1. GENERAL INTRODUCTION 1.1. Introduction the Brachyuran Crabs

1. GENERAL INTRODUCTION

1.1. Introduction The brachyuran crabs are most fascinating organisms among decapod crustaceans. They have broad hard carapace, massive chelate legs, bend abdomen and exhibit high degrees of adaptation to the environment. They show pelagic, benthic, intertidal, burrowing and terrestrial modes of life. These crabs found distributed in deep oceans as well as in shallow brackish backwater, estuaries and tidal pools and fresh water bodies. Decapod crustaceans are very common invertebrates inhabiting the marine environment. The diversity of the brachyuran crabs were abundant in littoral and deep sea regions (Kathirvel, 2008a and b). The life history, nutritive value, migration, feeding, growth and maturation, physiology and breeding behaviour of decapod crustaceans are of great interest to biologists.

In tropical water several types of breeding patterns exhibit in crustaceans. They are continuous breeders breed around the year, discontinuous breeders in relation to lunar phases, biannual breeders have two spawning periods in a year and annual breeders have one single breeding season in a year. The breeding pattern of the crabs of the same habitat varies in the season and may be discontinuous or biannual or annual breeders (Boolootian et al., 1959). The crabs belonging to the same species inhabiting different habitats have different types of breeding cycle (Rahaman, 1967). In crabs, the reproductive organs play an important role 2 in the synthesis and storage of organic materials needed for the production of gametes (Boolootian, 1965).

The “reproductive cycle” includes the series of events from the time of activation, growth and gametogenesis in the gonad to spawning of the gametes and recession of gonadal activity to a relatively substained resting level, until the gonad is once again activated. In continuous breeders the duration of reproductive cycle takes a period of few weeks or several months, and in annual breeders the reproductive cycle occurs during the whole period of the year (Giese, 1959).

The hepatopancreas in crustaceans is otherwise called as midgut gland, liver, digestive gland, hepatic caecum and hepatopancreatic caecum (Van Weel, 1970). The accumulation of food materials takes place in the hepatopancreas. This involves taking food from the environment and absorbing them in it. During the reproductive cycle, changes are seen in the accumulation of food materials in the storage organ, hepatopancreas. The series of changes which have taken place in the hepatopancreas is known as nutritive cycle and it can be correlated with reproductive cycle.

Reproductive and nutritional cycles, sex ratio and fecundity of crustaceans have been reported by many workers (Rahaman, 1967; Chandran, 1968; Diwan and Nagabhushnam, 1974). In decapod crustaceans, the ovaries are located either dorsally or dorsolaterally to the gut. The paired ovary is distinctly connected by a central bridge of ovarian 3 tissue assuming the shape of the letter „H‟ (Ryan, 1967; Ezhilarasi, 1978). The spermatheca looks like an inverted conical flask and is attached to the ovary at its mid portion of the posterior arm on each side through small duct. The maturation of ovaries has been classified into various stages. The colour of the ovary has been taken as a main criterion for classification. The ovary has been divided into four stages (Radhakrishnan, 1979) five stages (Rajendran, 1990) six stages (Ezhilarasi, 1978) and seven stages (Du Preez and Mclachlan, 1984b). The testes are paired organs which are also medially interconnected by a commissure so as to give the shape of the letter „H‟. The testis leads into long duct known as vasdeferens which can be divided into three functional regions namely anterior vasdeferens, mid vasdeferens and posterior vasdeferens (Joshi and Khanna, 1982)

The nutritional and reproductive cycle of decapod crustaceans show significant relationship. A clear cut inverse relationship is exhibited by the hepatic index to that of the gonadal index. A drop in the hepatic index, coinciding with or just preceding the rise of gonad index, suggests the possible transport of nutrients from hepatopancreas to gonad during maturation of gametes (Chandran et al., 1980; Rajendran, 1990).

In crabs, a slight deviation in the sex ratio may be due to migration of ovigerous females from deep water to offshore region during intensive breeding periods. It is a factors that the female decapod crustaceans known to produce more eggs as they grow larger. The total numbers of 4 eggs increase as length or width of carapace of the crab increases (Wenner et al., 1987).

The reproductive cycle of marine invertebrates seems to be influenced by exogenous and endogenous factors (Giese, 1959). Among the exogenous factors, the habitat is one of the important factors that play a major role in the breeding behaviour of marine organisms (Prasad and Tampi, 1953). The latitude is another factor which influences the reproductive cycle of tropical species (Pearse, 1965). The impact of temperature on the reproductive cycle was reported by Giese (1959). Orton (1920) stating that the marine animals of temperate and cold waters reproduce seasonally while the animals of tropical waters breed throughout the year. The changes in pH, salinity and DO of water influence the breeding of marine animals (Pillai and Nair, 1971; Joel and Raj, 1980; Rajendran, 1990). The rainfall, photoperiod and nutrients of water also influence the breeding activity of animals (Subramanian, 1963; Nagabhushnam and Faroogii, 1981).

The biochemical changes in relation to reproductive and nutritional cycles of crustaceans have been reported by many workers (Adiyodi, 1969; Adiyodi and Adiyodi, 1972; Rahaman, 1977). The fluctuation of biochemical components in gonad, hepatopancreas, muscle and heamolymph during the ovarian maturation strongly suggests their possible utilization during vitellogenesis (Kerr, 1969). The fact that the occurrence of very little biochemical reserves in the gonad prior to the 5 development of gametes formation suggests that there is the mobilization of organic material from the hepatopancreas to gonad. Further the fluctuation in biochemical component of the hepatopancras during the maturation of the gonad, strongly suggests the possible utilization of resources from the hepatopancreas (Pillai and Nair, 1973b). The biochemical component lipid and carbohydrate may be transferred from the hepatopancreas to the gonad as the animal matures but protein may be synthesized in the gonad (Rahaman, 1977). The nature of protein, sub units of protein with molecular weight and fatty acid composition varied during reproductive cycle (Wolin et al., 1973; Hoq et al., 2003).

The haemolymph proteins of marine invertebrates are unique in composition, as they do not contain immunoglobulin or albumin like protein. Studies on the biochemical composition and enzymes in the haemolymph of crab during moulting cycle have been undertaken by many workers (Adiyodi, 1969; Bedford, 1972; Kulkarni, 1983). Periodic shedding of the exoskeleton in crustaceans is called moulting. Life of crab consists of alternating periods of premoult, moult, post moult and intermoult. The crustaceans never achieve stability, moulting occurs at intervals throughout adult life. The periods between moults are known as molts. Changes occur in the size and biochemical composition of crab throughout the moulting cycle (Heath and Barnes, 1970).

The disease is mainly caused by microbes. Micro organisms are living things, larger in number, extreme diverse groups, lumped together 6 on the basis of one property. They are so small that they cannot been seen without the use of a microscope. It includes viruses, bacteria, fungi, protozoa and some algae. They are present everywhere on earth which includes biota, soil, water and atmosphere.

Fungi are filametous, non-photosynthetic eukaryotic microorganisms that have a heterotrophic nutrition. They are two types of heterotrophic fungi namely saprophytes and parasites. Saprophytes depend on dead organic matter whereas parasites live on or in the living bodies of other organism. In fungi reproduction is of three kinds, vegetative, asexual and sexual.

Bacteria are the exceedingly small unicellular prokaryotic cells whose single chromosome is not contained within a nuclear membrane. They live in the bodies of other organisms and their dead remains. The existences of microbes in various tissues of marine crabs have been reported by many workers (Childers et al., 1996; Leano, 2002; Kannathasan and Rajendran, 2010a; Najiah et al., 2010). Though works are available on reproductive and nutritional cycles of marine crustaceans, no work has been carried out on the economically important edible crabs in Nagapattinam coast. Hence the present investigation is aimed to study the physic-chemical parameters, survey, reproductive and nutritional cycles of marine crab Charbdis natator.

1.2. Objective of the study The present study has been planned with the following objects. ♦ To study the physico chemical characteristrics of marine environment of Nagapattinam coast. ♦ To study the biodiversity of brachyuran crabs collected from off shore, littoral and tidal zone in the study area. ♦ To isolate genomic DNA from tissues of Charybdis natator and to amplify the DNA by using the polymerase chain reaction (PCR). ♦ To analyse the 16S rRNA, 18S rRNA and COI gene sequences using BLAST. ♦ To construct the phylogenetic tree using MEGA-4 and find out the phylogeny of the crab. ♦ To study the morphometric characteristics of male and female crabs. ♦ To determine nutritional, reproductive and moulting cycles. ♦ To study the effect of physico chemical factors on reproductive and nutrition cycles. ♦ To determine the sex ratio and fecundity of the crab. ♦ To estimate the biochemical composition and enzymes of gonad, hepatopancreas and haemolymph related to reproductive and moulting cycles. ♦ To determine the protein profile by using SDS PAGE ♦ To characterize the organic and inorganic compounds of gonad and hepatopancreas by GC-MS. 8

♦ To isolate the microbial population in the various tissues of crab Charybdis natator.

1.3. Scope of the present study Decapod crustaceans are mostly marine, but many can tolerate brackish water and some are fresh water dwellers. These are found distributed in pelagic and benthic region of the oceans as well as in shallow brackish backwaters, estuaries and tidal pools. They rest and move on the substratum of the variety of riches ranging from the wave – beaten intertidal surface zone to virtually tideless intertidal areas of mangrove swamp, but swim temporarily for food capture or breeding. Crustaceans are of great use to man either directly or indirectly for his health and economic progress. Crustaceans, especially prawns, crabs, lobsters and shrimps are the important diet of man, with a great nutritive value. Fishes the main source of protein to man, in turn depend on crustaceans, for their food. They keep up the nutritional balance of fish life. Without them fishes would have perished.

The intertidal brachyuran crabs belong to the family of Grapsidae and Ocypodidae, which are commercially not important. However they play a dominant role in the marine foodweb. Most of the members of the above two families are either scavengers or herbivorous. Their role in recycling the nutrients and enhancing the rate of plant and animals are considerable. The burrowing members of these crabs also influence the soil conditions by digging numerous deeper burrows, which are often 9 more than half a meter in depth in the sand, as well as in muddy shores. These burrowers make the soil more aerated which obviously promotes the growth of seedling in the shore region. Moreover bottom feeding fishes like eels consume these intertidal crabs during high tides. During low tides when the beach is exposed they are fed upon by the aquatic birds.

Crabs, shrimps, prawns and lobsters are exported to foreign country in thousands of tones fetching crores of rupees worth of foreign exchange. In recent times the mechanized advancement leads to the bumper cropping of crustaceans both for export and local needs. There is a greater demand for the brachyuran crabs as it becomes a rich source of proteinous food. So possible ways to increase the population of crab species have to be explored either though culture or by stopping fishing activity during peak breeding seasons of crabs to increase population.

1.4. Description of the study area Brachyuran crabs used in the present study were collected from the offshore region of Nagapattinam which is located at Lat. 10046‟ NS and Long. 79051‟ EW on the mouth of river Kaduvayar, the branch river Cauvery in the south east coast of India. The Cauvery is one of the major perennial rivers in peninsular India which originate at Coorg district in Karnataka state and enters into south east coast of Bay of Bengal at Nagapattinam district. It travels about 750 km and carries a large amount of nutrients to the sea which probably promotes the species richness both 10 racially and individually. The Nagapattinam port is situated on the western bank of Kaduvayar which flows parallel to the coast in the northern direction for about 0.8km and debouches into the sea. This stretch is protected from the sea by narrow sand spit. There is a bar at the mouth of Kaduvayar having a depth of about 1.0m low tide (Fig.1 and Plate-1).

1.5. Fishery information Nagapattinam base serves twenty two fishing villages. Ten fishing villages are regularly landing their boats here. They are Seruthur, Vellankkani, Kallaru, Akkarapettai, Kichankuppam, Ariyanattutheru, Nambiar Nager, Samandampettai, Nagur and Santharapadi. While the mechanized fishing boats of these ten fishing villages of Nagapattinam move in and out Though the Kaduvayar river mouth and land their catch at Akkaraipettai where a small landing shed has been constructed by the Government of Tamil Nadu for the benefits of the fishery folk, those of the catamarans and other smaller crafts are landed on the shore of the respective villages and the crafts pulled out on the shore when not in use. There are about 845 mechanized fishing boats of 8 m and above in length fitted with an engine of 15 hp and above which operate on alternate days from Nagapattinam base and return from sea on the next day with a catch. During season, boats from other places land their catch in Nagapattinam base when the number of boats operated may swell to 1500 or ever more. Trawls, Gill nets and Long line are the gears used in these boats which are operated from 30-300 m depth. By a rough estimation it has been 11 calculated that about 50,000 tonnes of fish are landed by these mechanized fishing boats at Nagapattinam and marketed every year.

The traditional wooden catamarans of 3 or 4m long with or without Out Board Motors (OBM) numbering about 300 operate from these ten fishing villages which land about 18,000 tonnes of fish annually. In Nagapattinam marine, fish, crab and shrimp landed in the coast by capture fishing from the time immemorial.

1.6. Gear used for collection According to Prasad and Tampi (1953) different gears are used for the catching of crabs. Although different gears are used for fishing at Nagapattinam, the gears that are employeed in the present study are known as „trawl nets‟, „Sangu valai‟, „Arai valai‟, „Ponna number one valai‟, „Onney kal valai‟, „Ondarai valai‟, „Kattamati valai‟ and „Redaimati valai‟. These gears are used in mechanized fishing boats. In Fiber Reinforced Plastic (FRP) boats „Vala valai‟, „Disco valai‟, „Methappu valai‟, „Panija valai‟, „Eluvai valai‟, „Paddu valai ondarai‟, „Kavala valai‟, „Salangai valai‟, „Kola valai‟, „Surukku valai‟, „Pacha valai‟, „Nandu valai‟, „Beese valai‟, „Kala valai‟ and „Kola valai‟ are used. These gears are useful to catch the crab fisheries in the sandy region of the Nagapattinam coast.

2. ECOLOGY

2.1. Introduction The physico chemical parameters have an impinging effect on the aquatic organisms many physico chemical parameters vary 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 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 physiology 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, 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, 2010a; Santhosh Kumar and Perumal, 2011).

The physico chemical parameters of Bay of Bengal, India were influenced by monsoon rains. A comparative study of physico chemical 13 characteristic has been reported by many workers (Panikkar and Jayaraman, 1966; Sampathkumar, 1992; Soundaramanickam et al., 2008; Dronamraju et al., 2008; Mohamed Abubaker Sithik et al., 2009).

The temperature is the most important exogenous factor for the regulation of growth, metabolism and breeding of marine organisms. The factors other than temperature such as rainfall, humidity, salinity, pH, turbidity, DO, CO2, phosphate, nitrate, nitrite and photoperiod have a remarkable influence on the nutritional and reproductive cycle of marine crustaceans. The role of temperature and salinity on the breeding of animals have been extensively studied by earlier workers (Rahaman, 1967; Rangarajan and Marichamy, 1972; Raghavan and Antony Fernando, 1988; Rajendran, 1990; Rajaram et al., 2005; Soundarapandian et al., 2009; Azhagar et al., 2009).

Considerable informatios on the physico chemical parameters of the Bay of Bengal are available. However very few workers are available on the influence of rainfall, temperature, photoperiod, salinity, pH, DO,

CO2, phosphate, nitrate and nitrite on nutitional and reproductive cycle of marine crustaceans in the south east coast of India. Hence the present investigation is aimed to study the physico chemical parameters of the Nagapattinam coast at Bay of Bengal, India and find the pattern of breeding, sex ratio and fecundity of marine crab, Charybdis natator habitated in this environment.

2.2. Materials and methods Monthly seawater sample were collected in clean polythene containers, The sample was made from 8 a.m to 9 a.m. during low tide for a period of two year from January 2009 to December 2010. Samples were brought to the field laboratory and the different physico chemical characteristics of seawater were recorded using standard methods. Temperature was recorded using mercury filled Celsius thermometer at the time of sampling. The pH of the water was measured with help of Elico-model Lt- 10T pH meter. Salinity was recorded using Salinometer (Refractometer made in Japan). Turbidity of water is measured by using turbidity meter. The dissolved oxygen content of surface water was estimated by the modified Winkler‟s method (Winkler, 1888), CO2, inorganic phosphate and nitrite were estimated following the methods given by Strickland and Parson (1972). Nitrate was estimated in the method of Mullin and Reley (1955). Data on rainfall, atmospheric temperature, humidity and photoperiod pertaining to the site of collection Nagapattinam coast were obtained from the Meterological department of Chennai and Pune. The data were statistically analysed and presented.

2.3. Result The results of the present study are summarized in Table-1 in the data clearly indicates that there was a significant variation in physico chemical parameters during the study period. The variations in physico chemical parameters of Nagapattinam coastal water mainly depend on 15 monsoon rain and freshwater inflow. The fluctuation of these factors influence the reproductive and nutritional cycle of crabs.

2.3.1. Rainfall Based on the meterological data of the study area the entire year can be divided into four seasons namely post monsoon (January to March), summer (April to June), pre monsoon (July to September) and monsoon (October to December). The rainfall data at Nagapattinam coastal area showed a significant fluctuation during the study period. Monthly average rainfall ranged from 0.6 mm to 743.7 mm. There was no rainfall recorded during February and July in the year 2009 and February and April in 2010. During the study period of two years the maximum rainfall (743.7 mm) was recorded during November 2009 and minimum rainfall was recorded (0.6 mm) in March 2010. In both, the years havy rainfall experienced during north east monsoon season (Fig. 2).

2.3.2. Humidity The seasonal variation in humidity showed significant fluctuation. It varied from 66 to 92 per cent in the year 2009 and 73 to 93 per cent in the year 2010. It was found to be low (66%) in June 2009 and high (92%) in November 2009. The maximum humidity value (93%) was recorded in November 2010 and minimum (73%) in July 2010. In the present study, there was an indirect relationship noticed between temperature and humidity. In summer months temperature was found to be maximum when humidity was low and in monsoon it was reverse (Fig. 3). 16

2.3.3. Photoperiods The monthly mean day lengths in the present study showed a slight variation. Eventhough India is a tropical country, the day length showed fluctuation. It varied from 11.23 hours to 12.54 hours. It was found to be low (11.23 hrs) in December 2009 and high (12.53 hrs) in June 2009. Further, it was found to be maximum (12.54 hrs) in June 2009 and minimum (11.23 hrs) in December 2009 and 2010. The day length was found to be low during monsoon and post monsoon seasons and high during summer and pre monsoon seasons (Fig. 4).

2.3.4. Atmospheric and water temperature The monthly average of both atmospheric and water temperature showed significant fluctuation during the study period. The atmospheric temperature varied from 28.4 to 37.8°C in the year 2009 and 28.5 to 35.8°C in 2010. It was found to be the maximum (37.8°C) in June 2009 and minimum (28.4°C) in December 2009. The maximum atmospheric temperature (35.8°C) in June 2010 and minimum (28.5°C) in December 2010 was recorded. The data clearly indicates that there was a seasonal fluctuation noticed in the atmospheric temperature.

The water temperature ranged from 25.3 to 32.4°C in 2009 and 26.6 to 32.3°C in 2010. The maximum water temperature (32.4 and 32.3°C) was recorded in May 2009 and June 2010 and minimum (25.3 and 26.6°C) in December of both the years. There was a close relationship noticed between atmospheric and water temperature. The range of 17 atmospheric temperature was found to be always higher than water temperature (Fig. 5).

2.3.5. Turbidity The turbidity of water varied from 3.9 to 5.9 NTU in 2009 and from 3.6 to 5.8 NTU in 2010. The turbidity values were found to be minimum (3.9 NTU) in May 2009 and maximum (5.9 NTU) in August 2009. The highest value was recorded (5.8 NTU) in August 2010 and the lowest value (3.6 NTU) in June 2010 (Fig. 6).

2.3.6. Hydrogen ion concentration (pH) The pH of water showed alkaline range throughout the study period and fluctuated slightly. It varied from 7.34 to 8.60. The pH values were found to be low (7.34) in December and high (8.52) in June 2009. It was found to be the minimum (7.40) in December 2010 and the maximum (8.60) in April 2010. In both the years, minimum pH value was recorded during monsoon periods and maximum in summer season (Fig. 7).

2.3.7. Salinity The salinity of water showed wide fluctuation between seasons. It varied from 27.42 (November) to 36.00 ppt (June) with an average of 28.76 ppt in 2009. The minimum (27.42 ppt) salinity was recorded during monsoon and it slightly increased during post monsoon and reached its maximum (36.00 ppt) during summer. The lower value (26.98 ppt) was recorded in December 2010 and higher (36.2 ppt) in April 2010 (Fig. 8). 18

2.3.8. Dissolved oxygen (DO) The dissolved oxygen content of sea water showed a slight fluctuation. It varied from 4.8 to 6.10mg/1 in 2009 and from 4.0 to 6.9 mg/l in 2010. The DO values were found to be high (6.10 mg/l) in May 2009 and low (4.8 mg/l) in August 2009. The minimum value (4.0 mg/l) was recorded in September 2010 and maximum value (6.9 mg/l) in June 2010. The values of dissolved oxygen level fluctuated irrespective of seasons (Fig. 9).

2.3.9. Carbon-di-oxide (CO2)

The CO2 content of seawater showed a slight fluctuation. It varied from 3.41 to 5.9 mg/l in 2009 and from 3.55 to 5.5 mg/l in 2010. The CO2 values were found to be high (5.9 mg/l) in November and low (3.41 mg/l) in February 2009. The minimum value (3.55 mg/l) was recorded in March and maximum value (5.5 mg/l) in December 2010. In both the years the maximum CO2 value were recorded during monsoon seasons and minimum during post monsoon seasons (Fig. 9).

2.3.10. Phosphate (PO4) The seasonal fluctuation of phosphate showed a slight fluctuation.

It ranged from 1.41 to 3.12 g/ml in 2009 and from 1.45 to 3.8 1 g/ml in

2010. The PO4 value was found to be maximum (3.12 g/ml) in October and minimum (1.41 g/ml) in May 2009. The higher value (3.8 1 g/ml) was recorded in November and lower (1.45 g/ml) in June 2010 (Fig. 10).

2.3.11. Nitrate (NO3) The nitrate of water showed variation from 0.49 to 0.96 g/ml in

2009 and from 0.45 to 0.98 g/ml in 2010. The NO3 values were found to be high (0.96 g/ml) in November and low (0.49 g/ml) in June 2009.

The minimum value (0.45 g/ml) was recorded in May and maximum value (0.98 g/ml) in October 2010. The minimum nitrate value was recorded during summer season and maximum during monsoon season 2009 and 2010 (Fig. 10).

2.3.12. Nitrite (NO2) The nitrite of water ranged from 0.50 to 1.10 g/ml in 2009 and from 0.49 to 1.16 g/ml in 2010. The minimum value (0.50 g/ml) was estimated in May and maximum value (1.10 g/ml) in November 2009.

The higher value (1.16 g/ml) was recorded in October and lower value

(0.49 g/ml) in April 2010. The minimum nitrate value was recorded during summer and maximum during monsoon season in the years in 2009 and 2010 (Fig. 10).

2.4. Discussion Seasonal variations in environmental factors at Nagapattinam coast is mainly controlled by the spectacular regime of heavy rainfall during north east monsoon. The entire year can be divided into four distinct seasons namely post monsoon, summer, pre monsoon and monsoon. The study area experienced heavy rainfall during the monsoon season from October to December resulting in the lowering of temperature, salinity and 20 pH. The minimum rainfall was recorded during the summer months when the temperature was maximum and humidity minimum. Similar observations were reported by earlier workers (Somayajulu et al., 1987; Perumal, 1993; Sundaramanickam et al., 2008).

According to Subramanian and Mahadevan (1999) all the physico chemical parameters of Bay of Bengal were influenced by monsoon rain. The heavy rainfall was recorded during the north-east monsoon season and low rainfall during summer season (Perumal, 1993; 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, 2010a; Sankar et al., 2010). The rainfall had a remarkable influence on the reproductive cycle of marine organisms.

Temperature is considered as an important factor which influenced the growth, metabolism and reproductive activity of marine organism (Rahaman, 1967; Rajendran, 1990). In the present study 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 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 months when the rainfall and humidity was maximum. Similar observations were 21 reported by earlier workers (Rao et al., 1982; Soundarapandian et al., 2009).

According to Panikkar and Jayaraman (1966) 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 sea 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 (Raghavan and Antony Fernando, 1988; Dronamraju et al., 2008).

According to Kannan and Kannan (1996) 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 (Mohamed Abubaker 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.

The salinity of sea water is another important abiotic factor having a remarkable influence on the reproductive and nutritional cycles of 22 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 fresh water 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). 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).

In the present study, the seasonal average salinity registered high during summer and pre monsoon and low during monsoon and post monsoon period is in confirmation with the earlier report from Bay of Bengal (Soundaramanickam et al., 2008; Azhagar et al., 2009; Santhosh Kumar and Perumal, 2011). The average salinity values of the water varied from 30 to 33ppt and it showed a wide fluctuation of recording from 27 to 18ppt 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 33ppt respectively (Rajalakshmi Bhanu et al., 1981). The salinity varied from 33.9 to 34.8ppt at the off shore surface water and increased with depth (Rao et al., 1982).

The salinity of the sea at Port Novo coast varied between 26.5 and 34.5ppt and it increased during post monsoon and summer and began to decline during pre monsoon and monsoon month (Raghavan and Antony Fernando, 1988 and 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, 2010a).

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). 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 dissolved oxygen is one of the important biological factors which influence the bio-energetics of the aquatic organisms. In the present 24 study, dissolved oxygen level fluctuated irrespective of seasons, which may be due to wind velocity, rainfall and photosynthetic activities of phytoplankton. The DO level estimated in the present study agrees with earlier workers (Sridhar et al., 2006; Satpathy et al., 2007; Soundarapandian et al., 2009; Damotharan et al., 2010).

The CO2 is an important abiotic factor of aquatic ecosystem which influence the growth, metabolism and feeding. The CO2 contributors to the fitness of natural waters are derived from various sources such as atmosphere, respiration by organisms, bacteria decomposition of organic matter etc. In the present study, CO2 level showed slight seasonal fluctuation which agrees with earlier workers (Unni, 1972; Dronamraju et al., 2008). According to Unni (1972) the rate of changes in free CO2 concentration is considerably high due to decomposition of organic matter at the bottom. The maximum CO2 level was during monsoon season and the minimum during post monsoon season (Dronamraju et al., 2008).

In the present study, a slight seasonal fluctuation observed in phosphate level agrees with earlier workers (Sampathkumar, 1992; Kannathasan and Rajendran, 2010a; Sankar et al., 2010). 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 25 upwelling sea water into the creek, which in turn increased the level of phosphate (Nair et al., 1984).

In the present study, slight seasonal variation in nitrate content was observed agrees with earlier workers (Satpathy, 1996; Ashok Prabu et al., 2005; Mohamed Abubaker Sithik et al., 2009; Damotharan et al., 2010). The increase nitrates level in the sea was due to freshwater inflow and terrestrial run off during the monsoon season (Santhosh Kumar and Perumal, 2011). According to Rajaram et al., (2005) 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.

The nitrite is an essential factor of aquatic ecosystem. In the present study, the nitrite was maximum during monsoon season and minimum during summer season. Similar observarions were reported by earlier workers (Udaya Varma Therupad and Gangadhara Reddy, 1959; Mohamed Abubaker Sithik et al., 2009; Sankar et al., 2010). The seasonal variation in nitrite content could be attributed to the variation in phytoplankton in palk Bay (Kannan and Kannan, 1996).

The photoperiodicity is a well known fact in the animal world. Diurnal rythum is an important physiological event of the organism. Lengthening of photoperiod may induce gonadal activity in animals. In 26 the persent study maximum day length was recorded in summer and minimum in monsoon months. Similar observations were reported by earlier workers (Rajendran, 1990 and Kannathasan and Rajendran, 2010a). In the present study, 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 crabs.

3. TAXONOMY

3.1. Introduction The brachyuran crabs have discontinuous distribution, allometric growth pattern, polymorphism and sexual dimorphism. They have great degree of adaptation to different environments and also individual variability within the species. Hence the study of taxonomy in decapod crustacean is more difficult. But the synchronous observations of rigid exoskeleton make it easier. The taxonomy played a vital role in the study of crustacean phylogeny. The study relating to biology and development of portunid brachyuran crabs at south east coast of Bay of Bengal have been reported by many workers (Ameerhamsa, 1978; Saradha, 1998; Sethuramalingam and Ajmal Khan, 1991; Kannathasan and Rajendran, 2011a).

According to Leene (1938) and Stephenson and Campbell (1957) the portunid comprises a large number of species. The brachyuran crabs are worldwide in distribution. The crab fisheries are available throughout the years. Crab fishery resources from Indian waters have been reported by many workers (Sethuramalingam, 1983; Joel et al., 1985; Ravichandran and Kannupandi, 2004; Ajmal Khan et al., 2005; Ravichandran et al., 2007; Bandekar et al., 2011). Prasad and Tampi (1951) described the fishery and fishing method for swimming crab Neptunus pelagicus. Menon (1952) reported the crab fishery in Malabar Coast. Chhapgar (1962) demonstrated crab fishing at Bombay coast. 28

Observations on breeding biology of some crabs from south west coast of India have been made (Pillai and Nair, 1973a).

Decapod crustaceans are very common invertebrates inhabiting in the marine environment. The diversity of the brachyuran crabs was abundant in littoral and deep sea regions (Jayabaskaran, 1997; Jayabaskaran et al., 2000; Bertini et al., 2004; Kathirvel and Gokul, 2006; Krishnamoorthy, 2007; Kathirvel, 2008). The occurrence and distribution of brachyuran crabs from the south east coast of Bay of Bengal, India have been reported by the many workers (Radhakrishnan, 1979; Kathiresan, 2000; Ravichandran and Kannupandi, 2007; Balaji et al., 2007; Soundarapandian et al., 2008; Manokaran et al., 2008; Varadharajan et al., 2009). Biodiversity of many brachyuran crabs from other countries have been reported by earlier workers (Skilleter and Warren, 2000; Boschi, 2000; Quader, 2010). However little works are available on biodiversity of brachyuran crabs at Nagapattinam coast. Hence the present study is an attempt to assess the biodiversity of brachyuran crabs in Nagapattinam coast, the south east coast of the Bay of Bengal, India.

3.2. Materials and Methods The brachyuran crabs were collected from the littoral zone, tidal region and commercial fishing landing centre of Nagapattinam coast from January 2009 to December 2010. The crabs were characterized and segregated from consumable species, their numbers were recorded and 29 each individual was preserved in 5% neutralized formalin for further identification. The species were identified and classified according to earlier workers (Leene, 1938; Chhapgar, 1957; Stephenson and Campbell, 1957).

3.3. Result 3.3.1. Survey A primary survey of brachyuran crabs along a littoral zone, tidal region and fish landing centre of Nagapattinam coastal area indicates that the crab fishery resource showed significant result (Table 2). In the present study, there were a total of 133 species of brachyuran crabs belonging to 58 genera, 18 families, 16 sub families, and 8 super families recorded throughout the year with seasonal fluctuation in numbers (Fig. 11).

In the present study, crabs belonging to the family of Potunidae and Xanthidae were most dominant forms. Crab population was high during monsoon and pre monsoon months and gradually decreased during post monsoon and summer months. The crabs were landed from sea, both by „traweller‟ and „catamaran‟. The brachyuran crabs in most of the species were seen round the year. Out of these 133 species frequency of landing is restricted to 46 species. Nearly 25 species are found larger and they are edible. The list of crabs identified in the present study is as follows.

Super Family : PORTUNOIDEA (Rafinesque, 1815) Family : PORTUNIDAE (Rafinesque, 1815) Sub Family : PORTUNINAE (Rafinesque, 1815)

1. Genus: Charybdis (De Haan, 1833) Charybdis natator (Herbst, 1794) C. lucifera (Fabricius, 1798) C. feriata (Linnaeus, 1758) C. helleri (H. Milne Edwards, 1867) C. smithi (Macleay, 1838) C. affinis (Dana, 1852) C. granulata (Dana, 1852) C. anisodon (De Haan, 1835) C. annulata (Fabricius, 1798) C. truncata (Fabricius, 1789) C. vadorum (Alcock, 1899) C. variegata (Fabricius, 1798) C. orientalis (Dana, 1852) C. callianassa (Herbst, 1803) C. edwardsii (Leene and Buitendijk, 1952)

2. Genus: Portunus (Weber, 1795) Portunus pelagicus (Linnaeus, 1766) P. sanguinolentus (Herbst, 1783) P. gracilimanus (Stimpson, 1858) P. gladiator (Alcock, 1899) P. brockii (De Man, 1887) P. argentatus (A. Milne Edwards, 1861) P. hastoides (Fabricius, 1798) P. haanii (Stimpson, 1858)

3. Genus: Scylla (De Haan, 1833) Scylla serrata (Forskal, 1755) S. tranquebarica (Fabricius, 1797) S. olivacea (Herbst, 1796)

4. Genus: Thalamita (Latreille, 1829) Thalamita admete (Herbst, 1803) T. crenata (H.M. Edwards, 1834) T. danae (Stimpson, 1858) T. prymna (Herbst, 1803) 31

T. integra (Dana, 1852) T. sima (H. Milne Edwards, 1834)

Sub Family : PODOPHTHALMINAE (Borradaile, 1907)

5. Genus: Podophthalmus (Lamarck, 1801) Podophthalmus vigil (Fabricius, 1798)

Sub Family : CAPHYRINAE (Alcock, 1899)

6. Genus: Lissocarcinus (Adams and White, 1849) Lissocarcinus laevis (Miers, 1886) L. orbicularis (Dana, 1852)

Super Family : CALAPPOIDEA (De Haan, 1833) Family : CALAPPIDAE (Dana, 1852) Sub Family : CALAPPINAE (Alcock, 1896)

7. Genus: Calappa (Fabricius, 1798) Calappa calappa (Linnaeus, 1758) C. hepatica (Linnaeus, 1758) C. lophos (Herbst, 1782) C. philargius (Linnaeus, 1758) C. japonica (Ortmann, 1892) C. appa terraereginae (Ward, 1936) C. gallus (Herbst, 1803) C. clypeata (Borradaile, 1903) C. bicornis (Miers, 1884)

Sub Family : MATUTINAE (Macleay, 1838)

8. Genus: Matuta (Weber, 1795) Matuta lunaris (Forskal, 1775) M. banksii (Leach, 1817) M. planipes (Fabricius, 1798) M. victor (Fabricius, 1781)

Super Family : DROMIOIDEA (Alcock, 1899) Family : DROMIIDAE (Alcock, 1899)

9. Genus: Conchoecetes (Stimpson, 1858) Conchoecetes artificiosus (Fabricius, 1798) C. intermedius (Lewinsohn, 1984) Lauridromia indica (Gray, 1831)

10. Genus: Dromia (Fabricius, 1798) Dromia dehaani (Rathbun, 1923)

Super Family : RANINOIDEA (De Haan, 1833) Family : RANINIDAE (De Haan, 1833)

11. Genus: Ranina (Lamarck, 1801) Ranina ranina (Linnaeus, 1758)

Super Family : DORIPPOIDEA (De Haan, 1833) Family : DORIPPIDAE (White, 1847) Sub Family : DORIPPINAE (De Haan, 1833)

12. Genus: Dorippe (Fabricius, 1793 Dorippe frascone (Herbst, 1785) D. dorsipes (Linnaeus, 1764) Neodorippe callida (Fabricius, 1798)

Super Family : PAGUROIDEA Family : DIOGENIDAE

13. Genus: Diogenes (Henderson, 1893) Diogenes costatus (Henderson, 1893)

Super Family : XANTHOIDEA Family : CARPILIIDAE (Ortmann, 1893) Sub Family : POLYBIINAE (Ortmann, 1893)

14. Genus: Atergatis (De Haan, 1835) Atergatis florides (Linnaeus, 1767) A. integerimus (Larmack, 1801) A. subdentatus (De Haan, 1835)

15. Genus: Carplius (Leach, 1823) Carplius convexus (Forskal, 1775) C. maculatus (Linnaeus, 1758)

Family : XANTHIDAE (Macleay, 1838)

16. Genus: Actaea (De Haan, 1833) Actaea calculosa (H. Milne Edwards, 1834) A. avipes (Dana, 1852) A. savignyi (H. Milne Edwards, 1834)

17. Genus: Chlorodiella (Rathbun, 1897) Chlorodiella nigra (A, Milne Edwards, 1873)

18. Genus: Cymo (De Haan, 1835) Cymo melanodactylus (De Haan, 1835) C. andreossyi (Audouin, 1826)

19. Genus: Demania (Laurie, 1906) Demania baccalipes (Alcock, 1898)

20. Genus: Eriphia (Latreille, 1817) Eriphia sebana (Shaw & Nodder, 1803)

21. Genus: Galene (De Haan, 1835) Galene bispinosa (Herbst, 1783)

22. Genus: Halimede (De Haan, 1835) Halimede ochtodes (Herbst, 1783)

23. Genus: Leptodius (A, Milne Edwards, 1863) Leptodius exaratus (H.Milne Edwards, 1834) L. euglyptus (Alcock, 1898) L. crassimanus (H.Milne Edwards, 1867) L. sanguineus (H.Milne Edwards, 1834)

24. Genus: Liomera (Dana, 1852) Liomera margaritata (A, Milne Edwards, 1864) L. erythra (Lanchester, 1900)

25. Genus: Lophozozymus (A, Milne Edwards, 1864) Lophozozymus dodone (Herbst, 1801) 34

26. Genus: Ozius (H.Milne Edwards, 1834) Ozius rugulosus (Stimpson, 1858)

27. Genus: Platypodia (Bell, 1835) Platypodia granulose (Ruppell, 1830) P. cristata (A.Milne Edwards, 1835) P. semigranosa (Heller, 1861) 28. Genus: Pilodius (Dana, 1852) Pilodius areolatus (H.Milne Edwards, 1834)

29. Genus: Liagore (De Haan, 1833) Liagore rubramaculata (De Haan, 1835)

Family : PILUMNIDAE

30. Genus: Pilumnus (Leach, 1815) Pilumnus minutus (De Haan, 1835) P. tomentosus (Latreille, 1825) P. vespertilio (Fabricius, 1798)

Family : TRAPEZIIDAE

31. Genus: Trapezia (Latreille, 1825) Trapezia cymodoce (Herbst, 1801) T. ferruginea (Latreille, 1825) T. rufopunctata (Herbst, 1790)

Family : LEUCOSIIDAE (Dana, 1852) Sub Family : PHILYRINAE (Rathbun, 1937)

32. Genus: Arcania (Leach, 1817) Arcania erinaceus (Fabricius, 1798) A. heptacantha (De Haan, 1850)

33. Genus: Ixoides (Mac Gilchrist, 1905) Ixoides cornutus (Mac Gilchrist, 1905)

34. Genus: Ixa (Leach, 1815) Ixa cylindrus (Fabricius, 1777)

35. Genus: Iphiculus (Adams and White, 1848) Iphiculus spongiosus (Adams and White, 1848) 35

36. Genus: Myra (Leach, 1817) Myra fugax (Fabricius, 1798) M. coalita (Hilgendorf, 1878)

37. Genus: Philyra (Leach, 1815) Philyra globulosa (A. Milne Edwards, 1837) P. scabriscula (Fabricius, 1798)

38. Genus: Leucosia (Leach, 1798) Leucosia pubescens (Miers, 1877)

Family : MAJIDAE (Samouelle, 1819) Sub Family : PISIDAE (Alcock, 1895)

39. Genus: Doclea (Leach, 1814) Doclea ovis (Herbst, 1782) D. gracilipes (Stimpson, 1857) D. rissoni (Leach, 1815) D. canalifera (Stimpson, 1853)

40. Genus: Hyastenus (White, 1847) Hyastenus aries (Latreille, 1825)

41. Genus: Naxoides (A. Milne Edwards, 1865) Naxiodes mammillata (Ortmann, 1893)

42. Genus: Phalangipus (Latreille, 1825) Phalangipus hystrix (Miers, 1836)

43. Genus: Chorilibinia (Lockington, 1876) Chorilibinia andamanica (Alcock, 1893)

Family : PARTHENOPIDAE (Miers, 1879) Sub Family : PARTHENOPINAE (Miers, 1879)

44. Genus: Parthenope (Weber, 1795) Parthenope longimanus (Linnaeus, 1764) P. presnor (Herbst, 1790) P. echinatus (Linnaeus, 1764)

Super Family : HOMOLOIDEA (White, 1847) Family : HOMOLIDAE (White, 1847)

45. Genus: Homola (Leach, 1815) Homola megalop (Alcock, 1894)

Family : GECARCINIDAE (Dana, 1851)

46. Genus: Cardisoma (Latreille, 1825) Cardisoma carnifex (Herbst, 1794)

Family : OCYPODIDAE (Ortmann, 1894) Sub Family : MACROPHTHALMINAE (Dana, 1852)

47. Genus: Macropthalmus (Latreille, 1829) Macropthalmus (Mareotis) depressus (Ruppell, 1830)

Sub Family : OCYPODINAE (Dana, 1851)

48. Genus: Ocypode (Fabricius, 1798) Ocypode ceratophthalma (Pallas, 1872) O. cordimana (Desmarest, 1825) O. platytarsis (H. Milne Edward, 1852) O. macrocera (H. Milne Edward, 1834)

49. Genus: Uca (Leach, 1814) Uca annulipes (H. Milne Edwards, 1837) U. voccans, (Desmarest, 1823) U. forcipata (Adams & White, 1848) U. perplexa (H. Milne Edwards, 1837)

Sub Family : SCOPIMERINAE (Alcock, 1900)

50. Genus: Dotilla (Stimpson, 1858) Dotilla myctroides (H. Milne Edwards, 1852)

Family : TETRALIIDAE (Castro, Ng and Ahyong, 2004)

51. Genus: Tetralia (Dana, 1851) Tetralia rubridactyla (Garth, 1971)

Family : GRAPSIDAE (Dana, 1851) Sub Family : GRAPSINAE (Dana, 1851)

52. Genus: Grapsus (Lamarck, 1880) Grapsus albolineatus (Lamarck, 1818)

53. Genus: Metapograpsus (H. Milne Edwards, 1853) Metapograpsus messor (Forskal, 1775)

54. Genus: Varuna (Fabricius, 1798) Varuna literata (Fabricius, 1798)

Sub Family : PLAGUSIINAE (Dana, 1851)

55. Genus: Percnan (Gistel, 1848) Percnan planissimum (Herbst, 1804)

56. Genus: Plagusia (Latreille, 1806) Plagusia squamosa (Herbst, 1803)

Sub Family : SESARMINAE (Dana, 1852)

57. Genus: Nanosesarma (Tweedie, 1950) Nanosesarma minutum (De Man, 1887)

58. Genus: Sesarma (Say, 1817) Sesarma plicatum (Latreille, 1806) S. bidens (De Haan, 1835)

Out of these 133 species of crabs the specimens have been chosen for the systematic study from the genus „Charybdis‟. The present genomic name „Charybdis‟ was referred by Leene (1938) instead of its previous name „Goniosoma‟ since the name was preoccupied with the genus Arachnoidea. Charybsdis (De Haan, 1835) and Thalamita are said to be closely related. Chopra (1936) and Leene (1938) studied two other genera Portunus and Thalamita. Most of the species of Charybdis are Indo- 38

Pacific in distribution, many of them being endemic. Alcock (1898) divided the genus Charybdis into three sub genera namely Charybdis (Gonisoma) (Am. Edw.), Goniohellenus (Alcock) and Gonioeptunus (Ortmann), based mainly on six anterolateral spines of the cephalothorax along with other characters. Generally in Charybdis, the six anterolateral spines are dissimilar. They are divided into four or five well developed spines with one or two rudimentary ones. Leene (1938) observed this dissimilarity and the grouping of the six anterolateral spines of the cephalothorax and recognized this arrangement as a character of diagnostic value. Based on this, two new sub genera were added to the genus Charybdis namely Gonioinfradens and Goniosupradens (Leene, 1938). The former two sub genera have one large tooth loss while the later has atleast one tooth more. The genus Charybdis may be divided into five sub genera namely Charybdis, Gonioneptunus, Goniohellenus, Gonioinfradens and Goniosupradens.

3.3.2. Taxonomic characters The cephalothorax is hexagonal, moderately broad and depressed. At the anterior region having transverse ridges like progastric, mesogastric and metagastric regions. Similarly, ridges are found on the mesobranchial and epibranchial regions. Cervical groove is usually present along the transverse line which is oftern seen midway between the anteriolateral spines. The frontal side of the anterior border is cut in sex lobes. Out of these six lobes two of them are median, two submedian and other two 39 laterals. The inner supra orbital angle is distinctly separated from the frontal plane which is always excluded from the frontal teeth number.

The antero-lateral borders are oblique and slightly arched. It is larger than the postero lateral border. They cut into six to eight teeth including the outer orbital angles. But antero lateral borders are usually cut into six antero lateral spines. The posterior borders are almost straight and narrow. The upper border of the orbit usually has two notches or fissures. The lower border has a gap and the inner angle of this border is usually dentiform. The antennulae are folded transversely.

The „basal‟ antennal joint is short and broad. Its outer angle forms a lobe which usually fills the orbital hiatus and meets the front excluding the flagellum from the orbital hiatus. The epistome is sufficiently long. The chelipeds are massive and longer than other legs. The left and right cheliped have spines. The inner angle of the carpus is strongly spiniform. The outer angle usually armed with spinules. The dactylus strong and strongly denticulated.

The second to fourth pair of periopod is compressed in the natatory legs. The meropodites and the carpopodites shortened and broadened and the mesopodite usually has spine at a short distance proximal to the distal end of the posterior border. The dactylopodites are typically foliaceous for swimming. The abdomen in the both sexes is almost entirely concealed beneath the cephalothorax. 40

3.3.3. Systemetic position Kingdom : Animalia Phylum : Arthropoda Super class : Crustacea Class : Malacostraca Order : Decapoda Sub order : Pleocyemata Infra order : Brachyura Super family : Portunoidea Family : Portunidae Sub family : Portuninae Genus : Charybdis Species : Natator Scientific name : Charybdis natator

3.3.4. Morphological features The vernacular name is “Compi Nandu”. This is due to the presence of number of lines on the dorsal side of the carapace. They are originally dark red in colour with chocolate or red coloured markings. Cephalothorax in oval shape. There are six frontal teeth, two of them are medium, two sub medium and two laterals. The orbital cavity is broader and deeper. Hairs are present in linear fashion except in the last leg. Anterial borders are larger than posterolateral borders.

The chelipeds are stronger and stout. Chelae are more or less similar in size. The second to fourth pairs of periopods are adapted for and the walking last pair of legs are flattened for pelalogic adaptations. Carapace are very distinct, body colour light pink with numerous red spots on body and white spots on legs. The common name being „Red lined swimming crab‟ or „Coral swimming crab‟ (Plate- 2).

3.3.5. Geographical distribution In India the crab Charybdis natator is found distributed in both west and east coast and Andaman and Nicobar islands. Other areas in the Indo-pacific region of distribution are the south east coast of Africa, Madagascar, Arabian Gulf, Pakistan, Srilanka, Thailand, Indonesia, Malaysia, Philippines, Taiwan, Japan, Australia etc.

3.4. Discussion In the present study a total of 133 species of brachyuran crabs were recorded in the offshore region of Nagapattinam coast. The crabs were landed throughout the year but the total catches and number of species varied seasonally. The species composition and diversity were found to be high during monsoon and pre monsoon months and low during summer months. Similar observations were reported by earlier workers (Menon, 1952; Prasad and Tampi 1951; Ameerhamsa, 1978; Soundarapandian et al., 2008). The portunid comprises a large number of species inhabiting in various habitats (Leene, 1938; Stephenson and Campbell, 1957).

According to Kathiresan (2000) and Ravichandran et al., (2007) the minimum crab population was observed during summer and maximum monsoon months. The biodiversity of crabs were more in monsoon and less in summer (Ravichandran and Kannupandi, 2004). Most of the species of Charybdis are Indo-Pacific in distribution, many of them being endemic (Stephenson and Campbell, 1957). Chhapgar (1962) recorded the marine crab Portunus pelagicus throughout the year at Bombay coast. The brachyuran crabs Scylla serrata, Portunus sanguinolentus and Charybdis feriata were predominantly landed in Indian waters (Pillai and Nair, 1973a). Five species of marine crabs such as Scylla serrata, Portunus pelagicus, Portunus sanguinolentus, Podophthalmus vigil and Charybdis feriata were landed predominantly along the Parangipettai coast (Radhakrishnan, 1979).

According to Sethuramalingam, (1983) and Sethuramalingam and Ajmal Khan, (1991) the larvae and juvenile were found to be maximum during late post monsoon and summer and minimum during monsoon. Joel et al., (1985) demonstrated the distribution and zonation of 29 crabs in the Pulicat Lake. The diversity of brachyuran crabs was abundant in littoral and deep sea regions of Bay of Bengal (Jayabaskaran, 1997; Jayabaskaran et al., 2000; Kathirvel and Gokul, 2006). A total number of 38 brachyuran crabs were recorded in Pitchavaram mangroves of which most of them belonging to the family Grapsidae and Ocypodidae (Ajmal Khan et al., 2005). Biodiversity study of crabs in the Pichavaram 43 mangroves has shown that there are 46 species of crabs from the five different stations (Ravichandran and Kannupandi, 2007).

In Portunus sanguinolentus large numbers of berried females were recorded from December to May and July to August of the respective year (Saradha, 1998). In Moreton Bay Australia abundance and species richness of crab were observed in a subtrophical mangrove forest (Skilleter and Warren, 2000). A total of 1086 species belongs to 311 genera and 40 brachyuran families were reported from American sea (Boschi, 2000). In Brazil, 79 brachyuran crabs belong to 41 genera, 18 families under 9 super families were recorded (Bertini et al., 2004). At Chennai coast, 54 species of Portunid crabs are available around the year (Krishnamoorthy, 2007). Uca annulipes is the most abundant species among the crab species recorded at south east coast of India (Balaji et al., 2007). Janas choprai is first time recorded at Parangipettai coastal area (Manokaran et al., 2008). Kathirvel (2008a and b) recorded a total of 404 species of crabs belonging to 152 genera and 26 families available at Tamil Nadu coast. Further he reported 990 species of brachyuran crabs from Indian water. Out of this 990 species, 32 species attaining large size and are edible. Nearly 12 commercially important crabs were recorded from Arukattuthurai to Aiyammpattinam area, south east coast of Bay of Bengal (Varadharajan et al., 2009). A total of 16 species of edible crabs documented at Bangladesh coast (Quader, 2010).

There are about 15 species of crabs belonging to family Grapsidae and Ocypodidae distributed in Karwar mangrove environment (Bandekar et al., 2011). A total number of 90 brachyuran crabs were recorded at south east coast of Bay of Bengal (Kannathasan and Rajendran, 2011a). In the present study 133 species of crabs were recorded from Nagapattinam coast, Bay of Bengal, indicates that this place is suitable for crab inhabitation and propagation their species. The richness of biodiversity of crabs make the place is one of the important crab fishery centres.

3.5. MOLECULAR CHARACTERIZATION

3.5.1. Introduction The genus Charybdis is an important crustacean resource for commercial fisheries and aquaculture industries in the Indo-South east region. The crab Charybdis natator is distributed widely in the south east coast of Bay of Bengal (Kannathasan and Rajandran, 2011a). In recent years, several studies were carried out on taxonomy and phylogeny of brachyuran crabs using adult and larval morphological characters as well as molecular approaches (Abele et al., 1989; Wee and Ng, 1995; Geller et al., 1997; Yeo et al., 2007; Ahyong et al., 2007; Ng et al., 2008; Liu and Cui, 2009; Baeza et al., 2010). The 16S rRNA, 18S rRNA and COI gene were used in molecular analysis of crustaceans (Spears et al., 1992; France and Kocher, 1996; Zhao et al., 2002; Reuschel and Schubart, 2006). Kitaura et al., (1998) used 12S to 16S rRNA genes for molecular phylogeny and evolutionary significant of Ocypodid crabs. In terms of marine crustaceans, the COI gene has been used to analyse phylogenetic relationship of many taxa including 13 species in Penaeus, 6 species in 45

Gammarus 9 species in Cancer and Bresillidae shrimps (Harrison and Crespi, 1999).

In Grapsoid and Ocypodid crabs, the 16S rRNA gene were applied to study the molecular phylogeny (Schubart et al., 2000 and 2006; Stillman and Reeb, 2001; Kitaura et al., 2002; Quan et al., 2004). Schubart et al., (2001) observed the lack of divergence between mtDNA sequences of the swimming crabs Callinectes bocourti and C. maracaiboensis. A high degree of genetical differentiation as observed in various species of marine crabs such as Scylla serrata (Fratini and Vannini, 2002), Chaceon quinquedon (Weinberg et al., 2003), Carcinus maenas (Roman Palumbi, 2004), Callinectes bellicosus (Pfeiler et al., 2005), Betamorpho fusiformis (Raupach et al., 2007), Callinectes sapidus (Troedsson et al., 2008; Maja brachydactyla (Sotelo et al., 2008) and Tachypleus gigas (Ismail et al., 2011).

Molecular analysis of the taxonomic and distributional status for the hermit crab genera Toxopagurus and Isocheles have been reported (Montelatto et al., 2006). The phylogeny and biogeography of the crab genus Johora have been observed (Yeo et al., 2007). Klinbunga et al., (2010) reported the species identification of Portunus pelagicus with reference to molecular analysis. The phylogenetic inference from 18S rRNA gene sequences of horseshoe crab Tachypleus gigas has been documented (Ismail and Sarijan, 2011). The present study aims to analyse the molecular systematics of the brachyuran crab Charybdis natator from Nagapattinam coast.

3.5.2. Materials and methods Sample collection and DNA extraction Alive specimens of Charybdis natator were obtained from Nagapattinam coast. The crab muscle tissues were taken and immediately preserved in 100% ethanol at 20oC. The DNA was extracted by the standard phenol chloroform method (Sambrook et al., 1989) and visualized on 1.0% agrose gel.

16S rDNA and COI gene sequencing The 16S rRNA gene was amplified using primers forward (5‟- CGCCTGTTTAACAAAAACAT-3‟) and reverse (5‟-CCGGTCTGAAC TCAGATCATGT-3‟) method (Bouchon et al., 1994). The amplifications (total volume 50 µl) contained 1 ml DNA and 49 µl PCR-mix [reaction- mix: 40.3 µl sterile dH2O, 5 µl PCR-buffer (10x), 2 µl dNTP-Mix, 0.5 µl BSA-solution, 0.5 µl of each primer (forward and reverse, 10 pmol/µl), 0.2 µl Taq-Polymerase]. The cycling profile was as follows: 1 min at 92oC, 1 min at 52oC, and 1 min at 72oC for 35 cycles with an initial denaturation at step 92oC for 4 min, and a final extension step at 72oC for 5 min. The Cycle Sequencing reaction was performed in 5 µl total volume: 1 µl primer (5 pmol/ µl), 1-3 µl cleaned PCR-product, 1 µl Big

Dye Terminator, completed with dH2O. The cycle sequencing proﬁle consisted of 25 cycles: 20 s at 95oC, 15 s at 50oC, 2 min at 60oC. Automated sequencing was performed using an ABI Prism Genetic Analyzer 3100 (Applied Biosystems, USA).

The mitochondrial cytochrome oxidase subunit I sequences (COI) were amplified and sequenced with the universal primers forward (5‟- TTCTCCACCAACCACAARGAYATYGG-3‟) and reverse (5‟-CACCT 47

CAGGGTGTCCGAARAAYCARAA-3‟) method (Palumbi and Benzie, 1991) following similar procedures as for the 16S fragments.

18S rRNA gene sequencing The 18S rRNA fragments were amplified using the universal primers forward (5‟-ACCTGGTTGATCCTGCCAGT-3‟) and reverse (5‟- GATCCTTGCAGGTTCACCTAC-3‟) method (Palumbi and Benzie, 1991). The gene of 18S rDNA region was ampliﬁed via polymerase chain reaction (PCR) [reaction-mix: 5 µl of 10x PCR buffer with (NH4)2SO4

(Fermentas), 4-6.5 mM MgCl2 (Fermentas), 1 ng/ µl genomic DNA, 0.2 mM dNTPs 3.25 mM of each primer (18F35 and 18R1779), 0.02 U/ µl Taq DNA Polymerase (Fermantas)]. The primers applied were 18F35 and 18R1779 annealing to the conservative ends of the 18S rDNA (Struck et al., 2002). The PCR protocol was as follows: initial denaturation 98oC for 5 min and than 35 cycles with 1 min 94oC, 1 min 50oC, 2 min 72oC and 10 min at 72oC for final extension. The PCR products were analyzed by agarose gel electrophoresis and purified by HELINI PureFast PCR Clean up Kit. The purified PCR products were sequenced using BigDye v.3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The sequences were determined with the automatic ABI PRISM 310 Genetic Analyzer. Consensus sequence of 499bp segments of 16S rRNA, 1812bp of 18S rRNA and 699bp of COI gene was generated from forward and reverse sequence data using aligner software. The obtained gene fragments were aligned with the different gene fragments of other crabs by using BLAST (Basic Local Alignment Search Tool).

Data interpretation The Sequences from all genes were combined in to a single data set. The phylogenetic analyses were conducted in the computer program MEGA4 by applying Maximum parsimony (MP) and Neighbor joining (NJ). For the MP trees 1000 bootstrap replicates were analyzed by heuristic search with close Neighbor interchange (CNI). The evolutionary distances were computed using the Maximum composite Likelihood (ML) method (Tamura et al., 2007).

3.5.3. Result The results of DNA sequencing reveals that there were 499bp fragment of the partial 16S rRNA, 1812bp of 18S rRNA and 699bp of COI nucleotide sequence successfully isolated from the crab Charybdis natator genomic DNA (Plate 3-6). The BLAST alignment showed the high homology of nucleotide sequence to 16S rRNA gene of two different species of marine crabs Charybdis hellerii (93%) (Accession number FJ152142) and Charybdis japonica (93%) (Accession number HM237595). The nucleotide sequence to 18S rRNA gene having homology with two marine crabs Charybdis vadorum (99%) (Accession number EU284148) and Charybdis lucifera (98%) (Accession number HQ634241). The homology of nucleotide sequence to COI gene sequences of crabs Charybdis variegate (87%) (Accession number EU284142) and Charybdis japonica (87%) (Accession number EU586120). The tree view depicted that the partial 16S rRNA, 18S rRNA and COI gene of the samples studied shared the sister taxon with 16S rRNA, 18S rRNA and COI genus, with a bootstrap value of 1000- iteraction. The phylogenic position was determined by amplification of 16S rRNA, 18S rRNA and COI genes and using three tree building 49 methods. The topologies of the three phylogenetic trees indicate that the marine crab C. natator haplotypes were clustered into 10 clades in 16S rRNA, 8 clades in 18S rRNA and 12 clades in COI based on the sister pairs.

3.5.3.1. 16S rRNA The 16s rRNA gene sequence for the selected crab of C. natator revealed that the crab belongs to the genus Charybdis, order Decapoda, and family Portunidae. The result of C. natator showed 7% sequence variations with that of C. hellerii and C. japonica and it was submitted to Genebank with an accession number JF772863. The genetic divergence of C. natator compared with 32 close related species indicates cladistically informative within the ingroup and outgroups. The genetic divergence was found to be the highest (0.204) in Helicana wuana and lowest (0.052) in Charybdis hellerii. The overall result, the higher value (0.208) in Medaeus elegans and lower value (0.002) in Panopeus herbstu were obtained (Table-3).

3.5.3.2. 18S rRNA 18s rRNA sequence for the crab C. natator showed similar result as in 16S rRNA. The result of C. natator showed sequence variation at 1% with that of Charybdis accuta. Hence the sequences were submitted to Genebank with an accession number HQ676594. The genetic divergences of C. natator compared with 24 closed relate species showed significant results. The highest genetic divergence (0.011) with Portunus sanguinolentus and lowest genetic divergence (0.002) with Charybdis vadorum were found. The overall result, the higher value (0.208) in Hemigrapsus sanguineus and lower value (0.002) in Charybdis vadorum were observed (Table-4). 50

3.5.3.3. COI GENE The results of COI gene sequence also indicated that the crab comes under genus Charybdis, order Decapoda, and family Portunidae. The sequence of variations 13% with that of C. accuta and these sequences were submitted to Genebank with an accession number JF762864. The genetic divergence of C. natator was compared with 27 close related species of ingroup and outgroups. The results indicated that the highest genetic divergence (0.226) was with Uca boreal and lowest genetic divergence (0.147) with Charybdis japonica. The overall result, the higher value (0.258) in Sayamta sexpunctata and lower value (0.071) in Uca jocelynae were recorded (Table-5).

3.5.3.4. Phylogenetic analysis The phylogenetic trees of 16S rRNA, 18S rRNA and COI gene showed slightly different topologies (Fig. 12-14). The 16S rRNA, 18S rRNA and COI gene indicate trees that the genus Charybdis belong to monophyletic group. The overall tree topology suggested that the genus Charybdis is divided into main clades; clade C. natator, C. helleri, C. japonica and C. lucifera is one and C. ferriata, C. vadorum and C. acuta is another. In a tree the genus Charybdis, Portunus, Calliniectes and Scylla branching off first, followed by several clusters indicate the close relationship of family Portunidae. The family Portunidae, Xanthidae, Parthenopidae and Geryonidae have also shown phylogenitic relationship in decapod.

3.5.4. Discussion The molecular analysis of the 16S rRNA, 18S rRNA and COI gene are commonly used in assessing phylogenetic relationships among taxa. 51

The present study reveals phylogenetic relationships, evolutionary significance and genetic diversity of marine crab C. natator. The genomic and mtDNA were commonly used to study the phylogeney and genetic variability of brachyuran crabs by many workers (Spears et al., 1992; Stillman and Reeb, 2001). Phylogenetic relationships among grapsoid and ocypod crabs based on morphological and molecular analysis have been proposed (Schubart et al., 2000). In American grapsoid crab, 16S rRNA gene was used to investigate molecular phylogeny, taxonomy and evolution (Schubart et al., 2001; Kitaura et al., 2002; Schubart, 2006). In C. natator 499bp fragments of partial 16S rRNA nucleotides were successfully isolated from genomic DNA. This is in agreement with the previous workers (Spears et al., 1992; Stillman and Reeb, 2001; Yeo et al., 2007).

Kitaura et al., (1998) isolated 1416bp nucleotide sequence of mitochondrial 12S rRNA to 16S rRNA from 20 crab species of Oxypodidae. A total of 294bp of 16S mtRNA nucleotide sequences were isolated from crabs Callinectes boccurti and C. maracaiboensis (Schubart et al., 2001). Quan et al., (2004) isolated the 514 fragments of 16S rRNA from the swimming crab Portunus trituberculaus. The crab genera Loxopagarus, the 16S rDNA contained a total of 538bp nucleotide sequences including primer regions (Montelatto et al., 2006). In freshwater crab genera Jahora 560bp partial 16S rRNA nucleotide were successfully isolated by Yeo et al., (2007). In spider crab Maja brachydactyla the 16S rRNA alignment consists of 580bp nucleotide (Sotelo et al., 2008). In crab Xantho hydrophitatus, X. pilipes, X. poressa and X. sexdentatus nearly 527bp of 16S rRNA were isolated. Baeza et al., (2010) isolated a total of 550bp alignment of 16S rRNA from spider crabs. 52

In the present observation, 1812bp fragments of partial 18S rRNA nucleotides were isolated from genomic DNA of C. natator agrees with earlier observations (Troedsson et al., 2008 and Ismail and Sarijan, 2011). According to Spear et al., (1992) partial 18S rRNA and rDNA sequences ranging in length from 950 to 1179 nucleotides excluding gaps and unknown bases were obtained from 12 decapod crustaceans. Troedsson et al., (2008) observed 498bp of 18S rRNA gene from Hematodinium sp and C. sapidus. A total of 1832bp fragment of the partial 18S rRNA nucleotides were successfully isolated from T. gigas genomic DNA (Ismail and Sarijan, 2011).

In crab C. natator, 699bp fragments of COI gene nucleotides were isolated which confirms with previous workers (Frantini and Vannini, 2002; Klinbunga et al., 2010). In Cancer crab COI gene consists of 1072bp nucleotide sequences (Harrisson and crespi, 1999). Zhao et al., (2002) obtained 624bp section of COI gene from mitten crabs Eriocheir. In Scylla serrata a total of 535bp COI gene nucleotide sequence were isolated and identified from 24 different halotypes (Frantini and vannini, 2002). Reuschel and Schubart (2006) isolated 640bp of COI nucleotide sequences from four species of Xantho. Yeo et al., (2007) isolated 616bp COI nucleotide sequences from crab genus Jaharo. A total of 706bp in COI gene were amplified and sequenced from swimming crab P. pelagicus (Klinbunga et al., 2010).

The genetic divergence of Charybdis natator were found to be low (0.052) and high (0.204) in 16S rRNA and ranged from 0.002 to 0.011 in 18S rRNA and 0.147 to 0.226 in COI gene. Similar observations were reported by earlier workers (Raupach et al., 2007; Ismail et al., 2011). 53

The European crab Carcinus maenas and C. aestuarii showed the genetic divergence around 2.5 for 16S rRNA (Geller et al., 1997). In Betamorpha fusiformis, p-distance ranged from 0.0470 to 0.1440 across all 16S rRNA sequence and 18S rRNA p-distance ranged between 0.0032 to 0.0174 (Raupach et al., 2007). A maximum of 0.112 for p-distances within bathymetrically separated population of the lysianassid amphipod E. gryllus (France and Kocher, 1996). Abele et al., (1989) estimated a high rate of (0.9%) genetic divergence for the crustacean taxa Pentatomida and Branchiura. The pairwise estimation of sequence divergen from 0.5% to 15.5% for 18S rRNA (Spears et al., 1992). Weinberg et al., (2003) detected the genetic divergent value up to 0.029.

In cancer crabs pairwise distance ranged from 7.22 to 17.2 within group (Harrison and Crespi (1999). Zhao et al., (2002) observed sequence diversity of COI within the population of mitten crab Eriocheir ranged from 0.067% to 0.089%. In Carcinus maenas from the Atlantic and Mediterranean basins showed divergences around 11% for COI (Roman Palumbi, 2004). In Callinectes bellicosus, the genetic divergences between two clades were found to be low (1.1% COI) (Pfeiller et al., 2005). Ismail et al., (2011) observed genetic divergence between intrapopulation ranged from 0.111 to 0.903 and 0.375 to 0.955.

In the present investigation the phylogentic tree for 16S rRNA, 18S rRNA and COI gene indicates the genus Charybdis which is a monophyletic group that agrees with earlier workers (Wee and Ng, 1995; Ahyong et al., 2007; Baeza et al., 2010). According to Kitaura et al., (1998), the phylogenetic relation of Ocypodidae and Grapsidae are polypylotic subfamilies of these groups themselves were mostly 54 monophyletic. Ahyong et al., (2007) observed monophyletic clades in the topology of Podotremata. In the species of Mithrax - Mithraculus the overall topology tree indicates monophyletic clades (Baeza et al., 2010). The genus Charybdis together with Callinectes, Portunus, and Scylla had been previously included in the subfamily Portuninae of family Portunidae (Wee and Ng, 1995). Later most of the taxonomists classified genus Charybdis into subfamily Thalmitinae (Ng et al., 2008). This is also strongly supported by Liu and Cui, (2009) using 16S rRNA, COI and 18S rRNA gene. The phylogenetic tree information of marine crab C. natator indicates a close relationship with Charybdis japonica, C. hellirii, C. lucifera, P. pelagicus and C. sapidus then four species of Scylla. In the present study complete 16S rRNA, 18S rRNA and COI gene sequence available for C. natator, will provide information on both genomic and molecular analysis of this crab.

3.6. MORPHOMETRIC

3.6.1. Introduction Morphometric study deals with the relative growth pattern of various decapod crustaceans. The inverstigation relating to the development and morphometric of portunid, brachyuran crabs are available all over the world (Davidson and Marsden, 1987; Haefner 1990; Masunari et al., 2003; Kannathasan and Rajendran, 2010b). Haley (1969 and 1973) studied the relative growth and sexual maturity of the Taxas ghost crab Ocypode quadrata. In Corystes cessivelaunus, the relative growth showed distinct differences between pre and post puberty animals. Further crustaceans exhibit great differences between the growth rates of 55 males and females (Hartnoll, 1972 & 1978). In Ovalipes punctatus, the relative growth parameters are isometric (Du Preez and Mclachlan, 1984a).

Sumpton, (1990a) observed the morphometric growth and reproductive biology of Charybdis natator in Moreton Bay, Australia. The relative growth and size at maturity are the very important aspects for the better understanding of the brachyuran reproductive cycle (Chu, 1999; Costa and Soares-Gomes, 2008; Bello-Olusoji et al., 2006 and 2009). Allometeric analysis used to compare intraspecific variations among populations from different locations (Rosenberg, 2002) and variation on sexual maturity depends on environmental conditions (Oriola, et al., 2005; Dalabona et al., 2005). The length-weight relationships, morphometric and meristric features indicated positive allometric growth in some other crabs and a positive isometric growth in some other crabs (Duarte, et al., 2008). Omolara and Barakar (2009) documented the biology of swimming crab, Portunus validus. The reproductive biology of the blue crab Callinectes aminicola showed size difference between male and female (Omolara, 2010). Though information on morphomentric study are available an other crustaceans, there is a very little work on morphometric studies of genus Charybdis. Hence the present study is aimed to observe the basic aspects of morphometric of Charybdis natator from Nagapattinam, south east coast of Bay of Bengal.

3.6.2. Materials and Methods Marine crabs, Charybdis natator, were collected randomly from Nagapattinam coast. The collected specimens were kept in ice box and brought to the laboratory. The male and female sexes were identified based on the method of Barnes (1974). For morphometric studies the carapace length (CL) carapace width (CW) chelate leg length (CLL) chelae length (CHL) last leg length (LLL) abdominal length (AL) and abdominal width (AW) were measured using a vernier caliper. The width of the carapace was chosen as the basic character. All morphometric characters were analysed in the method of Frith and Brunemeister (1983).

3.6.3. Statistical analysis The total body weight (BW) of the crab was measured to the nearest 0.1g using a digital balance. The length-weight relationship was estimated using the equation W = a CWb where, W is total body weight of the crab (g) and CW is carapace width (mm). An „a‟ is intercept and „b‟ is the slope. The values of „a‟ and „b‟ were computed from the log transformed values of length and weight. i.e. Log W = log a + b log CW. The data were analysed statistically by means of least squares linear regression Y = a + bx.

3.6.4. Result The morphometric measurements of Charybdis natator are represented graphically in fig. 15-20. A total of 327 crabs, 152 males and 175 females were used in the present study. The regression equation 57 expressing the relative growth relationship is summarized in Table 6. From the data, the largest male had a carapace width of 130mm and the largest female with the carapace width 127mm were recorded. The smallest crabs recorded in the male had 54mm and in female 50mm. The maximum body weight, 270g in female and 355g in male was recorded. In the minimum weight group 40g was recorded in both male and female.

In C. natator there was no significant variation in weight between the male and female. However, the berried females showed slight variations due to the presence of egg mass in their abdomen. All relationships showed simple isometric growth, while only the plot of the abdominal width of the female showed a distinct discontinuity. There was a significant relationship between the relative growth of carapace width and carapace length in both the sexes.

There was a significant relationship between the relative growth of carapace width and abdominal length in both male and female. The propodus length of the left and right chelae was more or less same in the male and female of C. natator. The relative growth of chelipeds length and chelae length showed no significant differences. Similarly, there was no significant difference between relative growth of the propodus length between male and female. The right and left fifth legs in male and female were similar.

The regression equation for relative growth of carapace width and body weight, carapace width and carapace length, carapace width and abdominal length, carapace width and abdominal width, carapace width and chelipeds length, carapace width and last leg and chelipeds length and chelae length are represented as Y= a + bx.

3.6.5. Discussion The relative growth parameters measured in Charybdis natator are isometric. The carapace width and carapace length, carapace width and abdominal length are isometric in both the sexes. The propodus lengths of the left and right chela are more or less same. The abdomen is broader in female than in male shows sexual dimorphism. The abdominal width of the female shows an alternation in the growth rate. The increase in the abdominal width enables the female to carry and protect the eggs. Similar observations were reported by earlier workers (Halay, 1969 & 1973; Du Preez and Mclachlan, 1984; Haefner, 1990; Chu, 1999; Kannathasan and Rajendran, 2010b).

Haley (1969 and 1973) found distinct sexual dimorphism in abdominal width and increased rate of growth of the major chela in male Ocypode quadrata. The increase in width of the female abdomen showed sexual dimorphism. The relative growth of carapace width, carapace depth, eyestalk length and total length of the third ambulatory leg was isometric with no significant puberty related changes in Ocypode quadrate. The relative growth is both isometric and autometric equations. 59

If two dimensions maintain a constant ratio of size they are growing isometrically while the logarthimic values for these two dimensions are linear, they are growing allometrically. Further comparing the relative growth within genus Ocypode it varies between species.

According to Hartnoll, (1972 and 1978) in the burrowing crab Corystes cassivelaunus, the relative growth of the first pleopod, abdomen and propodus of the chela showed distinct differences between pre and post puberty animals and other parameters growing allometrically. In Ovalipes punctatus, the relative growth parameters are isometer. The propodus lengths of the left and right chela are the same, while propodus of the male chela is larger than that of female (Du Preez and Mclachlan, 1984a). In Ovalipes catharus males showed pronounced growth of the larger cheliped in the adult phase, while female whose abdominal width showed a high growth rate (Davidson and Marsden, 1987).

According to Sumpton (1990a) in Charybdis natator, positive allometry of the chela length was noticed in both the sexes. However in Portunus validus exhibited negative allometric growth in both sex (Omolara and Barakar (2009). In fiddler crabs Uca, the claws showed allometric trends in both shape and size (Rosenberg, 2002). The reduction of allometry level of the chelipeds may be related to the necessity of male to maintain body balance and which can prevent cheliped malformation (Masunari et al., 2003). Variation in sexual maturity depends on environmetal condition. In Callinectes pattidus and C. armatum the level 60 of allometry did not vary significantly at one site whereas it increased from juvenile to adult phase at other site (Oriola et al., 2005).

In Ucides cordatus, the carapace width and carapace length relationship was negatively allometry for male and isometric for female indicating the sexual dimorphism (Dalabona et al., 2005). According to Bello-Olusoji et al., (2006) the length weight relationship of Portunid crabs, Callinectes pallidus, was allometric. The presence of a highly developed chela in male Cardisoma guanhumi indicates sexual dimorphism between males and females (Duarte et al., 2008). In Uca rapax the relationship between carapace width and major cheliped length showed positive allometry in juveniles and negative one in adult male, but the females showed allometric positive growth in carapace width and abdomen width (Costa and Soares-Gomes, 2008). In Sudanonautes africanus, there was a strong correlation between the variables and strong relationship between the length and weight of male and females (Bello- Olusoji et al., 2009). In Callinectes amnicola, size difference was observed between male and female crabs (Omolara, 2010). In the present study, Charybdis natator both the male and female having propodus of chela are more or less the same size. The relative growths of carapace width and propodus length of chela are significant. 61

4. REPRODUCTIVE BIOLOGY

4.1. Reproductive and Nutritional cycles 4.1.1. Introduction The breeding pattern of marine invertebrate exhibits several types of breeding cycle (Stephenson, 1934). Giese (1959) formulated the definition the reproductive cycle, as a series of events from the time of activation of gametes until the spawning of the gametes and the recession of the gonadal activity until relatively resting level and including the resting period. The gametogentic activity of invertebrate organism could be either the result of a series of endogenous events such as hormones, biosynthesis of protein which build up inside the organism or the result of the operation of exogenous factors, the environment temperature, salinity, pH, photoperiod and food availability. The reproductive and nutritional cycles of marine crustaceans have been extensively studied by many workers (Chhapgar, 1957; Giese et al., 1958; Nagabhushnam and Kulkarni, 1977; Franz, 1986; Greco et al., 2000; Kannathasan and Rajendran, 2011b).

The reproductive and nutritional cycles of marine invertebrates is influenced by both exogenous and endogenous factors (Nagabhushnam and Faroogii, 1982; Rabalais and Cameron, 1985). Menon (1952) observed the annual breeding pattern in marine crab Neptunus sanguinolentus from the west coast of India. Prasad and Tampi (1953) reported that the crab Neptunus pelagicus breed intensively from 62

September to March. The reproductive cycle of five west coast crabs Pachygrapsus crassipes, Hemigrapus nudus, Pugettia producta, Emerita analoga and Petrolisthes cinctipes have been made by Boolootian et al., (1959) at Montery Bay. The normal breeding season for Rhithropanopeus harrisii was extended from May to September at North Carolina further the salinity and temperature influence on larval development (Costlow et al., 1966).

In tropical waters studies on the reproductive and nutritional cycles in relation to breeding periodicities of marine crabs Emerita protoricensis and Mysidium columbiae (Good body, 1965) Portunus pelagicus (Rahaman, 1967; Pillai and Nair, 1971; Radhakrishnan, 1979; Sumpton et al., 1994; Rajamani and Manickaraja, 1998) Charybdis variegate (Chandran, 1968) Portunus sanguinolentus (Radhakrishnan, 1979; Rasheed and Mustaquim, 2010). Charybdis cruciata, Charybdis hoplites pusilla, Scylla serrata, Mutate lunarus, Dorippe astute, Uca annulipes and Uca marionis nitidus (Pillai and Nair, 1973a) Barytelphusa cunicularis (Diwan and Nagabhushnam, 1974) Thalamita chaptali and Portunus spinipes (Sethuramalingam et al., 1980). Paratelphusa hydrodromous (Chandran et al., 1980; Kumara Pillai, 1981) have been reported.

Subramanian (1977) studied the sexual reproduction of the sand crab Emerita asiatica in the sand beaches of Madras coast, Bay of Bengal with reference to sexual differentiation, sex ratio, mating behavior and 63 spermatophore. Du Preez and Mclachlan (1984b) documented biology of the three spot swimming crab Ovalipes punctatus. Semiannual breeding pattern in burrowing sand crab Albunea symmysta have been observed (Subramanian and Panneer Selvam, 1985). The crab Rhithropanopeus harrisii breed intensively from April to September (Goy et al., 1985) at Neuse river estuary. Continuous breeding pattern in crab Ocypode macrocera have been observed (Nageswara Rao et al., 1986).

Recently the reproductive biology of Golden crab Geryon fenneri (Erdman and Blake, 1988), sand crab Sesarma intermedia (Kyomo, 1988), mud crab Scylla serrata (Prasad and Neelakantan, 1989a) marine crab Charybdis feriata (Rajendran, 1990) field crab Oziotelphusa senex senex (Dayakar and Ramana Rao, 1992), and other crabs Charybdis affinis (Chu, 1999) Thalamita crenata (Sigana, 2002a) have been studied.

The reproductive biology and relative growth in the spider crab Maja crispate have been made (Cormona-Suarez, 2003). The reproductive biology of mud crab Scylla serrata of the Sundarbans mangrove ecosystem (Ali et al., 2004) and tropical abalone Haliotis varia from Gulf of Mannar (Najmudeen and Victor, 2004) have been reported. Brante et al., (2004) observed different types of breeding patterns among the five species of brachyuran crabs at Chilean coast. The reproductive cycle of the crab Chasmagnathus granulatus was studied by (Ituarte et al., 2006).

Bezerra and Mathews (2007) studied population and reproductive biology of fiddler crab Uca thayeri from a tropical mangrove. Population and reproductive biology of the crab Uca burgersi in three subtropical mangroves have been reported (Benetti et al., 2007). Doi et al., (2008) observed the growth and reproduction of the portunid crab Charybdis bimaculata at Tokyo Bay. Population biology of the burrowing crab Neohelice granulate from a tropical mangrove has been studied (Gregati and Fransozo, 2009). The breeding cycle of Barytelphusa cunicularis (Pathre and Meena, 2010), Pagurus exilis (Terossi et al., 2010), Callinectes aminicola (Omolara, 2010) have also been reported.

It is known that the environment factors play a role on the reproductive cycle. The habitat influences on the reproductive cycles of the decapod crustaceans have been reported by many workers (Prasad and Tampi, 1953; Pearse, 1965; Rahaman, 1967; Radhakrishnan, 1979; Joel and Raj, 1980; Prasad and Neelakantan, 1989; Gregati and Fransozo, 2009). The relationship between rainfall and breeding activity in invertebrate animals, especially crustaceans Emerita asiatica (Subramanian, 1977), Barytelphusa cunicularis (Diwan and Nagabhushnam, 1974), Uca thayeri (Bezerra and Mathews, 2007), hermit crab Pagurus exilis (Terossi et al., 2010) have been observed. Hartnoll et al., (2010) observed that there was a close relationship between rainfall and the breeding activity of land crab Johngarthia lagostoma.

The temperature fluctuation in the sea influenced the reproductive cycle of marine organisms. The temperature in the sea shore may differ very much more than in deep waters but never as much as on land. The marine organisms living in bodies of water with high heat capacity essentially suffer against extremes of temperature because of their size (Giese, 1959; Brante et al., 2004; Ituarte et al., 2006). Orton (1920) proposed that temperature is the most important factor which regulates the breeding of marine invertebrates. Yonge (1940) observed that the spawning of marine species, at low Islets of Great Barrier Reef was influenced by temperature fluctuation. Kinne (1970) stated that the temperature and photoperiod influenced the reproduction of invertebrates animals. The effect of temperature and salinity on the gonad maturity in marine crabs Portunus pelagicus and Portunus sanguinolentus (Radhakrishnan, 1979). Charybdis feriata, Charybdis lucifera (Rajendran, 1990), Betaeus truncatus (Lardies and Wehrtmann, 2001), Macrophthalmus boscii and Uca chlorophthalmus (Litulo, 2005 and 2006) Chasmagnathus granulatus (Ituarte et al., 2006), Charybdis bimaculata (Doi et al., 2008), have been reported. The temperature, salinity, photoperiods influences the reproductive capacity of decapod crustaceans (Wenner et al., 1974; Rabalais and Cameron, 1985).

The salinity of sea water is one of the important abiotic factors having remarkable influence on the reproductive cycle of marine organism (Giese, 1959). An increase or decrease in salinity could be related to the breeding of marine and estuarine crabs (Pillai and Nair, 1971; Joel and 66

Raj, 1980; Nagabhushnam and Faroogii, 1982a). The influence of photoperiod on processes associated with molting and reproduction in Cray fish Orconectes nais (Rice and Armitage, 1974) crab Scylla serrata (Nagabhushnam and Faroogii, 1981) and Procambarus clarkia (Daniels et al., 1994) have been observed. Though there are many investigations on reproductive and nutritional cycle of marine crab, the work related to reproductive and nutritional cycles of crabs from south east coast of Bay of Bengal, is meagre. Hence, the present study is aimed to observe the reproductive and nutritional cycle of marine crab Charybdis natator in Nagapattinam, the south east coast of Bay of Bengal, India.

4.1.2. Materials and Methods For the present study, the brachyuran crabs Charybdis natator were collected monthly throughout the study period. The crabs were sexed and berried females were recorded separately. Care was taken to collect the crabs alive from the landing centre at Nagapattinam to the field station. They were kept in the mud pot with sea water which was aerated.

The animals were sacrificed and the colour of the gonads was observed, then the gonad and hepatopancreas were removed after taking the wet weight of the animal. The tissues of gonad and hepatopancreas were also weighed separately to the accuracy of milligrams. Several methods have been used to differentiate the period of maturation of testes and ovaries and hence the breeding activity of the animal. Some investigators have actually observed the liberation of gametes by the 67 animals and the inferences were made on the breeding pattern (Giese et al., 1958). Another method was to collect and count larvae, their abundance during different seasons (Menon, 1952). Third method was the microscopic examination of gonads as observed (Kerr, 1969; Dhas et al., 1981). Fourthly, the presence of eggs in brood pouches attached to swimmers has been exploited for determining the breeding season (Boolootian et al., 1959).

Fifthly, the gonad index has been used as the main criterion for determining the reproductive period of invertebrates. The gonads show cyclic morphological changes as seen by full growth, maturation and depletion. A significant change takes place in the size and colour of the gonads throughout the year. The body size and effect of it on the size of the gonads are correlated. The gonad index is often used to represent gonadal development or gonadal activity. The gonad index was obtained by several methods by many workers. Moore (1934) used the ratio of the gonad volume to the body volume multiplied by 100 as the gonad index while other investigators have taken the volume of gonad divided by the weight of the animal and multiplied it by 100 (Giese et al., 1958; Pearse, 1965). The method employed in the present investigation is somewhat modified and is similar to the one used by Kowalsky (1955) because it has been possible to discover the minor periods of activity besides the major reproductive periods. In this method the gonad index was obtained by dividing the weight of the gonad by the weight of the animal, multiplied by 100. This method has been used by several investigators (Giese, 1959; 68

Rahaman, 1967; Pillai and Nair, 1971; Chandran et al., 1980; Joshi and Khanna, 1982).

The method employed in determining gonad index in Ovalipes punctatus is described as wet mass of the egg expressed as a percentage of the total wet mass of the animal and gonadosomatic index is calculated as the percentage of wet weight of the gonads expressed as the percentage of total wet weight of the animal (Du Preez and Mclachlan, 1984b).

The weight of the hepatopancreas in relation to body weight of the crab is expressed as „hepatic index‟ this method was used to calculate hepatic index in Portunus pelagicus (Rahaman, 1967). However the nutritional cycle in Paratelphusa hydrodromous and P. Jacquementii was described as hepatopancreatic index (Chandran et al., 1980). In the present investigation the gonad and hepatic indexes were calculated by using the formula. Wet weight of the gonad Gonad index = 100 Wet weight of the animal

Wet weight of the hepatopancreas Hepatic index = 100 Wet weight of the animal

For the present investigation, the breeding season of the crabs was determined by plotting the percentage of ovigenous females against time (Subramanian, 1977). The various phases of the reproductive activity of 69 the animal were determined by observing colour changes in the ovary (Kerr, 1969) and by applying gonad index method (Giese, 1959; Rahaman, 1967; Chandran et al., 1980). The data were analysed statistically by mean and range.

4.1.3. Result The gonad and hepatic indexes of Charybdis natator are represented graphically in fig. 21 and 22.

4.1.3.1. Gonad index (GI) It is clear from the data that the gonad index in female Charybdis natator was maximum (3.921) in July and minimum (2.679) in May during 2009 and high (3.959) in July and low (2.675) in May during 2010. In the case of male it was high (1.161) in July and low (0.495) in May 2009 and the maximum value (1.157) in July and minimum value (0.501) in May 2010. There was a no significant variation in the male gonad index as against the female gonad index in all the months.

Among the population of C. natator collected every month, berried females occurred throughout the year in varying percentage. It is evident that the crab C. natator breeds continuously throughout the year. However, during July and December in both the year 2009 and 2010 the highest value of gonad index have been recorded suggesting the peak period of breeding activity (Table 7).

4.1.3.2. Hepatic indes (HI) In female C. natator, the hepatic index was found to be maximum (3.771) in November and minimum (2.398) in December 2009 and it was high (3.796) in November and low (2.472) in July 2010. In male the hepatic index exhibited an increasing trend (3.868) in June and a steep fall (2.759) in July 2009 and maximum (3.675) was recorded in November and minimum (2.772) in July 2010. In both the sexes the highest hepatic indices were recorded in the previous months of maximum gonad index and minimum exhibited during the peak breeding periods. In other words the highest hepatic indices are seen just before the peak breeding periods but the hepatic indices fall down during the month of peak breeding periods (Table 7).

4.1.4. Discussion In the present study an estimation of gonad index indicates that the crab C.natator is the continuous breeder breeding around the year. Further, a fall in the hepatic index coincides or just follows a rise of gonad index which indicates the possible utilization of reserved nutrients from hepatopancreas to maturing gonads. The clear cut inverse relationship exhibited by the hepatic index to that of the gonadal index suggests the possible transport of nutrients from hepatopancreas to gonads. The estimation of the gonad index and the hepatic index showed that the crab C. natator is the continuous breeder with two breeding peaks in a year. Similar observation have been reported by earlier workers (Menon, 1952; Pillai and Nair, 1973a; Nagabhushnam and Faroogii, 1982a; 71

Kyomo, 1988; Sumpton et al., 1994; Gregati and Fransozo, 2009; Kannathasan and Rajendran, 2011b).

Stephenson (1934) found that the tropical marine organisms exhibit several types of breeding cycle such as continuous breeders, around the year, biannual breeders, two spawning periods in a year and annual breeders with single breeding season. The crab Emerita protoricensis and Mysidium columbiae from Jamaica coast breed continuously (Good body, 1965). Portunus pelagicus breeds continuously with three peaks (Rahaman, 1967). Charybdis variegate breeds biannually with two distinct peaks (Chandran, 1968). Portunus pelagicus and Portunus sanguinolentus breed continuously around the year with two or three breeding peaks (Radhakrishnan, 1979; Rajamani and Manickaraja, 1998). Albunea symmysta in Bay of Bengal breed continuously with two distinct peaks (Subramanian and Panneer Selvam, 1985). The crab Ocypode macrocera breeds almost continuously throughout the year with higher breeding activity during certain months (Nageswara Rao et al., 1986).

The mud crab Scylla serrata breed continuously with two distinct peaks one in December to March and another in September to November (Prasad and Neelakantan, 1989a). Charybdis feriata breeds continuously with two breeding peaks one in July and another in December (Rajendran, 1990). The gonad development rate indicated that the crab Uca burrgerisi was reproducing continuously (Benetti et al., 2007).

However the other type of breeding pattern was also observed in some crustaceans. The annual breeding patterns have been recorded in Asterias forbesi (Franz, 1986). Barytelphusa cunicularis (Diwan and Nagabhushnam, 1974), Paratelphusa hydrodromous (Kumara Pillai, 1981), Geryon fenneri (Erdman and Blake, 1888) and Oziotelphusa senex senex (Dayakar and Ramana Rao, 1992). The marine species Portunus spinipes in Bay of Bengal breeds biannually while in the back water species of Thalamita chaptali breeding extends over several months of the year from February to September (Sethuramalingam et al., 1980). The freshwater crab Paratelphusa hydrodromous, and P. jacquement inhabiting in tropical region breed annually seems to have single breeding cycle (Chandran et al., 1980). In crab Maja crispate breeding season appeared to extend from May to September (Cormona-Suarez, 2003). In Pagurus exilis different types of breeding pattern was observed in two different geographical distributions (Terossi et al., 2010).

Studies on Portunus pelagicus (Rahaman, 1967) Charybdis variegate (Chandran, 1968) Uca annulipes and Portunus pelagicus (Pillai and Nair, 1971) Charybdis feriata (Rajendran, 1990) Charybdis affinis (Chu, 1999) Scylla serrata (Ali et al., 2004) and Haliotis varia (Najmudeen and Victor, 2004) indicated that ther was a relationship between hepatopancreas and gonads and the possible utilization of nutrients from hepatopancreas to gonads which confirmed the present study. The berried female was encountered throughout the year, in varying percentage clearly indicating that the marine crab C. natator breeds 73 throughout the year, with two distinct breeding peak periods. In Microphrys bicornutus ovigerous female were encountered throughout the year (Greco et al., 2000). In Thalamita crenata the continuous breeding was inferred from the percentage of ovigenous females (Sigana, 2002a). In Charybdis bimaculata ovigerous females were found in all the seasons except in winter (Doi et al., 2008). In Barytelphusa cunicularis ovigerous females were found throughout the year (Pathre and Meena, 2010). In Portunus sanguinolentus berried females were found in all the month except in June (Rasheed and Mustaquim, 2010).

According to Giese et al., (1958) the growth and maturation of gonads is said to be associated with certain physico chemical conditions such as temperature, salinity, pH, photoperiod, rainfall, abundance of food etc. The organism synchronises its reproduction with the favourable environmental conditions which thus can influence the reproductive process. The habitat plays an important role in the breeding behaviour of marine crustaceans as documented by many investigators (Prasad and Tampi, 1953; Litulo, 2006; Bezerra and Mathews, 2007; Omolara, 2010).

According to Boolootian et al., (1959) in tropical water, the annual reproductive cycles of Pachygrapsus crassipes, Hemigrapsus nudus, Pugettia producta, Emerita analoga and Petrolisthes cinctipes were varied as they live in the same habitat of Montery Bay. Pearse (1965) stated that latitude is an important factor in tropical species, it is found the reproductive activity of in Asteroid echinoid and holothuroid. The 74 breeding season of Rhithropanopeus harrisii in North Carolina was extended from May to September (Costlow et al., 1966) but the same species in Neuse river estuary, North Carolina breeds from April to September (Goy et al., 1985). Ovalipes punctatus showed two main breeding peaks covering a short period during summer and a more extensive period during winter (Du Preez and Mclachlan, 1984b). Geryon fenneri and holthuis breed annually from the south eastern coast (Erdman and Blake, 1988). Brante et al., (2004) observed different types of breeding patterns among the five species of brachyuran crabs.

In tropical water, the crab Neptunus sanguinolentus from the west coast of India breeds annually (Menon, 1952). The Portunus pelagicus in Bombay water breeds irregularly (Chhapgar, 1957). However Portunus pelagicus in Bay of Bangal breeds continuously around the year with two or three breeding peaks in a year (Radhakrishnan, 1979). According to Pillai and Nair (1971) the Portunus pelagicus in the south west coast of India, did not breed around the year but it extends to several months of the year. The tropical anomuran mole crab Emerita asiatica (Subramanian, 1977) and Emerita holthuisi (Nagabhushnam and Kulkarni, 1977) in the Bay of Bengal breed throughout the year. Nageswara Rao et al., (1986) observed that marine crab Ocypode macrocera from Vishakapatnam coast breeds continuously throughout the year.

Temperature is an important exogeneous factor influencing the reproduction of marine invertebrates. It is generally though that the marine 75 animals of temperate and cold water reproduce seasonally while those of tropical water breeds throughout the year (Orton, 1920). The tropical marine species usually exhibit reproductive periodicities having little relation with temperature fluctuation (Giese, 1959). According to Yonge (1940) the spawning of marine species at the low Islet of Great Barrier Reef were influenced by temperature fluctuations.

The temperature has some influence in regulating the annual reproductive cycle. Increased water temperature accelerated the maturity and egg laying in cray fish Orconectus virilis (Rahaman, 1967). In C. feriata an intensive breeding activity was found in July when a maximum temperature was recorded, indicating temperature influence on reproductive cycle (Rajendran, 1990). The temperature has been mentioned as one of the causes of the latitudinal change in the reproduction of Betaeus truncatus (Lardies and Wehrtmann, 2001).

According to Litulo (2005 and 2006) temperature is the main factor governing the breeding of Macrophthalmus boscii, Pilumus vespertilio, Uca annulipes and Uca chlorophthalmus. The breeding of estuarine crab Chasmagnathus granulatus could be related to higher temperature (Ituarte et al., 2006). In the present study temperature in summer was recorded maximum when one intensive peak was observed in Charybdis natator.

In the present investigation salinity at Nagapattinam coast varied from 26.98 to 35.12ppt. It was found to be maximum in summer and 76 minimum in monsoon. The maximum gonad index occurred when the salinity was increased during summer season. However, during winter months a second breeding peak is evident when the salinity was low. Similar observation was reported in Scylla serrata and Scylla tranquebarica (Joel and Raj, 1980). Fair and high saline conditions with plenty of planktonic food for the larvae are said to be favorable for the breeding activity in number of marine invertebrates (Giese, 1959). The planktonic larvae released at that time would definitely have a better rate of survival. The reduced salinity with sparse distribution of planktonic food was found to restrict breeding (Pillai and Nair, 1971). In Charybdis feriata the maximum gonad index occurred, when the salinity was increased during summer months. However, during winter months a second breeding peak evident when the salinity was low (Rajendran, 1990). In Betaeus truncatus salinity influenced the reproductive activities (Lardies and Wehrtmann, 2001).

In the present study at Nagapattinam coast a slightly less photoperiod during monsoon and post monsoon seasons and high during summer and pre monsoon seasons. The photoperiod was prolonged in summer months when the maximum gonad index recorded. The earlier reports confirm the results of the present investigation (Kinne, 1970; Wenner et al., 1974). According to Wenner et al., (1974), the reproductive capacity of a female is related to environmental photoperiod. The photoperiod and constant light increase was associated with moulting and reproduction in the cray fish Orconectes nais (Rice and Armitage, 1974) 77 and increased gametogenesis in the Scylla serrata (Nagaphushnam and Faroogi, 1981). Rabalais and Cameron (1985) stated that in Uca subcylindrica photoperiod influences the reproductive period of the species. The photoperiod induces the breeding activities of crab Procambarus clarkia (Daniels et al., 1994)

The relationship between rainfall and the breeding activity have been reported by many workers (Diwan and Nagabhushnam, 1974; Bezerra and Mathews, 2007; Hartnoll et al., 2010). In the present study, the coast of Nagapattinam, got heavy rainfall during monsoon season exactly the month of November. One peak of breeding activity of Charybdis natator was observed in monsoon month. Similarly, in Barytelphusa cunicularis breeding activity was found to be dependent on rainfall (Diwan and Nagabhushnam, 1974). In Uca chlorophthalmus reproductive patterns with peaks of breeding from March to December fluctuate according to the rainfall (Litulo, 2006). In Johngarthia lagostoma breeding, migrations extend from January to May, with peak migration in March related to rainfall (Hartnoll et al., 2010).

In the present investigation, the influence of environmental factors like rainfall, temperature, salinity and photoperiod, on the reproduction cycle revealed that Charybdis natator breed antagonistically. When the temperature, salinity and photoperiod recorded high in summer, the gonad index was maximum in the crab. Another peak of reproductive activity was found when there was heavy rainfall and minimum temperature, 78 salinity and photoperiod during monsoon. It is inferred that the crab breed intensively in the antagonistic environmental conditions, when the fluctuation in rainfall, salinity, temperature might be the triggering mechanism for the breeding pattern.

4.2. SEX RATIO

4.2.1. Introduction In sexually reproducing organisms, there are two types of individuals namely male and female. The information recording sex ratio of decapod crustaceans have been reported by many workers (Thampson, 1951; Prasad and Tampi, 1953; Kannathasan and Rajendran, 2011c). In a population of brachyuran crabs equall number of males and females are normally expected. In most of the species, the sex ratio is slightly deviated from the expected ratio or not significantly varied from the expected 1: 1 ratio (Radhakrishnan, 1979; Sethuramalingam et al., 1980; Litulo, 2005 and 2006). However, in some species the sex ratio is deviated from the expected ratio of 1:1 (Asakura, 1995; Ali et al., 2004). The differences in sex ratio and differential growth rate are influenced by many factors (Sigana, 2002a and b; Mzighani, 2005; Teixeira et al., 2009; Omolara and Barakar, 2009; Omolara, 2010).

The deviation in sex ratio is mainly due to migration of one sex and utilization of different habitats by the two sexes (Boolootian, 1965; Du Preez and Mclachlan, 1984b). The environmental factors such as 79 temperature, salinity, rainfall, water current and pH are also responsible for an apparent alternation of sex ratio (Wenner, 1972; Krajangdara and Watanabe, 2005; Oriola et al., 2005). The present study deals with sex ratio of marine crab Charybdis natator from Nagapattinam coast.

4.2.2. Materials and Methods Monthly random collection of male and female brachyuran crabs Charybdis natator were collected from the off shore region of Nagapattinam coast for a period of two years. A total of 1748 crabs were examined for the present study. Sex was determined using the method described (Barnes, 1974 and Kwei, 1978). The overall sex ratio was obtained using the χ2 test (King, 1997; Zar, 1999).

No. of the female crabs Sex ratio = No. of the male crabs

(O – E) 2 X2 = E

4.2.3. Result In the present investigation of the sex ratio of marine crab C. natator was thoroughly studied and the results are given in the Table 8. From the results it clearly indicates that there was a slight variation in the male and female number. Among the total 1748 crabs, the males were 864 and females were 884. The overall sex ratio of male and female was found to be 1:1.01 ratio. The chi-square ( 2) test analysis indicated that the value 80 has slightly deviated from the expected 1:1 ratio in both the years. The male individuals were abundant in post monsoon and monsoon seasons in the year 2009 and post monsoon and pre monsoon seasons in the year 2010. The females were dominant in summer and monsoon months in the year 2009 and 2010. In both the years, males were predominant in post monsoon month and females were found to be predominant in monsoon months when peak breeding occurred. The variation in sex ratio mainly depends on the migration of crabs for feeding and breeding (Fig. 23).

4.2.4. Discussion The occurrence of male and female individuals in a population of a particular species depends on many factors. Environment factors either directly or indirectly influence the sex ratio. In the present study, in marine crab, C. natator, there was a slight deviation observed in male and female number. The chi-square value for sex ratio had slight deviation from the expected 1:1 ratio. Similar observations were reported by earlier workers (Radhakrishnan, 1979; Sethuramalingam et al., 1980; Kannathasan and Rajendran, 2011c).

Boolootian (1965), while working on the crab Pachygrapsus crassipes found that during the reproductive season, ovigerous females migrated towards the water, whereas during non-reproductive season males and females were equally distributed. Differential normality between sex ratio and differential growth rates depended on migration of one sex and utilization of different habitats (Wenner, 1972). In marine 81 crab Portunus sanguinolentus, the sex ratio showed a slight deviation from the expected sex ratio of 1:1 whereas in Portunus pelagicus, the sex ratio was the nearest to expected 1:1 ratio (Radhakrishnan, 1979).

According to Sethuramalingam et al., (1980) in Thalamite chaptali and Portunus spinipes the sex ratio did not deviate from the expected 1:1 ratio. In Ovilipes punctatus, the number of females was significantly greater than the number of males (Du Preez and Mclachlan, 1984). In hermit crabs, sex ratio showed deviation from the expected 1:1 ratio. The females were predominant under natural condition because, males have a higher mortality due to their more intense competition for shell and mates (Asakura, 1995). In Thalamita crenata, the sex ratio was significantly deviated from the expected ratio 1:1 (Sigana, 2002a). In Scylla serrata the sex ratio was not significantly different from expected ratio of 1:1 (Sigana, 2002b). Ali et al (2004) observed an uneven number of males and females in mud crab Scylla serrata. The monthly sex ratio of Ranina ranina varied between 1:0.56 and 1:2.77 which was deviated from the expected ratio (Krajangdara and Watanabe, 2005). In Cardiosoma armatum and Callinectes pallidus the sex ratio showed a slightly deviated from the expected ratio (Oriola et al., 2005).

In Uca annulipes and Uca chlorophthlmus (Litulo, 2005 and 2006), there was a no significant differences in the sex ratio. In Callinectes amnicola, the sex ratio was found to be 1:1. 96 and not significantly differed from the expected ratio of 1:1 (Omolara, 2010). However, there 82 was a significant deviation observed in the sexes of Portunus pelagicus (Thampson, 1951; Prasad and Tampi, 1953). In crab Anadara antiguata (Mzighani, 2005), Portunus validus (Omolara and Barakar 2009) and Acanthonyx scutiformis (Teixeira et al., 2009) a significant deviation observed in male and female populations.

In the present study, a slight deviation in sex ratio was observed for 3 or 4 month in 2009 and 2010. The females were predominant during intensive breeding periods. During other months the female probably migrate to deeper water, resulting in the predominance of males causing change in the expected sex ratio. Thus it is inferred that a slight variation in sex ratio could be attributed to the migration of females in relation to breeding activity.

4.3. FECUNDITY

4.3.1. Introduction Fecundity study deals with the reproductive potentiality or egg production capacity of an organism or population. Fecundity is one of the most important parameters in studying the breeding pattern, reproductive strategies and potentiality of egg production (Efford, 1969; Heydson, 1969; Lopes et al., 2009). The study of fecundity provides information on the rate of replacement in natural population (Montes et al., 1987; Sumpton, 1990b). In crabs, fecundity is traditionally measured as the number of eggs produced in each clutch, and it is described as a function 83 of body size (Litulo, 2004; Morley et al., 2005; Kannathasan and Rajendran, 2010c). The number of eggs produced by females depends on many exogenous and endogenous factors. The total number of eggs per female significantly varies with reference to habitat (Barnes and Barnes, 1968; Sethuramalingam and Natarajan, 1982; Thurman, 1985; Wenner et al., 1987; Simon et al., 2003)

In most of the brachyuran crabs, the number of eggs increase with increasing carapace length and width. Fecundity was positively correlated to carapace length and body weight of the animal (Bird, 1978; Radhakrishnan, 1979; Du Preez and Mclachlan, 1984b; Haddon, 1994; Alexander and Fosca, 2001; Litulo, 2006; Alison et al., 2006; Tallack, 2007; Doi et al., 2008; Bello olusoji et al., 2009; Omolara, 2010). The fecundity of marine crabs vary to seasons (Jeffrey et al., 1991; Kobayashi 2001; Bas et al., 2007). The fecundity and brood size were highly correlated with carapace width (Arshad et al., 2006; Halawa, 2006; Rasheed and Mustaquim, 2010).

According to Fernando and Adilson (1997), the females of the same class have a wide amplitude of variation in total number of eggs. However, very low variation on fecundity was observed within the same size class (Litulo et al., 2005). The lack of relationship was noticed between environmental features and fecundity (Bas et al., 2007). The present study summarises the relationship between size class and number 84 of eggs in the marine crab Charybdis natator collected from Nagapattinam on the south east coast of Bay of Bengal, India.

4.3.2. Materials and methods Berried females of Charybdis natator were collected from fish landing centre of Nagapattinam. The specimens were individually packed in plastic bags and transferred to laboratory. The length and width of the carapace were measured to nearest millimeter. The weight of the animals were noted and eggs were removed by forceps and blotted with filter paper to remove the excess of water. Then eggs were weighed as accurately as possible nearest to 0.01mg. The samples were taken at different places and the weight of the sample was noted. The fecundity was estimated by the method of Kwei (1978). From the weight of the egg mass, total number of the eggs present in the brood was calculated using the formula (Zar, 1999).

Number of egg F = Total egg mass Weight Weight of the sample

The carapace width was chosen as the main reference dimension. The correlation and linear relationship between carapace width and total numbers of eggs were also calculated.

Fecundity (F) was related to carapace width (CW) and Body

Weight (BW) by the least square linear regression. Log10F = a + b log (CW) or (BW) described by (Parsons, 1988).

4.3.3. Result The data obtained in the present study are graphically represented in (Fig. 24 and 25). From the data it shows that the number of eggs in C. natator was related to size group of the crabs. In the minimum size class of carapace width 50 to 59mm having the mean wet egg mass weight was 8.667g with a mean total number of eggs 1, 93,229. In the maximum size class of carapace width 110 to 126mm having the mean wet egg mass weight was 34.464g with a mean total number of eggs per female was 7,68,043. The mean total wet weight of the egg per females as well as the mean total number of eggs per female were increased with increasing carapace width. Fecundity (F) was related to carapace width (CW) and

Body weight (BW) by the equation Log10F = a + b Log (CW) or (BW) respectively (Table 9).

4.3.4. Discussion An actual increase in total number of eggs appears to be related to both carapace width and body weight of the animal. In the present study, in Charybdis natator the total number of egg increased with increasing carapace width. Fecundity was positively correlated with carapace width. However, in some cases the same size class, there was a slight fluctuation 86 in the total number of eggs. This crab is a continuous breeder and subsequent broods may possibly be liberalised within the spawning period. The number of eggs may be more in the first time and may show a tendency to decline in the subsequent brood. This may also be a reason for the fluctuations in the total number of eggs irrespective of size during breeding period. Similar observations were reported by earlier workers (Barnes and Barnes, 1968; Morley et al., 2005; Kannathasan and Rajendran, 2010c).

The number of eggs increase with increasing body size (Heydson, 1969). Efford (1969) reported widely different values for the number of eggs produced equivalent size group in Emerita analoga. In grapsid crab, Sesarma catenata may carry up to 92,000 eggs, with an increase in the number of eggs as the crab grows larger (Bird, 1978). In Portunus pelagicus and P. sanguinolentus, the numbers of eggs were variable in relation to the width of the carapace (Radhakrishnan, 1979). In Ovalipes punctatus, large female can produce more eggs per brood. The total number of eggs per female increases with the size of the female (Du preez and Mclachlan, 1984b). In Uca subcyllindrica comparatively large ova, low fecundity and low per capita egg production are adaptations to habitate (Thurman, 1985).

Wenner et al., (1987) observed that the number of eggs in Emerita analoga increased as the length or width of carapace increased. The log body size and log fecundity relationship changed significantly with 87 seasons (Jeffrey et al., 1991). However, according to Haddon (1994) there was no significant relationship noticed between mean egg size and carapace width. There was a positive correlation observed between fecundity and body size of Thalamita chaptali and Portunus sinipes (Sethuramalingam and Natarajan, 1982), Callinectes similis (Montes et al., 1987), Charybdis natator (Sumpton, 1990b) and Callinectes ornatus (Fernando and Adilson, 1997).

According to Kobayashi (2001), the fecundity increased with the increasing carapace width and decreased in the later ovi position. Alexander and Fosca (2001) stated that the fecundity of all species was positively correlated to the size of the individuals. The higher number of eggs was produced by Portunus pelagicus during the spawning season (Simon et al., 2003). In Uca annulipes, the eggs number increased significantly with the increase in crab size (Litulo, 2004). The fecundity was observed to increase with body size, but reproductive allocation was found to differ significantly between species (Morley et al., 2005). In Macrophthalmus depressus, low variation on fecundity was observed with same size class (Litulo et al., 2005).

In Portunus pelagicus, the total number of eggs was highly correlated to carapace width (Arshad et al., 2006; Halawa, 2006). In crab Limulus polyphemus, larger female laid a higher percentage of the eggs (Alison et al., 2006). In Uca chlorophthalmus, brood size was positively associated with female size of the crab (Litulo, 2006). Significant 88 relationship was found in size and fecundity of crab Cancer pagurus and Necora puber (Tallack, 2007). In Chasmagnathus granulatus, the fecundity and biomass per egg were higher at the beginning as compared to the end of the reproductive season (Bas et al., 2007). The batch fecundity of Charybdis bimaculata ranged from 8,300 to 38,400 eggs per female and was positively correlated with body size (Doi et al., 2008).

According to Lopes et al., (2009) increased fecundity as well as improved embryo quality depends on size of the crab. In crab Sudanonautes africanus, there was no significant relationship noticed between egg size and carapace length (Bello Olusoji et al., 2009). In Portunus sanguinolentus, the number of eggs ranged from 2,72,000 and 1,395,000 in crabs having 63mm and 120mm respectively (Rasheed and Mustaquim, 2010). In Callinectes amnicola, the fecundity was positively correlated with the body weight of the animal (Omolara, 2010). In the present study, in C. natator, an increase in width of the carapace thus increases the number of eggs which coincides with the above findings. Further it suggested that the ovigerous female of large size is suitable for brood stocd collection and maintenance for production of more number of seeds in aquaculture practice. 89

5. BIOCHEMICAL COMPOSITION

5.1. Indroduction Decapod crustaceans are the rich sources of animal protein and good for human health. The proximate composition of gonad, hepatopancreas and haemolymph of crabs significantly varied during reproductive and nutritional cycle. The biochemical compositions in various species of decapod crustaceans have been studied by many investigators (Rahaman, 1977; Zafar et al., 2004). Haemolymph calcium fluctuation as related to environmental salinity during ecdysis of the blue crab, Callinectes sapidus has been reported (Haefner, 1964). Dean and Vernberge (1965) stated that in Callinectes sapidus the blood glucose level increased just before the spawning, but decreased in animals carrying an egg mass. The seasonal changes in the biochemical composition of various tissues of Callinectes sapidus has been reported by Kerr (1969). Giese (1969) observed that the glycogen level of the gonad showed the lowest value during spawning and the level rose again after spawning. The protein, carbohydrate and lipid metabolism in gonads and hepatopancreas of freshwater crab Paratelphusa hydrodromous in relation to reproduction and moulting have been studied (Adiyodi, 1969; Adiyodi and Adiyodi, 1970; 1972).

Kessel (1968) observed the mechanism of yolk protein synthesis and deposition in crustacean oocytes. Eyestalk factor affected the lipid metabolism and protein synthesis in crustacean hepatopancreas 90

(O‟Connor and Gilbert, 1968). Heath and Barnes (1970) stated that in Carcinus maenas, there was no defined seasonal change in biochemical composition of the hepatopancreas during reproductive cycle. Badawai (1971) observed the chemical composition of meat and waste portion of the red crab Portunus pelagicus. Lipid transport in the dungeness crab Cancer magister have been studied (Allen, 1972). Pillai and Nair (1973b) stated that the change in the biochemical constituents in the testis were not so pronounced as in the ovary in Uca annulipes, Portunus pelagicus and Metapenaes affinis. It indicates that the mobilization of organic components from hepatopancreas to gonads during peak breeding season. The yolk spheres developed in the egg primarily thorough micropinocytotic uptake of lipovitelline from the haemolymph (Wolin et al., 1973).

According to Diwan and Nagabhushnam (1974) in Barytelphusa cunicularis biochemical constituents increased during stages of gonadal maturity advance and all values fall considerably at the end of spawning. Rahaman (1977) stated that in marine crab Portunus pelagicus, the lipid and carbohydrate are transferred from hepatopancreas to gonads as the animal matured but protein is synthesised in the gonads. The seasonal variation in the total lipid content of Chorismus antarcticus has been reported (Clarke, 1977). Senthilkumar and Desai (1978) studied the haemolymph cation concentration of crab Neptunus pelagicus and Scylla serrata. Changes in the proximate composition during the ovarian cycle 91 of hermit crab Clibanarius longitarsus have been studied (Ajmal Khan and Natarajan, 1980).

Lipid content in hepatopancreas and gonad during reproductive cycle of mud crab Scylla serrata have been studied (Nagabhushnam and Faroogii, 1982b; George and Gopakumar, 1987). The biochemical composition of various tissue of crabs Menippe rumphii (Shyamasundari and Erri Babu, 1984), Ocypode macrocera (Nageswara Rao et al., 1986), Scylla serrata (Balasubramanian and Subramanian, 1987; Prasad and Neelakantan, 1989b; Zafar et al., 2004), Charybdis feriata (Rajendran, 1990), Charybdis affinis (Vasconcelos and Braz, 2001), Eriocheir sinensis (Xiao et al., 2001), Platyxathus patagonicus (Dima et al., 2009), Portunus sanguinolentus (Ravichandran et al., 2009), and Charybdis natator (Kannathasan and Rajendran, 2010d and 2011d) during reproductive and nutritional cycles have been reported.

Invivo recovary of glycogen metabolism in haemolymph and tissue of a freshwater field crab Barytelphusa querini have been observed (Venugopal et al., 1990). Anil and Suseelan (2001) studied experimental fettening of the green mud crab, Scylla oceanica. The effect of melathion on haemolymph glucose and lactate levels in the fiddler crab Uca pugilator have been studied (Tilden et al., 2001). The effects of reduced salinity on the biochemical composition of zoea of crustaceans have been observed (Gabriela et al., 2002). Thirunavukarasu (2005) studied biology, nutritional evaluation and utilization of mud crab Scylla tranqueberica. 92

The effect of unilateral eyestalk ablation on the biochemical changes of edible portunid crab Charybdis lucifera have been reported (Murugesan et al., 2008). A comparison of protein content in the haemolymph of brachyuran crabs has been documented (Rameshkumar et al., 2009a). Manivannan et al., (2010) studied the effect of feed on the biochemical composition of commercially important mud crab, Scylla tranquebarica.

The physiological, biochemical and enzymatic changes during the moulting cycle of decapod crustaceans have been studied by many investigators (Adiyodi, 1969; Philippenn et al., 2000; Ernest and Chang, 2000; Vijayavel and Balasubramanian, 2006). In crabs, moulting is the growth phenomenon which occurs several times throughout their lives (Adiyodi, 1969). During moulting cycle biochemical changes take place in various tissues of crabs (Griffin, 1968). Bedford (1972) studied the biochemical composition of the blood of the graphid crab Helie crassonace. The changes in the chemical composition of the common shore crab Carcinus maenas, during the moulting cycle have been reported (Spindler-Barth, 1976). Kulkarni (1983) observed changes in the biochemical composition of the sand crab Emerita holthuisi during moulting cycle. Ecdysteroid level during the moult cycle of the blue crab resemble those of other crustaceans (Cynthia et al., 1983).

In Callinectes sapidus ionic regulation and ATPase activities during the moult cycle have been studied (David et al., 1985). The significance of the variation in haemolymph copper free and bound 93 protein during aging and time of day in the crab Scylla serrata have been reported (Arumugam and Ravindranath, 1986). Daw (1987) studied metal regulation and moulting in the blue crab Callinectes sapidus. Mark and Ernest (1991) observed ecdysteroid in relation to the moult cycle of the American lobster. The chitobiose activities in epidermis and hepatopancreas of the fiddler crab Uca pugilator during moulting cycle have been observed (Zou and Fingerman, 1999). Ernest and Chang (2000) observed physiological and biochemical changes during the moult cycle in decapod crustaceans. The moult cycle of the crab Cancer antonnarius have been studied by Spaziani et al., (2008). In crab, Portunus sanguinolentus, the protein, carbohydrate and lipid contents found to be higher in hard shell and low in soft shell crab (Sudhakar et al., 2009).

The enzyme activies in various tissues of decapod crustaceans have been extensively studied by many workers (Ramanibai, 1986 Devi et al., 1993; Valarmathi and Azariah, 2002 and 2003; Vijayavel and Balasubramanian, 2006; Senthilkumar et al., 2007; Kannathasan and Rajendran, 2011d; Sreenivasan et al., 2011). Hepatopancreas acid phosphatase activities in the freshwater crab Oziotelphusa senex senex have been demonstrated (Reddy et al., 1984). Joshi and Kumar (2001) observed acid and alkaline phosphatase activity of different tissues of freshwater crab Paratelphusa masoniana. Activity of phosphatase in the hemocytes of estuarine crab Scylla serrata have been studied (Saha et al., 2009). The effect of heavy metal inducing enzyme activity of the crab Carcinus maenas have been reported (Hansen et al., 1992). 94

Though numerous information are available on biochemical composition of many crabs from different parts yet such a study is lacking in Charybdis natator related to biochemical composition and enzymes. The present investigation is aimed to study the biochemical constituents and enzymes in gonad, hepatopancreas and haemolymph of crab in relation to reproductive, nutritive and moulting cycles.

5.2. Materials and Methods Live and healthy brachyuran crab Charybdis natator were collected from the off shore region of Nagapattinam coast, The collected samples were taken immediately to the laboratory. The biochemical composition of gonad, hepatopancreas and haemolymph were thoroughly studied in various stages of reproductive and nutritive cycles. The stages of maturity of female gonads were apparently determined by their size and colour. The stages of ovary were classified based on the method outlined by Ezhilarasi and Subramanian (1980). There were immature (Stage-I), maturing (Stage-II), mature (Stage-III), ripe (Stage-IV) and spent (Stage- V). The moulting stage of crab can be divided into four stages namely moult (Stage-A), pre moult (Stage-B), post moult (Stage-C), and inter moult (Stage-D). The moult stage of crab was assessed using the methods of Hiatt (1948).

The animals were dissected to collect the tissues of gonad and hepatopancreas separately. The tissues were fixed in 5 percent trichloroacetic acid to stop the enzymatic actions (Giese et al., 1958). The excess solution was drained and the materials were blotted. The tissues 95 were kept in hot air oven and dried at 50-60°C. Care was taken to dry the tissues uniformly. Dried samples were weighed and powdered for estimation of biochemical components and the values were expressed in percentage of dry weight. Haemolymph was collected by cutting each walking legs of the animal with a fine sterile scissor. The biochemical components and enzymes composition of crab were determined by adapting to standard procedure.

Water content The percentage of water content was calculated based on the differences between wet weight and dry weight of the tissues (Rahaman, 1967).

Protein The tissue proteins of gonad and hepatopancreas was estimated in Lowry et al., method (Lowry et al., 1951) and haemolymph protein was estimated by Biuret method (Welchselbam, 1946).

Carbohydrate The total amount of carbohydrate present in the tissue sample was estimated by using Dubois et al., (1956) method.

Lipids The total amount of lipids in the sample was estimated by chloroform methanol method (Raymont et al., 1964). 96

Glucose The glucose was estimated by enzymatic colorimetric method (Schmidt, 1971).

Cholesterol The cholesterol was estimated by Enzy-colorimetric method (Fleg, 1973).

Triglycerides The triglyceride in haemolymph was estimated in Glycerol phosphate oxidase (GPO) method (Fossatip, 1982).

Enzymes Acid phosphosphatase (ACP), Alkaline phosphosphatase (ALP), Lactate dehydrogense (LDH), Serum glutamate oxalacetate transaminase (SGOT), Serum glutamate pyruvate transaminase (SGPT) were estimated in the method of King, (1965).

Statistical analysis For the present study, the data were statistically analysed by using the standard formula the mean, standard deviation and standard error of each and every parameters was calculated and tabulated.

5.3. Result 5.4. BIOCHEMICAL CHANGES OF REPREODUCTIVE CYCLES The classification of the ovary in reproductive cycles Stage I (Immature) In this stage, the ovary is prepubertal, reproductively inactive. The ovarian lobes are thin and translucent, closely adhering to the lobes of hepatopancreas. Only cluster of small primary oocytes are present. The ovary is restricted only to cephalothoracic region.

Stage II (Maturing) Reproductively active, ovary is slightly increased in size and acquired white to light yellow colour, distinctly seen from the lobes of hepatopancreas.

Stage III (Mature) Ovary becomes flexible. The anterior arms extend upto the proximal extremities and attains yellowish orange colour. Translucent. Bulged and covered by transpired connective tissue layers.

Stage IV (Ripe) The ovary is deep orange, lobulated oocytes are not very compact, full grown, extending into all available cephalothoracic space.

Stage V (Spent) In this stage the ovary is colouless flaccid, larger than immature and the arms extend up to abdomen. The ovary having few unspawned oocytes undergoes resorption.

The stages of moulting cycles Stage A (Newly moulted) In the stage, the exoskeleton is a soft membrane and parchment in nature. Endo and exocuticle deposition and mineration begins.

Stage B (Resently moulted) The exoskeleton is generally deformable without breaking. The parts the exoskeleton become rigid.

Stage C (Intermoult) The exoskeleton is completely rigid and branchiostegites. The endocuticle mineralization may still continue. The tissue growth is completed.

Stage D (Premoult) The epidermis separates from the membraneous layer and secretes a new epicuticle. The exocutile secretion begins. The old membranous layer degenerates to a gelatinous layer. It is the main period of resorption.

Stage E (Postmoult) The crab withdrawns from the old skeleton and takes up water rapidly

5.4.1. Water content The biochemical composition of ovary and hepatopancreas of Charybdis natator were studied the different stages of sexual maturity and are given in the Table 10. In the ovary, the percentage of water content ranged from 57.58 1.24 to 71.96 1.45% wet weight. It was found to be the maximum (71.96 1.45%) in stage II and minimum (57.58

1.24%) in stage IV and then increases (61.87 1.39%) in stage V. The water content in the ovary showed marked variation during reproductive cycle. An inverse relationship was noticed between the water content and gonad index. The ripe ovaries contain less water content than immature ovary. The percentage of water content is maximum during early stages of maturity and it is minimum when the gonad index is maximum (Fig. 26).

In the hepatopancreas of female, the percentage of water content ranged from 59.68 1.49 to 70.59 1.63% wet weight. It was found to be minimum (59.68 1.49%) in stage II and gradually increased to reach maximum (70.59 1.63%) in stage IV and then decreased (64.95

1.56%) in stage V. It was observed that the water content in the gonad decreased as the stages of maturity advanced, a simultaneous increase is seen in the hepatopancreas, showing an inverse relationship. The percentage of water content in gonad and hepatopancreas showed a slight fluctuation in relation to different stages of sexual maturity (Fig. 27).

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5.4.2. Protein The protein content in the ovary constitutes the highest percentage among the total organic compound in terms of dry weight. It ranged from

39.14 1.29 to 56.93 1.42 per cent dry weight. The protein content was found to be the minimum (39.14 1.29%) in stage 1 and it gradually increased as ovarian maturity advanced to reach the maximum (56.93

1.42%) in stage IV. The protein content in gonad increased as stages of maturity advanced. The maximum level of protein content was recorded when the gonad index was high (Fig. 26).

In the hepatopancreas, the protein content ranged from 25.80

1.25 to 41.97 1.36 per cent dry weight. The highest value (41.97

1.36%) was recorded in stage II and it decreased to reach the lowest value

(25.80 1.25%) in stage IV. There was a reciprocal relationship noticed in protein content between gonad and hepatopancreas. In gonad, the protein increased as the stages of maturity advanced, whereas at the same time in hepatopancreas it decreased (Fig. 27).

The data clearly indicates that the amount of protein content found in the haemolymph showed significant variation. It ranged from 5.14

0.43 to 7.28 0.68 g/dl. It was found to be minimum (5.14 0.43 g/dl) in stage 1V and maximum (7.28 0.68 g/dl) in stage I. It was inferred that the higher value of protein content was found in the haemolymph of marine crab at immature and mature stages and decreased in ripe and spent stages. Further, the result indicates that the protein content in the 101 haemolymph of the crab showed higher value when compared to other biochemical components (Fig. 28).

5.4.3. Lipid

The lipid content in the ovary ranged from 11.42 0.58 to 18.33

0.72 per cent dry weight. In ovary, the lower value of lipid content (11.42

0.58%) was recorded in stage I and it gradually increased to reach higher value (18.33 0.72%) at stage IV. From the data, it is inferred that the total lipid content increased in ovary during the ripening of gonads and it decreased during spawning and immature period (Fig. 26).

In the hepatopancreas of female, the lipid content raged from 24.15

0.74 to 33.49 0.81 per cent dry weight. It was found to be low (24.15

0.74%) in stage I and high (33.49 0.81%) in stage II. The lipid content is higher in the hepatopancreas than that of gonads. The lipid content in the hepatopancreas also fluctuated during reproductive cycle. As the sexual maturity advances, the lipid content increases in the gonad and decreases in the hepatopancreas (Fig. 27).

5.4.4. Carbohydrate The percentage of carbohydrate content of gonad and hepatopancreas is shown in Fig. 26 and 27. The carbohydrate in the ovary ranged from 3.73 0.31 to 6.03 0.53% dry weight. In the female gonad, it was found to be the maximum (6.03 0.53%) in stage I and gradually decreased to reach minimum (3.73 0.31%) in stage IV. From the data it 102 clear that there is a definite variation in the carbohydrate content of ovary during different stages of sexual maturity.

In the hepatopancreas, the carbohydrate content ranged from

3.87 0.34 to 6.81 0.46 per cent dry weight. It was found to be maximum (6.81 0.46%) in stage II and gradually decreased to reach minimum (3.87 0.34%) in stage IV. Like gonads the hepatopancreas have maximum value of carbohydrate at early stages of gonadal maturity and a decrease during development. Comparatively hepatopancreas has more carbohydrate content than gonads.

5.4.5. Glucose The amount of glucose found in the haemolymph of marine crab C. natator during the reproductive cycle showed significant fluctuation. It ranged from 54.85 1.03 to 67.2 1.08 mg/dl. The lower value (54.85

1.03 mg/dl) was recorded in stage V and the higher value (67.2 1.08 mg/dl) in stage I. The glucose content in the haemolymph decreased as gonadal maturity advanced indicating the utilization of energy for gametes production (Fig. 29).

5.4.6. Cholesterol In the present study the amount of cholesterol found in the marine crab C. natator during the reproductive cycle showed significant fluctuation. It ranged from 126 1.83 to 157 1.69 mg/dl. It was found to be minimum (126 1.83 mg/dl) in stage IV and maximum (157 1.69 103 mg/dl) in stage I. The cholesterol content in the haemolymph gradually decreased from immature stage to ripe stage, indicating mobilization of energy component for reproductive activity (Fig. 29).

5.4.7. Triglycerides The amount of triglycerides found in the haemolymph of marine crab C. natator during the reproductive cycle showed fluctuation. It ranged from 114 1.57 to 143 1.58 mg/dl. The lower value (114 1.57 mg/dl) was recorded in stage IV and the higher value (143 1.58 mg/dl) in stage III. During gonadial maturity the triglycerides content in the haemolymph considerably decreased (Fig. 29).

5.5. BIOCHEMICAL CHANGES DURING MOULTING CYCLE 5.5.1. Protein The amount of protein present in the haemolymph of marine crab Charybdis natator during the moulting cycle are give in Table 11. The data clearly indicates that the amount of protein content found in the haemolymph showed significant variation during moulting cycle. It ranged from 4.9 0.51 to 7.2 0.59 g/dl. It was found to be minimum

(4.9 0.51 g/dl) in moulting maximum (7.2 0.59 g/dl) in pre-moulting.

Further, the result indicates that the protein content of the crab showed higher value when compared to other biochemical components (Fig. 30).

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5.5.2. Glucose The amount of glucose present in the haemalymph of marine crab C. natator during the moluting cycle showed a slight variation. It ranged from 47.4 0.99 to 64.8 1.16 mg/d. It was found to be minimum (47.4

0.99 mg/dl) in moulted stage and maximum (64.8 1.16 mg/dl) in inter moulting stage (Fig. 31).

5.5.3. Cholesterol In the present study the amount of cholesterol was found in the marine crab, C. natator during the moulting cycle showed significant fluctuation.

It ranged from 138 1.33 to 158 1.57 mg/dl. It was found to be minimum (138 1.33 mg/dl) in moult stage and maximum (158 1.57 mg/dl) in pre-moult stage. The cholesterol content gradually increased from post-moult to pre-moult (Fig. 31).

5.5.4. Triglycerides The amount of triglycerides was found in the haemolymph of marine crab C. natator during the moulting cycle showed significant fluctuation. It ranged from 118 1.21 mg/dl to 146 ± 1.36 mg/dl. The lower value (118 1.21 mg/dl) was recorded in moult stage, and the higher value (146 1.36 mg/dl) in inter moult stage (Fig. 31).

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5.6. ENZYMES 5.6.1. Acid phosphatase (ACP) Acid phosphatase is non specific enzyme which hydrolysis phosphoric acid ester. In the present study, the acid phosphatase showed slight fluctuation in marine crab C. natator during the moulting cycle. It ranged from 2.4 0.18 to 3.3 0.22 IU/L. It was found to be low (2.4

0.18 IU/L) in moult stage and high (3.3 0.22 IU/L) in pre-moult stage. It increased in pre moult and decreased in moult stage (Fig. 32).

5.6.2. Alkaline phosphastase (ALP) Alkaline phosphastase is also a non specific enzyme which hydrolysis aliphatic, aromatic and heterocylic components. The alkaline phosphastase in haemolymph of marine crab C. natator during the moulting stages showed significant fluctuation. It varied from 67.9 1.52 to 85.6 1.59 IU/L. The minimum value (67.9 1.52 IU/L) was recorded in inter-moult stage, the maximum value (85.6 1.59 IU/L) in moult stage

(Fig. 32).

5.6.3. Lactate dehydrogenase (LDH) The enzyme lactate dehydrogenase found in the haemolymph of marine crab Charybdis natator during the moulting cycle showed a slight fluctuation. It ranged from 61.9 1.64 to 76.2 1.61 IU/L. It was found to be low (61.9 1.64 IU/L) in moult stage and high (76.2 1.61 IU/L) in pre-moult stage. It indicates that LDH considerably decreased in moult stage (Fig. 32). 106

5.6.4. Serum glutamate oxalacetate transaminase (SGOT) In the present study serum glutamate oxalacetate transaminase found in the haemolymph of marine crab C. natator during the moulting cycle showed slight variation. It ranged from 16.4 0.98 to 22.1 1.19

IU/L. The maximum value was recorded (22.1 1.19 IU/L) in premoult stage and the minimum value (16.4 0.98 IU/L) in the post moult stage

(Fig. 32).

5.6.5. Serum glutamate pyruvate transaminase (SGPT) In the present study serum glutamate pyruvate transaminase in the haemolymph of marine crab C. natator during the moulting cycle showed a slight variation. It ranged from 21.5 1.15 to 27.6 1.31 IU/L. It was found to be low (21.5 1.15 IU/L) in moult stage and high (27.6 1.31

IU/L) in inter-moult stage (Fig. 32).

5.7. Discussion Biochemical studies are very important from the nutritional point of view and bioenergitics of an organism. The biochemical constituents in animals are known to vary with season, size of the animal, stage of maturity and availability of food etc. Decapod crustaceans are rich sources of animal protein and good for human health. In the present investigation, biochemical composition of gonad and hepatopancreas of Charybdis natator are directly related to reproductive and nutritional cycle. The percentage of water content in the ovary and hepatopancreas of Charybdis natator showed significant trends during different stages of sexual 107 maturity. In the ovary the percentage of water content was found to be minimum during ripe stage than immature stage. The percentage of water content in the hepatopancreas fluctuated during reproductive cycle. The highest value of water content coincides with peak breeding activity of gonads. When the water content of the hepatopancreas increased, the lipid content decreased. An inverse relationship was noticed between water and lipid contents of the hepatopancreas. The present study agrees with earlier observations (Giese, 1969; Wolin et al., 1973; Pillai and Nair, 1973b; Kannathasan and Rajendran, 2011d).

According to Pillai and Nair (1973b) in Portunus pelagicus, the percentage of water content of the gonad showed low values during the month of higher gonad activity. In Uca annulipes, the water content of the ovary decreased as gonad index increased. In Portunus pelagicus, the gonad showed maximum water content in the immature stage and decreased as stages of maturity advanced (Rahaman, 1977). There was an increase in the water content of hepatopancreas of Scylla serrata during reproductive periods whereas during the same period it decreased in the ovary (Naghabhushnam and Faroogii, 1982b). In Charybdis feriata, the percentage of water content in the gonad was found to be minimum during peak breeding season (Rajendran, 1990).

In the present study, in C. natator, the percentage of protein content of the ovary showed higher values than hepatopancreas. The protein content of the gonad was comparatively higher than the lipids and 108 carbohydrates. The protein value in ovary and hepatopancreas varied during breeding cycle. The protein content of the ovary was found to be maximum in the ripe stage and minimum in immature stage of ovary. In the hepatopancreas there was a slight decrease in the protein content noticed during sexual maturity advance. An inverse relationship was noticed between gonad and hepatopancreas in protein content. The depletion of protein content in the hepatopancreas with ripening gonads may be due to a transfer of this material from hepatopancreas to gonads. Similar observation has reported by earlier workers (Griffin, 1968; Bedford, 1972; Mark and Ernest 1991; Vasconcelos and Braz, 2001; Vijayavel and Balasubramanian, 2006; Spaziani et al., 2008; Manivannan et al., 2010).

According to Kessel (1968) ovaries were capable of producing protein by biosynthesis. In crab Paratelphusa hydrodromous there was a protein yolk biosynthesis taking place during normal vitellogenesis (Adiyodi, 1969). Kerr (1969) determined in Callinectes sapidus that protein level was found to be high during vitelogenesis. In Portunus pelagicus the amount of protein was found along with the amount of sarcoplasmic fibrillar and stroma protein (Badawai, 1971). In Paratelphusa hydrodromous, there was a decreasing trend in the hepatopancreatic protein during vitellogenesis (Adiyodi and Adiyodi, 1972). In the gonad of Uca annulipes and Portunus pelagicus, the protein content was found to be higher during the reproductive periods and lower during non-reproductive period. The depletion of the protein level in the 109 hepatopancreas association with riping gonad may be due to transfer of this organic material from the hepatopancras to gonads (Pillai and Nair, 1973b).

In Barytelphusa cunicularis, the protein level was the highest during the period when the gonads were in developing condition and declined to minimum level during the spawning period (Diwan and Nagabhushnam, 1974). In Portunus pelagicus, the protein level was found minimum in immature gonads and it increased to reach maximum value in ripe gonads. In hepatopancras it decreased as stages of maturity advanced (Rahaman, 1977). In Clibaranris longitarsus, the protein level was found minimum during early stages of gonad and it increased to reach maximum value when the gonads were in developing condition (Ajmal Khan and Natarajan, 1980).

In Scylla serrata, the protein concentration of hepatopacreas did not show any significant relation with the reproductive activity. In the ovary the protein content decreased during reproductive period (Nagabhushnam and Faroogii, 1982b). In Menippe rumphii ovarian protein showed great fluctuations. It was found to be gradually increasing in gonad whereas a slight decrease trend was noticed in the hepatopancreas in the same period, which suggests a small amount of hepatopancreatic protein transport to the oocytes (Shyamasundari and Erri Babu, 1984). In Scylla serrata there was a progressive increase in the protein content of the ovary and decrease in the hepatopancreas as stages 110 of maturity advanced (Balasubramanian and Subramanian, 1987). In Scylla serrata, the protein content of body muscles showed higher value (Prasad and Neelakantan, 1989b). In Charybdis feriata, there was an increase in protein level in the gonad and decrease in hepatopancreas as stages of sexual maturity advance (Rajendran, 1990). In Scylla tranquebarica protein showed higher value than in other components (Thirunavukarasu, 2005). In Charybdis lucifera, the protein showed higher values in eyestalk ablated crabs (Murugesan et al., 2008).

In the present study carbohydrate content of the ovary and hepatopancreas of marine crab C. natator shows slight fluctuation during the course of reproductive cycle. The higher value of carbohydrate contents was recorded during early stages of gonadal development and the level decreased in ripe stages of the ovary. Similarly in the hepatopancreas the carbohydrate content are found to decrease as the gonadal maturity advance. This suggests that the stored carbohydrate content might be utilized for the formation of the reproductive elements and this accounts for the decrease in the carbohydrate content during the breeding season. The present study agrees with earlier investigators (Heath and Barnes, 1970; Pillai and Nair, 1973b; Rahaman, 1977).

In Paratelphusa hydrodromous, quantitative and qualitative fluctuations in the free sugars of the hepatopancreas were observed. The higher amount of sugar present in the hepatopancreas during early vitellogenesis but decreased as the ovarian growth advanced 111

(Adiyodi and Adiyodi, 1970). Heath and Barnes (1970) while working on marine crab Carcinus maenas found a large amount of glycogen in a spent stage which decreased considerably during development. In Portunus pelagicus, there was no significant change in carbohydrate level in ovary during development whereas in testis it decreased when the gonadal maturity advanced. In Uca annulipes the glycogen content increased in the ovary during gonadal growth (Pillai and Nair, 1973b). In Portunus pelagicus the maximum level of carbohydrate was found at the early stages of ovary and it decreased as development advanced (Rahaman, 1977). According to Ajmal Khan and Natarajan (1980) the fluctuation of glycogen value in hermit crab Clibanarious longitarus related to breeding cycle. In Scylla serreta, the glycogen content decreased in the hepatopancreas during the peak reproductive period and it increased in the gonads at the same period (Nagabhushnam and Faroogii, 1982b).

In Menippe rumphii more amount of glycogen was found to be recorded in the initial stage of ovary and hepatopancreas and it decreased simultaneously in both the organs in association with the ovarian growth (Shyamasundari and Erri Babu, 1984). Further, they also suggested that glycogen acted as a reserve food material and was utilized for the formation of gametes products. In Scylla serrata, the total carbohydrate was high during early ovarian stage, it declined to a very low level in the last stage whereas in the hepatopancreas the total carbohydrate was always high and did not show much fluctuation (Balasubramanian and Subramanian, 1987). In Barytelphusa cunicularis, the glycogen content of 112 the testis and ovary falls during spawning period and after spawning the level was increased till the complete growth of the gonad (Diwan and Nagabhushnam, 1974).

In the present study, lipid content of hepatopancreas and ovary of Charybdis natator showed significant fluctuation at different stages of sexual maturity. The hepatopancreas is known to be a storage organ for lipids and it is evident that considerable quantities of lipid being stored in the organ. The lipids are the most varing componenets of the gonad in relation to the stages of development. There was an indirect relationship noticed between ovarian lipids and the hepatopancreatic lipids with an increase in the lipid content of the ovary. There is simultaneous decrease in the lipids of hepatopancreas suggesting probable mobilization of hepatopancreatic lipids to the ovary at the time of vitellogeness. The present investigation agrees with earlier workers (Diwan and Nagabhushnam, 1974; Clarke, 1977; Spindler-Barth, 1976).

Eyestalk factors can affect lipid metabolism, protein synthesis, enzyme synthesis and ribonucleic acid synthesis in the crustacean hepatopancreas (O‟Connor and Gilbert, 1968). In Portunus pelagicus, Uca annulipes and Metapenaeus affinis there was a general increase in the lipid content during breeding season in female gonads whereas in testis it tends to show a slight decline as it ripens (Pillai and Nair, 1973b). In Barytelphusa cunicularis the total fat content increased in testis and ovary during the ripening of the gonads and the relative amount of 113 phospholipids increased with an increase in the size of the testis while neutral lipids fraction declined. This indicates the increased storage of phospholipids with an increase in the size of the testis (Diwan and Nagabhushnam, 1974).

According to Clarke (1977), lipid in Chorismus antarcticus, was in simultaneous increase in the ovary and a decrease in hepatopancreas. Thus the hepatopancreatic lipids are mobilized towards the ovary. In Portunus pelagicus, the lipid content was found higher in hepatopancreas than in gonads. In gonad, it gradually increased and reached the maximum when the gonadal maturity advanced (Rahaman, 1977). The gonad of Clibanarious longitarsus showed the lowest value of lipid content during ripe stage (Ajmal Khan and Natarajan, 1980). In Scylla serreta, the lipid content was found to be decrease in the hepatopancreas when the gonadal maturity advanced whereas it increased in the ovary during the period of peak reproductive activity (Nagabhushnam and Faroogii, 1982b). In Menippe rumphi there was a decrease in the lipid content of the hepatopancreas and an increase in ovary with the advent of vitellogenesis in the ovary (Shyamasundari and Erri Babu, 1984). ` In Scylla serrata the lipid content of the ovary increased gradually as the stages of maturity of gonad advanced. In hepatopancreas and haemolymph the total lipid level increased up to stage IV and declined during stage V. This decline corresponds to the high rate of lipid accumulation in the ovary (Balasubramanian and Subramanian, 1987). In 114

Scylla serrata assessed the lipid values in bodymeat fluctuation (George and Gopakumar, 1987). In muscular and haemolymph lipid concentration varied significantly during the ovarian development (Xiao et al., 2001). In Charybdis granulate protein and lipid content showed the higher and fluctuates during development (Gabriela et al., 2002). In Platyxanthus patagonicus the ovary, lipid and protein content were higher values in spring and summer and decreased to lower after spawning (Dima et al., 2009).

The protein, glucose, cholesterol and triglycerides content in the haemolymph of C. natator showed a marked variation during the reproductive cycle. The glucose content was found to be in decrease as the stages of maturity advanced. The cholesterol and triglycerides fluctuated irrespective of stages. It indicates that the biochemical components of haemolymph appear to be the most important sources of the energy which mobilized during breeding cycle. The results of the present study agree with earlier observations (Dean and Vernberge, 1965; Allen, 1972).

Haefner (1964) determined the Callinectes sapidus haemolymph calcium fluctuation as related to environmental salinity. In Carcinus meanas and Uca pugilator the difference in the haemolymph protein during the moulting cycle was found (Spindler-Barth, 1976). In Neptunus pelagicus and Scylla serrata the biochemical composition in the haemolymph showed a significant fluctuation (Senthilkumar and Desai, 1978). In Scylla oceanica the protein content varied in soft shell and hard 115 shell crab (Anil and Suseelan, 2001). In brachyuran crabs haemolymph protein varied among the species (Rameshkumar et al., 2009a). In Callinectes sapidus the blood glucose level increased just before the spawning period (Dean and Vernberge, 1965). In vivo recovery of glycogen metabolism in haemolymph and tissue of freshwater field crab Barytelphusa querini showed a significant result (Venugopal et al., 1990). In Uca pugilator haemolymph glucose and lactate level varied when the crab was exposed to melatonin (Tilden et al., 2001). In Portunus sanguinolentus the lipid concentration of muscle and haemolymph was found to be lower value than that of ovary and hepatopancreas (Ravichandran et al., 2009).

The protein metabolism in relation to moulting in the crab Paratelphusa hydrodromous was studied with reference to RNA activity and protein yolk biosynthesis during normal vitellogenesis (Adiyodi, 1969). In Carcinus maenas some changes in biochemical composition with season and during the moulting cycle (Heath and Barnes, 1970). In Callinectes sapidus the haemolymph and whole animals, ecdysteroid levels rose during premoult to a maximum at stage D, ecdysteroids declined rapidly from late premoult stage D2 Though post moult stage A increased slightly at post moult stage B, and returned to low levels where they remained during inter moult stage C (Cynthia et al., 1983). The high level of fat content during pre moult stage of Emerita holthuisi has been recorded (Kulkarni, 1983). In Callinectes sapidus no significant changes in haemolymph Na+ and K+ concentrations during moult stages 116 acclimated to high salinity, despite uptake of large amounts of water at moult (David et al., 1985). In Scylla serrata during growth the total quantity of copper free proteins in the haemolymph increased at high rate than copper bound protein (Arumugam and Ravindranath, 1986). In Ocypode macrocera the blood protein concentration was more during pre and intermoult stage but decreased in the post moult stage (Nageswara Rao et al., 1986).

According to Daw (1987) the Callinectes sapidus the concentration of haemocyanin, copper and zine in the haemolymph decreased significantly during moult cycle. In the fiddler crab Uca pugilator the chitobiase activity of the epidermis and hepatopancreas is regulated at least in part by the steroid moulting hormones (Zou and Fingerman, 1999). The ecdysis of decapods crustaceans is associated with a dramatic release of crustacean cardioative peptide into the haemolymph (Philippenn et al., 2000). In decapod crustaceans the biochemical, physiological and behavioral processes were influenced by the moult cycle (Ernest and Chang, 2000). According to Zafar et al., (2004) in Scylla serrata protein content significant fluctuated during moulting cycle. In crab Portunus sanguinolentus the protein, carbohydrate and lipid contents found to be higher in hard shell and low in soft shell (Sudhakar et al., 2009).

In the present investigation enzymes acid phosphatase, alkaline phosphatase, SGPT, SGOT and LDH in the haemolymph of C. natator showed fluctuation during the moulting cycle. Acid phosphatase increased 117 in premoult stage and decreased in moult stage. Alkaline phosphatase was found to be increase in moult stage. Lactate dehydrogenase decreased during moulting stage. Other enzymes SGPT, SGOT and LDH showed slight fluctuation during moulting cycle. The finding in the present investigation agrees with the earlier workers (Reddy et al., 1984; Joshi and Kumar, 2001; Saha et al., 2009; Kannathasan and Rajendran, 2010d). According to Ramanibai (1986) an increase was noticed in LDH activity in the muscle, gill and hepatopancreas of estuarine crab Scylla serrata after expouse to copper. In freshwater crab Oziotelphusa senex senex sumithion stress increased production and libration of acid phosphatase activity in the hepatopancreas (Reddy et al., 1984). Hansen et al., (1992) stated that the decrease or increase in the enzyme activity deponds on stress of the animals.

In Uca pugilator LDH activity increased in the abdominal muscles when the animal exposed to the cadmium (Devi et al., 1993). The decrease activity of acid phosphatase influence in the structure and integrity of cell organelles and physiological process of organism (Joshi and Kumar, 2001). The LDH levels were significantly elevated and SDH activity was suppressed in the muscle, gill and hepatopancreas tissues of the crab Sesarma quadratum when exposed to heavy metals (Valarmathi and Azariah, 2002 and 2003). Vijayavel and Balasubramanian (2006) observed that the enzymes of acid phosphatase, alkaline phosphatase, aspartate transaminase, alanine transaminase and lactate dehydrogenase were decreased in hepatopancreas and ovary compared to haemolymph of 118 the estuarine crab Scylla serrata. In freshwater field crab Spiralothelphusa hydrodroma the activity of alkaline phosphate was found to be decrease when the animal was exposed to sublethal concentration of toxicans (Senthilkumar et al., 2007; Sreenivasan et al., 2011).

In the present study, the biochemical constituents of ovary, hepatopancreas and haemolymph in the marine crab C. natator during reproductive, nutritional and moulting cycles showed significant result. The protein, carbohydrate, lipid and water content were found to fluctuate significantly but glucose, cholesterol and triglyceride changed slightly in the different stages of sexual maturity. Among the biochemical component, proteins have the highest value. It indicates that the crabs are the main sources of animal protein. Hence the present study suggested that the crabs are highly nutritive and proteinous item and suitable for human consumption.

6. ANALYSIS OF PROTEIN PROFILE

6.1. Indroduction Studies on the electrophoretic analysis of protein molecules of decapods crustaceans have been carried out by many investigators (Lee and Watson, 1995; Philippen et al., 2002; Chen et al., 2004 and 2007 Misnan et al., 2007; Kannathasan and Rajendran, 2001e). Protein is made up of different amino acid sub units which migrate into the polyacrylamide gel at different rates. The molecular movements depond 119 upon the molecular weight of aminoacids (Schagger and Jagow, 1987). The number of bands in SDS-PAGE gel is related to the size of the protein (Hoq et al., 2003). Hinsch and Cone (1969) stated that the major biochemical component in oocytes of crabs is lipoprotein which is lacking in immature ovary. Wolin et al., (1973) studied the uptake of yolk protein, lipovitelline by developing crustacean oocytes. In Callinectes sapidus, water soluble protein was determined by using SDS-PAGE (Dowdie and Biede, 1983).

The composition of yolk varies from species to species and even in the same species among the individuals. The variation in yolk composition mainly deponds on the proportion of water, protein and lipid (Adiyodi and Subramanian, 1983). In Portunus trituberculatus haemocyanin showed single band with a molecular weight of about 75,000 (Sun et al., 1989). Umetsu et al., (1990) studied protein nature of lactin from haemolymph of crab Charybdis japonica. Sakharov and Litvin (1992) observed substrate specificity of collagenolytic proteases from the hepatopancreas of Paralithodes camtschatica. In Cancer antennarius, serum contains high density lipoprotein (Spaziani et al., 1995). The yolk protein generally consists of carbohydrates, phospholipids and carotenoid components (Chen et al., 1999). Pateraki and Stratakis (2000) observed the synthesis and organization of vetellogenin and vitelline molecules in the land crab Potamon potamios.

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According to Tsukimura (2001), vitellogenin circulates in the haemolymph, while vitelline accumulates in the developing oocytes during the process of vitellogenesis. The molecular characterization and expression analysis of vitellogenin in the marine crab Portunus trituberculatus have been reported (Yang et al., 2005). Dai et al., (2006) demonstrated the purification and characterization of lactin from humoral fluids of Charybdis feriata. Rameshkumar et al., (2009a) observed two clean bands in the gel that represents proteins of 45kDa from haemolymph of female and 25kDa from haemolymph of male Charybdis lucifera.

In Portunus pelagicus, the selective staining reaction reveals that the vitellin in the oocytes is a lipo-glyco-caroteno protein with calcium moiety. The absence of vitellin in the immature and spent ovary is due to synthesis of vitellin in negligible quantity in these stages (Ravi and Mary, 2011).Though numerous information are available on separation of protein during vitelogenesis, yet such study is lacking in C. natator related to protein bands during ovarian development. Hence, the present study is aimed to observe protein nature in gonad, hepatopancreas, muscles and haemolymph of marine crab C. natator during the different stages of sexual maturity.

6.2. Materials and Methods Brachyuran, female crab C. natator of different sizes were collected from the off shore region of Nagapattinam and brought to laboratory in alive condition without any stress to the animals. The crabs 121 were sacrificed and the stages of sexual of maturiry were observed based on the colour and size of the gonad. The tissues of gonad, hepatopancreas and muscle of different maturity stages viz. immature, maturing, mature, ripe and spent were collected and washed in ice cold 100mM phosphate buffer containing 0.001% phenyl methyl sulfonyl flouride, which acted as a protease inhibitor. From the sample, 100mg of crude tissues of gonad, hepatopancreas and muscle were homogenized in 0.2ml of phosphate buffer saline. The homogenates were centrifuged at 1000rpm for 15 minutes at 4°C and the supernatant was collected and stored at 4°C. The haemolymph was collected by cutting each walking legs of the animal with a fine sterile scissors to avoid haemocyte degranulation and coagulation. The collected haemolymph was placed in sodium citrate buffer, pH 4.6 (2:1, V/V). Equal volume of physiological saline (0.85%, NaCl, w/v) was added to it. To remove haemocytes, the haemolymph was centrifuged at 2000rpm for 15min at 4°C. Supernatant were collected by aspiration and was stored at 4°C until further used.

Preparation of gel Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 10% polyacrylamide gel according to the method of Laemmli (1970).

Glass plates were rinsed and wiped with ethanol and were fixed on to the assembly using spacers and bull dog clips. The separating gel was prepared in a beaker (3.2 ml distilled water, 2.64 ml 30% acrylamide, 2ml 122

1.5 M Teris, 0.80 ml 10% APS, 0.004 ml TEMED, APS and TEMED were added just before pouring into the gel plate) and poured between gel plate up to the required level (in a mini gel about 7.8cms). Few ml of butanol was poured to overlay the separating gel and allow the gel to polymerize. After polymerization, the butanol was poured off and the gel was washed in distilled water. The stacking gel was prepared in a beaker (2.1 ml distilled water, 0.5 ml 30% acrylamide, 0.38 ml of 1.0 M Tris, 0.03 ml 10% SDS, 0.03 ml TEMED, APS and TEMED were added just before pouring), the comb was inserted and the stacking gel solution was poured. After polymerization of stacking gel the comb was removed and the wells were washed with distilled water. The assembly was filled with distilled water.

Preparation of sample 25µl of sample buffer was added to protein solution in a eppendoff. The wells were loaded with sample using a Hamilton syringe. The assembly was connected to the power pack. The electrophoresis was carried out at 20 mA till the samples reach the separating gel and then it was continued at 40 mA for 2-3 hours until the dye front reaches the end layer. After electrophoresis the gel was removed and stained with coomassive blue for 24 hours, and subsequently destained in acetic acid (2.3 hrs). The developed bands were then observed under illumination and photographed. The standard protein markers of known molecular weight (Sigma) such as phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), glyceraldehydes-3-phosphate dehydrogenase 123

(36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa) and ά- lactoalbumine (14.2 kDa), were loaded to find out the weight of the separated protein units.

Calculation The distance moved by the protein fraction and the distance moved by the gel front were used to calculate the RF value. Distance moved by the protein RF = Distance moved by the gel front

A graph was plotted by taking RF along X-axis and molecular weight along the Y- axis from which the molecular weight of the unknown protein was found out.

Statistical analysis The result obtained in the present investigation was subjected to statistical analysis like standard deviation (SD) following the procedure given by Zar (1999).

6.3. Results In the present investigation, the protein profile of crab C. natator were analysed by SDS-PAGE which showed siginificant result. The photographic evidences clearly indicates that there were seven protein bands observed in gonad, hepatopancreas, muscle and haemolymph during ovarian maturity (Fig. 7-11). 124

Stage-I (Immature)

In this stage, three bands were observed in haemolymph, muscle and two bands in gonad and hepatopancreas. The molecular weight of the protein was determined with callibiration curve phosphorylase b, bovine serum albumin, egg albumin, glyceraldehydes -3- phosphate dehydrogenase, carbonic anhydrase, trypsinogen and ά-lactoalbumine as standard and the results showed that the different proteins were present in various tissues of stages of sexual maturity. In haemolymph the protein got split in to three sub units of molecular weight 97.4, 45 and 24 kDa. The muscle proteins have three sub units of molecular weight 66, 45 and 29 kDa. In the hepatopancreas protein contains two sub units of molecular weight 45, 14.2 kDa, and gonad contains protein of two sub units of molecular weight 45 and 36 kDa. It indicates that the low molecular weight protein was found in hepatopancreas (Fig. 7).

Stage-II (Maturing) In this stage, four bands in haemolymph and gonad three bands in muscle and hepatopancreas were identified. In haemolymph the protein got split in to four sub units of molecular weight 97.4, 66, 45 and 29 kDa. The muscle protein have three sub units of molecular weight 45, 36 and 24 kDa The hepatopancreatic protein contains three sub units of molecular weight 45, 29 and 14.2 kDa. The gonad exhibits protein of four sub units of molecular weight 66, 45, 29 and 24 kDa. The higher molecular weight protein was observed in haemolymph and low molecular weight protein in hepatopancreas (Fig. 8). 125

Stage-III (Mature) In mature stage, five bands in haemolymph, three bands in muscle, gonad and hepatopancreas were observed. In haemolymph the five protein bands of molecular weight was 97.4, 66, 45, 29 and 24 kDa respectively and muscle three protein bands of molecular weight was 66, 45, and 29 kDa respectively. The hepatopancreatic protein showed three sub units of molecular weight 45, 24 and 14.2 kDa. The gonad expreses protein of three sub units of molecular weight 66, 45, 36 kDa each (Fig. 9).

Stage-IV (Ripe) In this stage, five bands in haemolymph, four bands were observed in muscle and gonad, three bands in hepatopancreas. In haemolymph the protein has five sub units of molecular weight 97.4, 66, 45, 36 and 24 kDa, and muscle protein has four sub units of molecular weight 66, 45, 36 and 24 kDa. In the hepatopancreas the protein attibuted three sub units of molecular weight 45, 36 and 14.2 kDa. The gonad protein contains four sub units of molecular weight 66, 45, 36 and 29 kDa (Fig. 10).

Stage-IV (Spent) In this stage, three bands were observed in haemolymph, muscle and two bands in gonad and hepatopancreas. In haemolymph the protein got split in three sub units of molecular weight 97.4, 45 and 24 kDa, the muscle protein has three sub units of molecular weight 45, 29 and 24 kDa. In the hepatopancreas the protein contains two sub units of molecular weight 45 and 14.2 kDa. In gonad the protein contains of three sub units 126 of molecular weight 45 and 24 kDa. The low molecular weight protein was found in hepatopancreas (Fig. 11).

6.4. Discussion In the present study, the array of protein in various tissues of marine crab C. natator showed significant results. The seven protein bands of molecular weight ranging from 14.2 to 97.4 kDa were identified. The protein nature in gonad and hepatopancreas significantly varied in different stages of sexual maturity. The high molecular weight fraction was identified in the haemolymph and gonad and low molecular weight fraction in the hepatopancreas. The lipoprotein was not expressed in immature and spent ovaries. Similar observation was reported by earlier workers (Schagger and Jagow, 1987; Chen et al., 1999; Kannathasan and Rajendran, 2011e). In Libinia emarginata, the lipoprotein was lacking at immature stage in female tissues. (Hinsch and Cone, 1969).

Wolin et al., (1973) observed that there were three protein bands of molecular weight 118, 105 and 83 kDa from developed crustacean oocyte. The yolk proteins occur as large molecular weight proteins of 300-500 kDa with carotenoid, sugar, carbohydrate, calcium and lipid moieties attached during biosynthesis. These characters distinguish the yolk proteins from other proteins like haemocyanin circulating in the haemolymph (Adiyodi and Subramanian, 1983). In Callinectes sapidus, water soluble fraction ranging in molecular weight between 130,000 and 12, 000 Daltons were identified (Dowdie and Biede, 1983). Sun et al., 127

(1989) observed that in Portunus trituberculatus the haemocyanin sub units have single band of protein with molecular weight ranging about 75,000. In Charybdis japonica, blood binding lactin from haemolymph gave a single protein band with molecular mass of 19 and 38 kDa (Umetsu et al., 1990).

The low molecular weight proteins of 24 to 36 kDa were found in the hepatopancreas of crab Paralithodes camtschatica (Sakharov and Litvin, 1992). In Cancer antennarius, serum having high density lipoprotein with three sub units of molecular weight 185, 100 and 84 kDa (Spaziani et al., 1995). In decapod crustaceans ovary, hepatopancreas, adipose tissue and haemocytes to be some of the sites for vitellin synthesis (Lee and Watson, 1995; Chen et al., 1999). Pateraki and Stratakis (2000) observed three major vitellin peptides of 115, 105 and 85 kDa in the ovary of land crab Potamon potamios. In decapods, the molecular weight range from 28.kDa to over 600 kDa vitellin is very common (Tsukimura, 2001). In Carcines maenas, different protein bands appear when the same tissues extracted in EDA, HCL, HAC and KC1 (Phillippe et al., 2002). Hoq et al., (2003) identified five different proteins of molecular weights such as 64, 61, 56.5, 49 and 36 kDa from the haemolymph of crab Scylla serrata.

According to Chen et al., (2004) vitellogenin had a native molecular mass of 520 kDa and the denaturing SDS-PAGE revealed two sub units of 97 and 74 kDa in the Chinese crab Erioheir sinensis. In Portunus trituberculatus, three major polypeptides of molecular weight 128

102, 100 and 85 kDa were found in the mature ovary (Yang et al., 2005). The purification and characterization of lactin from humoral fluids of Charybdis feriata showed single band (Dai et al., 2006). There were two major protein bands in the haemolymph of male and immature female of Scylla olivacea (Chen et al., 2007). Nineteen protein bands in the range of 15 to 138 kDa were identified from the crab Portunus pelagicus and Charybdis japonica (Misnan et al., 2007). In marine crab Charybdis lucifera there were two clear bands detected in the gel that represent proteins of 45 kDa from the haemolymph of female and 25 kDa in the haemolymph of male (Rameshkumar et al., 2009a).

In Portunus pelagicus, three sub units of vitelin molecular weight ranging from 94.2 to 103.5 kDa were mainly made up of lipo-glyco- caroteno protein with calcium moiety. The vitellin is absent in the immature and spent stage (Ravi and Mary, 2011). In the present study, in C. natator, applying SDS-PAGE revealed that there were seven protein bands of molecular sub units 97.4 to 14.2 kDa were identified. The haemolymph each contains six, protein bands and muscle, gonad and hepatopancreas each contain five bands at different stages of sexual maturity. Further, haemolymph and gonad have high molecular protein and hepatopancrease has low molecular weight protein. The characterization of protein is an important step in undastanding the process of vitellogenesis and mobilization of protein during reproductive and nutritive cycles.

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7. ANALYSIS OF BIOACTIVE COMPOUNDS

7.1. Introduction The fatty acids are found in both simple as well compound lipids. Many of the fatty acids are unsaturated and occur in both fat and oil. The nutritional quality of fish and fishery products are to a great extent associated with the content of essential fatty acids and organic and inorganic compounds (Lopes et al., 2009; Kaya et al., 2009). According to Allen (1971) the sea food is known as a rich source of polyunsaturated fatty acid. The higher mixed function, oxygenase activity was observed in female and male Callinectes sapidus (Singer and lee, 1977). The crab not only has a delicious taste and unique pleasant aroma but also a good nutritive value. The volatile components of crab are recorted as the most important determinant of the flavour quality, based on their concentration and recognition of threshold values (Matiella and Hsieh, 1990; Chung and Cadawallader, 1993; Chung et al., 1995; Spurvey et al., 1998). Hale (1988) demonstrated disposition of polycyclic aromatic compounds in the blue crab Callinectes sapidus. A low molecular weight lectin was isolated from haemolymph of Scylla serrata (Chattopadhyay and Chatterjee, 1993).

Jennifer et al., (1998) observed phospholipid composition of the granular amebocyte from horse shoe crab Limulus polyphemus. In green crab Cancinus maenas no significant difference was observed in the non - essential amino acids components (Skonberg and Perkins, 2002). The crab meat is an excellent source of minerals, particularly calcium, potassium and phosphorus (Gokodhu and Yerlikaya, 2003). The significant difference in the fatty acid composition of blue crab 130

Callinectes sapidus (Celik et al., 2004) and green crab Carcinus maenas (Naczk et al., 2004) have been reported. Ying et al., (2006) reported that there was a total of 18 fatty acid found in the ovary of Chinese mitten crab Eriocheir sinensis. Alava et al., (2007) stated that the percentage of lipid and fatty acids in mud crab Scylla serrata depends on its diet. The fatty acid profile of Eriocheir sinensis streamed dominated by monounsaturated fatty acids is similar type in the hepatopancreas of IV and V stage of ovarian maturation (Chen et al., 2007).

The occurrence and toxicity of hydrocarbon residues in crab Callinectes sapidus have been studied (Ololade et al., 2008). Mariniz et al., (2009) studied the fatty acid profile and total lipid content of crab Chionoectes opilio. Rameshkumar et al., (2009b) observed the antimicrobial peptide in the crab Thalamita crenata. The identification of characteristic aroma-active compounds in steamed mangrove crab Scylla serrata has been reported (Yu and Chen, 2010). The present study is aimed to estimate the lipid profile and organic and inorganic compounds of gonad and hepatopancreas of marine crab C. natator at different stages in sexual maturity.

7.2. Materials and Methods Female crab C. natator was collected from Nagapattinam, south east coast of Bay of Bengal and brought to the laboratory. The crab was scarificed and dissected out to observe the reproductive stage. The gonad and hepatopancreas were removed with forceps. The required quantity of tissue was weighed, transferred to flask, treated with ethanol until the tissue was fully immersed. The tissues were incubated El- electron impact ionisation along with sodium sulphate to remove the sediments and traces 131 of water in the filter paper. Before filtering, the filter paper along with sodium sulphate was wetted with absolute alcohol. The filtrate was then concentrated to 1ml by bubbling nitrogen gas into the solution. The extract contains both polar and non-polar components of the tissues and 1μl sample of the solutions was employed in GC-MS for analysis of different compounds.

GC-MS analysis GC-MS analysis of the ethanol extract of gonad and hepatopancreas of C. natator was performed using a Thermo GC-Trace ultra ver.5.0, thermo MS DSQ II and a Gas Chromatograph interfaced to a Mass Spectrometer (GC-MS) equipped with a Elite-1TR 5- MS capillary standard non polar column (30Mts, ID x 0.25mm x 0.25μm). For GC-MS detection, an electron ionization system was operated in electron impact mode with ionization energy of 70eV. Helium (99.999%) was used as carrier gas at a constant flow rate of 1ml/min, and an injection volume of 2μl was employed (split ratio of 10: 1). The injector temperature was maintained at 250°C, the ion-source temperature at 200°C, the oven temperature was programmed form 110°C (isothermal for 2min), with an increase of 10°C/min to 200°C, then 5°C/min to 280°C, ending with a 9min isothermal at 280°C. Mass spectra were taken at 70eV; a scan interval of 0.5 seconds and fragments from 45-450Da. The solvent delay was 0 to 2min and the total GC/MS running time 36min. The relative percentage amount of each component was calculated by comparing its average peak area to the total area.

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Identification of bioactive compounds Interpretation on mass-spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown components was compared with the spectrum of known components stored in NIST library. The name, molecular weight and structure of the components of the test materials were ascertained.

7.3. Result Chromatogram of GC-MS analysis of ovary and hepatopancreas of Charybdis natator at different stages of sexual maturity showed significant differences (Fig. 33-42). A total of 61types of bioactive compounds were found in the ovary and hepatopancreas. Out of them fourteen compounds were predomnently isolated and identified from the major peaks. The identified compounds have different peak area. Isopropyl acrylate (RT = 9.48min) which covered with 3.30% of spectrum area. 1-Heptanol (RT = 11.51min) with peak area 9.83%, Pentacosanoic acid, methyl ester (RT = 11.51min) with peak area 8.54%, 1-Tetradecanol (RT = 12.07min) with peak area 8.69%, 1-Hexadecanol (RT = 14.62min) with peak area 12.76%, 9-Octadecenoic acid (RT = 14.62min) with peak area 12.76%, Propane. 2-methox-2-methyl (RT = 28.54min) with peak area 12.76%, 1-Pentanol (RT = 9.82min) with peak area 7.31%, Heptanal (RT = 12.13min) with peak area 9.34%, Octadecanoic acid (RT = 5.14min) with peak area 11.55%, 1-Dodecanol (RT = 5.14min) with peak area 12.15%, 2,2-Bioxirane (RT = 12.06min) with peak area 11.83%, 1-Propanol, 2,2-dimethyl (RT = 22.85min) with peak area 9.83%, and Silane tetramethyl (RT = 28.77min) with peak are 8.36% were identified. Among the total bioactive compounds content the saturated, unsaturated, 133 mono saturated, mono unsaturated, poly saturated, poly unsaturated disaturated and fatty acid were found in different values. Some compounds were not detectable at certain stages of sexual maturity (Table 12).

7.4. Discussion In the present study the bioactive compounds of marine crab Charybdis natator were found to be saturated, unsaturated, monounsaturated, monosaturated, polysaturated, polyunsaturated and fatty acids. These compounds were significantly varied in ovary and hepatopancreas of different stages of sexual maturity. The result of the present study agrees with the previous workers (Allen, 1971; Chattopadhyay and Chatterjee, 1993; Chung et al., 1995; Jennifer et al., 1998; Lopes et al., 2009; Rameshkumar et al., 2009b). In Callinectes sapidus, mixed function oxygenase activity was found to be more in mature female then male during invitro assay (Singer and Lee, 1977).

According to Hale (1988), the highest concentrations of polycyclic aroma compound was detected in hepatopancreas, followed by ovary and muscle tissues in Callinectes sapidus. The long-chain aldehydes, such as pentadecanal and 2,3,10,14-tetramethylhexadecanal contribute little to the flavor because of their high boiling points and low volatility, but they may act as precursors for other important aroma compounds. Aldehydes, with 6-10 carbons were widely found in crab (Matiella and Hsieh, 1990; Spurvey et al., 1998). In Callinectes sapidus, the concentration of volatile compounds in a whole crab was much higher than those in crab meat (Chung and Cadwallader 1993).

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There were significant differences in mineral contents but no differences in the non essential amino acids composition and fatty acid in green crab Carcinus maenas (Skonberg and Perkins, 2002). In Callinectes sapidus and Portunus pelagicus there were significant difference in mineral content, aminoacids and fatty acids content (Gokodhu and Yerlikaya, 2003). There were significant differences in the fatty acid composition of green crab Carcinus maenas. Further, poly unsaturated fatty acids (PUFAS) were the predominant fatty acid accounting for about half of the total fatty acids. EPA and DHA were also the dominant fatty acids in these crabs (Naczk et al., 2004).

In crab Callinectes sapidus, the fatty acid profile showed significant difference between claw meat, breast meat and hepatopancreas. The percentage of total saturated fatty acid was higher in the hepatopancreas than the muscles (Celik et al., 2004). The fatty acid composition of the ovary and hepatopancreas of Eriocheir sinensis at different physiological stages showed significant differences. A total of 18 types of fatty acids were found in the ovary and hepatopancreas. Three of them were major fatty acids viz. oleic, palmitic and palmitoleic acids (Ying et al., 2006). In Chinese mitten crab Eriocheir sinensis, fatty acid profile consists of different fatty acids in varing percentage. The mono unsaturated fatty acid, which comprised about half (49.8%) of the total fatty acid while saturated and polyunsaturated fatty acids were apparoximately equal to 24.9 % and 23.9 % respectively (Chen et al., 2007).

According to Alava et al., (2007), fatty acid composition in the zoeae of Scylla serrata can be significantly influnced by brood stock diets. 135

In Callinectes sapidus, the average lipid content and fatty acid composition significantly varied during dry and wet seasons. The high lipid content enhances the chances of absorbing more hydrocarbon molecules especially those that are not easily degraded or eliminated (Ololade et al., 2008). The fatty acid profile of Chionoecetes opilio shells was dominated by poly unsaturated (39.87 %), monounsaturated (39.22 %) and saturated (20.98 %) fatty acid. The poly unsaturated fatty acid contains (11.63 %) of eicosalerraenoic, 10.97 % of docosahexaenoic (DHA) and 10.42 % of eicosapentaenoic acids (Mariniz et al., 2009).

The fatty acid composition of water crab Eriphia verrucosa was found to be 37.89 % saturated 29.41 % mono unsaturated and 20.05 % poly unsaturated fatty acids. Among the saturated fatty acids, palmitic acid was the dominant and it occupied 19.65% of the total fatty acids. Oleic acid was dominant mono unsaturated fatty acids (Kaya et al., 2009). In mangrove crab Scylla serrata there were 38 aroma-active components detected and 31 identified. The most important aroma were 2,3- butanedione 2,5-dimethylpyrazine, 3-methylbutanal, 2-acetyl-1-pyrroline and 2 – 2-acetylthiazole (Yu and Chen, 2010).

In the present investigation in the marine crab C. natator a total of 61types of bioactive compounds were found in the ovary and hepatopancreas at different stages of sexual maturity. Out of them fourteen compounds are predomnently isolated with significant difference during reproductive cycle. Hence, the study suggested that the bioactive compounds considerably mobilized from hepatopancreas to ovary during reproductive and nutritional cycles of crab.

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8. MICROBES

8.1. Indroduction The microbes are omnipresent on earth which includes biota, soil, water and atmosphere. They can easily enter into the biosphere from environment. Aquatic crustaceans always infected with large number of microbes into their body parts from water, sediments and food. There are several reports on microbial infection of crustaceans (Robert, 1988; Cockey and Tuu-Tyi, 1991; Kishio et al., 2000; Hernandez Roa et al., 2009; Kannathasan and Rajendran, 2010e).

According to Davis and Sizemore (1982), the species of Vibrio were predominant isolated from haemolymph and external carapace of blue crab Callinectes sapidus. The high levels of bacteria were isolated in the marine crab collected from the region of human habitation (Faghri et al., 1984). Sugita et al., (1987) isolated three types of anaerobic bacteria in the gut of coastal crustaceans. The isolation of Bdello vibrio in the gill tissues of blue crab Callinectes sapidus have been made by Kelley and Williams, (1992). The vibrio is the most common bacteria in gut of aquatic invertebrates (Harris, 1993). Prayitho and Latchford (1995) observed luminous bacteria related to photobacterium and Vibrio in crabs. Johnson and Lightner (1998) isolated rod shaped nuclear viruses from hepatopancreastic epithelium of decapod crustaceans. Eleven stains of new species of genus Kluyveromyces were isolated from sediments, clam and crab (Nagahama et al., 1999). 137

Ivanova et al., (1999) recorded sixteen representatives of the genus Bacillus from sea water and the marine crab Callinectes sapidus. In mud crab Scylla serrata, two species of Halophthoros bacteria (Leano, 2002) and filamentous bacteria from Kiwa hirsute (Gofferdi et al., 2008) have been made. Sixteen species of fungal flora and five species of bacteria from the marine crab Charybdis feriata have been isolated (Rajendran et al., 2008). The epibiotic and pathogenic bacteria were also predominantly found in marine crustaceans (Gofferdi et al., 2008; Uaboi-Egbenni et al., 2010). In Scylla serrata a total of 91 bacterial isolates consisting of 12 bacterial species were recorded (Najiah et al., 2010). However information on microbial infection of marine crabs during reproductive cycle is scanty. Hence the present investigation was undertaken for the isolation of fungi and bacteria from various tissues of marine crab Charybdis natator at different stages of sexual maturity.

8.2. Materials and Methods For the fungi and bacteria examination different reproductive stages (immature, matuting, mature, ripe and spent) of marine crabs Charybdis natator were collected from the off shore region of Nagapattinam. The specimen was washed with 70% ethanol before dissection. From each crab, 1g of tissue of carapace, gills, muscles, alimentary canal, hepatopancreas and gonads were taken aseptically and homogenized in tissue homogeniger. Then 0.2g of homogenized sample was transferred to a test tube containing 10ml of sterile water. 1ml of the dilution was serially diluted to obtain 10-1 to 10-5. 138

For bacteriological analysis 0.1ml of sample was spared into spred. Nutrient Agar (NA) medium and the petriplate were incubation at 30°C for 48 hours. All the purified isolats were observed for cell shape, spores by Gram staining. The isolated was then subjected to biochemical test such as methyl red (MR), voges proskauer (VP), indole, catalase, oxidase triple sugar iron (TSI), citrate utilization, urease utilization and nitrate test to conform the nature of bacteria. The bacterial identification followed by the Bergey's manual for Systematic Bacteriology (Holt et al., 1994; APHA, 1976).

For enumeration of fungi the diluted sample was taken with the help of micro pipette and inocubated into the centre of the potato dextrose agar medium (PDA) medium. The petri plates were turned upside down with addition of penicillin to reduce bacterial growth and then incubated at room temperature (25°C) for a 48-72 hours. The number of colonies in each samples was counted. Fungal colonies on potato dextrose ager plate were picked and stained with lactophenol cotton blue and identification was done based on the method of Gilman (1957) and Ellis (1971 & 1976).

8.3. Result The result of the present study on fungal flora in various reproductive stages marine crab Charybdis natator tissues are given the Table 13. From the result, clearly indicates that there were fifty species of fungal flora isolated from the tissues of carapace, gills, muscles, alimentary canal, hepatopancreas and gonads. The predominant species 139 isolated and identified in fungal flora were Aspergillus fumigatus, A. awamori, A. flavus, A. terreus, A. niger, A. ochraceaus, A. luchuensis, Curvularia lunata, Trichoderma viridae, T. koeningi, Trichoderma harzianum, Fusarium oxysporum, Penicilium jenthinellam and Verticillium sp (Plate-12) The total number of species varied among the tissues. Out of fifty species, thirty in carapace, twenty eight in gills, twenty five in hapatopancreas, twenty two in gonad, nineteen in muscle and thirty six in alimentary canal, were observed.

The existence of bacteria in various tissues of marine crab Charybdis natator is given in the Table 14. The result, clearly indicates that there were forty species of bacteria were isolated from carapace, gills, hepatopancreas, gonad, muscle and alimentary canal. The predominant bacterial species isolated were Aeromicrobium erythreum, Brachybacterium faecium, Cellulomonas cellasea, Marinococcus halophilus, Micrococcus halobius, Salmonella typhi, S. diversus, Sphaerobacter thermophilus, Rarobacter faecitabidus and Jonesia denitrificans (Plate-13). The total number of bacterial species varied among the tissues. Out of forty species, twenty six isolated from carapace, twenty four in gills, twenty one in hepatopancreas, eighteen in gonad, sixteen in muscles and thirty one in alimentary canal, were observed transparently.

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8.4. Discussion The result of the present study reveals that there were fifty species of fungi and forty species of bacterial flora isolated from various tissues of marine crab Charybdis natator collected from Nagapattinam coast. The number of isolates significantly varied among the tissues. Similar observation was reported by earlier workers (Johnson and Lightner, 1998; Aravindan and Sheeja, 2000; Leano, 2002; Hernandez Roa et al., 2009; Kannathasan and Rajendran, 2010e). The bacteria Vibrio parahaemolyticus, V.cholera, and V. vulnificus were predominantly isolated from haemolymph and the external carapace of blue crab Callinectes sapidus (Davis and Sizemore, 1982). A higher level of bacteria isolated from marine crabs was collected from region of human habitation (Faghri et al., 1984). Three types of anaerobic bacteria were isolated from gut of coastal crustaceans (Sugita et al., 1987). The occurrence of Trichomycetes was common in brachyuran crabs from Tampa Bay (Robert, 1988).

According to Cockey and Tuu-Tyi (1991), the control of bacterial quality is extremely difficult in crab meat. Kelley and Williams (1992) isolated Bdello vibrio from the gill tissues of blue crab Callinectes sapidus. Harris (1993) stated that the most common bacterial genera in the gut of aquatic invertebrates are Vibrio, Pseudomonas, Flavobacterium, Micrococcus and Aeromonas. Luminous bacteria related to photobacterium and Vibrio sp. were predominantly isolated from crustaceans (Prayitho and Latchford, 1995). Sixteen representatives of the genus Bacillus were isolated from sea water samples and marine crab 141

Callinectes sapidus (Ivanova et al., 1999). Eleven strains of a new species of the genus Kluyveromyces were isolated from sediments, clam and crab (Nagahama et al., 1999). Kishio et al., (2000) stated that Scylla serrata the larvae were infected by three species of fungi such as Lagenidium callinectes, Haliphthoros milfordensis and Halocrusticida baliensis spp. Two species of Halophthoros were isolated and identified from the spawned eggs of capture mud crab Scylla serrata (Leano, 2002).

A cluster of filamentous bacteria and fungi attached to the external surface of crab make a hairy structure and fungi (Gofferdi et al., 2008). Sixteen species of fungai and five species of bacteria predominantly isolated from ice stored marine crab Charybdis feriata (Rajendran et al., 2008). In the crab Kiwa hirsute the setae associated with epibiotic filamentous bacteria (Gofferdi et al., 2008). In swimming crab Callinectes sp the pathogenic bacteria such as Bacillus subtilis, Escherichia coli, Salmonella sp., Vibrio parahaemolyticus, Staphyloccus aureus, Micrococcus sp Streptococcus agalactiae and Proteus vulgaris were predominantely isolated (Uaboi-Egbenni et al., 2010). A total of 91 bacteria isolates consisting of 12 bacterial species were successfully recorded from mud crab Scylla serrata (Najiah et al., 2010).

In the present study, 50 species of fungi and 40 species of bacteria were isolated from various tissues of marine crab Charybdis natator. Out of which, more number of microbes were observed in alimentary canal and carapace which could be due to the decomposition of food and 142 environmental contamination. Only nineteen species in fungi and sixteen species in bacteria were detected from the muscles. It indicates less microbial contamination of muscles and is suitable for consumption because our edible portion was mainly the muscles of crab. Further there was no relationship noticed between microbial infection and reproductive cycle of crab. Hence it is suggested that the edible marine crabs should be collected from uncontaminated water which is more suitable for human consumption.

9. SUMMARY ►The present study deals with the reproductive and nutritional cycles of crab Charybdis natator (Decapoda: Portunidae) from January 2009 to December 2010. The study carried out from Nagapattinam (Lat. 143

100 46‟ NS and Long. 790 51‟ EW) on the south south east coast of India. ►The physico chemical parameters of Nagapattinam coastal water showed significant fluctuation during the study period. The rainfall was found to be the maximum (743.7mm) in November 2009 and minimum (0.6mm) in March 2010. ►The humidity of the study area showed fluctuates between seasons. It ranged from 66% (June) to 93% (November) during 2009 and from 73% (July) and 93% (November) during 2010. The photoperiod showed slight variation during the study period. It was found to be high (12.53hrs) in June and low (11.23hrs) in December 2009 and 2010 respectively. ►The difference in the atmospheric temperature was found to be higher than the water temperature. The atmospheric temperature varied from 28.4 to 37.8oC during 2009 and 28.5 to 35.8 oC during 2010. The water temperature ranged form 25.3 oC to 32.4 oC in 2009 and 26.6 to 323 oC in 2010. The turbidity of water showed slight fluctuated between seasons. It ranged from 3.9 to 5.9 NTU in 2009 and from 3.6 to 58.8 NTU in 2010. ►pH of the water showed alkaline range throughout the study period. It ranged from 7.34 (December) to 8.60 (June) in 2009 and 7.40 (December) to 8.60 (April) in 2010. The salinity of water showed wide fluctuations. It varied from 27.42 ppt (November) to 36.00 ppt (June) an average of 28.76 ppt during 2009 and from 27.42 ppt (December) to 36.2 ppt. in (April) 2010.

►The dissolved oxygen and Co2 content of sea water showed 144 slight fluctuation. DO varied from 4.8 to 6.10 mg/l in 2009 and from 4.0 to 6.9 in 2010. Co2 ranged from 3.41 to 5.9 mg/l in 2009 and from 3.55 to 5.5 mg/l in 2010. The phosphate, nitrate and nitrite did not show much fluctuation during the study period. ►A total of 133 species of brachyran crabs belongs to 58 genera, 18 families are available around the year with seasonal fluctuations. For the molecular characterization 499bp fragment of the partial 16S rRNA, 1812bp, 18S rRNA and 699bp COI nucleotide sequence were isolated and deposited in gene bank. The relative growth parameter measurements in C. natator were found to be isometric. The abdomen width of the female was broader than the male. ►The berried females were encounted throughout the year in varying percentage. The gonad showed at different degree of development in different months. The gonad index indicated that the crab Chaybdis natator breed continuously with two breeding peaks in a year. There was an inverse relationship noticed between gonad and hepatic indexes. ►The influence of environment factors like rainfall, humidity, temperature, salinity, turbidity, pH, DO and photoperiod etc. revealed that the crab C. natator breed antagonistically. The gonad index was maximum when temperature, salinity and photoperiod were recored high in summer. Another peak of gonad activity was found in monsoon when there was heavy rainfall and minimum temperature, salinity and photoperiod. It is inferred that the fluctuation of these factors might be the triggering mechanism for the breeding pattern. ►The sex ratio of C. natator was found to be slightly deviated 145 from the expected ratio of 1:1. The females were found predominant during the peak breeding months. The fecundity of crabs exhibited a direct relationship between carapace width and total number of eggs. The total number of eggs varied from 1, 93,229 to 7, 68,043. ►The biochemical composition of ovary, hepatopancreas and haemolymph at different stages of sexual maturity showed significant variation. The percentage of water content was found to be the maximum in early stages of maturity and minimum during peak breeding activity whereas in hepatopancreas an inverse relationship was noticed. The total protein and lipid content of the ovary showed increasing trend along with gonadal maturation. In the hepatopancreas and haemolymph protein, lipid, glucose, cholesterol and triglycerides were found to be the higher value in early stages and declined gradually as gonadal maturity advanced. The carbohydrate content was found to be more in ovary and hepatopancreas at early stage and declined gradually towards the advancement of gonadal maturity. The depletion of organic materials in the hepatopancreas and haemolymph with ripening of ovary is due to mobilization of these materials from hepatopancreas to ovary. ►The proximate composition in the haemolymph of crab showed significant variation during moulting cycle. The protein content varied from 4.9 0.51 to 7.2 0.59 g/dl. It was found to be the maximum in pre moulting stage and minimum in moulting stage. The amount of glucose showed a slight variation. The cholesterol ranged from 138 1.33 to 158

157 mg/dl and it gradually increased from post moult to pre moult. The triglycerides varied from 118 1.21 to 146 1.36 mg/dl. 146

►Enzymes showed a slight fluctuation during moulting cycle. Acid phosphate and LDH were found to be decreased whereas alkaline phosphatase increased during moulting. The SGOT was found to be the maximum in pre moulting and minimum in post moult. The SGPT ranged from 21.5 1.15 to 27.6 1.31 IU/L.

►Protein profile showed seven protein bands of molecular weight ranged from 14.2 to 97.4 kDa. The number of bands significantly varied among the tissues. The haemolymph contain protein bands of molecular weight 97.4, 66, 45, 36, 29 and 24 kDa, muscles 66, 45, 36, 29 and 24 kDa and hepatopancreas 45, 36, 29, 24 and 14.2 kDa. The number of protein bands were found to be minimum in immature and spent stages and the maximum in ripe stage of ovarian development. ►A total of 61 bioactive compounds isolated in gonad and hepatopancreas of C. natator at different stages of maturity. ►The microbes were found to be the 50 fungus and 40 bacteria in various tissues crab. In both microbes the higher population in alimentary canal and lower population in muscle. The predominantly isolated in 10 bacteria and 14 fungus of C. natator. ►There were 50 species of fungal flora and 40 bacteria isolated in various tissues of crab. More number of microbes isolated from alimentary canal and less in muscles.

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