Cell Culture Systems from Penaeus monodon: Development and Application

National Centre for Aquatic Animal Health, Cochin University of Science and Technology

Immune Response of Penaeus monodon to the Inactivated White Spot Syndrome Virus Preparation

National Centre for Aquatic Animal Health, Cochin University of Science and Technology

Marine Actinomycetes as Source of Antiviral Agents and as Probionts for Penaeus monodon Culture Systems

The present study revealed the importance of marine actinomycetes as a potent source of bio active secondary metabolites. The selected isolates were capable of protecting Peaneus monodon against WSSV infection. They also proved to be inhibitory to vibrios and is a rich pool of hydrolytic enzymes. Their capacity to proliferate in saline environments and their property of non-pathogenicity to prawns makes them good candidates to be applied as probionts in penaeid shrimp aquaculture. They also enhanced the immune status of shrimps challenged with WSSV and act as a good source of antioxidants. Exploitation of the potential for the prophylactic and therapeutic measures in aquatic animal health management would be highly rewarding. This work is a preliminary study targeting marine actinomycetes as a source of antiviral compounds and as probionts in Penaeus monodon culture systems. More work is needed to understand the nature and mode of action of the bioactive compound, the various aspects of immune and antioxidant responses under challenge and when exposed to pro active treatments, and the dose and frequency of application of such compounds under rearing conditions.

Biochemical effects of the pesticide Chlorpyrifos on the fish Oreochromis mossambicus (Peters)

Man uses a variety of synthetic material for his comfortable materialistic life. Thus human interactions may become harmful for various terrestrial and aquatic lives. This is by contaminating their habitat and by becoming a threat to organisms itself. Thus the application and dispersal of several organic pollutants can lead to the development of several mutated forms of the species when exposed to sublethal concentrations of the pollutants. Otherwise, a decrease in number or extinction of these exposed species from earth's face may happen. Pesticides, we use for the benefit of crop yield, but its persistence may become havoc to non-target organism. Pesticides reaching a reservoir can subsequently enter the higher trophic levels. Organophosphorus compounds have replaced all other pesticides, due to its acute toxicity and non-persistent nature.Hence the present study has concentrated on the toxicity of the largest market-selling and multipurpose pesticide, chlorpyrifos on the commonly edible aquatic organism, fish. The euryhaline cichlid Oreochromis mossambicus was selected as animal model. The study has concentrated on investigating biochemical parameters like tissue-specific enzymes, antioxidant and lipid-peroxidation parameters, haematological and histological observations and pesticide residue analysis.Major findings of this work have indicated the possibility of aquatic toxicity to the fish on exposure to the insecticide chlorpyrifos. The insecticide was found as effective to induce structural alteration, depletion in protein content, decrease in different metabolic enzyme levels and to progress lipid peroxidation on a prolonged exposure of 21 days. The ion-transport mechanism was found to be adversely affected. Electrophoretic analysis revealed the disappearance of several protein bands after 21days of exposure to chlorpyrifos. Residue, analysis by gas chromatography explored the levels of chlorpyrifos retaining on the edible tissue portions during exposure period of 21days and also on a recovery period of 10 days.

Nitrifying Bioreactors Integrated into Shrimp and Prawn Hatchery Systems: Molecular Characterization of the Nitrifying Consortia,Reactor Kinetics, Modeling and Validation

National Centre for Aquatic Animal Health, Cochin University of Science and Technology

Proteases from an Environmental Isolate of Pseudomonas Aeruginosa MCCB 123 and Their Applications

In the present study,heterotrophic protease producing bacterial isolates were screened for protease activity and a potent protease producing bacterial isolate was selected,identified and coded as Pseudomonas aeruginosa MCCB 123.The organism was capable of producing three different types of enzymes each having potential industrial applications.The non-toxic nature of the bacterial strain and the relatively non-toxic nature of three enzymes suggested their poetential application in various industries.Application of LasA protease and beta-1,3 glucanase in DNA extraction is a promising area for commercial utilization. LasB protease can find its potential application in detergent and tanning industries.As on today Bacillus sp.has been the source of commercial proteases,and the ones produced form P.aeruginosa 123 can pave way for making the industrial and biomedical processes more cost effective and refined.

Bioprocess Technology for Antagonistic Pseudomonas MCCB 102, 103,Micrococcus MCCB 104, and Evaluation of Synechocystis MCCB 114, 115 as Probiotics for the Management of Vibrio in Shrimp Culture Systems

National Centre for Aquatic Animal Health, School of Environmental Studies, Cochin University of Science and Technology

Biocontrol of Vibrio harveyi in Penaeus monodon larval rearing systems employing probiotics and vibriophages

National Centre for Aquatic Animal Health, School of Environmental Studies, Cochin University of Science and Technology.

Molecular genetic characterization of endemic red -tailed barb,Gonoproktoptems curmuca (Hamilton - Buchanan, 1807)

The family Cyprinidae is the largest of freshwater fishes and, with the possible exception of Gobiidae, the largest family of vertebrates.Various members of this family are important as food fish, as aquarium fish, and in biological research. In this study, a fish species from this family exclusively found in the west flowing rivers originating from the Western Ghat region — Gonoproktopterus curmuca — was taken for population genetic analysis.There was an urgent need for restoration ecology by the development of apt management strategies to exploit resources judiciously. One of the strategies thus developed for the scientific management of these resources was to identify the natural units of the fishery resources under exploitation (Altukov, 1981). These natural units of a species can otherwise be called as stocks. A stock can be defined as a panmictic population of related individuals within a single species that is genetically distinct from other such populations.It is believed that a species may undergo micro evolutionary process and differentiate into genetically distinct sub-populations or stocks in course of time, if reproductively and geographically isolated.In recent times, there has been a wide spread degradation of natural aquatic environment due to anthropogenic activities and this has resulted in the decline and even extinction of some fish species. In such situations, evaluation of the genetic diversity of fish resources assumes important to conservation.The species selected for the study, was short-listed as one of the candidates for stock-specific, propagation assisted rehabilitation and management programme in rivers where it is naturally distributed. In connection with this, captive breeding and milt cryopreservation techniques of the species have been developed by the National Bureau of Fish Genetic Resources, Lucknow. However, for a scientific stock-specific rehabilitation programme, information on the stock structure and basic genetic profile of the species are essential and that is not available in case of G. curmuca. So the present work was taken up to identify molecular genetic markers like allozymes, microsatellites and RAPDs and, to use these markers to discriminate the distinct populations of the species, if any, in areas of its natural distribution. The genetic markers were found to be powerful tools to analyze the population genetic structure of the red-tailed barb and demonstrated clear cut genetic differentiation between pairs of populations examined. Geographic isolation by land distance is likely to be the factor that contributed to the restricted gene flow between the river systems. So the present study emphasizes the need for stock-wise, propagation assisted-rehabilitation of the natural populations of red-tailed barb, Gonoprokfopterus curmuca.

Ecophysiology of nontuberculous Mycobacteria in marine aquaculture ponds

The world demand for fish and fishery products is increasing steadily and it is generally accepted that it will not be possible to meet the heavy demand with resources exploited from capture fishery alone. Now aquaculture is well established and fastdeveloping industry in many countries and is a major focus sector for development. During recent decades, aquaculture has gained momentum, throughout the world especially in developing countries. According to Food and Agricultural Oganisation (FAO, 2000), global aquaculture production was 26.38 tones in 1996 have reached 32.9 million tonnes during 1999. Only marine aquaculture sector has contributed 13.1 million tonnes during 1999.India is a major fish producing country. About one half of lndia’s brackish water lands are currently being utilized for farming in order to reduce the gap between supply and demand for fish. Aquaculture has become a major source of livelihood for people and its role in integrated rural development, generation of employment and earning foreign exchange, thereby alleviating poverty is being greatly appreciated around the world.Among the infectious agents, bacteria are becoming the prime causal organisms for diseases in food fishes and other marine animals. Sindermann, (1970) reported that bacterial fish pathogen most commonly found among marine fishes is species of Pseudomonas, Vibrio and Mycobacterium. These can be categorized into primary pathogens; secondary invaders that may cause systemic disease in immunocompromised hosts; and normal marine flora which are not pathogenic but may occur on body surfaces or even within the tissues of the host. I-Iigh density of animals in hatchery tanks and ponds is conducive to the spread of pathogen and the aquatic environment with regular application of protein rich feed, is ideal for culturing bacteria. Bacteria, which are normally present in seawater or on the surface of fish, can invade and cause pathological effects in fishes, which are injured or subjected to other environmental stresses.Mycobacteria except parasites are known as nontuberculosis mycobacteria (NTM), atypical mycobacteria or mycobacteria other than tuberculosis(MO'l'l"). This group of mycobacteria includes opportunistic pathogens and saprophytes. Environmental mycobacteria are ubiquitous in distribution and the sources may include soil, water, warm-blooded as well as cold-blooded animals. Disease caused by environmental mycobacterial strains in susceptible humans (Goslee & Wolinsky, 1976; Grange, 1987), animals and fishes are increasingly attracting attention. Greatest importance of environmental mycobacteria is believed to be their role in immunological priming of humans and animals, thereby modifying their immune responses to subsequent exposure to pathogenic species.

Haematology of Fenneropenaeus indicus in response to environmental alterations and microbial infections

Aquaculture has developed to become one of the fastest growing food producing sectors in the world.Today India is one among the major shrimp producing countries in the world.There are extensive and intensive shrimp culture practices. In extensive shrimp culture, shrimps are stocked at low densities (< 25 PLs m'2)in large ponds or tidal enclosures in which little or no management is exercised or possible. Farmers depend almost entirely on natural conditions in extensive cultures. Intensive shrimp culture is carried out in high densities (>200 PLs m'2). Much of the world shrimp production still comes from extensive culture.There is a growing demand for fish and marine products for human and animal consumption. This demand has led to rapid growth of aquaculture, which some times has been accompanied by ecological impacts and economic loss due to diseases. The expansion of shrimp culture always accompanies local environmental degradation and occurrence of diseases.Disease out breaks is recognised as a significant constraint to aquaculture production. Environmental factors, water quality, pollution due to effluent discharge and pathogenic invasion due to vertical and horizontal transmission are the main causes of shrimp disease out breaks. Nutritional imbalance, toxicant and other pollutants also account for the onset of diseases. pathogens include viruses, bacteria, fungi and parasites.Viruses are the most economically significant pathogens of the cultured shrimps world wide. Disease control in shrimp aquaculture should focus first on preventive measures for eliminating disease promoting factors.ln order to design prophylactic and proactive measures against shrimp diseases, it is mandatory to understand the immune make up of the cultivable species, its optimum culture conditions and the physico chemical parameters of the rearing environment. It has been proven beyond doubt that disease is an end result of complex interaction of environment, pathogen and the host animal. The aquatic environment is abounded with infectious microbes.The transmission of disease in this environment is extremely easy, especially under dense, culture conditions. Therefore, a better understanding of the immune responses of the cultured animal in relation to its environmental alterations and microbial invasions is essential indevising strategic measures against aquaculture loss due to diseases. This study accentuate the importance of proper and regular health monitoring in shrimps employing the most appropriate haematological biomarkers for application of suitable prophylactic measures in order to avoid serious health hazards in shrimp culture systems.

Studies on taxonomy and ecology of Mysidacea from the EEZ of India

Members of the order Mysidacea are important component in marine and estuarine plankton inhabiting all regions of the oceans. There are many brackish water species and few species occur in fresh water, some have become adapted to the specialized environments of caves and wells. They are omnivores, responsible for remineralisation of a substantial portion of the detritus in the water column. They form an important link in the food chain (between microbial producers and secondary consumers) and therefore play a major role in the cycling of energy within the aquatic ecosystem. In tropical and subtropical waters, swarms of mysids are exploited commercially and marketed as preserved cooked food. Mysids have been used in fish farming as live feed resource. They are also excellent experimental organism, extremely useful in the studies of potential impact of various pollutants in the aquatic environment. Mysids are also used in wood pulp effluent plants.Considering the significant role of mysids in the productivity of tropical and coastal ecosystems,the present study has been undertaken to extend our knowledge on the systematics, species composition, distribution,abundance and ecology of mysid fauna of the Indian EEZ and adjoining areas. The present study therefore will undoubtedly fumish valuable information on Mysidacea of the Indian waters.

Development of nitrifying and photosynthetic sulfur bacteria based bioaugmentation systems for the bioremediation of ammonia and hydrogen sulphide in shrimp culture

This thesis entitled Development of nitrifying ans photosynthetic sulfur bacteria based bioaugmentation systems for the bioremediation of ammonia and hydregen sulphide in shrimp culture. the thesis is to propose a sustainable, low cost option for the mitigation of toxic ammonia and hydrogen sulphide in shrimp culture systems. Use of ‘bioaugmentors’ as pond additives is an emerging field in aquaculture. Understanding the role of organisms involved in the ‘bioaugmentor’ will obviously help to optimize conditions for their activity.The thesis describes the use of wood powder immobilization of nitrifying consortia.Shrimp grow out systems are specialized and highly dynamic aquaculture production units which when operated under zero exchange mode require bioremediation of ammonia, nitrite nitrogen and hydrogen sulphide to protect the crop. The research conducted here is to develop an economically viable and user friendly technology for addressing the above problem. The nitrifying bacterial consortia (NBC) generated earlier (Achuthan et al., 2006) were used for developing the technology.Clear demonstration of better quality of immobilized nitrifiers generated in this study for field application.

Apptication of Biofloc Technology (BFI) in Ihe nunery rearing and farming of giant freshwater prawn, Macrobrachillm rosenbergii (de Man)

This thesis Entitled Application of Biofloc technology (BFT) In the Nursery Rearing and Farming of Giant Freshwater Prawn,Macrobrachium Rosenbergii(De Man). Aquaculture, rearing plants and animals under controlled conditions is growing with an annual growth rate of 8.3% in the period 1970-2008 (FAO, 2010). This trend of growth is essential for the supply of protein-rich food for ever increasing world population. But growth and development of aquaculture should be in sustainable manner, preferably without jeopardizing the aquatic environment.In the present study, the application of BFT in the nursery rearing and farming ofgiant freshwater prawn, M. rosenbergii, is attempted. The result of the study is organised into eight chapters. In the first chapter, the subject is adequately introduced. Various types of aquaculture practices followed, development and status of Indian aquaculture, present status of freshwater pravm culture, BF T and its use for the sustainable aquaculture systems, theory of BFT based aquaculture practices, hypothesis, objective and outline of the thesis are described. An extensive review of literature on studies carried out so far on biofloc based aquaculture are given in chapter 2. The third chapter deals with the application of BFT in the primary nursery phase of freshwater prawn. Several workers suggested the need for an intermediate nursery phase in the culture system of freshwater prawn for the successful production. Thirty day experiment was conducted to study the effect of BFT on the water quality, and animal welfare under the various stocking densities. The study concluded that stocking finfishes in biofloc-based monoculture system of freshwater prawns has the potential of increasing total yield. Prawns having a higher commercial value than finfishes besides ensuring economic sustainability. Results showed that prawn yield and survival was better in catla dominated tanks. Based on the results of the study, it is recommended to incorporate 25% rohu and 75% catla in the biofloc-based culture system of giant freshwater prawns. The results of the present study also recommend to stock relatively larger catla for biofloc-based culture system. Fish production was also higher in the 100% catla tank. When catla was added in higher percentages it should ensured that the hiding objects in the culture ponds shall be used in order to reduce the chance of cannibalism among prawns. rohu and catla equally have the ability to harvest the biofloc, catla consumes the planktonic contributes in the floc whereas rohu grazed on the bacterial consortium suspended in the water column. In Chapter 8, recommendations and future research perspectives in the field of biofloc based aquaculture is presented.

Characterization of polyhydroxyalkanoates accumulating vibrios from marine benthic environments and production studies of polyhydroxyalkanoates by Vibrio sp. BTKB33

The present work deals with the characterization of polyhydroxyalkanoates accumulating vibrios from marine benthic environments and production studies of polyhydroxyalkanoates by vibrio sp.BTKB33. Vibrios are a group of (iram negative, curved or straight motile rods that normally inhabit the aquatic environments.The present study therefore aimed at evaluating the occurrence of PHA accumulating vibrios inhabiting marine benthic environments; characterizing the potential PHA accumulators employing phenotypic and genotypic approaches and molecular characterization of the PHA synthase gene. The study also evaluated the PHA production in V:'hri0 sp. strain BTKB33, through submerged fennentation using statistical optimization and characterized the purified biopolymer. Screening for PHA producing vibrios from marine benthic environments. Characterization of PHA producers employing phenotypic and genotypic approaches.The incidence of PHA accumulation in Vibrio sp. isolated from marine sediments was observed to be high, indicating that the natural habitat of these bacteria are stressful. Considering their ubiquitous nature, the ecological role played by vibrios in maintaining the delicate balance of the benthic ecosystem besides returning potential strains, with the ability to elaborate a plethora of extracellular enzymes for industrial application, is significant. The elaboration of several hydrolytic enzymes by individuals also emphasize the crucial role of vibrios in the mineralization process in the marine environment. This study throws light on the extracellular hydrolytic enzyme profile exhibited by vibrios. It was concluded that apart from the PHA accumulation, presence of exoenzyme production and higher MAR index also aids in their survival in the highly challenging benthic enviromnents. The phylogenetic analysis of the strains and studies on intra species variation within PHA accumulating strains reveal their diversity. The isolate selected for production in this study was Vibrio sp. strain BTKB33, identified as V.azureus by 16S rDNA sequencing and phenotypic characterization. The bioprocess variables for PHA production utilising submerged fermentation was optimized employing one-factor-at-a-time-method, PB design and RSM studies. The statistical optimization of bioprocess variables revealed that NaCl concentration, temperature and incubation period are the major bioprocess variables influencing PHA production and PHA content. The presence of Class I PHA synthase genes in BTKB33 was also unveiled. The characterization of phaC genes by PCR and of the extracted polymer employing FTIR and NMR analysis revealed the presence of polyhydroxybutyrate, smallest known PI-IAs, having wider domestic, industrial and medical application. The strain BTKB33 bearing a significant exoenzyme profile, can thus be manipulatedin future for utilization of diverse substrates as C- source for PHA production. In addition to BTKB33, several fast growing Vibrio sp. having PHA accumulating ability were also isolated, revealing the prospects of this environment as a mine for novel PHA accumulating microbes. The findings of this study will provide a reference for further research in industrial production of PHAs from marine microorganisms .