1. Introduction

1. INTRODUCTION

Utilization of marine resources for human consumption has increased rapidly in worldwide. Seafood products are currently in high demand as they are considered healthy, nutritional and possess medicinal values. The oceans offers a large biodiversity of fauna and flora which is estimated to be over 5,00,000 species, which are more than double of the land species (Anand et al., 1997; Kamboj, 1999). There are approximately 5,000 species of Sponges, 11,000 species of Cnidarians, 9,000 species of Annelids, 66,535 species of Molluscs and 6,000 species of Echinoderms (Laxmilatha et al., 2006).

Molluscs are widely distributed throughout the world and have many representatives such as slugs, whelks, clams, mussels, oysters, scallops, squids, and octopods in the marine and estuarine ecosystems. Among the Molluscs, 50,000 species of Gastropods, 15,000 species of bivalves and 600 species of Cephalopods have been reported (Alfred et al., 1998). Molluscs occupy a variety of habitats ranging from mountains fresh waters to sea. These are more abundant in the littoral zones of tropical seas. Gastropods and bivalves constitutes 98 per cent of the total populations of molluscs.

Molluscs are delicious and protein rich food among the sea foods (Jagadis, 2005). Bivalves belong to Molluscs, which is the second largest phylum among the invertebrates. They have been exploited worldwide for 2 food, ornamentation, pearls etc. for the the human welfare. Bivalves of orders, Orterids, Mytulids and Pectinids are harvested for food globally. The bivalves in the coastline could form an important source of food, raw material for village industries, indigenous medicine etc. and it is widely used as a cheaper food source for coastal area people. Shell fish such as mussel and bivalves contain approximately 20 to 28 per cent calories of fat. Bivalves also provide high quality protein with all the dietary essential aminoacids for maintenance and growth of the human body. For this reason, bivalves could be considered a low fat, high protein food that can be included in a low-fat diet (King et al., 1990).

In India, 5,070 species of Molluscs have been recorded, of which 3,370 species are from marine environment (Venkataraman and Wafar, 2005; Ramachandran et al., 2011). These are included among the economically important edible species of bivalves such as oysters, Crassostrea madrasensis and C. gryphoides, bivalvas, Meretrix casta, M. meretrix, Paphia malabarica and Villortia cyprinoides and green mussel, Perna viridis (Chatterji et al., 2002). In Indian coastal marine ecosystems, 17 species of bivalves have been exploited (Ramachandran et al., 2011).

Molluscs with rich diversity of marine organisms assume a great opportunity for the discovery of new bioactive compounds. Thus, the marine environment is an exceptional reservoir for bioactive natural products, many of which exhibit structural features that are not found in terrestrial natural products (Joshua, 1999). A wide variety of bioactive 3 substances are being isolated and characterized from the food that is derived from the marine environment, several with great promise for the treatment of human and fish diseases. There is a vital interest in discovering new antimicrobial compounds with fever environmental and toxicological risks and the resistance developed by the pathogens (Challeram et al., 2004; Periyasamy et al., 2012).

The marine environment comprises complex ecosystem with a plethora of organisms and many of these organisms are known to possess bioactive compounds as a common means of defense (Indap and Pathare, 1998). The marine natural products have been investigated predominantly for their antimicrobial, cytotoxic, anti-leukamic, anti-tumour, anti-viral and anti-inflammatory properties (Kamiya et al., 1984; Anand et al., 1997; Anand and Edward, 2001; Naganuma et al., 2006; Liyan Song et al., 2008; Chandran et al., 2009; Shanthi et al., 2011; Ramasamy and Balasubramanian, 2012; Periyasamy et al., 2012). The marine ecosystem remains as untapped resources for discovery of many drugs and contemporary experimental studies which indicates that pharmacologically active substances could be isolated from marine organisms (Naganuma et al., 2006; Liyan Song et al., 2008).

In the last two decades alone, structures of over 6,500 marine natural products have been elucidated (Kamboj, 1990; Wright, 1998; Naganuma et al., 2006). More than 100 pure compounds of known and new structural types have been isolated and characterized (Kamboj, 1999; Ramasamy and Balasubramanian, 2012). The screening of marine 4 organisms, especially bivalves for therapeutic drugs are of greater interest now-a-days. They have been recognized as a potential source of antibacterial and antifungal substances. The potential of marine bivalves as sources of biologically active products remain largely unexplored. Therefore, a broad screening of bivalves for bioactive compounds is necessary.

The Indian bivalves have protein content ranging from 5-80 per cent, carbohydrate 3-24 per cent, fats 0.5 – 3 per cent, calcium 0.04 – 1.84 per cent, phosphorus 0.1 – 0.2 per cent and iron 1-29 mg/100 g of the fresh weight (CSIR, 1992). Fatty acids in marine invertebrates have been studied in many habitats because of their significance in human life (Ackman, 2000). Bivalves have a great importance because of their fatty acid components and their variability in different areas. The polyunsaturated fatty acids (PUFA) have been recognized as effective factors in human health and nutrition (Bruckner, 1992). The quality of protein is usually assessed by its amino acid composition. The amino acid composition in turn is helpful in assessing the nutritive value of an organism. Bivalves, Anadara granosa and Meretrix casta commonly occur in intertidal areas of Indian coasts, particularly in South East and West coast of India (Salaskar and Nayak, 2011; Ramasamy and Balasubramanian, 2011). As bivalves form good protein food, a comprehensive knowledge of their biochemical constituents during different seasons of the year would be valuable for large-scale exploitation from natural resources and to promote culture. Cyclical changes in 5 biochemical composition of bivalves tissue and mainly studied to assess the nutritive status of an organism.

Several species of bivalves are found in the estuaries, coastal and backwaters of India coastal ecosystems (Laxmilatha et al., 2006) which are exploited for their meat and shells. Information on the distribution and exploitation of bivalves from India was reported very earlier also (Rao, 1963; Jones, 1968; Alagarswami and Narasimham, 1973). Studies on the resource characteristics, exploitation and biology of bivalves from Karnataka, Kerala and Andra Pradesh were made by Harkantara (1975), Rao (1984), Nayar et al. (1984), Rao and Rao (1985), Achery (1988), Laxmilatha and Narasimham (2002), Laxmilatha et al.(2006) and Ramasamy and Balasubramanian (2011). Laxmilatha et al. (2006) felt that the existing potential for bivalve culture and distribution was immense and stressed the need for organizing culture programs and exploitation of distribution to augument production. At present there is a great demand for bivalves meat as a delicacy in developed countries (Appukuttan, 1996). Considering the demand for meat and shells by the various industries in Tamil Nadu State, India, from the coastal ecosystems of Muthupet estuary and Adirampattinam coastal waters of South East of India, a broad screening of bivalves for the distribution, and seasonal variation in physico-chemical characteristics, status and elucidation of bioactive compounds, nutritional quality, biochemical compositions in whole body of bivalves and their antibacterial activity against bacterial pathogens is necessary.

Estuaries are highly productive, dynamic and unique ecosystems providing food, transport, recreation etc. and estuaries play a predominant role in the socio-economics of the coastal regions by providing valuable resources like fishes, bivalves, molluscs, crabs, shrimps etc. Water quality studies are important and have been taken up because they play a key role in aquaculture. The maximum production of bivalves is obtained, when physico-chemical factors are at optimum level. Therefore, water quality is a permanent factor in an ecosystem productivity. The reproductive cycle of bivalves influenced by exogenous or endogenous or both factors. Among exogenous factors, the habitat is one of the factor for breeding behaviour of bivalve organisms. The rainfall, temperature, photoperiod, humidity, salinity, pH, dissolved oxygen, free CO2, solids, hardness and nutrients of water also influence the breeding, distribution and biology of the bivalves (Ramasamy and Balasubramanian, 2011).

Though works are available on biodiversity of bivalves, seasonal variations happen in physico-chemical characteristics of coastal waters of South East coast of India and their elucidation of bioactive compounds, and nutritional quality. Most of the studies carried out so far are related to anti-bacterial activity against bacteria and fungal pathogens. However, there is no work on economically important bivalves like A. granosa and M. casta from Muthupet estuary and Adirampattinam coastal waters of South East coast of India, and their elucidation of structural bioactive compounds and nutritional quality and their antibacterial activity against pathogenic bacteria are important.

Therefore, the present study was carried out on bivalves, Anadara granosa Linn. and Meretrix casta (Chemnitz) from Muthupet estuary and Adirampattinam coastal waters of South East Coast of India, which is an effort to document the edible bivalves distribution, water quality status and the often neglected socio-economic life based on it and their structural elucidation and determination of bioactive compounds, nutritional quality and their antibacterial activity against five pathogenic bacteria.

The following are the specific objectives of the study:

 to determine the seasonal variations of physico-chemical characteristics and the distribution of bivalves, Anadara granosa (Linn.) and Meretrix casta (Chemnitz) from Muthupet estuary and coastal waters of Adirampattinam, South East Coast of India,

 to determine the biochemical composition of various parts of the tissues of the two test bivalves in relation to seasonal changes,

 to investigate the antibacterial activity of the whole body tissue extracts of A. granosa and M. casta against bacterial pathogens and

 to determine the structural elucidation, characterization and identification of the bioactive compounds from the whole body tissues of A. granosa and M. casta.

Scope of the problem

 The exploitation of bivalves has been observed to be only as a subsistence occupation, but the growing demand for protein food and multiple uses of the Molluscan shell in lime-based chemical 8

industries have created tremendous awareness of the benefits of exploiting and developing these resources.

 Local people for food exploit these highly nutritious molluscs, shell fish bivalves resources and shells which are transferred to small scale industries for preparation of lime.

 The general features of the coast, estuarine ecosystems, hydrobiological conditions, ecological associations and distribution of bivalves and magnitude of standing stocks, nutritive quality and bioactive compounds of medicinal value and their antibacterial activity against pathogenic bacteria by using the whole body tissue extracts of A. granosa and M. casta have been used for research and socio-economic values of people.

 Nutritional quality of bivalves are studied in many habitats because of their significance in human life and the polyunsaturated fatty acids (PUFA) have been recognized as effective factors in human health and nutrition, especially for cardio vascular diseases.

 This study is a part of our overall efforts to understand and estimate the distribution of bivalves and ecosystem values as well as the magnitude of goods and services that the estuary and coastal waters contributes towards human welfare. These studies will also helpful in preserving the biodiversity of marine organism.

2. REVIEW OF LITERATURE

This chapter reviewed the bivalves in a brief manner under the following headings: (a) Physico-chemical characteristics of coastal waters and estuaries (b) Distribution and diversity of bivalves (c) Biochemical compositions of bivalves (d) Antibacterial activity of bivalves (e) Bioactive compounds of bivalves

2.1. Physico-chemical characteristics of coastal waters and estuaries The physico-chemical parameters have an impinging effect on the aquatic organisms and many parameters varied with temporal and spatial variations. These factors are either exogenous or endogenous or both. These parameters might affect the life activities and the distribution of organisms. All the factors are interrelated even to one another. Variation in one may affect the other. Considerable data of information are available on physico-chemical factors and their impact on the general distribution, diversity and reproductive biology of the organisms (Unni, 1972; Rao et al., 1982; Sridhar et al., 2006). The hydrobiological study is a pre-requisite in any aquatic system for the assessment of its potentialities and to understand the realities between its different trophic levels and food webs (Rajalakshmi Bhanu et al., 1981; Nair et al., 1984; Sasamal et al., 1985; Kannan and Kannan, 1996; Satpathy et al., 2007). The seasonal variations of the hydrobiological factors in the Bay of Bengal, South coast 10 of India have been reported by many workers (Udaya Varma Thirupad and Gangadhara Reddy, 1959; Somayajulu et al., 1987; Perumal, 1993; Satpathy, 1996; Subramanian and Mahadevan, 1999; Ashok Prabu et al., 2005; Damotharan et al., 2010; Sankar et al., 2010; Kannathasan and Rajendran, 2010; Santhosh Kumar and Perumal, 2011).

2.1.1. Rainfall Subramanian and Mahadevan (1999) have reported that the physico-chemical parameters of Bay of Bengal was influenced by monsoon rain. The heavy rainfall was recorded during the north-east monsoon season and low rainfall during summer season (Damotharan et al., 2010). At Nagapattinam coastal area, the peak values of rainfall were recorded during the monsoon month of October and November (Kannathasan and Rajendran, 2010; Sankar et al., 2010). The rainfall had a remarkable influence on the reproductive cycle of marine organisms.

2.1.2. Temperature Temperature is considered as an important factor which influenced the growth, metabolism and reproductive activity of marine organism (Rahaman, 1967; Rajendran, 1990). Kannathasan (2011) reported that the seasonal variation in atmospheric and water temperature was found to be associated the intensity of rainfall and humidity existed at that time. The range of variation in the atmospheric temperature was slightly more than the surface water temperature. The atmospheric and water temperature were found to be the maximum in summer months when the rainfall and humidity were low. The temperature was found to be low during monsoon 11 months when the rainfall and humidity was maximum. Similar observations were reported by earlier workers (Rao et al., 1982; Soundarapandian et al., 2009; Kannathasan and Rajendran, 2010).

Panikkar and Jayaraman (1996) reported that the surface water temperature in Bay of Bengal normally ranged from 27 to 29°C except in shallow areas near the coast. The temperature of east coast varied from 27.3 to 29.1°C which may be due to the diurnal variation of sea surface (Rao et al., 1982). Somayajulu et al. (1987) observed that the surface temperature value decreased from the pre monsoon to post monsoon. Both atmospheric and water temperature were found to be maximum during summer season and minimum during monsoon season (Dronamraju et al., 2008).

Kannan and Kannan (1996) found that the low temperature in post monsoon at Palk Bay was due to cloudy sky and heavy rainfall during that period. The atmospheric temperature is always higher than surface water temperature (Sithik et al., 2009). At Ayyampattinam coast, the surface water temperature varied from 25.5 to 33.4°C (Santhosh Kumar and Perumal, 2011). Humidity always correlated with temperature. The temperature was high during summer months when the humidity was found to be low. During monsoon, humidity was found to be the maximum when temperature was low.

2.1.3. Salinity The salinity changes in a locality are influenced by many factors like freshwater inflow, rainfall, temperature, tide, water current, mixing evaporation and precipitation etc. Similar observations were reported by earlier workers (Rangarajan and Marichamy, 1972; Rao et al., 1982). The salinity of sea water is another important abiotic factor having a remarkable influence on the reproductive and nutritional cycles of marine crustaceans (Soundarapandian et al., 2009). A wide seasonal fluctuation in salinity was recorded during the study period. The maximum value of salinity was recorded during summer and minimum during monsoon. The minimum salinity during monsoon could be due to heavy rainfall and influx of freshwater from land after the monsoon rain and discharge of water from the rivers (Kannan and Kannan, 1996; Dronamraju et al., 2008; Mohamed Abubaker Sithik et al., 2009).

Kannathasan (2011) had reported that the seasonal average salinity recorded high during summer and pre-monsoon and low during monsoon and post monsoon period. The average salinity values of the water varied from 30 to 33 ppt and it showed a wide fluctuation recorded from 27 to 18 ppt at the head or even less during monsoon season and 31 ppt in the south part of the Bay of Bengal (Panikkar and Jayaraman, 1966). The lowest and highest salinity obtained in the field were 26 to 33 ppt respectively (Rajalakshmi Bhanu et al., 1981). The salinity varied from 33.9 to 34.8 ppt at the off shore surface water and increased with depth (Rao et al., 1982).

The salinity of the sea of Port Novo coast varied between 26.5 and 34.5 ppt and it increased during post monsoon and summer and began to decline during pre monsoon and monsoon month (Sridhar et al., 2006). The turbidity of sea water is caused by many factors such as dissolved and suspended solids, dust particles, clay and silts, water current, tide and planktonic organisms. The turbidity place an important role in the productivity of the sea thereby controlling other physical factors. In the present study, turbidity showed slight seasonal variation which agrees with earlier observation (Kannathasan and Rajendran, 2010).

2.1.4. pH In the north western Bay of Bengal the surface sea water was more alkaline and mono mesohaline in nature (Sasamal et al., 1985). The pH of Chennai coastal water showed slight fluctuation, it was found to be minimum during north east monsoon and maximum during summer (Subramanian and Mahadevan, 1999). The hydrogen ion concentration (pH) is an another important hydrobiological parameter which influence the growth, metabolism and proximate composition of aquatic organism. The variation in pH of the water was less pronounced throughout the study period. The slight seasonal fluctuation in pH was mainly due to rainfall and freshwater inflow. Similar observations have been reported by earlier worker (Soundaramanickam et al., 2008).

2.1.5. Dissolved oxygen The DO level was also estimated by other earlier workers by coastal waters of South east coast of India (Sridhar et al., 2006; Satpathy 14 et al., 2007; Soundarapandian et al., 2009; Damotharan et al., 2010). The dissolved oxygen is one of the important biological factors which influence the bio-energetics of the aquatic organisms. Kannathasan (2011) reported that the dissolved oxygen level fluctuated irrespective of seasons, which may be due to wind velocity, rainfall and photosynthetic activities of phytoplankton.

2.1.6. Free CO2 According to Unni (1972) found that the rate of changes in free

CO2 concentration is considerably high due to decomposition of organic

matter at the bottom. The CO2 is an important abiotic factor of aquatic ecosystem which influence the growth, metabolism and feeding. The CO2 contributes to the fitness of natural waters are derived from various sources such as atmosphere, respiration by organisms, decomposition of

organic matter etc. CO2 level showed slight seasonal fluctuation (Unni, 1972; Dronamraju et al., 2008).

2.1.7. Inorganic phosphate At Madras coast the minimum phosphate was estimated during the post monsoon and maximum during pre monsoon season (Udaya Varma Thirupad and Gangadhara Reddy, 1959). The recorded high concentration of inorganic phosphates during monsoon season might possibly be due to intrusions of upwelling sea water into the creek, which in turn increased the level of phosphate (Nair et al., 1984). A slight seasonal fluctuation observed in phosphate level reported by earlier workers (Sampathkumar, 1992; Kannathasan and Rajendran, 2010; Sankar et al., 2010). 15

2.1.8. Nitrate Rajaram et al. (2005) found that the low values of nitrite content observed during non-monsoon period may be due to its utilization by phytoplankton as evidenced by high photosynthetic activity and the dominance of nitrite sea water having a negligible amount of nitrate. Kannathasan (2011) reported that the slight seasonal variation in nitrate content was observed at Nagapattinam coastal waters and also by many workers in Bay of Bengal (Satpathy, 1996; Ashok Prabu et al., 2005; Mohamed Abubaker Sithik et al., 2009; Damotharan et al., 2010). The increased nitrates level in the sea was due to freshwater inflow and terrestrial run off during the monsoon season (Santhosh Kumar and Perumal, 2011).

2.1.9. Nitrite The seasonal variation in nitrite content could be attributed to the variation in phytoplankton in Palk Bay (Kannan and Kannan, 1996). The nitrite is an essential factor of aquatic ecosystem. Kannathasan (2011) reported that the nitrite was maximum during monsoon season and minimum during summer season. Similar observations were also reported by earlier workers in coastal waters of Bay of Bengal (Udaya Varma Therupad and Gangadhara Reddy, 1959; Sithik et al., 2009; Sankar et al., 2010).

2.1.10. Photoperiod The photoperiodicity is a well known fact in the animal world. Diurnal rhythm is an important physiological event of the organism. 16

Lengthening of photoperiod may induce gonadal activity in animals. Kannathasan (2011) reported that maximum day length was recorded in summer and minimum in monsoon months. Similar observations were also reported by earlier workers (Rajendran, 1990; Kannathasan and Rajendran, 2010). Kannathasan (2011) has reported that most of the physico-chemical parameters were found in optimum level and showed seasonal fluctuation. The fluctuations of these factors have influence the reproductive, nutritional and moulting cycles of marine organisms including bivalves.

The seasonal variations in physico-chemical parameters of coastal waters of Pondicherry (Ananthan, 1995), Pitchavaram mangrove estuary (Ashok Prabu et al., 2008), Coramandal coast (Govindasamy and Kannan, 1991; Govindasamy et al., 2000), Palk Bay (Kannan, 1992), Gulf of Mannar and Palk Bay (Palanichamy and Rajendran, 2000), Cuddalore and Uppanar estuary (Pillai, Mathavan, 1994; Soundarapandiyan et al., 2009), Vellar estuary (Seenivasan, 1998; Rajasekar, 2003), mangroves of Kach- Gujarat (Saravanakumar et al., 2008), Agnitheertham and Kothadaramar temple, Rameswaram coast (Sithik et al., 2009), Muthupet mangrove estuary (Srilatha et al., 2012). Coral reef and seagrass ecosystems of Palk Bay (Sridhar et al., 2008) and Parangipettai coast (Sundaramanickam et al., 2008) of Bay of Bengal, South East Coast of India were reported.

Hussain Dar et al. (2012) studied that the seasonal variations in physico-chemical characteristics of Adirampattinam mangrove region of back waters for four seasons. The physico-chemical parameters such as 17 rainfall, turbidity, DO, EC, solids, salinity, pH, chlorides, fluorides and BOD exhibited high values were observed in monsoon season and the low values were observed in summer season.

Atmospheric and surface water temperature are the important environmental factors. During summer, solar radiation and clear sky enhance the atmospheric temperature whereas in monsoon season, rainfall and cloudy sky reduced the atmospheric temperature and consequently the water temperature fall into the minimum (Govindasamy and Kannan, 1991). Temperature is basically important for its effect on chemical and biological reactions takes place in water and in organisms also and inhabiting aquatic media. It will depend upon season, time of sampling and ambient temperature etc. (Bhagwan et al., 2004).

Salinity is one of the important factor which profoundly influence the abundance and distribution of the biological organisms in estuarine environment and inshore waters (Hussain Dar et al., 2012). The lower salinity was recorded during the monsoon season was due to heavy rainfall and large quantity of freshwater inflow. Water salinity positively correlated with temperature and pH and negatively correlated with DO (Hussain Dar et al., 2012). Similar trend in the salinity values were also observed from various parts of coastal waters and estuarine ecosystems of South East Coast of India (Satpathy, 1996; Palanichamy and Rajendran, 2000; Sulochana and Muniyandi, 2005; Srilatha et al., 2012).

The water quality characteristics of aquatic environment arise from a multitude of physico-chemical and biological interactions (Dezuane, 1979; Dee, 1989). Humidity influences the evaporation rate of salinity which in turn affects the salinity (Sridhar et al., 2008; Sithik et al., 2009) in Palk Bay. Dissolved oxygen can be removed from the water by discharges of the oxygen demanding wastes other inorganic reductants like hydrogen sulphide, ammonia, ferrous, nitrate and other oxidable substances tends to decrease DO in water. The low DO concentration observed during summer could be described to the higher salinity of the water and higher salinity of the water and higher temperature (Hussain Dar et al., 2012; Srilatha et al., 2012). DO concentration varies according to many factors, the main factors are due to photosynthesis and respiration by organisms, BOD and COD values were similar to the DO concentration in Adirampattinam coastal waters (Hussain Dar et al., 2012).

2.2. Distribution and diversity of bivalves Several species of bivalves, found in the estuaries and coastal waters of India. The distribution and exploitation of bivalves from estuaries, backwaters and coastal waters of South East Coast of India were reported very earlier by Rao (1963), Jones (1968), Alagarswami and Narasimham (1973), Rasalam and Sebastian (1973). Studies on the resource characteristics, distribution, diversity and biology of bivalves from Karnataka, Kerala and Andhra Pradesh were made by Harkantara (1975), Nayar et al. (1984), Rao and Rao (1985), Achary (1988), 19

Narasimham et al. (1984), Laxmilatha and Appukuttan (2002) and Laxmilatha et al. (2006).

A rapid survey was carried out to assess the bivalve resources and potential stock at Chettuva estuary, Kerala. The estuary harbours are estimated standing stock of 378 t bivalves, Meretrix casta was the dominant species followed by Villorita cyprinoides. Hydrological and sediment characteristics of the Chettuva actuary influence the distribution of bivalves (Laxmilatha et al., 2006).

Harkantra and Rodrigues (2004) reported that higher species diversity was observed at salinity tine sane and high sedimentary biochemical parameters of total organic carbon (TOC), total organic nitrogen (TON) and carbon of biopolymeric fraction (c-BPF) sites.

Varadharajan et al. (2010) investigated that the seasonal abundance of macro benthic composition and diversity of species along the South East Coast of India. The results indicated that polychaetes are dominated followed by bivalves. Among the bivalves, Anadara granosa, A. veligers, Cardium setosum, Meretrix casta, M. meretrix, M. veligers and Paphia textile are commonly available in Arukkattuthurai to Aiyyampattinam coastal backwaters of South East Coast of India.

Chandran et al. (1982) and Prabadevi and Ayyakannu (1989) were also observed the Anadara granosa, Meretrix casta, M. meretrix and Penaeus semisulcatus from Coleroon estuary and Vellar estuary. 20

Temperature is an important ecological factor, which influence the distribution of bivalves. High temperature 35°C, recorded in premonsoon season influence the distribution of bivalves. The salinity also considered to be a dominant limiting factor (Varadharajan et al., 2010).

Tanyaros and Tongnunui (2011) reported that the environmental variables influenced the distribution and abundance of estuarine bivalve, Meretrix casta in Trang Province, Southern Thailand. The results of the study showed that salinity and concentrations of total suspended solids were the water quality parameters most closely related to population densities of estuarine bivalves. Among bottom sediment parameters, pH and ferrous and ferric iron concentrations gave the strongest correlations to bivalves densities.

The influence of environmental parameters on population density and distribution of bivalves has been reported by other workers also (Franz, 1976; Boonruang and Janekarn, 1983; Absalao, 1991; Baron and Clavier, 1992; Soares-Gomes and Pires-Vanin, 2005; McLeod and Wing, 2008).

Also, a population of the bivalves, M. casta from the estuarine ecosystem in Trang Province, Thailand was reported by Songrak et al. (2009). The estuarine bivalve habitat was found to contain a high percentage of sand. The OM content in bottom sediment was low when compared with the OM content in the soils of mangroves estuary (Lovelock, 2008) and seagrass beds (Taryaros, 2009). 21

Changes in water salinity affect a wide variety of biochemical and physiologic processes in marine bivalves. An increase or decrease in salinity often results in increase or decrease of free amino acid (FAA) levels in the tissues of marine bivalves, which are often monitored as a stress indicator (Powell et al., 1982; Lee et al., 2004). Variations in FAA in two marine bivalves, Macoma balthica and Mytilus sp. have been related to salinity changes by Kube et al. (2007). The effects of salinity changes on the feeding physiology and growth of bivalves had been reported by Sara et al. (2008).

The pH values in sediment during the rainy season were lower than 6.7. When pH is lower than 6.8 – 6.9, calcium loss to the external environment exceeds gains (Hunter, 1990; Vindograv et al., 1993). The pH and iron concentrations in bottom sediments were most closely related to the distribution and abundance of estuarine bivalves (Vindograv et al., 1993).

Venkataraman and Wafar (2005) reported that the distribution and diversity of bivalves from coastal and marine ecosystems of South East Coast of India.

Arnaud et al. (2000) studied the population of the Calafia pearl oyster, Pinctada mazatlanica from American Pacific coasts, considered endangered because of overfishing and/or alteration to coastal area. They have assessed genetic variability and the pattern of population structures collected from Mexico to Panama. 22

Ramachandra et al. (2011) reported that the distribution and diversity of bivalves of Aghanashini estuary, West Coast of India. The eight species of edible bivalves viz., Paphia malabarica, Katelysia opima, Meretrix meretrix, M. casta, Villorita cyprinoides, Arca granosa, Crassostrea sp. and Perna viridis were observed from Aghanashini estuary. P. malabarica was very commonly available bivalve followed by Katelysia opima, Meretrix meretrix and M. casta.

Several species of bivalves found in the estuaries and backwaters of India, are exploited for their meat and shells. Information on the distribution and exploitation of clams from India was provided by Rao (1963), Jones (1968), Alagarswami and Narasimham (1973) and Rasalam and Sebastian (1973). Biological aspects of important clam species were dealt with by Durve (1970). Silas et al. (1982) felt that the existing potential for bivalve culture was immense and stressed the need for organizing culture programme to augment production. Studies on the resource characteristics, exploitation and biology of bivalves from Karnataka, Kerala and Andhra Pradesh were made by Harkantara (1975), Narasimham et al. (1984), Nayar et al. (1975), Rao (1984), Rao and Rao (1985), and Achary (1988), Laxmilatha and Appukuttan (2002).

Salaskar and Nayak (2011) reported that quality aspects of oysters (Crassostrea madrasensis) and mussel (Perna viridis) in the Kali estuary, Karwar, India were examined in different seasons over a period of 13 months. Eco-physiological parameters and nutritional quality parameters 23 of oysters and mussel (composition of protein, carbohydrate, lipid and ash content) were determined at different seasons of the year.

Varadharajan et al. (2010) reported that the benthic communities are important to marine ecosystem and form important food source for most of the marine organisms especially bivalves. The estimation of benthic production would serve as a useful index for assessing the fishery potentials, interaction, pollution and interdial ecology.

Roberts (1999) has been studied that bivalves belong to Mollusca which is the second largest phylum among the invertebrates. They have been exploited worldwide for food, ornamentation, pearls, etc. throughout human history wrong way of writing reference and its everywhere in this paper. Mainly, bivalves of orders ostreids, mytilids and pectinids are harvested for food globally. Annual harvests of bivalves for human consumption represent about 5 per cent by weight of the total world harvest of aquatic resources.

Tanyaros and Tongnunui (2011) reported that the interrelationship between abiotic parameters and biotic structures. Estuarine ecosystems are ideal for examining such interactions such interactions due to their wide range of abiotic parameters. This is usually accompanished by inferential analysis of empirical data by multivariate techniques, and by conducting manipulative experiments (Brown et al., 2000; Edgar and Barrett, 2002). Work on the influence of environmental parameters on population densities of bivalves has been performed elsewhere (Franz, 1996; 24

Boonruang and Janekaran, 1983; Absalao, 1991; Baron and Clavier, 1992; Soares-Gomes and Pires-Vanin, 2005; McLeod and Wing, 2008).

Harkantra and Rodrigues (2004) reported that the interrelationship between environmental parameters and benthic community structures (Mannino and Montagna, 1997; Edgar and Barrett, 2002; Brown et al., 2000), modeling of the ecosystem (Longhurst, 1978) and anthropogenic impacts (Frouin, 2000). Estuaries form an ideal ecosystem to examine such interactions between the abiotic and biotic factors due to their wide range of these parameters (Mannino and Montagna, 1997; Brown et al., 2000; Edgar and Barrett, 2002). Several studies have examined relative influence of environmental parameters on species diversity, biomass and population density of soft bottom macrofauna in temperate estuaries (Longhurst, 1978; Zajac and Whitlatch, 1982; Mannino and Montagna, 1997; Brown et al., 2000; Frouin, 2000 and Edgar and Barrett, 2002) and more than the tropical estuarine systems (Harkantra, 1975; Parulekar et al., 1980; Harkantra and Parulekar, 1985; Parulekar et al., 1986).

2.3. Biochemical compositions of bivalves Molluscs are delicious and protein rich food among the sea foods (Jagadis, 2005). The bivalves in the coastline and in estuaries could form an important source of food, raw material for village industries and indigenous medicine and also widely used as a cheaper food source for coastal area people (Babu et al., 2012). Bivalves contains approximately 20 to 28% calories of fat and also provide high quality protein with all the dietary essential aminoacids (King et al., 1990). Fatty acids in marine 25 bivalves were reported in many habitats because of their significance in human life (Ackman, 2000).

Recently, Babu et al. (2012) reported that the biochemical composition of different body parts of Gafrarium tumidum collected from Mandapam, South East Coast of India. Tissues of different body parts such as mantle, viscera and foot were analyzed. Biochemical constituents like protein, carbohydrate and fat were estimated in different body parts and the percentage of protein was 61.74%, carbohydrate 32.64% and lipid 14.37%. The fatty acids of bivalve were analysed by GC-MS. In the fatty acids, polyunsaturated fatty acids (PUFA) were found to be dominant, containing 6.99%, whereas saturated fatty acids (SFA) contributed 5-19% followed by monounsaturated fatty acids (MUFA) which contributed 2.15% and palmitic acids (0.71%) were the dominant saturated fatty acid.

The biochemical compositions of other marine bivalves such as oyster and scallops, which typically high protein content and low levels of fat and cholesterol reported by Pearson (1977). The fat and cholesterol content of number of bivalves ranged between 1.3 and 2.3% (oyster and scallops) 0.33 and 0.59% (Mussels and Cockles).

The seasonal variation in biochemical constituents of different body components of Meretrix meretrix were reported by Jayabal and Kalyani (1987). The results showed 24.82% protein, 13.53% carbohydrates and 7.6% fat. Seasonal variations in biochemical 26 compositions of different soft body parts of Sunetta scipta has reported by Rajan et al. (1990).

Babu et al. (2009) estimated a total of 19 amino acids in the protein of Bursa spinosa (6.8%). Ajaya (2002) observed amino acid content in the bivalve molluscs Perna virdis, Crassostrea madrasensis and Meritrix casta, a total of 18 amino acids were recorded. The total amino acid in the P. virdis was 95.76% among them essential was 47.28%.

Shanmugam et al. (2008) have recorded 36 individual fatty acids were identified. Among them, the saturated fatty acids were the dominant (35.28%).

Murphy et al. (2003) observed that the freeze dried and frozen samples of green lipped mussels of Perna canaliculus, among the 30 individual fatty acids, polyunsaturated fatty acids were found to be dominant. The majority of marine bivalves are rich sources of long chain PUFA (Murphy et al., 2002).

Salaskar and Nayak (2011) investigated that the nutritional quality aspects of oysters, Crassostrea madrasensis and mussel (Perna virdis) in the Kali estuary, Karwar were examined in different seasons over a 13 month period. Seasonal variations were observed in the nutrient content, with particular regard to protein 52.33% (September 2003) to 63.86% (June 2004), carbohydrate 14.01% (February 2004) to 25.24% (October 2003), lipid 8.46% (October 2003) to 18.77% (February 2004) in oyster’s 27 whereas in mussels, protein 57.39% (March 2004) to 66.51% (November 2003), carbohydrates 14.69% (November 2003) to 26.81% (March 2004) and lipid 8.09 (February 2004) to 12.62% (November 2003). A low level of fat was detected in the edible meet of oysters and mussels. Nutritional quality of meat in both oysters and mussel species was very high, during summer season.

2.4. Antibacterial assay of bivalves The first attempt to locate antimicrobial activity in marine organisms was initiated around 1950’s (Berkholder and Burkholder, 1958). Since this time, a large number of marine organisms from a wide range of phyla have been screened for antimicrobial activity (Shaw et al., 1976). Many of these organisms have antimicrobial properties, although most of the antibacterial agents that have been isolated from marine sources have not been active enough to complete with classical antimicrobial activity against microorganisms (Rinehart et al., 1981). The presence of antimicrobial activity in molluscs has been reported from the mucus of the giant snail, Achantina fulica (Kubota et al., 1985; Iguchi et al., 1982). The methanol extracts of Anadara granosa showed the highest activity against E. coli and the lowest activity against S. typhi (Srinivasan, 2008). The maximum antibacterial inhibition zone was exhibited from acetone extract of Trochus tentorium against S. typhi. The maximum antibacterial inhibition zone was exhibited from acetone extract of Trochus tentorium against human pathogen S. pneumonia (Anbuselvi et al., 2009).

Periyasamy et al. (2012) have reported that the antimicrobial activities of the tissue extracts of Babylonia spirata from Thazhangudu, South East Coast of India, against nine bacterial and three fungal pathogens. The maximum inhibition zone (12 mm) was observed against Pseudomonas aeruginosa in the crude ethanol extract of B. spirata and the minimum inhibition zone (2 mm) was noticed against Staphylococcus aureus in the crude methanol extract of B. spirata.

Ramasamy and Balasubramanian (2012a) reported that the antibacterial activity of Anadara granosa against pathogenic bacteria. The highest activity was observed against Pseudomonas aeruginosa of the crude ethanol extract.

The antibacterial activities of ethanol extracts of gastropods, B. spirata and Turbo brunneus showed the maximum activity against E. coli, K. pneumoniae, Proteus vulgaris and Salmonella typhi (Anand et al., 1997).

The maximum antibacterial activity against S. aureus and E. coli by Trochus radiates was reported by Elezabeth et al. (2003). The antibacterial activity was reported in four bivalves against few bacterial pathogens and found that extracts showed significant activity against Bacillus subtilis (Jayaseli et al., 2001). The antimicrobial activity from the gill extraction of Perna viridis (Chandran et al., 2009), Meretrix meretrix and M. casta (Sugesh, 2010), Crossostrea madrasensis (Annamalai et al., 2007) were reported. 29

The methanol extract of Sepia officinalis showed the maximum inhibition zone against E. coli and Lactobacillus vulgaris and the minimum inhibition zone was recorded against S. paratyphi (Reddy, 2008). The antibacterial activities of marine gastropod, Murex virgineus was reported by Lenin (2011).

Recently, Ramasamy and Balasubramanian (2012) found that the whole body extracts of the Meretrix casta and Tridacna maxima with different solvents were assayed for antibacterial activity against ten human pathogenic bacteria. The ethanol and crude extracts exhibited broad antibacterial activity. Highest activity was exhibited against E. coli and S. typhi by M. casta crude extract of ethanol. Ethanol extract of T. maxima exhibited highest activity against S. aureus and E. coli.

Shanthi et al. (2011) reported that the bioactive potential of Tonna galea from Gulf of Mannar against seven different bacterial pathogens. The highest activity was exhibited against Vibrio cholera in the ethanol crude extract. The promising bioactive compounds, Benzenedicarboxylic acid was the main antibacterial activity against V. cholera.

The presence of antimicrobial activity in bivalves has been reported from the mucus of the giant snail, Achatina fulica (Kubota et al., 1985). Santhana Ramasamy and Murugan (2005) reported that the potential antimicrobial activity of marine molluscs from Tuticorin, South East Coast of India against forty biofilm bacteria.

Anderson and Beaven (2001) reported that the anti-Bacillus megaterium activity measured in unfractionated plasma withdrawn from three common US East coast bivalve molluscs, an oyster Crassostrea virginica and the mussels, Geukensia demissa and Mytilus edulis. The activities of the plasma samples from these bivalves were also measured against a C. virginica pathogen Perkinsus marinus. Strong anti-B. megaterium activity was measured in plasma from C. virginica and M. edulis, but was not detected in G. demissa. Bactericidal activity was found in hemocyte extracts from all bivalves in their studies, suggesting a cellular origin of cytotoxic humoral factors.

Shanthi et al. (2011) reported that the compound responsible for antibacterial activity, the potent fraction was subjected to GC-MS analysis. The broadest antibacterial activity was noted in fraction 1 and fraction 5. The highest activity was exhibited against Vibrio cholerae (0139) in the crude extract. The column purified Hexane: Chloroform fraction showed higher activity against Vibrio cholerae 0139 (8 mm) and maximum inhibition zone was exhibited against Aeromonas hydrophila (10 mm). These promising results were confirms the presence of Benzenedicarboxylic acid. Mono (2-ethlexyl) ester, squalene and methyl 3-bromol-1-adamantaneacte by GC-MS may be related to mollusc defense mechanism and antimicrobial activity against these pathogens.

Santhana Ramasamy and Murugan (2005) reported that Methanol; Water (1:1); Methanol: Dichloromethane (1:1) and acetone extracts of 31 molluscs comprising 77 whole body, four inks, four operala, 10 egg masses and 10 digestive glands were screened for antimicrobial activity on marine biofilm bacteria. The methanol: water (1:1) whole body extracts of Nerita albicilla and Nerita oryzarum showed broad spectral inhibitory activity against 93 and 95 per cent of the 40 biofilm bacteria.

Chellaram et al. (2004) have studied the whole body extracts of the winged oyster, Pteria chinensis obtained with different solvents were assayed for antibacterial activity using agar well diffusion technique against human and fish pathogens. The acetone and chloroform crude extracts exhibited broad antibacterial activity. Highest activity was exhibits against Klebsilla pneumoniae (5 mm) and Staphylococcus epidermidis (5 mm) by the crude extract of acetone and against Salmonella paratyphi B (5 mm) by the chloroform extract.

Roch et al. (2003) studied the cultivated bivalve molluscs – mainly Pacific cupped oyster, Crassostrea gigas, American cupped oyster, C. virginica, and native oyster, Ostrea edulis, blue mussel, Mytilus edulis, and Mediterranean mussel, M. galloprovincialis – suffer from various infectious diseases (caused by protozoa, bacteria, and viruses) that threaten their production.

Jayaseeli et al. (2001) studied that four filter feeding bivalves, Donax faba, Disinia modesta, Circe scripta and Gafrarium pectinatum were screened for antibacterial activity. The whole body ethanol, heptane and water extracts were prepared and tested against nine pathogens, viz. 32

Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Escherichia coli, Bacillus shigella flexneri. The ethanol extracts of Circe scripta showed prominent activity against Bacillus subtilis. The ethanol extract of Gafrarium pectinatum exhibited activity against Proteus vulgaris.

Morvan et al. (1997) studied that tachyplesin 1 is an antimicrobial peptide extracted from hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus. In vitro activity of tachyplesin was studied against bivalve pathogens: the oyster parasites, Bonamia ostreae, the intra- haemocytic parasite of the flat oyster Ostrea edulis and Perkinsus marinus, the histozoic parasite of the Eastern oyster Crassostrea virginica, and the bacterium vibrio P1, pathogenic for the clam, Tapes philipinarum.

2.5. Bioactive compounds assay Melson and Cowgill (1976) found that the comparative study on paramyosin isolated and purified from the molluscan bivalves, Crassostrea virginica and Mercenaria mercenaria, the whelk, Busycon contrarium and the chiton, Acanthopleura granulate.

Suzuki et al. (2000b) reported that the arginine kinase (AK) isolated from the radula muscle of the gastropod molluscs Cellana grata (subclass Prosobranchia) and Aplysia kurodai (subclass Opisthobranchia), respectively, by ammonium sulfate fractionation, Sephadex G-75 gel filtration and DEAE-ion exchange chromatography. 33

Brown et al. (1998) reported that the a full-length clone with sequence similarity to genes in the cytochrome P450 super family was isolated from a cDNA library prepared from female Mercenaria mercenaria gonadal tissue. This clone was isolated while screening an expression library with an antibody prepared against a peptide sequence within the ligand-binding region of the murine Ah receptor.

Reunova et al. (1997) reported that the ganglia of marine bivalve mollusks contain two types of neurons which secrete regulatory peptides. Some neurosecretory cells deposit lipids in the perikaryon, while others deposit polysaccharides. The cytoplasmic inclusions are probably involved at the energetic and structural levels in the production of neurosecretory material of lipoprotein and glycoprotein nature.

Gardner et al. (1996) found out that the seven polymorphic and four monomorphic allozyme loci were assayed from nine wild populations and one cultured population of the endemic New Zealand greenshell mussel, Perna canaliculus.

Vega-Villasante et al. (2002) isolated a raw extract of langostilla pleuroncodes planipes, obtained by mechanical pressing of the whole organism, was analysed as a potential feed ingredient or additive for cultured marine species. The lyophilized red crab extract possessed: an enzymatic activity of protease, trypsin, amylase, and lipase; no inhibition of serine-proteases; antioxidant capacity against lipoperoxidation and 34 superoxide ions produced by xanthine oxidase reaction and activity of insulin-like peptides.

Odintsova et al. (2000) discovered the possibility of a morphofunctional myogenic differentiation of larval mussel cells in vitro. The shape and extensive cytoskeletal network of the cultured contracting cells mimic largely those of smooth muscle cells in vivo.

Suzuki et al. (2000a) have analysed the heterodont clam Calyptogena kaikoi, living in the cold-seep area at a depth of 3761 m of the Nankai Trough, Japan, has abundant hemoglobins and myoglobins in erythrocytes and adductor muscle, respectively. Two types of hemoglobins (Hb I and Hb II) were isolated, and the complete amino acid sequences of Hb I (145 residues) and Hb II (137 residues) were obtained with combination of cDNA and protein sequencing. The amino acid sequences of C. kaikoi Hbs I and II differed from homologous chains of the congeneric clam Calyptogena soyoae in eight and five positions, respectively.

Chen et al. (2000) studied adenosine deaminase was purified from the adductor muscles of the scallop Patinopecten yessoensis and the round clam Mactra chinensis to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Henry (2000) has studied the APGWamide-related neuropeptides, predicted by the cDNA of the APGWamide precursor of Mytilus edulis, 35 have been sought by means of HPLC and electrospray mass ionization. The three predicted peptides KPGWamide, RPGWamide and TPGWamide were detected in the three main muscles and surprisingly an ion at m/z 429 corresponding to the gastropod peptide APGWamide was also demonstrated.

Ohtani et al. (1995) have studied that thirteen bioactive peptides including FLRFamide were isolated from the bivalve mollusk Meretrix lusoria. The actions of the peptides were examined on several muscles.

Le-Moine et al. (1997) have studied that the digestive enzyme alpha-amylase in Pecten maximus has been purified from the digestive gland, where it is present as two isoforms. In order to gain information on its structure and regulation, a digestive gland cDNA library, constructed in lambda phage zap II (Stratagene, La Jolla, Calif., U.S.A.), was screened with a shrimp alpha-amylase cDNA probe. One 0.02 per cent of the clones were positive, and the longest clone, having a size of 1700 bp and identical to that of the mRNA, was fully sequenced. It contains the complete cDNA coding frame for one of the amylase isoforms of P. maximus.

Mahmood-Alam and Qasim (1997) reported that the cytotoic activities of a toxin isolated by successive chromatographic steps of conventional and HPLC techniques from contaminated shellfish Perna viridis. The toxin induced marked cellular degeneration in tissues of heart, 36 lungs, kidneys and most notably in liver, which is suggested to be its target organ.

Rhodes and Van-Beneden (1997) studied the Ribosomal proteins contribute to the regulation and activity of ribosomes, and hence, the translational activity of the cell. Aberrant expression of ribosomal proteins has been linked to certain pathological conditions such as neoplasms.

Ramasamy and Balasubramanian (2012) have reported that comprehensive inventory of the bioactive compounds produced by Anadara granosa during full growth has been established by whole tissue using organic solvent system and subjecting in to gas chromatography mass spectrum. Twelve bioactive compounds of which ester, from and ketone were identified. The most representative bioactive compounds of Anadara granosa were employed in the antibacterial activity against pathogenic bacteria and the study pertaining to the identification of active- principles and the antimicrobial activities.

Kawashima et al. (2007) have reported that the sterol composition of muscle and viscera tissues of the marine bivalve Megangulus zyonoensis investigated. Among 13 different sterols identified by gas chromatography mass spectrometry of their trimethylsilyl ether derivatives, cholesterol was the most abundant sterol, followed by 24-methylenecholesterol, in both muscle and viscera tissues.

Gueguen et al. (2006) have reported in invertebrates, defensins were found in arthropods and in the mussels. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunitycharacterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters.

The sterol composition of three different populations of Ruditapes decussatus from three localities close to each other, but where different environment conditions prevail, was investigated over a period of 14 months. Sterols of adult clams were isolated by thin layer chromatography and identified by gas chromatography/mass spectrometry. In all samples, the major sterol component was cholesterol (>40% of total sterols); other sterols identified were 24-norcholesta, 5.22-dienol, 22-cis- dehydrocholesterol, 22-trans-dehydrocholesterol, brassicasterol, 24- methylene-cholesterol, campesterol, stigmasterol, -sitosterol and

isofucosterol (Pazos et al., 2005).

Midorikawa et al. (2004) foundout that in the bivalves such as clams Meretrix spp., green mussels Perna viridis, and arkshells Anadara granosa and A. antiquate six organotin compounds monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), monophengltin (MPT), diphenyltin (DPT) and triphenyltin (TPT) were analysed in these samples using GC-MS.

The total synthesis campaigns towards complex heterocyclic natural products are a prime source of inspiration for the design and execution of complex cascade sequences, powerful reactions, and efficient synthetic strategies and highlighted selected examples of such innovations in the course of our total syntheses of diazonamide A, azaspiracid-1, thiostrepton 2, 2’epi-cytoskyrin A; and nughlosin, abyssomicin C, platensimycin, and uncialamycin (Nicolau and Chen, 2008).

Song et al. (2008) have reported that two purified proteins G-6 and G-4-2 were obtained from Arca subcrenata using the homogenization, salting out with ammonium sulfate, ion exchange chromatography and gel filteration chromatography techniques.

Mayer et al. (2007) have reported that 166 bioactive compounds extracted from a diverse group of marine animals, algae, fungi and bacteria which have Antihelmintic, antibacterial, anticoagulant, antimalarial, antiplatelet, antiprotozoal, antituberculosis or antiviral activities. Additionally 45 bioactive compounds were shown to have significant effects on cardiovascular, immune and nervous system as well as possessing anti-inflammatory effects.

Patel and Chikhalia (2006) studied the 3, 4-dimethoxy-1[{2- aryl/alkyl amino]-2-oxoethyl} amino-ethylbenzene-4a-0 and 2-[{2-(3,4- dimethoxy phenyl ethyl amino)-2-oxoethyl}amino] -4,6-diaryl pyrimidines 59.0 have been synthesised and tested for their antibacterial and anti HIV activities against different microorganisms. The structural 39 configuration of novel synthesised compounds has been established on the basis of elemental analysis, HNMR, IR and mass spectral data.

Fukushima et al. (2003) have reported that the trace elements in soft tissues of marine bivalves were determined by neutron activation analysis (NAA) and photon activation analysis (PAA). Elemental levels of Ag, As, Br, Co, Cu, Fe, I, Mn, Ni, Rb, Se and Zn in the organs of giant ezoscallos, rock oysters and giant cramp were obtained. The metal-bound proteins were extracted from the mantles and hepatopancreases of rock oysters.

Kawashima et al. (2007) have reported that the sterol composition in muscle and viscera tissues of the marine bivalve Megangulus zyonoensis was investigated. Among 13 different sterols identified by gas chromatography-mass spectrometry of their trimethylsilyl ether derivatives, cholesterol was the most abundant sterol, followed by 24-methylenecholesterol, in both muscle and viscera tissues.

Babu et al. (2012) studied that the tissues of different body parts such as mantle, viscera and foot were analyzed. Biochemical constituents like protein, carbohydrate and fat were estimated in different body parts and the percentage of protein was 61.74%, carbohydrate 32.64% and lipid 14.3%. The fatty acids of bivalve were analyzed by gas chromatography/ mass spectrometry. In the fatty acids, polyunsaturated fatty acids (PUFA) were found to be dominant, contributing 6.99% (2.34, 2.67 and 1.98% mantle, viscera and foot, respectively), whereas saturated fatty acids 40

(SFA) contributed 2.75% (0.92, 0.93, 0.90%). Stearic (0.81%) and palmitic acids (0.71%) were the dominant saturated fatty acid in viscera and oleic acid (0.93%) was the monounsaturated fatty acid found in the viscera. Linoleic (1.38%) and -linolenic acids (1.07%) were the dominant polyunsaturated fatty acid in viscera. 3. MATERIALS AND METHODS

3.1. Description of the study area 3.1.1. Adirampattinam Coast (Station I) Adirampattinam is located along the South East Coast of India in Thanjavur district of Tamil Nadu State (Lat.10°18’ N, Long. 79°52’ E) bounded by a part of the Bay of Bengal on the nearest and Palk Strait on the South West. It embraces a vast swamp area and coastal waters. It is located 65 km south of Thanjavur and 40 km south of Thiruthuraipoondi in the Tamil Nadu State, on the South East Coast of India (Plate Ia; Fig.2a). This study was carried out over a period of 12 months during January 2011 to December 2011.

The Adirampattinam represents a mixed ecosystem influenced by both freshwater and seawater (Fig.1 and 2). The extensive mud flats of the swamp are subjected to variation in water quality. During the monsoon time, the whole swamp area is covered by freshwater from land run-off and discharges from various places and enduring this period pumping brine for salt production is temporarily suspended. During summer (April to June) penetration of seawater into the swamp area results in high saline conditions (40 ppt) and in the salt pan reservoirs the salinity goes upto 70 ppt.

3.1.2. Muthupet (Lagoon) (Station II) Muthupet is one of the regions in Tamil Nadu coast which has fairly well developed salt marsh mangrove complexes. Muthupet is 42 situated 400 km south of Chennai city and lies close to Point Calimere on the South East coast Peninsula India (Latitude 10°4’ N; Longitude 79°51’ E). It is at the south end of cauvery delta covering an area of approximately 1803 ha (Shanmugasundaram, 1985) of which only 4 per cent is occupied by well-grown mangroves. The rivers Paminiyar, Koraiyar, Kilaithankiyar, Marakkakoraiyar and other tributaries of the river cauvery flow through Muthupet and other adjacent villages at the tail end to form a lagoon before meeting the sea. The northern and western borders of the lagoon are occupied by muddy silt ground (Plate Ib; Fig.2b).

Muthupet reserve forest covers the lagoon, river, creeks and the mud flats. The study area (lagoon) is a spectangular natural creation which is nearly 6-7 km from Muthupet town and can be reachable only by boat. The lagoon is shallow with the average of 1 m depth. Bottom of the lagoon is formed by silt clay substratum. The tidal fluctuations can be observed well with the exposure of oyster beds and roots during low tide. The tidal fluctuations play a major role in dispersing the mangrove seeds. Mangroves mostly cover the lagoon shore on western side which are submerged with high tide. The salinity is the major environment factor controlling zonation of Muthupet mangroves forest. Avicenna marina is the conqueror of the forest which is found as a single dominant species. The southern side (mudflat) separates the lagoon from adjacent sea that also leaves a mouth of lagoon with seasonally opened shallow water ways. The width of mudflat is increased from lagoon mouth to eastern direction. The mudflat looks like a desert in summer, but the presence of dead 43 gastropods under the surface soil layer and the erosion of soil at the centre of mudflat reveal submergence of mudflat during flood. There is a difference between the lagoon shore and seashore of the same mudflat in the aspect of distance of mangroves from fluctuating water level in lagoon side but not in seashore. The reason may be the difference in the nature of fine silt deposition that carried by the rivers. The density of mangroves in eastern side of Muthupet lagoon is comparatively lower than other areas. Tamil Nadu forest department excavated about 12 canals across the mudflat to enhance the water movement between sea and the lagoon.

3.2. Collection of water samples Collection trips were undertaken to station I and II at a monthly interval over a period of one year from January 2011 to December 2011. Four seasons have been recognized in calendar year viz., Pre-monsoon (July – September), Monsoon (October – December), Post-monsoon (January – March) and Summer (April to June). Throughout the study period sampling of water and sediments were carried out on the basis of the last week of every month. Sampling of water was done usually during the morning hours between 8.30 – 10.30 a.m. The samples were collected first week of every month between 9.00 to 10.00 a.m. Water samples were collected from the study sites, just below the surface and transferred to the pre-cleaned polypropylene containers. After collection, all the samples were immediately brought to the laboratory and filtered through the filter paper before nutrient analysis.

3.3. Physico-chemical parameters assay The physico-chemical parameters such as air and surface water temperature, light penetration, pH, salinity, dissolved oxygen, nitrate, nitrite, silicates, ammonia and inorganic phosphates were analysed from the water samples.

Rainfall data were obtained from the meteorological unit of the Regional Meteorological Station, Chennai, Tamil Nadu. India.

The atmospheric and water temperatures were measured by using a centigrade thermometer and pH was measured by using digital pH meter (Elico pH 13 model).

pH of the water samples were determined by immersing a commercially available direct – reading electric pH meter in the water and pH values were read directly from the digital screen. Nitrate, nitrite, ammonia, reactive silicates and inorganic phosphates were estimated by the following method of Strickland and Parsons (1972).

DO concentration was measured using the modified Winkler’s method as described by Strickland and Parsons (1972). The DO concentration was estimated by the standard volumetric Winkler’s method. The water samples were collected in narrow mouthed glass stoppered amber coloured bottles, without air bubbles. One ml of 40 per cent manganous chloride solution was added to each sample followed by the addition of one ml of alkaline iodide solution for fixing the oxygen in 45 the field. In the laboratory one ml of concentrated sulphuric acid was added to dissolve the manganous hydroxide precipitate. After acidification 50 ml of sample was transferred into a conical flask and added 2 or 3 drops of 1 per cent freshly prepared starch for producing blue colour. Then it was titrated against 0.025 N sodium thiosulphate solution. The titration was stopped when the blue colour disappeared. The amount of dissolved oxygen was calculated as ml/l.

Salinity was estimated by using ‘ATAGO’ hand refractometer at the site itself. The salinity of the water sample was determined by measuring of chlorinity. Five ml of the sample was taken in the conical flask and 3 drops of 5 per cent potassium chromate was added for indicating purpose. Then it was titrated against 0.1 N silver nitrate solution. The titration was concluded at the first appearance of brick red colour.

Titre value (ml)  N of AgNO  1000  35.5 Chlorinity = 3 Volume of the sample (ml)

Salinity (ppt) = 0.03 + (1.805  chlorinity of the sample).

Salinity of the samples were expressed in ppt (Klein, 1973).

3.3.1. Statistical analysis for water quality status Pearson’s correlation analysis was used to assess the relationship between physico-chemical parameters and diversity of bivalves. The data were computed and analysed using Statistical Package for Social Sciences (SPSS) software version 15.0 was used for these analysis.

3.4. Distribution of bivalves 3.4.1. Sediment analysis Sediments were collected from both stations I and II for a period of January 2011 to December 2011 using a Peterson grab, transferred to clean Polybags, transported to the laboratory and air dried. Totally 15 samples were analysed for each station. The percentage composition of sand, silt and clay in the sediment samples were determined by the combined sieving and Pipette Method of Krumbein and Pettijohn (1938). The texture of the sediment was ascertained by plotting the values on a textural triangle deviced by U.S. Department of Agriculture (Anonymous, 1951).

3.4.2. Population studies A survey was carried out to assess the bivalves from two study sites. Meretrix casta was collected from the study site, Muthupet estuary – Station II: Anadara granosa was collected from Adirampattinam coastal waters - Station I: the study period from January 2011 to December 2011. A bivalve drag was used to collect bivalve samples. The drag was made from stainless steel with an opening, 35 cm in width and 15 cm in height.

The net (mesh size 1.8  1.8 cm) was connected to the tail of the drag.

Bivalves were collected at both stations by drawing the drag by hand for a distance of 15 m. The numerical density of bivalves were calculated as No/m2 and biomass as g/m2. The number of bivalves were then counted and bivalve density calculated per square meter. The bivalves were identified using the standard references (Sathiyarmoorthy, 1952) and also confirmed with Zoological Survey of India. 47

3.4.3. Statistical analysis Pearson correlation analysis was used to access the different sediment composition in Station I and Station II. The different population characteristics of M. casta and A. granosa (Density and Biomass) were tested using ANOVA. The data were computed and analysed using Statistical Package for Social Sciences (SPSS) Software.

3.4.4. Systematic position and biology of the study materials Systematic positionof Meretrix casta Phylum - Mollusca Class - Bivalvia Order - Eulamellibranchiata Family - Veneridae Genus - Meretrix Species - casta

Systematic position of Anadara granosa Phylum – Mollusca Class – Bivalvia Order – Arcoida Family – Arcidae Genus – Anadara Species – granosa

3.4.5. Biology of M. casta (Plate IIa and b) M. casta is characterized by a thick ovate and smooth cell, devoid of any external sculpture. The outer surface is pale yellow, tinged with dark gray posteriorly. The front margin is evenly rounded but the lower part of the hind margin is somewhat angular. The lunule is not well defined and the ligament is short. The linge margin is thick and has three cardinal teeth. A tooth is present in front of the cardinals on the left valve and a corresponding depression on the right valve (Jonas Gunasekaran et al., 2003).

3.4.6. Biology of A. granosa (Plate III a and b) The shell of A. granosa is a medium size, fairly thick, ovate, convex, inflated, equivalve and in equilateral dorsal margin, straight anterior and rounded, sloping ventrally, posterior lud obliquely rounded and ventral margin concave. The ligamental area is narrow, rhomboidal with dark complete chevaron markings and anterior part not covered with ligament periostra cum brown and smooth. Hind line is straight. Inner shell is white in colour, ventral, anterior and posterior margins of valves with crenulations of ribs faintly visible (Narashimham, 1968).

3.5. Biochemical compositions assay 3.5.1. Collection and extraction of samples The bivalves, M. casta and A. granosa were collected from Adirampattinam coastal waters and Muthupet estuary, South East coast of India, respectively, at monthly intervals during the period of January 2011 to December 2011. The live specimens were brought to the laboratory and 49 thoroughly cleaned of encrusting material, washed and kept in clear aerated seawater for at least 24 hrs. These specimens were sorted into different size groups and used for biochemical studies. The size range from 25 to 35 mm (both test samples) were selected and the meat was separated and used for the biochemical studies. Five to ten bivalves were selected and weighed accurately. Then the bivalves were opened and excess water blotted with filter paper from tissue. The whole body of both bivalves were dissected to remove the different body components viz., mantle, gill, gonad, siphon, adductor muscles, foot and digestive gland. These component parts were separately weighed and kept it in oven at 60°C for 24 hours. The dried tissue samples were weighed to constant weight, powdered and then the dried powdered extract were used to determine biochemical composition (Plate IV and V).

3.5.2. Proximate compositions 3.5.2.1. Total carbohydrate For the estimation of total carbohydrate content, the procedure of Carroll et al. (1956) was adopted using anthrone reagent. 100 mg of both test dried samples were taken and homogenized in 2 ml of 10 per cent TCA in a glass tissue homogenizer. The homogenates were transferred to centrifuge tubes and were centrifuged at 3000 rpm for 5 minutes. The supernatants were decanted to test tube. To each test tube, 10 ml of anthrone reagent (0.05% anthrone in 72% sulphuric acid containing 1.0% thiourea) was added with constant shaking of the test tubes. One holed rubber cork fitted with glass tubing served as air condenser which also served to prevent the entry of water while heating in 50 boiling water bath the tube were immersed to the level of liquid in the tubes and kept for 15 minutes after which they were removed and immersed in cold water bath on reaching room temperature the content of the tubes were transferred to 1 cm cuvettes and read at 620 nm in Spectronic 21 after adjusting the system with the reagent blank prepared with 2 ml of 10 per cent TCA. Standard was run along with the experiment with 0.1 mg glucose in 2 ml of 10 per cent TCA. The concentration of carbohydrate in the samples were calculated with the standard value as given below.

Calculation

OD of sample 0.1  vol. of extract  100 % wt of carbohydrate  OD of standard Weight of tissue

= mg of carbohydrate / 100 mg of dry tissue where 0.1 = mg of glucose in 2 ml of the standard solution

3.5.2.2. Estimation of total protein The Folin-Ciocalteu phenol method of Lowry et al. (1951) was adopted for the estimation of total proteins in the tissues of both test samples. The dry tissue of both samples weighing 10 mg was thoroughly homogenized with 1 ml of deproteinizing agent (10% TCA) by keeping the tubes in ice. All samples were centrifuged for 20 min at 3000 rpm. The precipitate obtained was used for protein estimation. The precipitate was dissolved in 2 ml 1 N NaOH and to 1 ml of this solution, freshly prepared 5 ml alkaline reagent was added. This was kept at room temperature for 10 min after which 0.5 ml of 1 N Folin-Ciocalteu reagent was added and 51 mixed rapidly. A standard stock solution was prepared using bovine serum albumin crystals at a concentration of 25 mg/5 ml NaOH. Different dilutions in the range of 0.25 to 2.5 mg/ml were prepared from this stock solution, the alkaline reagent and Folin-phenol reagent was added as in the case of tissue samples. A blank was prepared with 1 ml 1 N NaOH and treated the same way as earlier mentioned. All the test tubes were kept for 30 min at room temperature, the blue colour developed, and the O.D was evaluated against the blank at 660 nm:

Standard value  OD of sample % composition of protein =  100 Weight of tissue

3.5.2.3. Estimation of total lipid The chloroform-methanol extract procedure of Folch et al. (1956) was used for extracting lipid from the various body parts of both test samples separately. The lipid content was estimated gravimetrically by following Folch et al. (1956) method. The lipid was extracted from 500 mg of powdered oven-dried tissue with 5 ml of chloroform; methanol (2:1) mixture added. The mixture was filterd by a microfilter. This extract was taken in a preweighed beaker and oven dried. Beaker was reweighed with lipid. The difference in weight was taken as total lipid content and the percentage was calculated as follows. weight of lipid % weight of lipid =  100 weight of tissue

3.5.3. Enzyme assay 3.5.3.1. Protease The total protease activity was measured by the casein digestion method by using the amino or carboxyl groups separated from a protein substrates (Mahadevan and Sridhar, 1996) in both test samples separately.

To the test tube having 10 ml of 1 per cent casein solution, 5 ml of 0.1 M phosphate buffer (pH 7), and 5 ml of enzyme extract were added. This mixture was incubated at 30°C in a water bath for 1 h. From this, 1 ml of sample was drawn to a test tube and neutralized using 0.1 N NaOH. One ml of ninhydrin was added to the sample, and mixed thoroughly. The content of the last tube was heated for 20 min in a boiling water bath and 5 ml of distilled water was added to the mixture. The purple colour thus formed was measured at 570 nm. A blank was maintained with 1 ml of distilled water instead of animal extract.

3.5.3.2. Lipase The lipase was measured by the method of Sadasivam and Manickam (1996) in both test samples. This method estimates the quantity of fatty acids released per unit time by measuring the quantity of NaOH required to maintain pH constant. The milliequivalent of alkali consumed was taken as measure of the enzyme.

Twenty ml of vegetable oil emulsion was taken into 5 ml of phosphate buffer (pH 7.0) in a 500 ml beaker, and kept in the top of a magnetic stirrer-cum-hot plate and stirred the contents slowly, at a 53 temperature of 35°C and pH of 7.0 and 0.5 ml of enzyme extract was added, immediately recorded the pH and set the times on. At an interval of 30 min as the pH drops, about 0.2 unit of the content was tested to bring using 0.1 N NaOH to the initial value. The volume of alkali consumed was recorded at the regular interval. The enzyme activity was defined as the amount of enzyme released one milliequivalent of free fatty acids perminute per of sample. Specific activity is expressed as milliequivalent/min/mg protein. Activity (meq/min/g cample).

Volume of alkali consumed  Strength of alkali = Weight of sample in g  Time in min

3.6. Antibacterial assay 3.6.1. Collection and extraction of samples Live specimens of bivalves (M. casta and A. granosa) were collected at a depth of 6 m in Muthupet estuary and Adirampattinam coastal waters, South East Coast of India, respectively. They were immediately brought to the laboratory and their soft bodies were removed by breaking the shells. The whole body muscle of the both the test samples (30 g) were cut into small pieces and the tissue samples were used for extraction using different solvents such as ethanol, methanol, chloroform and water. The extracts were cold steeped over night at -18°C and filtered with Whatman No.1 filter paper. The filtrate was poured in previously weighed petriplate and evaporated in dryness in rotary evaporator (Becerro et al., 1994; Wright, 1998). The dried crude extracts were used for antibacterial assay against human bacterial pathogens. 54

3.6.2. Pathogenic bacteria used Five human pathogenic bacteria were obtained from Government Hospital in Thanjavur, Tamil Nadu, India.

 Escherichia coli

 Salmonella paratyphi

 Klebsiella pneumoniae

 Salmonella typhi

 Staphylococcus aureus

3.6.3. Disc diffusion assay Bivalves, M. casta and A. granosa crude extracts were tested for inhibition of bacterial growth against human bacterial pathogens. Bacterial assay was carried out by the disc diffusion technique followed by Kelmen et al. (2001). Bacterial strains were inoculated in sterile nutrient broth and incubated at 37°C for 24 h. Pathogens were swabbed on the surface of the Muller Hinton agar plates and disc (Whatman No.1 filter paper with 9 mm diameter) impregnated with 50 L of both bivalves extracts placed on the

surface, separately. Control discs were with water and solvents to assess the effect of water and solvents on pathogens. The plates were incubated at 37°C for 24 h and the antibacterial activity was measured accordingly based on the inhibition zone around the disc impregnated with bivalves extracts.

3.7. Bioactive compounds assay 3.7.1. Gas Chromatography – Mass Spectrometer (GC-MS) method for analyzing bioactive compounds in both test samples (Williams and Fleming, 1987) GC-MS plays a key role in the analysis of unknown components of plant origin. GC-MS ionizes compounds and measures their mass numbers. Ionization method includes EI (Electron Ionization) and CI (Chemical Ionization). Typically, the CI method is used. The EI method produces ions by colloiding thermal electrons emitted from a filament with sample gas molecules. This method provides high stability in ionization and the obtained mass spectra show good reproducibility. The EI method provides good results for quantitative analysis of compounds.

Gas chromatography technique involves the separation of volatile compounds in a test sample using suitable capillary column coated with polar or non-polar or intermediate polar chemicals. Elite-1 column (100% Dimethyl poly siloxane) is a non-polar column used for the analysis of phytocomponents in medicinal plants and pesticide residues. Elite-5 column (5% phenyl and 95% methyl poly siloxane) is an intermediate column used for the estimation of animal tissues and food grains. An inert gas such as Hydrogen or Nitrogen or Helium is used as a carrier gas. The components of test sample is evaporated in the injection port of the GC equipment and segregated in the column by adsorption and desorption technique with suitable temperature programme of the oven controlled by software. Different components are eluted form the column based on the boiling point of the individual components. The GC column is heated in 56 the oven between 60 to 270°C. The time at which each component eluted from the GC column is termed as Retention Time (RT). The eluted component is detected in the Mass detector. The spectrum of the unknown component is compared with the spectrum of the known components stored in the NIST library and ascertains the name, molecular weight and structure of the components of the test materials in GC-MS study. Bioactive compounds from bivalves tissues were analyzed in the GC-MS study.

Sample extraction The two bivalves samples were extracted separately in methanol (1 gm/10 ml). One-gram sample was dissolved in methanol and heated to 50°C for 5 min and then filtered in a filter paper along with 1-2 gm sodium sulfate to remove the traces of moisture from the filtrate. The filtrate was concentrated to 1 ml of bubbling nitrogen gas into the solution.

Sample analysis in GC-MS One microlitre of sample was injected into the GC column fitted with Elite-1 capillary column (100% Dimethyl Polysiloxane) with the dimension of 30 m x 0.25 mm IDx 1 m df. The GC column used was a non-polar column. The carrier gas used was helium gas at the rate of 1 ml per minute. The split used in the injector was 10:1. The injector temperature maintained was at 250°C throughout the experiment. In the GC programme, the oven temperature was kept at 110°C for 2 min and the temperature was raised to 280°C at the rate of 5°C per minute. The 57 holding time after reaching the temperature of 280°C was nine minutes. The total GC programme was worked out to be 45 min. The equipment used was Perkin Elmer-Clarus 500 GC.

The software used in the above GC MS programme was Turbo mass gold ver.5.1. The library used in analyzing the spectrum of unknown components was NIST (National Institute of Standards and Technology, USA) of ver. 2.1. The EI source used was 70 mV. The mass numbers (m/z) monitored was 45-450. The total programme time in the MS was 46 minutes.

3.8. Extraction and separation of compounds (Mercado Luis et al., 2005) 3.8.1. Processing of whole body tissue The purification procedure is summarized as follows. Frozen whole body tissues from 20 specimens were crushed to homogeneity in liquid nitrogen. The resulting whole body tissue powder (40 g) was suspended in cold acetic acid 11% (1:10) in order to solubilize cationic molecules, followed by Vortex-mixing for 5 min. The homogenate was sonicated for

3  30 sec at 11 resonant macrosonic synthesis (RMS) in ice and incubated with mild agitation at 4°C overnight. The crude extract was centrifuged at 11,000  g, 35 min at 4°C and the pellet was discarded. The

supernatant was called acid extract (AE) and further shaken at 37°C for 1 h to favour sugar hydrolysis. Tris-base crystals were slowly added to raise the pH to a value of 3.0. In order to enrich cationic peptides, four batches of 100 ml each of AE were sequentially mixed overnight with a 58 sulfoethyl (SE) Sephadex C-50 cation-exchange beads (BioRad) with mild agitation at 4°C in a ratio of 1.5 ml gel slurry/25 ml AE. The mixture was centrifuged at 1,500  g 10 min and resuspended in 1% acetic acid 0.1 M

NaCl (pH 3.0). Putative peptides were recovered from beads with mild agitation for 1 h in the presence of 1% acetic acid 1 M NaCl (pH 3.0). The eluate (6 ml) was applied onto a sep-pak C18 vac cartridge (Waters Associates) equilibrated in acidified water (0.05% trifluoroacetic acid in UPW – Ultra Pure Water). After a wash with acidified water, the peptides were eluted, flow 1 ml/min, with 5, 20, 40, 60 and 80% CAN, to obtain several hydrophobic fractions. The samples obtained were lyophilized and tested for antibacterial activity.

3.8.2. Separation of components using HPLC The test samples of both bivalves tissue extracts separately (0.5 ml) were injected in to the RP-18 octadecylsilyl silica (DDS) column (25  1 cm, i.e.) with LC-UV detector (Bioanalytical system, USA) and -1 monitored of 254 nm. The flow rate was adjust to 1.5 l min . The fractioned samples were collected in vials.

3.9. Antibacterial assay using HPLC fraction The HPLC fractions (purified compounds) from the bivalve extracts of A. granosa and M. casta were tested for their antibacterial activity against K. pneumoniae and S. paratyphi. Sterile paper discs (5 mm in diameter) were soaked with each fraction (70 l) and dried completely. The discs were placed on agar medium in which the test pathogens had been plated previously. The inhibition zones (mm) formed around paper discs were measured to find out the antibacterial activity against the pathogen tested. 59

3.10. Structure and identification of bioactive compounds by using Fourier Transform Infrared Spectroscopy (FTIR) The lyophilized samples were mixed with 100 mg of dried potassium bromide (Kbl) and compressed to prepare as a salt disc. The disc was then read spectrophotometrically (bio-rad FTIR-40-model, USA). The frequencies of different components present in each sample were analysed. 4. RESULTS

4.1. Environmental parameters In the present study the important physicochemical parameters such as rainfall, atmospheric temperature, surface temperature, pH, salinity, dissolved oxygen, inorganic phosphate, nitrate, nitrites, reactive silicate, ammonia and photoperiod were analysed from the station I and station II and the results were summarized.

4.1.1. Rainfall The rainfall data of the study area are shown in Fig.3. Rainfall varied from 4 to 96.2 mm during the study period and minimum was recorded (4 mm) during premonsoon in July 2011 and maximum (96.2 mm) during premonsoon in August 2011 at Station I. The rainfall was recorded minimum of 7.2 mm during post monsoon in January 2011 and maximum of 64 mm during monsoon in November 2011 at Station II. The rainfall was totally absent in the months of February and June 2011 at Station I and February and July 2011 at Station II of the study period.

4.1.2. Atmospheric temperature Atmospheric temperature data of the study are shown in Fig.4. It ranged from 26 – 31°C during the study period and minimum of 26°C was recorded during December 2011 and maximum of 31°C during April, May 2011 at Station I. In Station II, minimum of 28°C was recorded 61 during January, December 2011 and maximum of 31°C was recorded during May 2011.

4.1.3. Surface water temperature Water temperature data are shown in Fig.5. It ranged from 27 - 31°C during study period and minimum was recorded (27°C) during December 2011 and maximum was recorded (31°C) during May, July 2011 at Station I. Minimum was recorded (29°C) during January 2011 and maximum was recorded (32°C) during February, May and June 2011 at Station II.

4.1.4. Hydrogen ion concentration (pH) The pH values are shown in Fig.6. It varied between 7 and 8.5 during the study period. At station I, the minimum value (7) was recorded during the months of January, February, April, September, November and December 2011. At Station II, the minimum pH (7) was recorded during the months of January to April and August to December, 2011. The maximum value (8.5) was recorded during summer in May 2011 at Station I, and 7.5 was recorded during May, June and July 2011 at Station II.

4.1.5. Salinity Salinity values are given in Fig.7. It ranged from 30-36 ppt during the study period. Minimum salinity was recorded as 30 ppt during January, March, October 2011 and maximum was recorded as 36 ppt during June 2011 at Station I. At Station II, minimum salinity was 62 recorded as 30 ppt during January, March 2011 and maximum was recorded as 35 ppt during May, June 2011.

4.1.6. Dissolved oxygen (DO) Dissolved oxygen concentrations are depicted in Fig.8. It varied between 2.2 to 5.4 mgl-1 during the study period. Dissolved oxygen concentration was minimum 2.2 mg.l-1 during June 2011 and maximum concentration was 3.7 mg.l-1 during November 2011 at Station I and minimum concentration was 2.4 mg.l-1 during April 2011 and maximum concentration was 5.4 mg.l-1 during November 2011 at Station II.

4.1.7. Inorganic phosphate Inorganic phosphate concentrations are shown in Fig.9. It varied between 0.5 and 3.5 mgl-1. Concentration of phosphorus was minimum as 0.5 mgl-1 during March 2011 and maximum was recorded as 2.2 mgl-1 during December 2011 at Station I. Minimum concentration was observed as 0.9 mgl-1 during May and June 2011 and maximum concentration was observed as 3.5 mgl-1 in December 2011 at Station II.

4.1.8. Nitrate Dissolved nitrate concentrations are given in Fig.10. It ranged from 2.8 to 8.6 mgl-1. The minimum value was recorded as (2.8 mg mgl-1) during May 2011 and maximum value recorded was 8.0 mgl-1 during October 2011 at Station I. Minimum value was recorded as 3.5 mgl-1 during March and May 2011 and maximum was recorded as 8.6 mgl-1 during October 2011 at Station II. 63

4.1.9. Nitrites

The nitrite of water ranged from 0.64 to 2.85 g/ml in January

2011 to December 2011 (Fig.11). The minimum value 0.64 g/ml was estimated in July 2011 and maximum value (9.5 g/ml) in December 2011 at Station I. The minimum nitrite value (2.5 g/ml) was recorded during summer and maximum (8.7 g/ml) in monsoon season during the study period at Station II.

4.1.10. Reactive silicates Silicate concentration fluctuated between 2.15 and 6.24 mgl-1 (Fig.12) at both stations. The minimum value was recorded during summer season at Station I (2.15 mgl-1) in April 2011 and in Station II (2.16 mgl-1) in April 2011. Maximum silicate was recorded during the post monsoon season in January 2011 at Station I (6.24 mgl-1) and in Station II (6.16 mgl-1) during August 2011.

4.1.11. Ammonia Ammonia level varied from 0.42 to 1.92 mgl-1 (Fig.13). Minimum value was recorded during post-monsoon season at both stations and the maximum was recorded during the summer season at June 2011 at Station I. The minimum value of 0.42 mgl-1 was estimated in March 2011 and maximum value (1.92 mgl-1) was estimated at May 2011 in Station II.

4.1.12. Photoperiod The monthly mean light lengths in the present study showed a slight variation (Fig.14). Even though India is a tropical country, the day 64 length showed fluctuation. It varied from 11.14 to 12.56 hours during the study period (Fig.14). It was found to be low (11.25 hrs) in March 2011 and high (12.51 hrs) in May 2011 at Station I. Maximum (12.5 hrs) was found to be in August 2011 and minimum (11.14 hrs) day length showed in March 2011 at Station II. The day length was found to be low during monsoon and post monsoon seasons and high during summer and pre-monsoon seasons at both stations.

4.1.13. Correlation studies The Pearson co-efficient of correlation studies (Table 1 and 2) revealed that the physico-chemical features in Station I showed the significant positive correlation with the atmospheric temperature, water temperature, salinity with M. casta abundance and negative correlation with rainfall, DO and nutrients. In station II, also a similar correlation pattern could be observed with the population density of A. granosa. Surface water temperature and salinity were showed significant negative correlation with rainfall, dissolved oxygen, nitrate, nitrites, reactive silicates, phosphates and ammonia. pH showed a positive correlation with salinity.

Station I

 Surface water temperature was positively correlated with air temperature (r = 0.837**).

 Salinity was positively correlated with air temperature (r = 0.697*), surface water temperature (r = 0.594*) and pH (0.450*). 65

 Inorganic phosphate was positively correlated with rainfall (r = 0.449*) and DO (r = 0.425*).

 Inorganic phosphate was negatively correlated with salinity (r = -0.720**) (Table 1).

Station II

 Surface water temperature was positively correlated with air temperature (r = 0.856**).

 pH was positively correlated with air temperature (r = 0.724*).

 Salinity was positively correlated with surface water temperature (r = 0.547) and pH (r = 0.456*).

 Inorganic phosphate was positively correlated with rainfall (r = 0.428*), salinity (0.626*) and dissolved oxygen (r = 0.438*).

 Salinity was negatively correlated with atmospheric temperature (r = 0.499*).

 Nitrate was negatively correlated with atmospheric temperature (r = 0.412*) (Table 2).

4.2. Distribution of bivalves 4.2.1. Sediment analysis In station I, the percentage of sand, silt and clay fluctuated between 59.32 and 73.46 per cent, 13.90 to 22.56 per cent and 12.64 to 19.72 per cent respectively during the study period and the type of soil was sandy loam (Table 3).

In station II, the percentage of sand fluctuated from 77.12 to 88.72 per cent, the silt showed 7.30 to 16.20 per cent and the clay was 1.32 to 7.98 per cent (Table 4) during the study period and the type of soil was sandy loam.

4.2.2. Population density of bivalves The highest population (64 No/m2) density of A. granosa was recorded during the month of August 2011 and the lowest population density (28 No/m2) was recorded during the month of December 2011 at Station II whereas the highest population (84 No/m2) density of M. casta was recorded during the month of May 2011 and the lowest population density (50 No/m2) was recorded during the month of November 2011 at Station I (Table 5; Fig.15).

Dendrogram of cluster analysis based on Muthupet estuarine, bivalve A. granosa population density is shown in Fig.17 and the bivalve M. casta population density is shown in Fig.18 based on Adirampattinam coast.

4.3. Biomass of bivalves The highest biomass value (1030 g/m2) was recorded in A. granosa which was collected during September 2011 and the lowest biomass (207.65 g/m2) was recorded during in March 2011 at Station II and in M. casta, the highest biomass value was 815.0 g/m2 during in August 2011 and the lowest biomass (357.65 g/m2) was recorded during in March 2011 at Station I (Fig.16). 67

4.3.1. Statistical analysis 4.3.1.1. Correlation studies The Pearson coefficient of correlation studies (Table 3.1 and 4.1) revealed the sediment analysis at Station I and II showed the significant negative correlation with silt and clay and positive correlation with clay and silt.

Station I Silt negatively correlated with sand (r = -0.900**). Clay negatively correlated with sand (r = -0.769**). Clay positively correlated with silt (r = 0.413) (Table 3.1).

Station II Silt negatively correlated with sand (r = -0.758**). Clay negatively correlated with sand (r = -0.741**). Clay positively correlated with silt (r = 0.123) (Table 4.1).

4.3.1.2. Analysis of variance for population density and biomass from Station I and Station II One way ANOVA was performed for population density and biomass revealed the variation in population (F = 153.670) and available biomass (F = 51.711) between the stations (Table 5.1). 68

4.4. Biochemical composition of bivalves The proximate composition of the various parts of the tissues of M. casta and A. granosa was estimated at monthly intervals and given in Tables 5 and 6).

4.4.1. Total carbohydrate Whole body tissue In matured animals of M. casta, the carbohydrate content was determined in whole body of the bivalve which varied from 2.5 (August 2011) to 5.6 per cent (April 2011) at monthly intervals with an average of 4.1 per cent (Table 5). In A. granosa, the carbohydrate content varied from 4.51 (Jan.2011) to 7.92 per cent (April 2011) with an average of 6.16 per cent (Table 6).

Gonad The carbohydrate content of gonad of M. casta varied from 2.8 (August 2011) to 6.9 per cent (April 2011) at monthly intervals with an average of 4.0 per cent (Table 5). In A. granosa, the carbohydrate content varied from 4.84 (August 2011) to 7.52 per cent (May 2011) with an average of 6.11 per cent.

Mantle In M. casta, the carbohydrate content of mantle varied from 1.2 (August 2011) to 5.2 per cent (April 2011) at monthly intervals with an average 3.5 per cent (Table 5). In A. granosa, the carbohydrate content varied from 3.21 (August 2011) to 9.84 per cent (March 2011) at monthly intervals with an average of 5.48 per cent (Table 6). 69

Adductor muscle The carbohydrate content of adductor muscle (M. casta) was determined and it varied from 0.8 (August 2011) to 3.5 per cent (April 2011) at monthly intervals with an average of 2.39 per cent (Table 5). In A. granosa, the carbohydrate content varied from 2.88 (August 2011) to 5.86 per cent (April 2011) with an average of 4.30 per cent (Table 6).

Digestive gland In matured individuals of M. casta the carbohydrate content was determined in digestive gland showed variations from 1.3 (August 2011) to 5.1 per cent (April 2011) with an average 3.33 per cent (Table 5). In A. granosa, it varied from 3.34 (August 2011) to 7.74 per cent (April 2011) at monthly intervals with an average of 5.32 per cent (Table 6).

Foot In M. casta, the carbohydrate content was determined in foot of the bivalve which varied from 0.3 (August 2011) to 2.5 per cent (April 2011) at monthly intervals with an average of 1.57 per cent (Table 5). In A. granosa, the carbohydrate content varied from 2.32 (August 2011) to 4.87 per cent (April 2011) in A. granosa with an average of 3.55 per cent (Table 6).

Gill In M. casta, the carbohydrate content varied from 0.34 (August 2011) to 2.50 per cent (May 2011) with an average of 1.63 per cent (Table 5) in the gills. In A. granosa, it varied from 2.15 (November 2011) 70 to 4.89 per cent (May 2011) at monthly intervals with an average of 3.37 per cent (Table 6).

Siphon In matured animals of M. casta, the carbohydrate content was determined in siphon of the bivalve which varied from 1.02 (Oct.2011) to 3.51 per cent (April 2011) with an average of 2.7 per cent (Table 5). In A. granosa, the carbohydrate content varied from 3.00 (October 2011) to 5.89 per cent (April 2011) with an average of 4.09 per cent (Table 6).

4.4.2. Total protein Whole body tissue In matured samples of M. casta, the protein content was determined in whole body of the bivalve which varied from 48.4 (October 2011) to 66.02 per cent (June 2011) at monthly intervals with an average of 56.29 per cent (Fig.19). In A. granosa, the protein content varied from 50.45 (October 2011) to 68.32 per cent (June 2011) with an average of 55.68 per cent (Fig.20).

Gonad The matured animals of M. casta, the protein content was determined in the gonad which varied from 48.40 (December 2011) to 64.20 per cent (March 2011) at monthly intervals with an average 55.35 of per cent (Fig.19). In A. granosa, the protein content varied from 49.98 (December 2011) to 64.83 per cent (April 2011) with an average of 54.89 per cent (Fig.20). 71

Mantle In M. casta, the protein content was determined in mantle of the bivalve which varied from 46.20 (December 2011) to 58.3 per cent (June 2011) at monthly intervals with an average of 52.58 per cent (Fig.19). In A. granosa, the protein content varied from 47.60 (December 2011) to 60.56 per cent (June 2011) with an average of 55.11 per cent (Fig.20).

Adductor muscle In matured individuals of M. casta, the protein content varied from 49.82 (September 2011) to 61.32 per cent (June 2011) at monthly intervals with an average of 55.93 per cent (Fig.19). In A. granosa, the protein content varied from 51.76 (April 2011) to 60.51 per cent (July 2011) with an average of 54.76 per cent (Fig.20).

Digestive gland In M. casta, the protein content was determined in digestive gland of the bivalve which varied from 50.86 (October 2011) to 65.56 per cent (June 2011) with an average of 55.60 per cent (Fig.19). In A. granosa, the protein content varied from 51.00 (November 2011) to 61.63 per cent (May 2011) at monthly intervals with an average of 55.53 per cent (Fig.20).

Foot In matured samples of M. casta, the protein content was determined in foot of the bivalve which varied from 46.32 (December 2011) to 56.30 per cent (May 2011) at monthly intervals with an average 72 of 50.44 per cent (Fig.19). The protein content varied from 47.86 (December 2011) to 66.02 per cent (July 2011) in A. granosa with an average of 53.13 per cent (Fig.20).

Gill In M. casta, the protein content of gill varied from 39.74 (February 2011) to 53.62 per cent (July 2011) with an average of 46.13 per cent (Fig.19). In A. granosa, it varied from 41.24 (February 2011) to 55.83 per cent (July 2011) at monthly intervals with an average of 47.19 per cent (Fig.20).

Siphon In matured individuals of M. casta, the protein content was determined in siphon of the bivalve which varied from 47.52 (October 2011) to 53.62 per cent (May 2011) with an average of 51.02 per cent (Fig.19).

In A. granosa, the protein content varied from 49.54 (September 2011) to 55.91 per cent (April 2011) with an average of 52.67 per cent (Fig.20).

4.4.3. Total lipid Whole body tissue In matured samples of M. casta, the lipid content was determined in whole body which varied from 1.6 (September 2011) to 3.8 per cent (June 2011) at monthly intervals with an average of 3.39 per cent (Fig.21). 73

In A. granosa, the lipid content varied from 3.07 (August 2011) to 6.12 per cent (June 2011) with an average of 4.71 per cent (Fig.22).

Gonad The lipid content of gonad tissue of M. casta varied from 1.4 (August 2011) to 3.8 per cent (May 2011) at monthly intervals with an average 2.85 per cent (Fig.21). In A. granosa, the lipid content varied from 3.07 (August 2011) to 6.31 per cent (May 2011) with an average of 4.00 per cent (Fig.22).

Mantle The lipid content of mantle tissue of M. casta varied from 1.5 (September 2011) to 3.7 per cent (June 2011) at monthly intervals with an average 2.65 per cent (Fig.21). In A. granosa, the lipid content of mantle tissue varied from 3.81 (October 2011) to 6.43 per cent (June 2011) at monthly intervals with an average of 4.69 per cent (Fig.22).

Adductor muscle In M. casta, the lipid content was determined in the adductor muscle and it varied from 0.9 (August 2011) to 3.5 per cent (May 2011) at monthly intervals with an average of 2.19 per cent (Fig.21). In A. granosa, the lipid content varied from 2.87 (August 2011) to 6.1 per cent (June 2011) with an average of 4.19 per cent (Fig.22).

Digestive gland In matured samples of M. casta, the lipid content was determined in digestive gland of the bivalve is which varied from 1.2 (August 2011) to 4.2 per cent (May 2011) with an average 2.78 per cent (Fig.21). In A. granosa, it varied from 3.32 (August 2011) to 6.45 per cent (May 2011) at monthly intervals with an average of 4.66 per cent (Fig.22).

Foot In M. casta, the lipid content was determined in foot of the bivalve which varied from 1.3 (October 2011) to 3.8 per cent (April 2011) at monthly intervals with an average 2.67 per cent (Fig.21). The lipid content varied from 3.33 (October 2011) to 6.30 per cent (April 2011) in A. granosa with an average of 4.70 per cent (Fig.22).

Gill In M. casta, the lipid content of gill varied from 0.5 (August 2011) to 3.2 per cent (April 2011) with an average of 1.98 per cent (Fig.21). In A. granosa, it varied from 2.87 (September 2011) to 5.84 per cent (July 2011) at monthly intervals with an average of 4.07 per cent (Fig.22).

Siphon In matured animals of M. casta, the lipid content was determined in siphon of the bivalve which varied from 0.7 (September 2011) to 3.3 per cent (May 2011) with an average of 2.05 per cent (Fig.21). In A. granosa, the lipid content varied from 2.63 (September 2011) to 5.67 per cent (May 2011) with an average of 4.02 per cent (Fig.22). 75

4.4.4. Analysis of averages of proximate composition 4.4.4.1. Average composition of carbohydrate in M. casta The average composition of carbohydrate values in M. casta were observed as 4.1, 4.0, 3.5, 2.39, 3.33, 1.57, 1.63 and 2.7% in whole body, gonad, mantle, adductor muscle, digestive gland, foot, gill and siphon, respectively. The maximum was observed in whole body tissue (4.1%) and minimum was observed in foot (1.57%) (Table 5).

4.4.4.2. Average composition of carbohydrate in A. granosa The average composition of carbohydrate values in A. granosa were found out as 6.16, 6.12, 5.48, 4.30, 5.32, 3.55, 3.37 and 4.09% in whole body, gonad, mantle, adductor muscle, digestive gland, foot, gill and siphon, respectively. The maximum of carbohydrate was observed in whole body (6.16%) and minimum was observed in gill (3.37%) (Table 6).

4.4.4.3. Average composition of proteins in M. casta The average composition of protein values in M. casta were observed as 56.29, 56.10, 55.10, 55.93, 55.60, 50.44, 46.13 and 51.02% in whole body, gonad, mantle, adductor muscle, digestive gland, foot, gill and siphon, respectively. The maximum level observed in whole body tissue (56.10%) and minimum was observed in gill (46.13%) (Fig.19).

4.4.4.4. Average composition of proteins in A. granosa The average composition of protein values in A. granosa were observed as 55.68, 54.25, 55.10, 55.04, 55.96, 53.13, 47.19 and 52.67% in 76 whole body, gonad, mantle, adductor muscle, digestive gland, foot, gill and siphon, respectively. The maximum level was observed in whole body tissue (55.68%) and minimum was observed in gill (47.19%) (Fig.20).

4.4.4.5. Average composition of lipid in M. casta The average composition of lipid values in M. casta were observed as 3.39, 2.85, 2.65, 2.19, 2.78, 2.67, 1.98 and 2.05% in whole body, gonad, mantle, adductor muscle, digestive gland, foot, gill and siphon, respectively. The maximum was observed in whole body tissue (3.39%) and minimum was observed in gill (1.98%) (Fig.21).

4.4.4.6. Average composition of lipid in A. granosa The average composition of lipid values in A. granosa were observed as 4.71, 4.00, 4.69, 4.19, 4.66, 4.70, 4.07 and 4.02% in whole body, gonad, mantle, adductor muscle, digestive gland, foot, gill and siphon, respectively. The maximum was observed in whole body (4.71%) and minimum level of lipid was observed in siphon (4.02%) (Fig.22).

Nutritional quality of meat in both bivalves was very high, during summer season. Protein was the major organic constituent found in both bivalves. Carbohydrate was the second major organic component found. The present studies indicate inverse relation between water and protein content. In the present investigation, A. granosa and M. casta of coastal waters and estuaries show very high calorific values throughout the year.

4.4.5. Enzymes: Protease and lipase assay from the tissue of bivalves Protease In matured individuals of M. casta, the protease content was determined in gut of the bivalve varied from 0.08 (Dec.2011) to 0.22% (July 2011) and at monthly intervals with an average of 0.12%. In A. granosa, the protease content varied from 0.07 (Jan.2011) to 0.22% (July 2011) with an average of 0.14% (Fig.25 and 26).

The protease content of digestive gland was determined in the bivalve M. casta which varied from 0.06 (Jan.2011) to 0.24% (July 2011) and at monthly intervals with an average of 0.14%. In A. granosa, the protease content varied from 0.07 (Jan.2011) to 0.21% (June 2011) with an average of 0.14% (Fig.23 and 24).

Lipase In matured individuals of M. casta, the lipase content was determined in gut of the bivalve which was varied from 0.08% (Jan. 2011) to 0.22% (July 2011) at monthly intervals with an average of 0.14%. In A. granosa, the lipase content varied from 0.06 (Jan.2011) to 0.21% (June 2011) with an average of 0.13% (Fig.25 and 26).

The lipase content of digestive gland of M. casta varied from 0.06 (Jan.2011) to 0.21% (May 2011) at monthly intervals with an average of 0.13% whereas in A. granosa, the lipase content varied from 0.08 (Jan.2011) to 0.24% (July 2011) with an average of 0.15%.

4.5. Antibacterial activity of bivalves against bacterial pathogens In the present study, various solvent extracts of the two bivalves, M. casta and A. granosa were tested for their effect on five pathogenic strains of bacteria causing diseases in human beings. The bivalves, M. casta and A. granosa, the whole body tissue extracts were prepared separately using methanol, ethanol, chloroform and water solvents and screened for antibacterial activity against the bacteria, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi and Salmonella paratyphi.

The inhibition zones of ethanol, methanol and chloroform extracts as compared with aqueous (water) extract against the specific test organisms (Plate VI and VII). The maximum inhibition zone (9 mm) was observed against Klebsiella pneumoniae followed by E. coli, S. aureus in the crude ethanol extract of A. granosa and the minimum inhibition zone (6 mm) was noticed against S. paratyphi (Table 9). The maximum inhibition zone (9 mm) was observed against Klebsiella pneumoniae, followed by E. coli, S. aureus in the ethanol extract of M. casta and the minimum inhibition zone (2 mm) was noticed against S. paratyphi (Table 8). The methanol extracts were able to produce a zone of 8.5 mm against K. pneumoniae and E. coli and 7.5 mm of S. aureus and S. typhi. However, only slight activity was shown by the crude extract of chloroform. The zone of inhibition in mm recorded for the both bivalves extracts in the present study is compared with the standard drug, Ciprofloxacin. The bivalves ethanol extracts is nearing the effect of 79 standard drug ciprofloxacin which causes the inhibition of 14 mm (Table 8 and 9).

4.6. Identification of bioactive compounds from two bivalves GC-MS analysis were made to identifie the bioactive compounds of two bivalves namely M. casta and A. granosa.

4.6.1. GC-MS analysis of ethanol extract of M. casta and A. granosa The potent ethanol extract of the two bivalves, A. granosa and M. casta were subjected to analysis by using GC-MS, which were carried out in GC clarus 500 perk in Elmer system. The following compounds were identified from the mass spectra analysis (Table 10 and 11).

4.6.1.1. GC-MS analysis of M. casta In GC-MS analysis, the ethanol extract of M. casta showed four prominent peaks (Table 10; Fig.27) with retention time 14.13, 17.92, 21.81 and 28.03 mins. The peaks with retention time 14.13 mins corresponds to 7, 12-dihydro-6, 7-bis (4-hydroxyphenyl)-6H-[1,2,4] triazolo [1’,5’:1, 2] pyrimido [5,4-c] chromen-2-ol with 4.13 per cent of peak area; 17.92 mins corresponds to 2-nonadecanone 2,4- dinitrophenylhydrazine with 8.61 per cent of peak area; 21.81 mins corresponds to 3-pyridinecarboxylic acid, 2,7, 10-tris (acetyloxy)-1, 1a, 2, 3, 4, 6, 7, 10, 11, 11a-decahydro-1, 1, 3, 6, 9-pentamethyl-4-oxo-4a, 7a- epoxy-5H-cyclopental [a] cyclopropa [f] cycloundecen-11-yl ester with 3.96 per cent of peak area and 28.03 mins corresponds to 9, 12, 15- octadecatrienoic acid, 2-1-ethyl ester with 83.30 per cent of peak area. 80

4.6.1.2. GC-MS analysis of A. granosa In GC-MS analysis, the ethanol extract of A. granosa showed 12 prominent peaks (Table 11; Fig.28) with retention time 13.91, 16.60, 17.29, 17.65, 19.75, 20.42, 20.79, 24.33, 24.54, 24.86, 25.54 and 26.20 mins. The peaks with retention time 13.91 min corresponds to 3-phenyl-2, 3-dihydrobenzo(b)furon-2-ol with 2.15 per cent of peak area; 16.60 min corresponds to methyl5-(2-phenyl propionyl) hexanoate with 0.45% peak area; 17.29 min corresponds to n-Hexadecanoic acid with 4.31 per cent of peak area; 17.65 min corresponds to Hexadecanoic acid ethyl ester with 2.70 per cent of peak area; 19.75 min corresponds to octadecanoic acid, methyl ester with 0.36 per cent of peak area; 20.42 min corresponds to octadecanoic acid with 3.59 per cent of peak area; 20.79 min corresponds to octadecanoic acid, ethyl ester with 3.42 per cent of peak area; 24.33 min corresponds to methanone, [1,4-dimethyl-7-(1-methylethyl)-2- azulenyl] phenyl, with 3.17 per cent of peak area; 24.54 min corresponds to methanone, [2-(1-methyl ethyl) phenyl] phenyl with 2.96 per cent of peak area; 24.86 min corresponds to 4, 7, 10, 13, 16, 19-docosahexaenoic acid, methyl ester (all-z) with 0.95 per cent of peak area; 25.54 min corresponds to phenol, 2,4-bis (1-phenylethyl) with peck 9.57 per cent of peak area; 26.20 min corresponds to 1,2-benzenedicarboxylic acid, with 66.35 per cent of peak area.

4.6.2. HPLC fractionation of ethanolic extracts of M. casta and A. granosa The ethanolic extract of M. casta and A. granosa were subjected to analysis by HPLC and which yielded two major peaks each. Of them, the 81 major peak showed retention time as 3.050 and 3.508 mins for A. granosa and 3.208 and 3.783 mins for M. casta (Fig.29 and 30).

4.6.2.1. Antibacterial activity of HPLC fractionation of M. casta and A. granosa Among the four fractions in A. granosa, peak 2 showed highest antibacterial activity against K. pneumoniae with the inhibition zone of 16 mm diameter. The minimum inhibitory zone effect was observed in peak 1 against S. paratyphi.

Among the four fractions in M. casta, the peak 2 showed highest antibacterial activity against K. pneumoniae with the inhibition zone of 16 mm diameter. The minimum inhibitory effect was observed in peak 1 against S. paratyphi.

4.7. Detection of different functional groups by FTIR technique from the M. casta and A. granosa from HPLC fractions FTIR spectral analysis The lyophilized samples of M. casta and A. granosa (10 mg) were mixed with 100 mg of dried potassium bromide (kbr) and compressed to prepare as a salt disc. The disc was then read spectrophotometrically (Bio-Rad FTIR 40-model, (USA)). The frequencies of different components present in both samples were analysed separately (Table 11).

The FTIR spectra of the lyophilized sample of M. casta of the 8 peaks were at 2860.27, 1484.93, 1342.47, 1218.14, 1152.18, 933.63, 82

899.25 and 860.39 cm-1 whereas the spectra of the sample of A. granosa showed all peaks with very close values at 3388.75, 2958.90, 2925.32, 1643.07, 1539.73, 1402.05, 673.97, 615.76, 536.99 and 465.75 cm-1 (Fig.31-34). As per the FTIR study, the following functional groups have been identified from these compounds.

M. casta – peaks 1 and 2 The FTIR showed the presence of Hetrocyclicamine, C-H asymmetric stretch-lipids and carbonateions (Fig.31 and 32).

A. granosa – peaks 1 and 2 The FTIR showed the presence of Hetrocyclicamine, methylene C-H and secondary amine (Fig.33 and 34).

The FTIR analysis reveals the presence of bioactive compounds signals of different ranges. Four chemical components in each test organism were identified in FTIR analysis with the sample extract of A. granosa and M. casta. Among these compounds, Heterocyclic amine, Secondary amine, methylene OH and asymmetric stretch-lipids were identified and are known antibacterial compounds. The compound is a good antioxidant.

The research shows that the medicinal value of the bivalves, A. granosa and M. casta whole body tissue may be due to high quality of antibacterial compounds. The present study also reveals that the species of 83

A. granosa and M. casta show antibacterial activities against the pathogenic bacterial forms. It indicates that they possess potential pharmacological action. However, some novel and uncharacterized mechanism of action that might ultimately benefit the ongoing global search for clinically useful antibacterial agents need to be explored to explain the characteristic of antibacterial activity of both bivalves. It could also be added that the composition of marine bivalves is a nutritional assurance to millions of malnourished people. In spite of this variability, the nutritional quality of the bivalves is generally good, especially just before gamete release (pre-monsoon), when the concentrations of nutrients is as its maximum. 5. DISCUSSION

5.1. Environmental parameters 5.1.1. Physico-chemical characteristics of Adirampattinam coastal waters and Muthupet estuarine environment The physico-chemical parameters have an impinging effect on the aquatic organisms and are of many parameters varied with temporal and spatial variations. These factors are either exogenous or endogenous or both. These parameters might affect the life activities and govern the distribution of biological organisms. All the factors are interrelated even to one another. Variation in one may affect the other. Considerable data on information are available on physico-chemical factors and their impact on the distribution, physiology and reproductive biology of the organisms (Sridhar et al., 2006; Varadharajan et al., 2010; Srilatha et al., 2012).

The hydrobiological parameters played very important role in the life of aquatic organisms. The water quality study is a pre-requisite in any aquatic organism for the assessment of its potentialities and to understand the realities between its different trophic levels and food webs (Sesamal et al., 1985; Kannan and Kannan, 1996; Sathpathy et al., 2007; Hussain Dar et al., 2011). The seasonal variations of the hydrobiological factors in the Bay of Bengal, South East Coast of India have been reported by many workers (Satpathy, 1996; Ashok Prabu et al., 2005; Sundaramanikam et al., 2008; Sithik et al., 2009; Damotharan et al., 2010; Sankar et al., 2010; Kannathasan and Rajendran, 2010). In the present study, the physico- 85 chemical parameters of coastal waters of Adirampattinam (Station I) and estuary of Muthupet (Station II) were considered throughout the year from January 2011 to December 2011 at monthly intervals. The estuarine representatives are subjected to both diurnal and seasonal changes in these physico-chemical parameters. The impact of this will be more on resident species particularly bivalves and mussels (Salaskar and Nayak, 2011).

The rainfall data of both the study areas varied from 4 to 96.2 mm. Minimum (4 mm) was recorded in pre-monsoon season (July 2011) and maximum (96.2 mm) was recorded in monsoon season (October 2011) at Station I. The minimum rainfall of 7.2 mm and maximum of 64 mm were recorded during monsoon season in November 2011 at Station II. The rainfall was totally absent in the months of February and June 2011 at Station I and February and July 2011 at Station II. Similar observations were made by Hussain Dar et al. (2011) and Srilatha et al. (2012).

The temperature is the most important exogenous factor for the regulation of growth, metabolism and breeding of marine organisms (Rajendran, 1990). The factors other than temperature such as salinity,

pH, DO, CO2 and nutrients have a remarkable influence on the nutritional and reproductive cycles of marine biological organisms. Atmospheric temperature ranged from 26-31°C during the study period at both stations. The minimum (26°C) was recorded during December 2011 and maximum (31°C) during April, May 2011 at Station I. In Station II, minimum (28°C) was recorded during January, December 2011 and maximum (31°C) was recorded during May 2011. Water temperature ranged from 24-32°C 86 during the study period and the minimum (27°C) was recorded during December 2011 and the maximum (31°C) was recorded during May 2011 at Station I. The minimum (29°C) was recorded during January 2011 (Post monsoon) and maximum (32°C) recorded during February, May and June, 2011 (Summer) at Station II. These findings are in accordance with that of Srilatha et al. (2012). The decrease of the water temperature increases the capacity of oxygen to dissolve in water as observed by Nedumaran et al. (2001). Lakshmilatha et al. (2006) reported that the water temperature is the most significant factor which influence the distribution of bivalves in marine and estuarine environment. Kannathasan and Rajendran (2010) found that the surface water temperature at Nagapattinam coastal waters normally 27 to 29°C, which may be due to diurnal variation of sea surface (Rao et al., 1982). Somayajulu et al. (1987) observed that the surface water temperature value decreased from the pre-monsoon to post monsoon. Both atmospheric and water temperature were found to be maximum during summer season and minimum during monsoon season (Dronamraju et al., 2008; Hussain Dar et al., 2011; Srilatha et al., 2012).

Kannan and Kannan (1996) reported that low temperature in post monsoon at Palk Bay was due to cloudy sky and heavy rainfall during that period. Similarly in the present study also, that low at monsoon and post- monsoon seasons at both stations. At Ayyampattinam coast, the surface water temperature varied from 25.5 to 33.4°C (Santhosh Kumar and Perumal, 2011). The low temperature was recorded during post-monsoon season only.

The salinity of coastal water is an another important abiotic factor having a remarkable influence on the reproductive and nutritional cycles of marine molluscs, particularly bivalves (Harkantra and Rodriques, 2004; Ramachandra et al., 2011). A wide seasonal fluctuation in salinity was recorded during the study period. Salinity values ranged from 30 to 36 ppt during the study period (Fig.7). The maximum (36 ppt) value of salinity was recorded during June 2011 (Summer) and minimum (30 ppt) during January, March 2011 post monsoon and monsoon season at Station I. In station II, the salinity was recorded as minimum (30 pt) during monsoon and maximum (35 ppt) at summer season. A similar observation was observed by Padmavathi and Sathya Narayan (1999) and Govindasamy et al. (2000). The minimum salinity during monsoon season could be due to heavy rainfall and influx of freshwater from land after the monsoon rain and discharge of water from the rivers (Kannan and Kannan, 1996; Dronamraju et al., 2008; Sithik et al., 2009). The salinity changes in a locality are also influenced by many factors like freshwater inflow, rainfall, temperature, tide water current, mixing, evaporation and precipitation.

In the present study, the seasonal average salinity registered high, during summer and premonsoon seasons and low during monsoon and post monsoon period is in conformation with the earlier reports from Bay of Bengal, India (Soundaramanickam et al., 2008; Santhosh Kumar and Perumal, 2011; Srilatha et al., 2012). The average salinity values of the water varied from 11 to 36 ppt and it showed a wide fluctuation of recording from 18 to 27 ppt at the head or even less during monsoon 88 season and 31 ppt in the South part of the Bay of Bengal (Kannathasan and Rajendran, 2010). The lowest and highest salinity obtained in the study areas 11 to 36 ppt respectively. The salinity varied from 33.9 to 34.8 ppt at the off-shore surface water and increased with depth (Rao et al., 1982). Salinity shows direct relationship with pH (Bhave and Borgse, 2007). Salinity is an important ecological factor that determines the distribution of bivalves in inshore waters estuaries and salt marshes. The role of salinity in bivalves distribution was emphasized by many workers (Laxmilatha et al., 2006; Varathanarajan et al., 2010; Tanyaros and Tongnunui, 2011). The salinity of the sea at Port Novo Coast, South East Coast of Tamil Nadu, varied between 26.5 and 34.5 ppt and it increased during post monsoon and summer and began to decline during premonsoon and monsoon season (Sridhar et al., 2006).

The hydrogen ion concentration (pH) is an another important hydrobiological parameter which influence the distribution, growth, metabolism and proximate composition of aquatic organisms. The variation in pH of the water was less pronounced throughout the study period. In this study, the pH values varied from 7 – 8.5 (Fig.10). The minimum pH (7.0) was recorded during the months of January, February, April, September, October, November and December 2011 and the maximum (8.5) was recorded during May and June 2011 at Station I and the minimum pH (7.0) was recorded during monsoon and post monsoon and the maximum (7.5) was recorded during summer at Station II. The high pH recorded during summer season could be due to the increased temperature coupled with salinity (Hussain Dar et al., 2011; Srilatha et al., 89

2012). The slight seasonal fluctuation in pH was mainly due to rainfall and freshwater inflow. Similar observations have been reported by Soundaramanickam et al. (2008) in Pitchawaram estuary. The pH of Chennai coastal waters also showed slight fluctuation, it was found to be minimum during north east monsoon and the maximum pH during summer (Subramanian and Mahadevan, 1999).

The dissolved oxygen (DO) is one of the important biological factor which influence the bioenergetics of the aquatic organisms. In the present study, DO concentrations varied between 2.2 – 5.4 mg.l-1 during the study period (Fig.12). DO was minimum (2.2 mg.l-1) during June 2011 in summer season and maximum (3.7 mg.l-1) during November 2011 in monsoon season at Station I and the minimum concentration was 2.4 mg.l-1 during April 2011 and the maximum concentration 5.4 mg.l-1 during November 2011 at Station II. The DO level fluctuated during summer and monsoon seasons, which may be due to wind velocity, rainfall and photosynthetic activities of biological organisms. The DO level estimated in the present study also agrees with earlier workers (Sridhar et al., 2006; Satpathy et al., 2007; Soundarapandian et al., 2009; Damotharan et al., 2010). Subramanian and Kannan (1988) have also reported higher level of DO during monsoon season in Tuticorin coast in Gulf of Mannar but contradicting report at Coovam estuary have been noticed high DO concentration during monsoon season.

In the present study, phosphorus concentration varied between 0.5 and 3.5 mg.l-1 (Fig.13). P-concentration was recorded minimum 90

(0.5 mg.l-1) during March 2011 and maximum (2.2 mg.l-1) was recorded during December 2011 at Station I. The minimum concentration was observed as 0.9 mg.l-1 during May, June 2011 and maximum concentration was observed as 3.5 mg.l-1 during December 2011 at Station II. Similar observations were already made by many earlier workers (Prema and Subramanian, 2003; Sakthivel, 2004; Senthilkumar, 2008; Hussain Dar et al., 2011; Srilatha et al., 2012). In contrast at Madras Coast, the minimum phosphate was observed during the post-monsoon season and maximum during pre-monsoon season (Subramanian and Mahadevan, 1999). The recorded high concentration of inorganic phosphates during monsoon season at both stations in the present study might possibly be due to intrusions of upwelling sea water into the creek, which in turn increased the level of phosphate (Nair et al., 1984). The increased nitrates and nitrites level in the sea was due to freshwater inflow and terrestrial run off during monsoon season (Santhosh Kumar and Perumal, 2011).Rajaram et al. (2005) found that the low values of nitrite content observed during non-monsoon period may be due to its utilization by phytoplankton as evidenced by high photosynthetic activity and the dominance of nitrite in sea water having a negligible amount of nitrate. In the present study, high concentrations of nitrate was observed during monsoon season at both stations, as a result of monsoonal flow of freshwater and land runoff. In the present study, the nitrate concentration ranged from 2.8 – 8.0 mg.l-1 (Fig.14) at station I and at Station II, the maximum value was recorded as 8.6 mg.l-1 during October 2011 and the minimum was recorded as 3.5 mg.l-1 during March and May 2011 in summer season. 91

In the present study, the nitrate, nitrite and ammonia concentrations ranged from 2.8 to 8.0 mgl-1, and 0.64 to 9.5 mgl-1 and 0.42 to 1.92 mgl-1 respectively at Station I and in Station II, the nitrate, nitrite and ammonia

-1 -1 -1 ranged from 3.5 to 8.6 mgl and 0.56 to 2.85 mgl and 0.38 to 1.92 mgl respectively. The maximum value of nitrite was recorded as 2.84 mgl-1 during October 2011 and the minimum was recorded as 0.64 mgl-1 during February 2011 in postmonsoon season at station I and the maximum value

was recorded as 2.85 ml-1 during monsoon season and the minimum

value was recorded as 0.56 ml-1 during post monsoon season at

Station II.

The nitrite content estimated in the present study also agrees with earlier workers (Hussain Dar et al., 2011; Srilatha et al., 2012). The

maximum amount ammonia were recorded as 1.84 ml-1 and as 1.92 ml-

1 during summer season and the minimum were recorded as 0.42 ml-1 and as 0.42 ml-1 during postmonsoon season at both stations respectively.

The ammonia concentration estimated in the present study also agrees with earlier workers in Adirampattinam coastal waters by Hussain Dar et al. (2011) and Muthupet estuary by Srilatha et al. (2012).

In the present study, the reactive silicates ranged between 2.15 and

6.24 ml-1. The silicate concentration was recorded minimum 2.15 ml-1

and maximum 6.24 ml-1 during monsoon and postmonsoon seasons,

respectively. The reactive silicates estimated in the present study also 92 agrees with earlier workers (Hussain Dar et al., 2011; Srilatha et al., 2012).

5.2. Distribution, population density and biomass of bivalves Most of the molluscs, particularly bivalves possess broad tolerance to varied environmental conditions, and are able to survive through most of the seasons. However, there may be only one or two accessions during the year when they might be really dominant (Thajuddin, 1991). This phenomenon known as seasonal variation or succession was studied in considerable detail to answer the questions of the interaction of the bivalves with the environmental conditions. In the present study, the high levels of nutrients that occurred during the monsoon and premonsoon seasons are utilized by the population of bivalves and there is an increase in the population of bivalves, A. granosa and M. casta at Station II and Station I, respectively in summer seasons. The location specific variation in abundance of A. granosa and M. casta were recorded at Muthupet estuary and Adirampattinam coastal waters, respectively in the present study. Similarly, the location specific of bivalves have been demonstrated for Mytilus edulis (Incze et al., 1980), Mya arnaria and Crassostrea gigas (Brown and Hartwick, 1988).

Many reports are available on the bivalves wealth in different environments around the world (Absalo, 1991; Baron and Clavier, 1992; Tanyaros and Tongnunui, 2011). In this study, the bivalves diversity, particularly A. granosa and M. casta were recorded seasonally around the 93 year 2011 from Muthupet estuary and Adirampattinam coastal waters respectively.

The influence of environmental parameters on population densities of bivalves has been performed elsewhere (Absalo, 1991; Baron and Clavier, 1992; Soares-Gomes and Pres-Vanin, 2005; McLead and Wing, 2008; Tanyaros and Tongnunui, 2011). A population of the bivalve, Meretrix casta from estuarine ecosystems in the coastal area of Trang Province, Thailand has been studied (Songrak et al., 2009; Tanyaros and Tongnunui, 2011).

Tanyaros and Tongnunui (2011) reported that the water quality parameters appeared to have the greatest influence on the abundance and population structure of the estuarine bivalve, M. casta was due to salinity and TSS. Changes in water salinity affect a wide variety of biochemical and physiologic processes in marine bivalves. An increase or decrease in salinity often results in the increase or decrease of free amino acid levels in tissues of marine bivalves, which are often monitored as a stress indicator (Powell et al., 1982; Lee et al., 2004). The effects of salinity changes on the feeding physiology and growth of bivalves had been reported by Sara et al. (2008). TSS concentration was the most important factor influencing the abundance of estuarine bivalves, reported by Tanyaros and Tongnunui (2011).

In general, the distribution of bivalves are influenced by physicochemical and biological characteristics, prevailing in the 94 environment. In the present study, bivalves, Anadara granosa was recorded highest abundance in the Muthupet environs whereas Meretrix casta was recorded predominance in coastal waters of Adirampattinam. Similar species were observed in Coleroon estuary by Prabha Devi and Ayyakannu (1989). Vellar estuary by Chandran et al. (1982) and Aiyyampattinam and Arukkattuthurai by Varadharajan et al. (2010). Salinity and temperature is an important ecological factors which influence the distribution of bivalves. High temperature 35°C recorded in summer season influence the distribution of both bivalves in both stations predominantly. Low temperature recorded in December influence higher faunal density. Low density of bivalves recorded in November due to heavy downpour, which caused drastic fluctuations in the sampling stations. Positive relationship between the abundance of bivalves and concentration of organic carbon in sediments had been documented by Parulekar et al. (1975) and Prabha Devi (1994). The salinity also considered to be a dominant factor, in the distribution of bivalves in Dammar Cornice and half moon Bay of Arabian Gulf by Anvar Batcha (1997). pH and DO did not play any considerable role in bivalves abundance in the present study in both stations. Similar findings were also reported by Prabha Devi and Ayyakannu (1989), Prabha Devi (1994) and Varadharajan et al. (2010).

In the present study, station I (Adirampattinam) have less species of M. casta during the months of November and January 2011. This is due to abundance of salt ponds and also salt refinery industries wastes that drained in the shore environment. Because of this the population of 95 bivalves diversity is disturbed. So the present study conforming that pollution in the intertidal coastal areas directly affecting the species diversity of bivalves as well as indirectly the fishery potential.

M. casta prefers high salinity and sandy bottom (Laxmilatha et al., 2006) and have salinity ranging from 20 to 33 ppt due to its proximity to the sea. The average density of M. casta was 51 No/m2 in Zone II and 12 numbers per sq.m. in zone VI, in Chettuva estuary reported by Laxmilatha et al. (2006). In the present study, the average maximum density of M. casta was 86 No/m2 in Station I (Adirampattinam off-shore coastal waters) during summer season.

The average biomass of M. casta, denoted by the total weight in grams per sq.m. was highest in Chettuva estuary (674.75 g/m2) reported by Laxmilatha et al. (2006). In the present, the average biomass of M. casta denoted by the total weight in g/m2 was highest in Adirampattinam coastal waters (756.67 g/m2) during the premonsoon season. The total average biomass of M. casta (609.16 g/m2) was recorded in Station I. The overall total biomass of the coastal waters of Adirampattinam was 7309.92 g/m2.

The bivalve, A. granosa prefers high temperature and sandy bottom (Ramasamy and Balasubramanian, 2011) and have temperature ranging from 35 to 38°C due to its proximity to the sea. The average density of A. granosa was 54 No/m2 in Muthupet estuary (Station II) during the summer season. The average biomass of A. granosa was highest in 96

Muthupet estuary (815.0 g/m2) during the premonsoon season. The Adirampattinam and Muthupet estuary remains saline for most part of the year (summer months 2011 ) and therefore offers immense scope for bivalves, M. casta and A. granosa respectively, culture in both stations.

Several species of bivalves, found in the estuaries, coastal and backwaters of India, are exploited for their meat and shells. Information on the distribution and exploitation of bivalves from India was reported by very earlier workers also (Rao, 1963; Jones, 1968; Alagarswami and Narashimham, 1973).

Recently, Laxmilatha et al. (2006) reported that the distribution of bivalve resources of the Chettuva estuary of Kerala. Studies on the resource characteristics, exploitation and biology of bivalves from Karnataka, Kerala and Andhra Pradesh were made by Rao (1984), Archary (1988), Narashimham et al. (1984), Laxmilatha and Appukkuttan (2002). In this study, the diversity and distribution of bivalves from Adirampattinam coast and Muthupet estuarine were studied seasonally.

The bivalve distribution in a unit area (25  25 cm) was taken by

demarking the area of bivalve bed with a quadrant. About 2” sample within the quadrant area was collected for analysis. Laxmilatha et al. (2006) reported that in the Chettuva estuary is dominant in Meretrix casta and to some extent Villorita cyprinoides. In the present study, the M. casta was recorded seasonally, throughout the year in Adirampattinam coastal off-shore regions. Salinity and the substrate influence the distribution of bivalve, M. casta in Chettuva estuary reported by Laxmilatha et al. 97

(2006). Srilatha et al. (2012) reported that the physico-chemical status of Muthupet estuary. The water quality parameters showed seasonal variation and the variations recorded may be due to the environmental fluctuations in relation with season. In the present study also the water quality parameters showing seasonal variations recorded due to the environmental fluctuations in relation with seasons and also with distribution of bivalves due to high level of salinity and temperature.

Significantly higher species diversity was observed at high salinity, fine sand and high sedimentary biochemical parameters of total organic carbon, total organic nitrogen and carbon of biopolymeric fraction (Harkantra and Rodrigues, 2004).

5.3. Seasonal variations in biochemical composition of bivalves Cyclic changes in biochemical composition of animal tissues are mainly studied to assess the nutritive status of an organism. Molluscs are delicious and protein rich food among the sea foods (Jagadis, 2005). The bivalves in the coastline could form an important source of food, raw material for village industries, indigenous medicine etc. and it is widely used as a cheaper food source for coastal area people. The bivalves are the rich sources of animal protein and good for human health. The biochemical studies are very important from the nutritional point of view and bioenergetics of an organism. The biochemical constituents in bivalves are known to vary with season, size of the organism, stage of maturity, and availability of food (Babu et al., 2012). Due to importance and abundance of the commercial bivalve species, Anadara granosa and 98

Meretrix casta commonly distributed in Muthupet estuary and Adirampattinam coastal waters, respectively; this study investigated the nutritive value of biochemical compositions and enzymes of A. granosa and M. casta. It is found that the seasonal changes have greater influence on meat quality and weight.

Biochemical constituents (proximate composition like protein, carbohydrate and lipids) were estimated in the different body tissues of the bivalves, A. granosa and M. casta. Among the body fractions, whole body tissue recorded the highest protein, lipid and carbohydrates. Investigation on biochemical compositions in different parts of an animal would be more informative than estimation in the entire body for studies (Giese, 1969; Babu et al., 2012). Biochemical compositions in different body fractions were mainly influenced by reproductive cycle and secondarily by food availability. Accumulation of protein, carbohydrate and lipid will be high during proliferation of gonads (George, 1980). Percentage of these constituents will increase during maturation of gonads (Ansell, 1974). In the present study, the percentage of proteins, carbohydrates and lipids were recorded from eight body fractions. Among the eight body fractions, whole body tissue portion recorded high protein, carbohydrates and lipids. The digestive gland acts as a storage site in most of the bivalves (George, 1980). The variation in the biochemical constituents in digestive gland indicated that this organ acts as probable storage site in both bivalves, A. granosa and M. casta.

The biochemical compositions of A. granosa and M. casta tissues are similar to those of other marine bivalves, such as oyster and scallops, Gafrarium tumidum (Babu et al., 2012) which typically high protein content and low levels of fat and cholesterol. The fat and cholesterol content of number of bivalves including oyster, scallops, mussels, cockles, ranged between 1.3 and 2.3% and 0.33 and 0.59% respectively (Pearson, 1977; Babu et al., 2012). The seasonal variation in biochemical constituents of different body components of Meretrix meretrix were also studied by Jayabal and Kalyani (1987), the results showed 24.82% protein, 13.53% carbohydrate and 7.26% fat. The results of the species, A. granosa and M. casta showed the same trend in the present study. Biochemical constituents of many species of bivalves have been analysed by Rajan et al. (1990), Jagadis (2005) and Babu et al. (2012). Salaskar and Nayak (2011) found that the nutritional quality of the oyster, Perna viridis was generally good, especially just before gamete release (pre monsoon) when the concentration of the nutrients was as its maximum. A low level of fat was detected in the edible meat of oysters and mussels. Protein is the major biochemical constituent of bivalve meat. In the present study, the highest protein content was recorded in whole body tissues of both bivalves during summer and premonsoon seasons. In the case of bivalves, it was noticed that as temperature increases protein content also increases. During monsoon season gradual fall in protein content can be seen. While in case of oyster and mussels two peaks can be seen during post monsoon (66.51%) and pre-monsoon (63.54%) (Salaskar and Nayak, 2012).

100

5.4. Antibacterial activity of bivalves against pathogenic bacteria The marine environment is a huge source to discover bioactive natural products. There is a vital interest in discovering new antimicrobial compounds with fewer environmental and toxicological risks and no resistance developed by the pathogens (Chellaram et al., 2004). Many classes of bioactive compounds from molluscs exhibiting anti-tumour, anti-leukemia, anti-bacterial and antiviral activities have been reported worldwide (Kamiya et al., 1984; Anand et al., 1997; Rajaganapathy et al., 2000; Periyasamy et al., 2012; Ramasamy and Balasubramanian, 2012). Among the molluscs, bivalves have pronounced pharmacological activities or other properties which are useful in the biomedical arena. The potential of marine bivalves as a source of biologically active products is largely explored in India. Therefore, the present study was carried out to evaluate the antibacterial activity of the five solvents tissue extracts of bivalves, A. granosa and M. casta against five pathogenic bacterial strains. Several marine molluscan possessed broad spectrum of antimicrobial activities affecting the growth of bacteria, fungi and yeasts (Rajaganapathi et al., 2000; Anand et al., 2001; Anderson and Beaven, 2001).

There is a growing interest in marine natural products or marine secondary metabolites. In the present study, the ethanol, methanol, chloroform and aqueous crude extracts of bivalves, A. granosa and M. casta showed activity against five bacterial strains. The maximum zone of inhibition was recorded in Klebsiella pneumoniae strain followed by Escherichia coli and minimum zone of inhibition was observed in 101

Salmonella paratyphi and S. typhi of both bivalves with ethanol tissue extracts.

Similar results were reported in the antibacterial activities of bivalves, A. granosa (Ramasamy and Balasubramanian, 2012a), Meretrix casta and Tridacna maxima (Ramasamy and Balasubramanian, 2012b), Babylonia spirata (Periyasamy et al., 2012) and Turbo brunneus (Elezabeth et al., 2003; Anand et al., 1997) against human bacterial pathogens. These investigations support the present findings of the antibacterial activity of tissue ethanol extraction of A. granosa and M. casta. Similarly, the antibacterial activity was reported in four bivalves against few pathogens and found that the ethanol extracts showed significant activity against Bacillus subtilis (Jayaseli et al., 2001). The maximum antibacterial activity was recorded in Pseudomonas aeruginosa strain and minimum zone of inhibition was observed in Streptococcus pneumoniae by using the ethanol extracts of Babylonia spirata (Periyasamy et al., 2012). This study also colloborates the results of the present investigation. The antimicrobial activity from the gill extraction of Perna viridis (Chandran et al., 2009), Meretrix meretrix and M. casta (Sugesh, 2010), antibacterial activities of green mussel, Perna viridis and edible oyster (Crassostrea madrasensis) (Annamalai et al., 2007), antibacterial activities of bivalve Anadara granosa (Srinivasan, 2008) and Teucrium polium (Darabpour et al., 2010) were reported. The difference in the antibacterial activity found in bivalves extract may depend on extracting capacity of the solvents and the compounds extracted (Anand et al., 2001). 102

The methanol extract of Hemifusus pigilinus possessed the highest activity against E. coli and the lowest activity was observed against Klebsiella oxytoca. In the present study, ethanol extract of A. granosa and M. casta possessed the highest activity against K. pneumoniae and E. coli. Srinivasan (2008) found that the methanol extracts of Anadara granosa showed the highest activity against E. coli and the lowest activity against S. typhi. In the present study, the ethanol extracts of A. granosa showed the highest activity against Klebsiella pneumoniae followed by E. coli and the lowest activity against S. paratyphi and S. typi. The methanol extract of Sepia officinalis showed the maximum inhibition zone against E. coli the maximum inhibition zone against E. coli and minimum inhibition zone was recorded Salmonella paratyphi (Reddy, 2008). The highest activity was exhibited against E. coli and S. typhi by M. casta crude extract of ethanol and Tridacna maxima exhibited highest activity against S. aureus and E. coli (Ramasamy and Balasubramanian, 2012).

Antibacterial activity of common marine molluscs from Parangipettai coast was studied and reported that the methanol extract of molluscs exhibited significant activity against E. coli (Anand et al., 1997). This finding corroborates the results of the present study, since the ethanol extract of A. granosa and M. casta showed pronounced activity against Klebsiella pneumoniae and E. coli. The inhibitory action of the methanol extract of Tonna galea showed pronounced activity against E. coli (Shanthi et al., 2011). Antibacterial activity of opercular extract of Chicoreus rasmosus and Pleuroploca trapezium against six bacterial pathogens was reported (Murugan and Ayyakannu, 1997). The methanolic 103 extract of Chicoreus virginens and C. ramosus experimentally analyzed and observed the broad spectrum antibacterial activity of body tissue extract (Santhana Ramasamy and Murugan, 2005). The antibacterial activity of a marine mollusc, Babylonia spirata was screened against bacterial pathogens (Thilaga, 2005). Similar results were also reported in chloroform extract of Pterai chinensis which inhibited eight fish pathogens and the acetone extract in the same molluscs showed broad spectral activity against all the fish pathogens tested (Chellaram et al., 2004). The antibacterial activities of ethanol extracts of Babylonia spirata and Turbo brunneus was observed maximum activity against K. pneumoniae, E. coli, Proteus vulgaris and Salmonella typhi (Anand et al., 1997). This study corroborates the results of the present investigation. Very similar to this maximum antibacterial activity against Staphylococcus aureus and E. coli on Trochus radiatus was reported (Elezabeth et al., 2003).

The present study revealed that the species of A. granosa and M. casta showed antibacterial activities against the pathogenic bacterial strains. So they possess potential pharmacological action. However, some novel and uncharacterized mechanisms of action that might ultimately benefit the ongoing global search for clinically useful antibacterial agents need to be explored to explain the characteristic of antibacterial activity of A. granosa and M. casta. The present study indicates the whole body extraction of A. granosa and M. casta would be a good source of antibacterial agents and would replace the existing inadequate and cost effective antibiotics. 104

Water, ethanol, methanol and chloroform extracts of bivalves used in the present study showed significant antibacterial activity compared with other solvents. It is worthy to note that the product from natural source is good for health and devoid of side effects. In the present investigation, the whole body tissue extraction showed that the antibacterial activity was subjected to HPLC to estimate the number of bioactive and bioeffective antibiotics. GC-MS and FTIR analysis reveals the bioactive compounds signals at different ranges. The research shows that the medicinal value of the bivalves, A. granosa and M. casta tisues may be due to high quality of antibacterial compounds.

As the bivalves resources are rich and varied in Adirampattinam coastal waters and Muthupet estuary, South east coast of India, there exist a great potential for the extraction of bioactive compounds of medical importance at a lower cost.

5.5. Identification of compounds from bivalves Most of the pathogens are increasingly resistant to the major classes of the routinely used antibiotics. There is an urgent need for the discovery of new and novel antibacterial drugs to effectively combat not only the drugs resistance but also the new disease producers, hence the search for active drugs from alternative sources including marine environment, obviously becomes imperative. Marine invertebrates offer good source of potential antimicrobial drugs (Bansemir et al., 2006; Jayaraj et al., 2008). Among the invertebrates, the molluscs are very good source for biomedical important products (Shenoy, 1998). 105

Many classes of molluscs particularly bivalves exhibit bioactive compounds like anti-tumour, anti-leukemic, antibacterial, cytotoxic, anti-inflammatory, and antiviral properties (Anand et al., 2001; Rajaganapathi et al., 2000). Discovered bioactive compounds in molluscs are identified as specific types of activities (Balcazar et al., 2006; Blunt et al., 2006). Proteins and glycoproteins with antibacterial activity have been demonstrated in the digestive organs of various molluscs (Iguchi et al., 1982; Pakashi, 2001). In the present study, interpretation on mass spectrum GC-MS was conducted using the database National Institute Standard and Technology (NIST) have more than 62,000 patterns. The following compounds were identified from mass spectra analysis.

For the susceptibility of the above mentioned pathogens, the following compounds 7,12-dihydro-6,7-bis (4-hydroxyphenyl-6H-[1,2,4] triazolo [1’,5’:1,2] pyrimido [5,4-c] chromen-2-ol, 2-nonadecanone 2,4-dinitrophenylhydrazine, 3-pyridinecarboxylic-4-oxo-4a, 7a-epoxy-5H- cyclopenta [a] cyclopropa [f] cycloclondencen-11-yl ester and 3-phenyl- 2,3-dihydrobenzo [b] furan-2-ol, phenol, 2,4-bin (1-phenylethyl), 1,2- benzenedicarboxylic acid, diisooctyl ester were identified from GC-MS in M. casta and A. granosa respectively.

In the present study, the magnitude of inhibition of the ethanol extract of A. granosa and M. casta possibly reveal the presence of antibacterial compounds of the four peak fractions, using HPLC, the number of peak fractions were peak 1 and peak 2, K. pneumoniae and 106

E. coli were the most susceptible pathogens in concern with the A. granosa and M. casta ethanol extract.

In the present investigation, the whole body tissue ethanol extraction of both bivalves by using FTIR analysis, the structural compounds were identified. They are methylene C-H asymmetric / symmetric stretch, aliphatic iodo compounds, heterocyclic amine, N-H stretch, secondary amine, N-H Bead, polysulfide, carbonate ion, C-O stretching-polysaccharides, CH asymmetric stretch lipids.

The present study showed that the bivalve A. granosa and M. casta possess the antibacterial compounds acts as the potent inhibitor of pathogens. 6. SUMMARY

The ocean covers about 70 per cent of the Earth’s surface and contains an extraordinary diversity of life. Interest in the understanding of the marine organisms has been accelerated in recent years with growing recognition of their importance in human life. The marine ecosystem remains as an untapped resource for discovery of many drugs. Molluscs are widely distributed throughout the world and have many representatives. Among the molluscs, 15,000 species of bivalves have been reported. The bivalves have been recognized as potential sources of antimicrobial substances and biologically active products. Therefore, a broad screening of bivalves for bioactive compounds is necessary. The screening of bivalves for therapeutic drugs are of greater interest now-a-days. Bivalves provide a high quality proximate compositions and enzymes with all the dietary essential aminoacids for maintenance and growth of the human body. The basic and fundamental requirement for initiating bivalves is first to enumerate the available bivalves. Particularly, Anadara granosa and Meretrix casta from coastal waters of marine environment and estuary of South East Coast of India, assume much significance. For instance, a strain/species, which is highly seasonal and restricted in its distribution may pose problems, if round the year abundance and cultivation is attempted, while a strain that is both versatile as well as constant in its distribution may be more easily amenable to round the year cultivation. Therefore, the present investigation was carried out to determine the seasonal variations in physico-chemical parameters 108 and distribution of bivalves, A. granosa and M. casta from Muthupet estuary environ and coastal waters of Adirampattinam, South East Coast of India, for a period of one year (from January 2011 to December 2011).

For this study, two stations were chosen, one is the Adirampattinam coastal environ (Station I) and another from Muthupet estuary environ (Station II).

Station I – The marine coastal zone is situated at light house on the eastern side of Adirampattinam coast and Station II – the mangrove zone is situated at a lagoon formed by the rivers of Paminiyar, Koraiyar, Kidaithankiyar, Marakkakoriar and other tributaries of the river Cauvery flowing through Muthupet and other adjacent villages. This study mainly focuses attention on the influence of meterological and physico-chemical factors on seasonal distribution and abundance of bivalves, M. casta and A. granosa from coastal waters of Adirampattinam and Muthupet estuary environ, respectively. In addition, laboratory studies were carried out to assess the nutritional status and structural identification of bioactive compounds from bivalves using GC-MS, HPLC and FTIR analysis and also to test the antibacterial activity of both bivalves against five pathogenic bacteria. From these studies, the following significant findings were made and given below:

 The physico-chemical parameters of the study areas varied not only between Station I and Station II but also during the study period around the year. 109

 The rainfall varied from 4 to 96.2 mm during the study period. The maximum rainfall (96.2 mm) was recorded during in August 2011 and the minimum (4 mm) during premonsoon in July 2011 at Station I. In Station II, the maximum (64 mm) was recorded during monsoon in November 2011 and the minimum (7.2 mm) was recorded during postmonsoon in January 2011.

 The humidity of the study areas showed fluctuation between seasons. It ranged from 64 to 90% in station I whereas in station II it was 64 to 86%.

 The difference in the atmospheric temperature was found to be higher than the surface water temperature. The atmospheric temperature ranged from 26 to 34°C during the study period at both stations. The minimum air temperature (26°C) was recorded during monsoon in December 2011 and maximum of 32°C during summer in May and premonsoon in June and July 2011 at Station I. In station II, minimum of 27°C was recorded during December 2011 and maximum of 34°C was recorded during June 2011.

 The surface water temperature ranged from 27 to 31°C at station I whereas in station II ranged from 24 to 32°C, during the study period. The minimum (26°C) was recorded during monsoon in November 2011 and maximum (31°C) was recorded during premonsoon in July 2011. In station II, the minimum (24°C) was recorded during monsoon in October 2011 and maximum (32°C) was recorded during summer in May and June 2011.

 The photoperiod showed slight variations during the study period. It was found to be high (12.51 hrs) in September 2011 and low 110

(11.25 hrs) in March 2011 at station I whereas high (12.56 hrs) in August 2011 and low (11.14 hrs) in March 2011 at Station II.

 pH of the water from both stations showed acidity, neutral to alkaline and alkaline ranges throughout the study period. It ranged from 7.0 to 8.5 during the study period at both stations.

 At station I, the minimum value (7) was recorded during the months of January, February, September, November and December 2011. At station II, the minimum pH (7) was recorded during the months of February and November 2011. The maximum value (8.5) was recorded during summer in May 2011 at station I and 7.5 was recorded during May, June and July 2011 at station II.

 The low pH, salinity and temperature during monsoon were found to associate with low density of bivalves distribution and abundance. Higher pH, salinity and temperature during summer season coincided with high density of bivalves distribution and abundance at both stations.

 The salinity of water showed wide fluctuations at both stations. It ranged from 11 to 36 ppt during the study period. Minimum salinity was recorded as 24 ppt during April 2011 and maximum 36 ppt in June 2011 at station I. In station II, it ranged from 11 to 29 ppt during the study period. The minimum salinity was recorded during monsoon season and the maximum was recorded during summer and post-monsoon seasons could have favoured the abundance of bivalves at both stations. 111

 During the study period, Muthupet estuary was found to possess maximum number of A. granosa when compared to coastal water zone which possesses lower number of bivalve, M. casta.

 The population density, biomass and distribution of bivalves were recorded during post-monsoon and summer. The maximum abundance occurs throughout the season in both stations. This could be due to abundant terrigenous deposits and lot of nutrients as land-run-off from monsoonal rain.

 The dissolved oxygen (DO) showed significant fluctuation at both stations. DO varied from 1.4 to 4.6 mgl-1 during the study period at both stations. The minimum concentration was 2.2 mgl-1 during June 2011 and maximum concentration was 4.5 mgl-1 during August and October 2011 at station I. DO content was recorded maximum in post monsoon and the lowest during monsoon was recorded at station II. The minimum concentration was recorded as 1.4 mgl-1 during April 2011 and maximum 4.6 mgl-1 was recorded during October 2011 at station II.

 The inorganic phosphates, nitrates, nitrites, reactive silicates and ammonia showed much fluctuations during the study period at both stations. Nutrients are abundant during monsoon at both stations due to monsoonal flow of freshwater and land-run-off.

 The total phosphorus concentration varied between 0.2 and 5.5 mgl-1 during the study period. The minimum was recorded as 0.2 mgl-1 during May 2011 and maximum was recorded as 5.2 mgl-1 during December 2011 at station I. In station II, the minimum was 112

observed as 0.89 mgl-1 during April and June 2011 and maximum concentration was recorded as 5.5 mgl-1 during December 2011.

 The nitrate, nitrite and ammonia concentrations ranged from 2.5 to

9.5 ml-1, 0.64 to 2.85 ml-1 and 0.46 to 1.92 ml-1 respectively

during the study period at both stations.

 The reactive silicates ranged from 2.15 to 6.24 ml-1 during the

study period at both stations.

 The nutritional quality parameters of bivalves, A. granosa and M. casta (proximate composition of protein, carbohydrate and lipid) were determined at monthly intervals of the different seasons of the year, January 2011 to December 2011.

 Seasonal variations on the nutrient content of the whole body tissue were observed with particular regard to protein 50.45% (October 2011) to 68.32% (June 2011), carbohydrate 4.51% (August 2011) to 7.73% (May 2011), lipid 3.27% (August 2011) to 6.05% (April 2011), the enzyme lipase 0.06% in gut (January 2011) to 0.21% (June 2011) and protease 0.07% in gut (January 2011) to 0.22% (July 2011). In A. granosa whereas in M. casta, protein 48.4% (October 2011) to 66.02% (June 2011), carbohydrate 2.5% (August 2011) to 5.6% (April 2011), lipid 1.6% (September 2011) to 3.8% (June 2011), lipase 0.08% in gut (January 2011) to 0.22% in gut (July 2011), protease 0.08% (December 2011) to 0.22% (July 2011).

 The maximum average percentage of protein, carbohydrate and lipid were observed in the whole body tissues of A. granosa as 55.68, 6.16 and 4.71% respectively. Whereas in M. casta, 56.29% 113

of protein, 4.10% of carbohydrate and 3.39% of lipid was recorded. In A. granosa, the enzymes protease and lipase have maximum value in digestive gland such as 0.22 (July 2011) and 0.24 (July 2011) in digestive gland respectively. In M. casta, the protease and lipase have maximum value in digestive gland such as 0.24 (July 2011) and 0.22 (July 2011) in gut respectively.

 In spite of this variability, the nutritional quality of the A. granosa was generally good, especially during premonsoon season, when the concentrations of nutrients is at its maximum. A low level of fat was detected in both bivalves.

 The antibacterial activity of both bivalves were tested against five pathogenic bacteria.

 The nutrient agar plates were incubated for 24-48 hrs and observed for clear zone of inhibition and was measured in mm.

 The ethanolic whole body tissue extracts of both bivalves exhibited maximum antibacterial activity against Klebsiella pneumoniae followed by E. coli with inhibition zone of 9 and 7.5 mm respectively, this is nearing the inhibition zone value of 14 mm produced by the standard antibiotic drug, Ciprofloxacin. Therefore, the ethanolic extracts of both the bivalves were potent to be the source of antibacterial compounds.

 The ethanolic tissue extracts of both bivalves exhibited the minimum antibacterial activity against Salmonella paratyphi and S. typhi, with inhibition zone of 8 mm and 6 mm respectively.

 The structural chemical bioactive compounds of both bivalves, A. granosa and M. casta were analysed using GC-MS, HPLC and 114

FTIR analysis. The antibacterial compounds identified were phenolics, alkaloids and sterols.

 In GC-MS analysis, the ethanol extract of A. granosa showed 12 prominent peaks with retention time 13.91, 16.60, 17.29, 17.65, 19.75, 20.42, 20.79, 24.33, 24.54, 24.86, 25.54 and 26.20 mins. The structural compounds were identified as: 4,7,10,13,16,19- Docosahexaenoic acid, 3-phenyl-2, 3-dihydrobenzo(b)furan-2-ol, n-Hexadecanoic acid, Hexadecanoic acid, phenol, 2,4-bis (1-phenylethyl) and 1,2-benzenedicarboxylic acid.

 The ethanol extract of M. casta showed 4 prominent peaks with retention time 14.13, 17.92, 21.81 and 28.03 mins. The structural compounds were identified as: 7, 12-dihydro-6, 7-bis (hydroxyphenyl)-6H-[1,2,4] triazoio [1’,5’:1, 2] pyrimido [5,4-c] chromen-2-ol, 2-naxadecanone 2,4-dinitrophenylhydrazine, 3- pyridinecarboxylic acid, 2,7, 10-tris (acetyloxy)-1, 1a, 2, 3, 4, 6, 7, 10, 11, 11a-decahydro-1, 1, 3, 6, 9-pentamethyl-4-oxo-4a, 7a- epoxy-5H-cyclopental [a] cyclopropal [f] cycloundecen-11-yl ester, 9, 12, 15-octadecatrienoic acid, 2-1-ethyl ester.

 In HPLC analysis, the ethanol extract of both M. casta and A. granosa showed two major peaks. The major peak showed retention time as 3.050 and 3.508 mins for A. granosa and 3.208 and 3.783 mins for M. casta.

 Among the four fractions in A. granosa, and M. casta, the peak 2 fraction showed highest antibacterial activity against K. pneumoniae followed by E. coli. 115

 In FTIR analysis, the ethanol extract of A. granosa showed 8 major peaks with retention time and the presence of the compound, heterocyclicamine, methylene C-H and secondary amine were identified.

 In M. casta, the FTIR showed the presence of heterocyclimine, C-H asymmetric stretch lipids and carbonate ion were identified.

CONCLUSIONS This study is concluded with the observations, that the marine and estuary environs are rich in bivalves distribution and the bivalves, A. granosa and M. casta constitute an important component of the marine and estuary environment. Therefore, it is necessary to conserve the marine bivalves for enrichment of diversity in general and the bivalves, A. granosa and M. casta in particular. The bivalves are of significance in the marine and estuary ecology as well as nutritional value and marine biotechnology as a source of high value bioactive products like antibacterial activity, and medicinal importance. The whole body tissue extraction of A. granosa and M. casta would be a good source of antibacterial agents and would replace the existing inadequate and cost effective antibiotics. The proximate compositions of marine bivalves is a nutritional assurance to millions of malnourished people. As the bivalves resources are rich and varied in South East Coast of India; there exists a great potential for the extraction of bioactive compounds of medicinal importance at a lower cost.