Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays

Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1 ), tryptose phosphate broth (2.95 g l 1), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 lgml 1 chloramphenicol, 100 lgml 1 streptomycin and 100 IU ml 1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-20-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals

Nitrification in a packed bed bioreactor integrated into amarine recirculating maturation system under different substrate concentrations and flow rates

BACKGROUND: A packed bed bioreactor (PBBR) activated with an indigenous nitrifying bacterial consortia was developed and commercialized for rapid establishment of nitrification in brackish water and marine hatchery systems in the tropics. The present study evaluated nitrification in PBBR integrated into a Penaeus monodon recirculating maturation system under different substrate concentrations and flow rates. RESULTS:Instantnitrificationwasobservedafter integration ofPBBRinto thematuration system.TANandNO2-Nconcentrations were always maintained below0.5 mg L−1 during operation. The TANandNO2-N removalwas significant (P < 0.001) in all the six reactor compartments of the PBBR having the substrates at initial concentrations of 2, 5 and 10 mg L−1. The average volumetric TAN removal rates increased with flow rates from 43.51 (250 L h−1) to 130.44 (2500 L h−1) gTAN m−3 day−1 (P < 0.05). FISH analysis of the biofilms after 70 days of operation gave positive results with probes NSO 190 ((β ammonia oxidizers), NsV 443 (Nitrosospira spp.) NEU (halophilic Nitrosomonas), Ntspa 712 (Phylum Nitrospira) indicating stability of the consortia. CONCLUSION: The PBBR integrated into the P. monodon maturation system exhibited significant nitrification upon operation for 70 days as well as at different substrate concentrations and flow rates. This system can easily be integrated into marine and brackish water aquaculture systems, to establish instantaneous nitrification

Mass production of nitrifying bacterial consortia for the rapid establishment of nitrification in saline recirculating aquaculture systems

Two distinct nitrifying bacterial consortia, namely an ammonia oxidizing non-penaeid culture (AMO NPCU-1) and an ammonia oxidizing penaeid culture (AMOPCU-1), have been mass produced in a nitrifying bacterial consortia production unit (NBCPU). The consortia, maintained at 4 C were activated and cultured in a 2 l fermentor initially. At this stage the net biomass (0.105 and 0.112 g/l), maximum specific growth rate (0.112 and 0.105/h) and yield coefficients (1.315 and 2.08) were calculated respectively, for AMONPCU-1 and AMOPCU-1 on attaining stationary growth phase. Subsequently on mass production in a 200 l NBCPU under optimized culture conditions, the total amounts of NH4 ?–N removed by AMONPCU-1 and AMOPCU-1 were 1.948 and 1.242 g/l within 160 and 270 days, respectively. Total alkalinity reduction of 11.7–14.4 and 7.5–9.1 g/l were observed which led to the consumption of 78 and 62 g Na2CO3. The yield coefficient and biomass of AMONPCU-1 were 0.67 and 125.3 g/l and those of AMOPCU-1 were 1.23 and 165 g/l. The higher yield coefficient and growth rate of AMOPCU-1 suggest better energy conversion efficiency and higher CO2 fixation potential. Both of the consortia were dominated by Nitrosomonas-like organisms. The consortia may find application in the establishment of nitrification within marine and brackish water culture systems.

Molecular characterization of the nitrifying bacterial consortia employed for the activation of bioreactors used in brackish and marine aquaculture systems

The addition of commercial nitrifying bacterial products has resulted in significant improvement of nitrification efficiency in recirculating aquaculture systems (RAS). We developed two nitrifying bacterial consortia (NBC) from marine and brackish water as start up cultures for immobilizing commercialized nitrifying bioreactors for RAS. In the present study, the community compositions of the NBC were analyzed by universal 16S rRNA gene and bacterial amoA gene sequencing and fluorescence in situ hybridization (FISH). This study demonstrated that both the consortia involved autotrophic nitrifiers, denitrifiers as well as heterotrophs. Abundant taxa of the brackish water heterotrophic bacterial isolates were Paenibacillus and Beijerinckia spp. whereas in the marine consortia they were Flavobacterium, Cytophaga and Gramella species. The bacterial amoA clones were clustered together with high similarity to Nitrosomonas sp. and uncultured beta Proteobacteria. FISH analysis detected ammonia oxidizers belonging to b subclass of proteobacteria and Nitrosospira sp. in both the consortia, and Nitrosococcus mobilis lineage only in the brackish water consortium and the halophilic Nitrosomonas sp. only in the marine consortium. However, nitrite oxidizers, Nitrobacter sp. and phylum Nitrospira were detected in both the consortia. The metabolites from nitrifiers might have been used by heterotrophs as carbon and energy sources making the consortia a stable biofilm.

Biochemical and molecular characterization of pathogenic vibrios from hatcheries and aquaculture farms

Vibrio are important during hatchery rearing. aquaculture phase and post-harvest quality of shrimps. Vibrio spp are of concern to shrimp farmers and hatchery operators because certain species can cause Vibriosis. Vibrio species are of concern to humans because certain species cause serious diseases.With the progress in aquaculture, intensive systems used for shrimp aquaculture create an artificial environment that increases bacterial growth. To maintain the productivity of such an intensive aquaculture, high inputs of fish protein have to be employed for feeding together with high levels of water exchange and the massive use of antibiotics/ probiotics / chemicals. It seems that the combination of these conditions favours the proliferation of vibrios and enhances their virulence and disease prevalence. The risk of a microbial infection is high, mainly at larval stages. The effect and severity are related to Vibrio species and dose, water, feed, shrimp quality and aquaculture management.Consumption of seafood can occasionally result in food-bome illnesses due to the proliferation of indigenous pathogens like Vibrio.Of the l2 pathogenic Vibrio species, 8 species are known to be directly food associated. Strict quality guidelines have been laid by the importing nations, for the food products that enter their markets. The microbiological quality requirement for export of frozen shrimp products is that V.cholerae, V.parahaemolyticus and V. vulnificus should be absent in 25g of the processed shrimp (Export Inspection Council of India, 1995). The mere presence of these pathogenic Vibrios is sufficient for the rejection of the exported product.The export rejections cause serious economic loss to the shrimp industry and might harm the brand image of the shrimp products from the countiy.There is a need for an independent study on the incidence of different pathogenic vibrios in shrimp aquaculture and investigate their biochemical characteristics to have a better understanding about the growth and survival of these organisms in the shrimp aquaculture niche. PCR based methods (conventional PCR, duplex PCR, multiplex-PCR and Real Time PCR) for the detection of the pathogenic Vibrios is important for rapid post-harvest quality assessment. Studies on the genetic heterogeneity among the specific pathogenic vibrio species isolated from shrimp aquaculture system provide; valuable information on the extent of genetic diversity of the pathogenic vibrios, the shrimp aquaculture system.So the present study was undertaken to study the incidence of pathogenic Vibrio spp. in Penaeus monodon shrimp hatcheries and aquaculture farms, to carry out biochemical investigations of the pathogenic Vibrio spp isolated from P. monodon hatchery and. aquaculture environments, to assess the effect of salt (NaCl) on the growth and enzymatic activities of pathogenic Vibrio spp., to study the effect of preservatives, and chemicals on the growth of pathogenic Vibrio spp. and to employ polymerase chain reaction (PCR) methods for the detection of pathogenic V ibrio spp.Samples of water (n=7) and post-larvae (n=7) were obtained from seven Penaeus monodon hatcheries and samples of water (n=5), sediment (n=5) and shrimp (n=5) were obtained from five P. monodon aquaculture farms located on the East Coast of lndia. The microbiological examination of water, sediment, post-larvae and shrimp samples was carried out employing standard methods and by using standard media.The higher bacterial loads were obtained in pond sediments which can be attributed to the accumulation of organic matter at the pond bottom which stimulated bacterial growth.Shrimp head. (4.78 x 105 +/- 3.0 x 104 cfu/g) had relatively higher bacterial load when compared to shrimp muscle 2.7 x 105 +/- 1.95 x 104 cfu/g). ln shrimp hatchery samples, the post-larvae (2.2 x 106 +/- 1.9 x 106 cfu/g) had higher bacterial load than water (5.6 x 103 +/- 3890 cfu/ml).The mean E.coli counts were higher in aquaculture pond sediment (204+/-13 cfu/g) and pond water (124+/-88 cfu/ml). Relatively lower Escherichia coli counts were obtained from shrimp samples (12+/-11 to 16+/-16.7 cfu/g). The presence of E.coli in aquaculture environment might have been from the source water. E.coli was not detected in hatchery waters and post-larvae.

Bioeconomic Evaluation of Semi-intensive Shrimp Farming in Kerala

Penaeid shrimps are, perhaps, the most important fishery resource of the coastal waters of our country. Their exceptionally tasty. protein-rich flesh tops any seafood in foreign exchange earnings. No wonder, the demand of shrimp, the "Pinkish Gold of the Sea" (MPEDA. 1992). is increasing in the world market. The study of the growth of an organism is important in understanding the conditions under which optimum growth occurs. It is also important in getting an insight into the various factors that influence growth. Studies on the growth pattern of commercially important species of shrimp and of the factors that influence their growth rate are essential for the successful cultivation of shrimps.

Development of Bioreactors for Nitrifying Water In Closed System Hatcheries Of Penaeid And Non-Penaeid Prawns

In the present study the development of bioreactors for nitrifying water in closed system hatcheries of penaeid and non-penaeid prawns. This work is an attempt in this direction to cater to the needs of aquaculture industry for treatment and remediation of ammonia and nitrate in penaeid and non-penaeid hatcheries, by developing nitrifying bacteria allochthonous to the particular environment under consideration, and immobilizing them on an appropriately designed support materials configured as reactors. Ammonia toxicity is the major limiting factors in penaeid and non-penaeid hatchery systems causing lethal and sublethal effects on larvae depending on the pH values. Pressing need of the aquaculture industry to have a user friendly and economically viable technology for the removal of ammonia, which can be easily integrated to the existing hatchery designs without any major changes or modifications. Only option available now is to have biological filters through which water can be circulated for the oxidation of ammonia to nitrate through nitrite by a group of chemolithotrophs known as nitrifying bacteria. Two types of bioreactors have been designed and developed. The first category named as in situ stringed bed suspended bioreactor(SBSBR) was designed for use in the larval rearing tanks to remove ammonia and nitrite during larval rearing on a continuous basis, and the other to be used for nitrifying freshly collected seawater and spent water named as ex situ packed bed bioreactior(PBBR). On employing the two reactors together , both penaeid and non-penaeid larval rearing systems can be made a closed recirculating system at least for a season. A survey of literature revealed that the in situ stringed bed suspended reactor developed here is unique in its design, fabrication and mode of application.

Extraction of Glucan from Acremonium Diospyri and Its Application in Macrobrachium Rosenbergii Larval Rearing System Along With Bacterians as Microspheres

Present work is aimed at development of an appropriate microbial technology for protection of larvae of macrobrachium rosenbergii from disease and to increase survival rate in hatcheries. Application of immunostimulants to activate the immune system of cultured animals against pathogen is the widely accepted alternative to antibiotics in aquaculture. The most important immunostimulant is glucan. Therefore a research programme entitled as extraction of glucan from Acremonium diospyri and its application in macrobrachium rosenbergii larval rearing system along with bacterians as microspheres. The main objectives of the study are development of aquaculture grade glucan from acremonium diospyri, microencapsulated drug delivery system for the larvae of M. rosenbergii and microencapsulated glucan with bacterian preparation for the enhanced production of M. rosenbergii in larval rearing system. Based on the results of field trials microencapsulated glucan with bacterin preparation, it is concluded that the microencapsulated preparation at a concentration of 25g per million larvae once in seven days will enhance the production and quality seed of M. rosenbergii.

STUDIES ON TAXONOMY, DISTRIBUTION, ECOLOGY AND REPRODUCTIVE POTENTIAL OF ROTIFERS FROM SELECTED CENTRES IN COCHIN BACKWATER SYSTEM, KERALA

The studies were conducted in nine stations with varying ecological characteristics along Cochin backwaters and adjoining canals. Many workers opined that the distribution of rotifers is cosmopolitan. The significance of rotifers as first food for early larvae was indicated by Fujita. Aquaculture is a fast growing field in fisheries sector and it is gaining more importance as the fish landings and supply are getting irregular. A consistent supply of fish/shellfish can only be achieved through aquaculture. The success of any culture activity depends on the timely production of seeds of finfishes/shellfishes. The availability of wild seed is seasonal and erratic. So, a dependable source of seed of fishes and shellfishes is possible only through large scale production in hatchery. A successful seed production activity depends on the availability of a variety of suitable live feed organisms in sufficient quantities at the proper time for use in the larval stages. As the live feeds promote high growth rates, easy digestion, assimilation and the quality of not contaminating the culture water when compared to other artificial feeds, make the culture of live feed organisms the principal means of providing food for the larvae of finfishes and shellfishes. Rotifers are considered to be an excellent and indispensable food for larvae of many finfishes and crustaceans. It (1960) was the first to culture Brachionus plicatilis for feeding marine fish larvae, and now it is being extensively used as live feed in hatcheries all over the world. They are a group of microscopic organisms coming under the Phylum Rotifera which comprises of about 2000 species. Their slow swimming habits, ability to tolerate a wide range of salinities, parthenogenetic mode of reproduction and ability to get enriched easily, make rotifers an ideal live feed organism. The major factors such as temperature, salinity and food that influence the reproductive potential and thereby the population size of rotifer, Salinity is one of the most important aspect influencing the reproductive rate of rotifers. The feed type and feed concentration play a vital role in influencing the reproductive rate of rotifers. For culture of rotifers, the commonly used micro algae belong to Chlorella, Nannochloropsis, Isochrysis and Tetraselmis. While some studies have suggested that, algal diet has little effect on reproductive rates in 1979 while using the rotifer, Brachionus plicatilis as feed for the larvae of red sea bream, Pagrus major. It is generally accepted that rotifers play a pivotal role in the successful rearing of marine fish larvae.

Development of Safety and Quality Management System in Shrimp Farming

Shrimp Aquaculture has provided tremendous opportunity for the economic and social upliftment of rural communities in the coastal areas of our country Over a hundred thousand farmers, of whom about 90% belong to the small and marginal category, are engaged in shrimp farming. Penaeus monodon is the most predominant cultured species in India which is mainly exported to highly sophisticated, quality and safety conscious world markets. Food safety has been of concem to humankind since the dawn of history and the concern about food safety resulted in the evolution of a cost effective, food safety assurance method, the Hazard Analysis Critical Control Point (HACCP). Considering the major contribution of cultured Penaeus monodon to the total shrimp production and the economic losses encountered due to disease outbreak and also because traditional methods of quality control and end point inspection cannot guarantee the safety of our cultured seafood products, it is essential that science based preventive approaches like HACCP and Pre requisite Programmes (PRP) be implemented in our shrimp farming operations. PRP is considered as a support system which provides a solid foundation for HACCP. The safety of postlarvae (PL) supplied for brackish water shrimp farming has also become an issue of concern over the past few years. The quality and safety of hatchery produced seeds have been deteriorating and disease outbreaks have become very common in hatcheries. It is in this context that the necessity for following strict quarantine measures with standards and code of practices becomes significant. Though there were a lot of hue and cry on the need for extending the focus of seafood safety assurance from processing and exporting to the pre-harvest and hatchery rearing phases, an experimental move in this direction has been rare or nil. An integrated management system only can assure the effective control of the quality, hygiene and safety related issues. This study therefore aims at designing a safety and quality management system model for implementation in shrimp farming and hatchery operations by linking the concepts of HACCP and PRP.

Carbon/Nitrogen Ratio Optimization and Periphyton Development on the Production and Sustainability of Penaeus Monodon(Fabricius)in Extensive Culture System

Controlling the inorganic nitrogen by manipulating carbon / nitrogen ratio is a method gaining importance in aquaculture systems. Nitrogen control is induced by feeding bacteria with carbohydrates and through the subsequent uptake of nitrogen from the water for the synthesis of microbial proteins. The relationship between addition of carbohydrates, reduction of ammonium and the production of microbial protein depends on the microbial conversion coefficient. The carbon / nitrogen ratio in the microbial biomass is related to the carbon contents of the added material. The addition of carbonaceous substrate was found to reduce inorganic nitrogen in shrimp culture ponds and the resultant microbial proteins are taken up by shrimps. Thus, part of the feed protein is replaced and feeding costs are reduced in culture systems.The use of various locally available substrates for periphyton based aquaculture practices increases production and profitability .However, these techniques for extensive shrimp farming have not so far been evaluated. Moreover, an evaluation of artificial substrates together with carbohydrate source based farming system in reducing inorganic nitrogen production in culture systems has not yet been carried-out. Furthermore, variations in water and soil quality, periphyton production and shrimp production of the whole system have also not been determined so-far.This thesis starts with a general introduction , a brief review of the most relevant literature, results of various experiments and concludes with a summary (Chapter — 9). The chapters are organised conforming to the objectives of the present study. The major objectives of this thesis are, to improve the sustainability of shrimp farming by carbohydrate addition and periphyton substrate based shrimp production and to improve the nutrient utilisation in aquaculture systems.

“Development of lymphoid cell culture system from Penaeus monodon and molecular approaches for its transformation

Unveiling the molecular and regulatory mechanisms that prevent in vitro transformation in shrimp remains elusive in the development of continuous cell lines, with an arduous history of over 25 years (Jayesh et al., 2012). Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. In addition, the regulatory mechanisms that prevent in vitro transformation and carcinoma in shrimps might provide new leads for the development of anti-ageing and anti-cancer interventions in human (Vogt, 2011) and in higher vertebrates. This highlights the importance of developing shrimp cell lines, to bring out effective prophylactics against shrimp viruses and for understanding the mechanism that induce cancer and ageing in human.. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. With this backdrop, the research described in this thesis attempted to develop molecular tools for induced in vitro transformation in lymphoid cells from Penaeus monodon and for the development of continuous cell lines using conventional and novel technologies to address the problems at cellular and molecular level.

Development of Zero Water Exchange Shrimp Culture System Integrated With Bioremediation of Detritus and Ammonia- Nitrogen

Aquaculture is one of the fastest growing food sectors in the world. Amongst the various branches of aquaculture, shrimp culture has expanded rapidly across the globe because of its faster growth rate, short culture period, high export value and demand in the International market. Indian shrimp farming has experienced phenomenal development over the decades due to its excellent commercial viability. Farmers have adopted a number of innovative technologies to improve the production and to maximize the returns per unit area. The culture methods adopted can be classified in to extensive, modified extensive and semi intensive based on the management strategies adopted in terms of pond size, stocking density, feeding and environmental control. In all these systems water exchanges through the natural tidal effects, or pump fed either from creek or from estuaries is a common practice. In all the cases, the systems are prone to epizootics due to the pathogen introduction through the incoming water, either brought by vectors, reservoir hosts, infected tissue debris and free pathogens themselves. In this scenario, measures to prevent the introduction of pathogen have become a necessity to protect the crop from the onslaught of diseases as well as to prevent the discharge of waste water in to the culture environment.The present thesis deals with Standardization of bioremediation technology for zero water exchange shrimp culture system

Development of a cell culture system from the ovarian tissue of African catfish Clarias gariepinus

A growth medium with Leibovitz-15 L-15.as the base, supplemented with foetal bovine serum 10% vrv., fish muscle extract 10% vrv., prawn muscle extract 10% vrv., lectin concanavalin A. 0.02 mg mly1., lipopolysaccharide 0.02 mg mly1., glucose D 0.2 mg mly1., ovary extract 0.5% vrv.and prawn haemolymph 0.5%. has been formulated with 354"10 mOsm for the development and maintenance of a cell culture system from the ovarian tissue of African catfish, Clarias gariepinus. For its subculturing, a cell dissociationrextracting solution, composed of equal portions of trypsin phosphate versene glucose TPVG. containing 0.0125% wrv.trypsin and 25% vrv.non-enzymatic cell dissociation solution 1 and 2, has also been developed with which the cell culture can be passaged 15 times after which they cease to multiply and consequently perish. The cell cultures can be maintained for 12–15 days without fluid change between the passages. This is the first report of a cell culture system from the ovarian tissues of African catfish

Penaeus monodon larvae can be protected from Vibrio harveyi infection by pre-emptive treatment of a rearing system with antagonistic or non-antagonistic bacterial probiotics

This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non-antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having su⁄cient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB111under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen.These inhibitory propertieswere further con¢rmed in the larval rearing systems of P. monodon. All these four probionts signi¢cantly improved larval survival in long-term treatments as well as when challengedwith a pathogenic strain ofV. harveyiMCCB111. We could demonstrate that Pseudomonas MCCB 102 andMCCB103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems throughactive antagonism of vibrios,whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme-supporting modes of action.