INCIDENCE OF PARASITIC INFECTION

Transcription

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 20 th 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. 1

2 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 2

3 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 digenean 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. 3

4 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 4

5 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 5

6 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 6

7 (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 7

8 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. 8

9 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 9

10 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. 10

11 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. 11

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15 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 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 15

16 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 16

17 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 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 17

18 troughs. Changes in survival number of snails in both the trough was noted down daily at normal room temperature 27±2 0 C. 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 18

19 was measured with the help of calibrated scale and weight by one pan electronic balance in terms of mg ± S.D. 19

20 OBSERVATIONS AND RESULTS i.frequency of larval trematode parasitic infection in the snail Lymnaea acuminata normal sized healthy snails measuring 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 Maximum infection 50 to 60 % was found in the month of September respectively in 2009 and 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 3 rd to 5 th 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) 20

21 C. Post phase of patency: During 6 th and 7 th day of patency, the frequency of cercarial release decreases compared to peak phase of patency. Generally in the present study, after 7 th 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 % 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 7 th day, number of cercaria released is given in the observation table 3. From the table it is observed 21

22 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 6 th and 7 th 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 % at the end of 7 th day. Whereas the normal snails group shows 83.54% at the end of 7 th 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. 22

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. 23

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 90 0 to the body and the cercaria with the acetabulum attached underneath. 24

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. 25

26 Table 1 Frequency of larval trematode infection in the snail Lymnaea acuminata during two subsequent years, from January 2009 to December Month and year Frequency of infection in % Month and year Frequency of infection in % January January 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 December

27 Table 2 Different phases of patency in the freshwater snail, Lymnaea acuminata. Period of Each Phase of patency Phases of Patency patency in in days Days 1 Initial phase of 2 patency Peak phase of patency Post phase of patency

28 Table 3 Cercarial release during patency period in Lymnaea acuminata Period of patency in days Number of cercaria released Mean ± S.D. 1 st 1009 ± 25 2 nd 1400 ± 15 3 rd 2000 ±27 4 th 2400 ± 30 5 th 2200 ± 20 6 th 1600 ±15 7 th 800 ±12 28

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 Plagiorchisvespectilionis Echinostome sp Pseudoechinoparyphium sp Trichobilharziaocellata Diplostomumhepaticum Total

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. 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± ±9 500±6 9700±53 30

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 %

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 32

33 33

34 34

35 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. 36

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 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 37

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 5 th 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 4 th or 5 th 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 38

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. 39

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. 40

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 41

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. 42