of 10%

formalin were observed under binocular microscope for their

identification. After identification of particular type of cercaria, percentage

of snails got infected with particular type of trematode larvae was

calculated.

vi. Effect of infection on Survival of snail during patency period

The survival of both infected and non-infected snail also studied by

keeping batches of infected and non-infected normal sized snails in

separate trough with sufficient dechlorinated tap water and ad libitum as

food material with hydrophytes collected from snail‘s habitat provided,

during patency period. The water changed after every 24 hrs. in both

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troughs. Changes in survival number of snails in both the trough was

noted down daily at normal room temperature 27±20C.

vii. Host size dependence larval trematode infection and cercarial

release during patency period

In order to determine number of cercariae released by different sized

snail host of L. acuminata were collected from Kham River, Aurangabad.

Immediately after getting to laboratory, were washed in order to remove

mud particles and algal material attached if any. Different sized animals

such as 5 ± 1mm, 10 ± 2mm,15 ± 1mm and 20 ± 1mm shell length were

sorted out and maintained in 100 ml dechlorinated water taken in 250 ml

capacity glass beakers. Separately observations were made in order to

detect naturally infected snails. 5-6 specimens of different sizes selected

were maintained individually for release of cercaria. Naturally infected

snails start release of cercaria under laboratory condition. After identifying

cercarial release by each sized snails continued for cercarial count for total

period of patency. Counting of cercaria was done as usual method

mentioned earlier. Total number of cercaria released by each sized animals

was calculated. Average count of cercaria by5 ± 1mm, 10 ± 2mm, 15 ±

1mm and 20 ± 1mm sized snails was calculated for total 7 days period of

patency.

viii. Effect of larval trematode parasitic infection on shell size and

weight of the snail during patency period

Non- infected snails when collected randomly along with infected

one are small in size (18±1mm shell length) compared to parasitized ones

(20±1mm shell length). Continuous observations were made on

measurement of shell length and weight of animals in both infected and

non-infected snails during different phases of patency. Shell length in mm

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was measured with the help of calibrated scale and weight by one pan

electronic balance in terms of mg ± S.D.

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OBSERVATIONS AND RESULTS

i.Frequency of larval trematode parasitic infection in the snail

Lymnaea acuminata

70-80 normal sized healthy snails measuring 20-22 mm shell

length were sorted and maintained individually in 250 ml beaker, during

every month of the two years study period. The frequency of trematode

larval infection observed in percent is depicted in the table 1. No infection

was observed during summer months i.e. from February to May during

both the years of study. Snails start getting invaded by trematode larval

pathogens from June onwards. Heavily infection was observed in the

month of September and October, 2009 and 2010. Maximum infection 50

to 60 % was found in the month of September respectively in 2009 and

2010.

ii.Period of patency in the snail Lymnaea acuminata

Naturally infected snail specimens were identified in the laboratory

and cercarial release period was observed and mentioned in the form of

observation table 2. The total period of patency was observed and lasts for

seven days‘ time period. Depending upon the number of cercariae released

by theinfected snails, the total period of patency can be divided into three

phases-

A. Initial phase of patency:

Since the cercarial release starts with emergence of few cercaria

during first two days.

B. Peak phase of patency:

From the 3rd

to 5th

day of cercarial release, is the peak phase of

patency because during these days there is enormous number of cercarial

release by the snail L. acuminata. (See table 2)

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C. Post phase of patency:

During 6th

and 7th

day of patency, the frequency of cercarial release

decreases compared to peak phase of patency. Generally in the present

study, after 7th

day of cercarial release, there is no further release of

cercaria observed.

iii.Different types of trematode larval pathogens (cercariae) found in

the snail L. acuminata

Various type of larval trematode pathogens got invaded in the

naturally infected L. acuminata is shown in the form of observation table

4. In all some six types of cercaria have been identified during study

period. Of the total parasitized snails 43.25 % snails were found invaded

by, cercaria of Fasciola hepatica and least number of infected snails 2.33

% were found invaded by the cercaria of Diplostomumhepaticum.

iv. Number of cercariae released during period of patency

During patency period large number of cercariae released from the

infected snail. The cercariae released from the snail were kept separately

without snail host to check their viability. The cercaria could not survive

more than 24 hrs. in laboratory conditions and die in the beaker. The

mortality rate was found to be higher among infected snail than the normal

snail. Again the behavior of the snail observed, the infected snail was slow

as compare to non-infected one, which shows active movement. The

freshly released cercariae get used for their identification after killing by

formalin.(Table 3)

v. Frequency of cercaria released during period of patency:

From the start of period of patency to the 7th

day, number of cercaria

released is given in the observation table 3. From the table it is observed

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that from first day of patency, to completion of peak phase of patency there

is continues increase in the cercarial release (1009 ± 25 to 2400 ± 30)

observed. The data included in the table is average of three infected snails

along with standard deviation. Again on 6th

and 7th

day there is decrease in

the number of cercarial emergence from the parasitized snails. Maximum

numbers of cercariae (2400 ± 30) were released on fourth day of patency

i.e. during peak phase of patency period. Least number of cercariae were

released (800± 12) on last day of patency.

vi. Effect of infection on Survival of snail during patency period

Gradual decrease in the percentage of snail‘s survival form both

infected and normal group showed in table 6. Mortality rate was high in

infected snails than non-infected group. At beginning both groups show

same survival percentage, but in infected group during post phase of

patency the mortality rate got increases and survival rate decreases upto

65.62 % at the end of 7th

day. Whereas the normal snails group shows

83.54% at the end of 7th

day of patency.

vii. Snail host size dependence cercarial release during period of

patency in Lymnaea acuminata

The number of cercaria released by naturally infected, different

sized snails of L. acuminata is summarized in the observation table 5.

Total number of cercaria released by small sized (5±1mm shell length)

snail is 2300 ± 27 during total period of patency. Medium sized snails (10

± 1mm and 15 ± 2 mm shell length) released respectively 5100 ± 39 and

7400 ± 35 cercaria. Maximum numbers of cercaria were released by the

snail size having 20 ± 2 mm shell length released 9700 ± 53 cercariae.

Number of cercaria invaded in snail is directly proportional to the size of

snail hostLymnaea acuminata.

23

viii. Effect of larval trematode infection on shell size and weight of the

snail during patency period

Effect of trematode parasitism on shell length and total body weight

of Lymnaea acuminata observed during patency period and is summarized

in the observation table 7. There is a slight variation in the shell size of

non-infected and naturally infected snails. The control snails weight

remain constant (18±1mm) while the infected snail shows gradually

increase in shell length (20±1mm to 23±1mm) and body weight (230±2 mg

to 237±2 mg) respectively.

Following different types of cercariae were found invaded in the

snail L. acuminata-

1. Cercarial form of Fasciola hepatica.

2. Cercarial form of Plagiorchissp.

3. Cercarial form of Echinostome sp.

4. Cercarial form of Pseudo echinoparyphium sp.

5. Cercarial form of Trichobilharziaocellata.

6. Cercarial form of Diplostomumhepaticum.

The distinguishing features of these cercariae are as follows:

1. Fasciola hepatica – (Linnaeus 1758)

a. The cercaria has a heart shaped body and bears a long tail.

b. Mouth is surrounded by oral sucker.

c. Most of the adult organs are present.

d. Rudiments of reproductive organs are also present.

24

2. Plagiorchis sp.-(Müller in 1780)

a. The cercaria displays a total length of approximately 450µm.

b. The swimming behavior of cercariae is with snaking, crawling

movements.

c. Mouth is surrounded by oral sucker, ventral sucker also present.

d. The tail is short, not longer than the body length.

3. Echinostome sp. –(Rudolphi 1809)

a. A good feature of cercariae is of its tail with finger like pulled out end.

b. The total length of cercaria is approximately 780 µm.

c. The intestinal branches reach the end of the body.

d. The mature rediae of the parasite contain up to 30 cercariae in various

stages of development.

4. Trichobilharziaocellata – (La Valette 1855)

a. The furcocercaria is with its body approximately 800 µm, very large

and easy to distinguish with naked eye.

b. Good features for recognition are bifurcated tail hence called as

furcocercus, is considerably longer than the body length.

c. The bifurcated parts being half as long as tail stem.

d. The pharynx and oral sucker fuse together in to head organ.

e. In the process of movement they take up a typical position towards

lights, hence photo positive, in which the tail is held at about 900

to the

body and the cercaria with the acetabulum attached underneath.

25

5. Diplostomumhepaticum – (Retzius 1786)

a. It is also furcocercariae, with body length approximately 400 µm.

b. It is identified easily by its typical swimming position. The cercariae

hangs motionless with their bodies bent in the water and let themselves

be carried along by the current, interrupted to only by a short and fast

swimming movements.

c. It is having a strong arming of oral sucker and acetabulum.

6. Pseudoechinoparyphium sp. –

a. It is a representative of Echinostomatidae family. Total length of the

cercaria measures approximately 1.2 mm; is the largest found

echinostome cercaria to date.

b. The most striking feature of cercaria is strongly developed intestinal

system, which appears to be subdivided.

c. The tail exhibits no fin edge.

26

Table 1

Frequency of larval trematode infection in the snail Lymnaea

acuminata during two subsequent years, from January 2009 to

December 2010.

Month and

year

Frequency of

infection in %

Month and

year

Frequency of

infection in %

January 2009 05 January 2010 07

February 02 February Nil

March Nil March Nil

April Nil April Nil

May Nil May Nil

June 09 June 11

July 30 July 15

August 35 August 25

September 50 September 60

October 49 October 45

November 20 November 39

December

2009 10

December

2010 13

27

Table 2

Different phases of patency in the freshwater snail, Lymnaea

acuminata.

Period of

patency

in days

Phases of Patency

Each Phase of

patency in

Days

1

2

Initial phase of

patency 2

3

4

5

Peak phase of

patency 3

6

7

Post phase of

patency 2

28

Table 3

Cercarial release during patency period in Lymnaea acuminata

Period of patency in days

Number of cercaria

released

Mean ± S.D.

1st 1009 ± 25

2nd 1400 ± 15

3rd 2000 ±27

4th 2400 ± 30

5th 2200 ± 20

6th 1600 ±15

7th 800 ±12

29

Table 4

Incidence of different trematode larval infection to the

snailLymnaea acuminataduring infection period.

Sr.Number Cercaria of following

trematode

Infected snails in

%

1 Fasciola hepatica 43.25

2 Plagiorchisvespectilionis 20.84

3 Echinostome sp. 15.62

4 Pseudoechinoparyphium

sp. 10.41

5 Trichobilharziaocellata 07.55

6 Diplostomumhepaticum 02.33

Total 100.00

30

Table5

Snail host size dependence cercaria release during period of patency in Lymnaea acuminata.

Snail size in mm

Number of Cercariae released during patency period in days Total ± S.D.

1 2 3 4 5 6 7

5±1 200±1 200±2 400±3 600±4 500±5 300±5 100±7 2300±27

10±1 300±2 500±3 700±2 1100±9 1300±7 700±8 500±5 5100±39

15±2 800±3 700±2 900±5 2000±7 1400±7 1000±8 600±6 7400±35

20±2 900±4 1100±6 2400±8 2200±9 1600±11 1000±9 500±6 9700±53

31

Table 6

Effect of parasitic infection on mortality of host snail, Lymnaea

acuminata.

Patency

period in

days

Survivalist of non-infected

snails in %

Survivalist of Infected snail

in %

1 98.21 97.23

2 96.43 95.51

3 96.35 94.32

4 94.16 92.34

5 93.67 88.11

6 89.52 78.70

7 83.54 65.62

32

Table 7

Effect of trematode parasitism on shell length and total body weight of

Lymnaea acuminata observed during patency period.

Phases of Patency

Period

Body shell length

(mm) Body weight (mg)

Control 18±1mm 225±2

Initial phase 20±1mm 230±2

Peak phase 22±1mm 245±3

Post phase 23±1mm 237±2

33

34

35

36

DISCUSSION

Many aquatic snails act as an intermediate host for larvae of

trematode parasites. Some part of the life cycle is being spent within the

body of snails as primary host. The adult form causes a number of

dreadful diseases to man and his domesticated animals. In Japan the

numbers of people at risk (600 million), infected (200 million) and dying

(20,000 per year) from Schistosome infections (Oliveira et al. 2004).

From economical point of view worldwide losses due to Fasciolasis are

conservatively estimated as some US $ 3.2 billion per annum (Piedrafita

et al. 2004). Parasitic Platyhelminthes are important economically and

socially and deserve much more attention. They deserve the attention not

simply because they are fascinating animals that provide challenges to

understanding associations of animals,but as model systems for study of

any field of biology.

At the habitat of Lymnaeid snails which act as an intermediate host,

free swimming miracidia of Fasciola hepatica approach toward these

intermediate hosts by increasing their rate of change of direction, when

they enter the areas of snails. A sort of chemosensory stimulus is being

released into the water through mucus secretion from L. acuminata

(Neuhans, 1941). These trematode larval pathogens possess a remarkable

ability to discriminate among snail‘s species while approaching them. This

has been reported by investigators working with miracidia of liver fluke, F.

hepatica (Thomas 1883 and Neuhans, 1941 and 1953). Also it has been

experimentally proved by Chipev (1993), by varying the number of

introduced miracidia of F. hepatica in the vicinity of non-host and target

snails, concluded that there must be a species - specific chemo orientation

is involved at the time of infestation to primary intermediate snail host.

37

One of the most characteristic features of trematode-mollusc

interactions is their specificity. Despite their fact that aquatic environments

are usually inhabitated by several snail species, each host species is

normally found infected with specific larval trematode and never with

others, despite the array of trematode miracidia present in the environment.

This use of a restricted group of hosts by the parasite is a phenomenon,

known as ―host specificity‖. In the present investigation the intermediate

snail host L.acuminata, most of the time got invaded by only one type of

cercaria.

The occurrence of multiple infections in nature may provide a

possible mechanism for intermediate host switching, the process by which

a trematode transfers to a new host. The miracideal penetration of the

wrong host would normally result in elimination of the incompatible

parasite, but if the invading parasite survives by overcoming the snail

internal defence system, the infection may persist. The same possibility

cannot be ruled out for present incidences of occurrence of two types of

cercariae found invaded in the body of naturally infected snail L.

acuminata by Plagiorchis and Echinostomum. Such type of double

infection may interfere with snail internal defence system,one factor that

might facilitate hosts shifts, in this way, there is presence of other digenean

species infecting the same snail host. A suppressive effect from one

parasitic infection may allow other parasites to eventually adapt to a new

host.

During two years of study from the point of incidences of cercarial

infestation in naturally infected snail L. acuminata, found heavily invaded

by larval trematodes during post monsoon period i.e. during September

and October 2009 and 2010. Choubisa (2008) while screening freshwater

gastropod snail from the point of larval trematode infection in the tribal

region of Southern Rajasthan, reported that the most favourable season for

38

cercarial infection was found during late rainy or prewinter season. During

this season more than 95 % matured snail species found release of different

kinds of cercaria like the present intermediate snail host Lymnaea from

Aurangabad.

Total length of the cercarial shedding period is called as period of

patency. The shedding period begins with a ―flushing – out of larvae from

the naturally infected snail body. In L. acuminata cercarial release starts

with moderate number of cercariae on first two days with gradual

increasing order up to 5th

day, which is peak phase of cercarial shedding.

Similar pattern of larval shedding has been observed by Elon and George

(1954) while working on experimentally infested positive snail

Physagyrina in the laboratory. Usually a moderately large number was

released on the first day followed by more or less steady increase in

number the next 2 or 3 days, culminating or attaining peak initial peak

number on 4th

or 5th

day. In the present investigation ―flushing – out‖

period of the number of cercarial release for further two to three days

declined to establish a lowest level of count is referred as post phase of

patency.

L. acuminata is found distributed both lentic and lotic type of water

body and is having a surface dwelling habit. May be because of this fact it

is found invaded by diversified species of trematode cercarial larvae has

been reported by Choubisa (2008) in various genera of the family

Lymnaeidae. Many workers have reported the diversity of the snails of

lentic habitats and their pathogenic cercarial fauna from different

geographical areas. He also found that the larval digenean infection in

surface dwelling snails from the lentic environment especially in the

perennial ponds or reservoirs was higher than those of bottom dweller

species in the same habitat. The present snail L. acuminata may be due to

surface dwelling habit shows maximum diversity of trematode cercarial

39

larvae. In the life cycle of the digenetic trematodes the radial stage is a

very important step, it could represent a form of resistance to unfavorable

environmental conditions, raising generations of daughter redia during

intramolluscan development. Location and structure of rediae of

Echinostomaperaensei collected from intermediate host Lymnaea

collumella (Pinheiro et al. 2004) are similar to the present finding on redia

present in Lymnaea acuminata. The mature rediae collected from L.

acuminata are colorless and are located in the peripheral region of the

digestive gland.

Randomly collected snail species of L. acuminata, size ranging

from 5-22 mm shell length were found naturally infected by various types

of larval trematodes after getting crushed the animals. It has been observed

that there is a specific relationship between size of the snail and infestation

by pathogens of larval trematodes during intense infestation period i.e. in

the months of September and October of two subsequent years of the

study. The numbers of cercariae shed from the naturally infected snails

were quantified in the laboratory. Small sized infected snails released less

number of cercariae compared with moderately sized (20 mm shell length)

infected snails during their total period of patency. Age of the mollusc is

probably a controlling factor for many species of miracidia. Among the

blood flukes, Schostosomatidae, Spirorchidae and in Paragonimuskellikotti

(Troglotrematidae), also in Clinostomummarginatum (Clinostomatidae)

many experiments have shown that young snails from a few days to a few

weeks are most readily penetrated by miracidia. Miracidia of some species

rarely or never penetrates snails over 2-3 months of age. But this limitation

does not appear to exist in certain other species. In the present

investigation it has been observed and confirmed that number of larval

trematodes emerged is directly proportional to the size of the snail host.

40

The analysis of prevalence in relation to maturity and age produces a

more complicated picture for two reasons - First, high prevalence of

trematode infections in large sized host have to be attributed to host

maturity, because the trematode larvae exclusively invaded the

reproductive organs of the adult individuals. The gonadal tissue was

completely replaced by sporocysts or rediae, which corresponds with

Ankel‘s (1962) findings. Second, the trematode prevalence in mature

individual increased with size suggested by Probst and Kube, (1999).

Ballabeni (1995) hypothecated experimentally parasite induced gigantism

in hermaphrodite mollusc Lymnaea peregra infected with the trematode

Deplostomumphoxini. The present pond snail L. acuminata infected in

nature also show slight difference in their shell size when observed during

period of patency. The parasitically infected snail suffers total reproductive

inhibition caused by parasites like the prediction derived by Minchella

(1985) and Ballabeni (1995). Due to parasitic infection by larval trematode

there is increased host mortality rate, may be because of this fact there is

no gigantism resulted, though there is slightly increased in shell length of

infected snails than normal non-infected host snails. It is well known that

trematode infection influence the growth rate of molluscs. Chernin (1960)

and Pan (1963 and1965) reported that experimental infections with

Schistosomamansoni accelerated the growth rate of Australorbisglabratus.

The effect of infection can however be influenced by the age of the

host. Thus significant in shell size and body weight occur in Lymnaea

stagnalisonly if the trematode infection is established at an early stage

(Joosse, 1964). Away from the results of Pan (1965) gigantism is observed

in the present investigation on naturally infected L. acuminatasnail

compared to non-infected normal host snails.

Whitlock et al. (1977) made a comparison of two laboratory method

of maintaining field collected Lymnaea tomentosa for the production of F.

41

hepaticametacercariae. It has been observed that the number of

metacercariae recovered per snail dissected increase with in initial shell

length up to 8 mm. Naturally infected various sized of snails of L.

acuminata showed increased number of cercaria with increase in size of

snails.

Many workers have reported the diversity of the snails of lentic

habitats and their pathogenic cercarial fauna from different geographical

areas (Pandy and Agrawal, 1978; Choubisa and Sharma, 1983). The

digenean trematode Diplostomumspathaceum is well known parasite in

fishes where it occurs as metecercercaria in the eye lenses of the host and

often causes parasitic cataract (Sheriff et al.1980). While screening the

present freshwater snail L.acuminata from the point of larval trematode

infections, it has been observed that the cercarial stage of

Diplostomumwere found invaded in the body of snails collected from

Salim Ali Lake Aurangabad. Fish host may be the definitive host of this

trematode larval pathogen because many fishes are the inhabitants of this

lake as associate animals.

Lake water inhabitants of snail L. acuminata in the present study

found heavily infected with cercarial larval trematodes compared with

snail species collected from lotic water dwellers.

Wealth of information is available on morphology and various types

of larval trematode stages such as furcocercous cercaria were described by

Azim (1935), Xiphidiocercaria by Azim (1936) EL- Gindy and Hanna

(1963) and Sakla and Khalifa (1981) and Pleurolophocercous cercariae

were described by Khalifa et al. (1977) and Fahmy et al. (1986) in Egypt.

Recently Yousif and his co-workers (2010) described morphology of new

eleven types of cercariae from prosobranch snail, Melanoidestuberculatain

Egypt. Of these cercariae they have describe two new types of cercariae

which were released from M. tuberculatus. Based on variations in

42

superficial morphological characteristic features some ten different types

of cercarial forms have been noticed in the naturally infected present

intermediate host snail L. acuminata. Similar type of observations were

made by Cort (1914), while working on larval trematodes, some 14 new

species of cercaria have been collected from freshwater snails from

different localities throughout the United States and from various

ecological situations.

The emergence of two types of cercariae from single snail host is

referred as a case of double infection. Such type of incidence occurred

rarely in present study. Two types of furcocercariae have been found

infested in L. acuminata at one or two occasions of naturally infected

snails may be due to identical host- specificity of two different digenetic

trematode parasites. Two types of furcocercariae namely Trichobilharzia

and Diplostoma have been found infesting different snail specimens of L.

acuminata, collected from same locality the city Aurangabad (M.S.).

These two furcocercous cercaria never found infesting together in the same

animal during two years of study period. Similar type of infection have

been also observed by Mukharjee (1966); Jain (1970);Pande and Agrawal

(1978) and Choubisa (1986b).

The present study provides to estimate larval trematode parasites

among lymnaeids snails and their zonotic importance in animal or human

health.