Introduction

Aquaculture is the fast growing sector in the global food production (Subasinghe et al., 1998) due to the combination of a strong increasing demand for seafood products and depletion of fish stocks in the world’s oceans. Asia produced 80% of fish production at the rate of $ 38.855 billion (FAO, 1996). About 70% of fish production in China alone was through aquaculture in the year of 2002 (FAO, 2002). The world population consumed about 16% of animal protein, derived from fish and the fish is the main source of animal protein for about one billion people worldwide. Consumption of fish in the worldwide increased from 40 million tons during 1970, to more than 130 million tons during 2000. Further, an additional 40 million tons of seafood will be required by 2030 (FAO, 2000).

Protein-rich alternative feed ingredient is needed for better aqua feeds. An ingredient species has certain properties such as easy to handle shipping and storage, wide availability and competitive price. A number of species has been tested as alternative protein sources, such as single cell proteins, animal by-products including bacterial single cell proteins and microalgae (El-Sayed, 1994; Mazurkiewicz, 2009). It has been found that the algae can be used to improve the color, flavor and quality of aqua feeds. It is cultivated and commercialized worldwide due to its nutritional characteristics including high concentration of protein (~65%), vitamins and minerals salts (Sakthivel and Kathiresan, 2015). A number of works have tested the utilization of microalgae in the diet of aquaculture species (Allam, 2007; Tredici et al., 2009; Ayoola, 2010; Guedes and Malcata, 2010; Robin, 2012; Koye, 2013, Abdulrahman, 2014). Some of the reports revealed the beneficial growth or survival effect of different algal diets in shrimp culture systems (Behanan, 1990; Albentosa et al., 1997). High dietary protein of marine algae provides best growth for juveniles (Knuckey, 2001).

Cyanobacteria are used as food supplement because of their biochemical composition and easy digestibility. A number of studies have documented the nutritional excellence of dried cyanobacterial diets in shrimp and fish (Allam, 2007; Ayoola, 2010; Koye, 2013; Wood et al., 1991; Subramanian et al., 1992; Palaniselvam and Kathiresan, 1998; Sivakumar et al., 2011; 2014). Studies have shown that diets containing fish-based ingredients are generally performed better in terms of growth and feed efficiency than diets containing alternative plant based sources (Moyan et al., 1992; Webster et al., 1992; Kikuchi, 1999). In spite of efforts to replace microalgae by inert feed, aquaculturists are even now dependent on the production and use of microalgae as live food for commercially important molluscs, fish and crustaceans (Pauw and Pruder, 1986). Algal concentrates made by centrifugation are fed to bivalves and prawn larvae with successful results (Souza et al., 2000; Heasman et al., 2000; Robert et al., 2001).

The marine cyanobacteria have been tested for their nutritional values for hybrid Tilapia fish fry and found that most of the strains of Phormidium valderianum are acceptable as single-ingredient feed based on nutritional quality and non-toxic nature (Mitsui et al., 1981). The prawns are mainly predators feeding on organisms that are utilizing the benthic filamentous cyanobacteria like Spirulina sp., as a primary carbon source (Stoner and Zimmerman, 1988). The Spirulina species have gained commercial importance as a source of proteinaceous food and aquatic food (Vonshak et al., 1988). Nutritive value of marine cyanobacteria in prawn diet has been studied by Subramanian (Subramanian, 1998). Marine cyanobacterium, Phormidium tenue was tested as supplementary feed for two species of shrimp Penaeus monodon and P. semisulcatus (Palaniselvam and Kathiresan, 1998). The marine cyanobacterium Phormidium valderianum BDU 30501 was proved to be an aquaculture feed (Thajuddin and Subramanian, 2005) along with Synechococcus sp. and Phormidium sp., as feed for shrimp Penaeus monodon (Sivakumar et al., 2011, 2014). Although filamentous forms of cyanobacteria are well studied for dietary potential, unicellular marine forms are only little studied. Hence, the present work focused on Synechocystis salina as a supplementary feed for the shrimp Penaeus monodon.

Materials and methods

Composition of shrimp feed

The cyanobacterial species, Synechocystis salina was isolated from mangrove sediment and cultured under laboratory conditions using Marine Nutrient (MN) medium (Rippka et al., 1979). The MN medium was prepared by mixing seawater (750 ml), distilled water (250 ml), magnesium sulphate (0.04 g), sodium nitrate (0.75 g), sodium carbonate (0.02 g), dipotassium hydrogen orthophosphate (0.02 g), calcium chloride (0.02), citric acid (0.003 g), ferric ammonium citrate (0.003 g), ethylene diamine tetra acetic acid (0.000 5 g) and A5 micronutrients (1 ml). A5 micronutrients were prepared by adding distilled water (500 ml), boric acid (1.43 g), manganous chloride (0.95 g), zinc sulphate (0.111 g), sodium molybdate (0.008 6 g) and copper sulphate (0.039 5 g). The subculture of actively growing cells was maintained from stock cultures. Ten days old sub culture (500 ml/day) was added as additional feed directly to 20 L of experimental shrimp tank. The commercial feed without addition of cyanobacteria was used as control diet.

Feeding experiment of shrimp

The tiger shrimp Penaeus monodon popularly cultured in the aqua farms was used as the test organism. The active and healthy shrimps were visually sorted out and stocked for three days for acclimatization to laboratory conditions. The acclimatized animals were kept starved for 3 days prior to the feeding experiment. Ten animals of the same length (5.13 ± 0.44 cm) and weight (1.14 ± 0.28 g) were used for each treatment. Before stocking, the shrimps were measured for total length and wet weight. The experiment was conducted for 15 days in a 40 L plastic tank containing 20 L of filtered seawater. The water salinity 30 ± 2 g l^-1, temperature 29 ± 1℃, pH 8.0 ± 1 and dissolved oxygen 5 ± 0.5 mg l^-1 are maintained till the end of the experiment. The dissolved oxygen was maintained by using air-stone aerator, by adding freshwater and lime to maintain water salinity and pH. The feed was given at 10% of animal body weight. The fecal matter and excess of feeds were collected daily before water exchange, dried and weighed. For each treatment, triplicates were maintained. The parameters such as mean weight, production, relative growth rate, assimilation efficiency and food conversion efficiency were calculated (Crip, 1971). Then, the shrimps were scarified for the determination of carbohydrate (Dubois et al., 1956), protein (Lowry et al., 1951) and lipids (Folch et al., 1957).

Results

Growth of shrimps

The growth of shrimp in terms of length was higher (1.38 cm) in the shrimp fed with unicellular cyanobacterium Synechocystis salina than that in the control shrimp (1.18 cm) for 15 days experiment (Fig. 1). The physicochemical characters of all the experimental tank waters were well within the normal limit even after addition of marine cyanobacterial diets. Survival of the shrimp was recorded at 90 % in both control and cyanobacterial fed shrimps.

Effect of cyanobacterial diets on nutritional budget in shrimps

The parameters such as consumption (39%), fecal output (20%), assimilation (44%), metabolism (74%), and assimilation efficiency (11%) decreased due to the cyanobacterial supplementary feed to the shrimps (Fig. 2a-2e). The feed increased Gross Conversion Efficiency (46%) and Net Conversion Efficiency (63%) in the shrimps (Fig. 2f, 2g). However, the feed decreased Conversion Efficiency by 30% and Food Conversion Ratio by 43% in the shrimps (Fig. 2h & 2i). The feed increased gross conversion efficiency (78.91%) and decreased food conversion ratio (44.15%) in the shrimp.

Effect of cyanobacterial diets on biochemical composition of shrimps

Proximate composition of shrimp after the experimental period is shown in Figure 3a-3c. The cyanobacterial feed significantly increased the percentage of carbohydrate (31%) and lipid (13%), but protein level showed not much variation in the body tissue of the shrimp.

Discussion

Attempts have been made in utilizing different types of marine cyanobacteria for a variety of aquaculture applications. In one such study, 91 strains of nitrogen-fixing marine cyanobacteria have been tested for their nutritional value in Tilapia fish and found that majority of the strains are acceptable as single-ingredient feeds. Twenty-two strains of these marine cyanobacteria belonging to the genera Nostoc, Anabaena and Calothrix have supported the growth equivalent to those of fish feed. Very high growth rates of Tilapia fish using cyanobacteria with seawater, in indoor as well as outdoor cultures have been observed (Mitsui et al., 1983). The cyanobacterial feed enhanced the gross conversion efficiency and net conversion efficiency. However, it reduced the consumption, assimilation and metabolism. Food conversion ratio in the range from 1.48 to 1.81 is considered to be excellent (Mitsui et al., 1982). The food conversion ratio (FCR) was 0.25 in the cyanobacterial species Phormidium tenue treated as against 0.276 in commercial treated shrimp (Palaniselvam and Kathiresan, 1998).  Supplemented feed with Spirulina platensis power improved that feed conversion ratio and growth rates in common carp (Ibraheam et al., 2013, Abdulrahman 2014). The growth and feed utilization of Oreochromis niloticus were obtained at 5.0 g fresh culture of Spirulina platensis /kg diet (Al-Koye, 2013). In the present study, the food conversion ratio was 1.71 and it was 44.15% lower than control (Fig. 2i).

The proximate compositions of the cyanobacterial fed shrimp were higher for lipid (13%) and carbohydrate (31%) than controls (Fig. 3a, 3c). Proximate composition of shrimp particularly total content was significantly high in shrimp fed. This findings support by earlier studies when shrimp are fed with microalgae feed (Souza and Loneragan, 1999; Tobias-Quinitio and Villegas, 1982). However, in the present study, protein content in shrimp tissue was not significantly changed due to cyanobacterial feed (Fig. 3b). A similar observation has also been made in edible flesh of shrimp fed with different type of microalgal diets (Sivakumar et al., 2011). The cost involved in the production of cyanobacterium was less as compared to that in the commercial feed and hence, the usage of cyanobacterium in shrimp feed is advantageous. 

Microalgae grown to late-logarithmic growth phase typically contain 30 to 40% protein, 10 to 20% lipid and 5 to 15% carbohydrate (Brown et al., 1997; Renaud et al., 1999). In the algal culture of stationary phase, the proximate composition can change significantly; for example when nitrate is limiting, carbohydrate levels can double at the expense of protein (Harrison et al., 1990; Brown, 1993). There seems no strong correlation between the proximate composition of microalgae and nutritional value, although algal diets with high levels of carbohydrate are reported to produce the best growth in juvenile oyster (Ostrea edulis) (Enright et al., 1986) and polyunsaturated fatty acid (PUFAs) are also present in adequate proportions in larval scallops (Patinopecten yessoensis) (Whyte and Bourne, 1989). 

In the present study, the unicellular cyanobacterial feed increased the length growth of shrimp (16.95%) as compared to the control (Fig. 1a). This is in accordance with earlier studies with Phormidium valderianum (Thajuddin and Subramanian, 2005). The cyanobacterial pellet feed tested with the freshwater prawn Macrobrachium malcomsonii has shown that there is a 2-fold growth of the prawn, as compared to the live feed control (Subramanian, 1998). Similarly, the Phormidium sp., fed shrimp Penaeus monodon exhibits significant growth (Sivakumar et al., 2011). Microalgae supplementing in fish diet improve the growth and maturation of Mekong Giant Catfish (Meng-Umphan and Saengkrachang, 2008; Ungsethaphand and Hangsapreuke, 2008; Tongsiri, 2010). 

The present study has concluded that Synechocystis salina is a suitable supplementary feed for aquaculture shrimps based on increased gross conversion efficiency, decreased food conversion ratio and higher levels of carbohydrate and lipid in the cyanobacteriun-fed shrimps.

Acknowledgments

The authors are thankful to authorities of Annamalai University for providing facilities and to University Grants Commission (UGC) under Centre with Potential for Excellence in Particular Area (CPEPA) for financial support.

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