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.

Development of Innovative Low Cost Larviculture Technologies of the Giant Fresh Water Prawn,Macrobrachium Rosenbergii(De Man)

School of Industrial Fisheries, Cochin University of Science and Technology

Reducing Vibrio load in Artemia nauplii usingantimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture

We propose antimicrobial photodynamic therapy (aPDT) as an alternative strategy to reduce the use of antibiotics in shrimp larviculture systems. The growth of a multiple antibiotic resistant Vibrio harveyi strain was effectively controlled by treating the cells with Rose Bengal and photosensitizing for 30 min using a halogen lamp. This resulted in the death of > 50% of the cells within the first 10 min of exposure and the 50% reduction in the cell wall integrity after 30 min could be attributed to the destruction of outer membrane protein of V. harveyi by reactive oxygen intermediates produced during the photosensitization. Further, mesocosm experiments with V. harveyi and Artemia nauplii demonstrated that in 30 min, the aPDT could kill 78.9% and 91.2% of heterotrophic bacterial and Vibrio population respectively. In conclusion, the study demonstrated that aPDT with its rapid action and as yet unreported resistance development possibilities could be a propitious strategy to reduce the use of antibiotics in shrimp larviculture systems and thereby, avoid their hazardous effects on human health and the ecosystem at large.

Standardization of Optimum Conditions for the Production of Glucosamine Hydrochiloride from Chitin

The study entitled standardization of optimum conditions for the production of glucosamine hydrochloride from chitin. Shellfish processing industries around the world turn out a significant quantity of head and shell as industrial waste. The waste must be removed immediately to prevent the contamination to the processing environment. The technique that are available for their disposal include ocean dumping, incineration or disposal of landfill sites. In this thesis the techniques and methods are used to process glucosamine hydrochloride from crustacean processing waste. Chitin is a nitrogenous polysaccharide, which is white, hard, inelastic, found in outer skeleton of insects, crabs, shrimp and lobsters and in the internal structures of other invertebrates. Glucosamine can be considered as a nutraceutical product by virtue of its properties. It is important for healthy skin, and plays a major role in the healing of surgical incisions and skin wounds. Deproteinisation of shrimp shell had significant effect on quality of chitin. Demineralization is also influences chitin quality. Solvents used for glucosamine hydrochloride affects the final yield and purity.

INVESTIGATIONS ON THE STRESS EFFECTS OF PETROLEUM HYDROCARBONS ON METAPENAEUS DOBSON (MIERS)

The present investigation has addressed the effects of PHC contaminated culture medium on the morphology, physiology and behaviour of shrimps. The shrimp Metapenaeus dobsoni is an important member of the crustacean animal community abounding the oil contaminated benthic regions of Cochin backwater system. Since it is known that true pollutants can disrupt the sustainability of ecosystems by its effect on species, populations and communities,a representative species was used for the study. The results discussed in this work is bound to help in understanding the ecotoxicant resistance that the animal may display under toxic conditions compared to dynamic steady-state systems in nature.

Production of Alkaline Protease by Free and Immobilised Cells of VIBRIO sp. Under Different Fermentation Systems and Its Application on Deproteinisation of Prawn Shell Waste for Chitin Recovery

This thesis presents a detailed account of a cost - effective approach towards enhanced production of alkaline protease at profitable levels using different fermentation designs employing cheap agro-industrial residues. It involves the optimisation of process parameters for the production of a thermostable alkaline protease by Vibrio sp. V26 under solid state, submerged and biphasic fermentations, production of the enzyme using cell immobilisation technology and the application of the crude enzyme on the deproteinisation of crustacean waste.The present investigation suggests an economic move towards Improved production of alkaline protease at gainful altitudes employing different fermentation designs utilising inexpensive agro-industrial residues. Moreover, the use of agro-industrial and other solid waste substrates for fermentation helps to provide a substitute in conserving the already dwindling global energy resources. Another alternative for accomplishing economically feasible production is by the use of immobilisation technique. This method avoids the wasteful expense of continually growing microorganisms. The high protease producing potential of the organism under study ascertains their exploitation in the utilisation and management of wastes. However, strain improvement studies for the production of high yielding variants using mutagens or by gene transfer are required before recommending them to Industries.Industries, all over the world, have made several attempts to exploit the microbial diversity of this planet. For sustainable development, it is essential to discover, develop and defend this natural prosperity. The Industrial development of any country is critically dependent on the intellectual and financial investment in this area. The need of the hour is to harness the beneficial uses of microbes for maximum utilisation of natural resources and technological yields. Owing to the multitude of applications in a variety of industrial sectors, there has always been an increasing demand for novel producers and resources of alkaline proteases as well as for innovative methods of production at a commercial altitude. This investigation forms a humble endeavour towards this perspective and bequeaths hope and inspiration for inventions to follow.

Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps

The constitutive production of AMPs in shrimps ensures that animals are able to protect themselves from low-level assaults by pathogens present in the environment. As these molecules play important roles in the shrimp immune defense system, the expression level of these AMPs are possible indicators of the immune state of shrimps. The present study also indicates the antiviral property of AMPs, especially ALF, stressing the importance of their up-regulation through the application of immunostimulants/probiotics as a prophylactic strategy in aquaculture. The present study shows that shrimp defense system is equipped enough to evade WSSV infection to a certain extent, when the animals were maintained on marine yeast and probiotic diet, whereas the control diet fed group succumbed to WSSV infection. This study reveals that marine yeast and probiotic supplemented diet can delay the process of WSSV infection and confer greater protection to the animals. Particularly, the protection conferred by marine yeast, C. haemulonii S27 and Bacillus MCCB101 were highly promising imparting greater hope to the aquaculture community to overcome the prevailing disease problems in aquaculture. It may be inferred from the present study that up-regulation of AMP genes could be effected by the application of immunostimulants and probiotics. Also, AMP expression profile could be used as an effective tool for screening immunostimulants and probiotics for application in shrimp culture. Ultimately, it is likely that no single compound or strategy will provide a solution to the problem of disease within aquaculture and that, in reality, a suite of techniques will be required including the manipulation of the rearing environment, addition of probionts as a matter of routine during culture, and the use of immunostimulants and other supplements during vulnerable growth phases. Finally, the development of good management practices, the control of environmental variables, genetic improvement in the penaeid species, understanding of host-virus interaction, modulation of the shrimp immune system, supported by functional genomics and proteomics of this crustacean, as a whole suggests that the control of WSSV is not far.

Cellular and humoral factors involved in defense mechanisms of Fenneropenaeus indicus (H. Milne Edwards, 1837)

The present investigation revealed three types of circulating haemocytes in the haemolymph of F. indicus: hyalinocytes, small-granule haemocytes, and large-granule haemocytes. Intermediate stages indicate the maturing process of a single cell. The presence of enzymes such as peroxidase, phenoloxidase and acid phosphatase in the haemocytes, and the substantial production of oxygen radicals during phagocytosis show that the haemocytes are capable of mounting a fme cellular defense mechanism. The enzyme activities of the serum and the presence of agglutinins in the serum, which may act as opsonins, agglutinate foreign particles and augment phagocytosis, confirm the presence of a superior humoral immune system in F. indicus.Bacterial infection caused considerable variations in the cellular and humoral factors, such as the number of circulating cells and haemagglutinating activity, especially in the initial hours of infection. The total haemocyte count, haemagglutination titer and phenoloxidase enzyme showed significant reductions on bacterial presence and could be used as indicators of bacterial infection.The number of circulating cells showed drastic fluctuation on exposure to pollutants. Nuvan at low concentrations was able to produce changes in the haemolymph factors and in the tissue organization, which implies that the animal is under stress and is easily prone to infections. Exposure to nuvan resulted in significant variation in all of the cellular and humoral factors, especially, the total haemocyte count, percentage of small granule haemocytes, phagocytic activity and the haemagglutinating activity, which might be good indicators of pesticide pollution. Heavy metal exposure caused significant increase in total haemocyte count and reduction in phenoloxidase enzyme activity Even changes in the physio-chemical parameters, such as salinity caused fluctuations in the defense factors, indicating stress in this euryhaline species. The dietary incorporation of a commercial immunostimulant containing P-l,3 glucan resulted in stimulation of some of the humoral defense factors of F indicus, but was time dependent. The modulations, on exposure to various external factors, in the cellular and humoral factors, especially, total haemocyte count, phagocytic activity, haemagglutinating activity and the phenoloxidase and acid phosphatase enzymes suggest that these parameters could be used as indicators of the health status of F indicus, which assist in better monitoring and effective health management of this important cultured species.

“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.

Application of Biofloc Technology (BFT) in the nursery rearing and farming of giant freshwater prawn, Macrobrachium rosenbergii (deMan)

In India a study conducted by CIFE and CIBA (1997), concluded that shrimp farming does more good than harm and it is not eco-unfriendly (Krishnan and Birthal, 2002). Upsurge in coastal aquaculture activity induced by high profitability is reported to have caused adverse impacts on coastal ecosystems and social environments (Parthasarathy and Nirmala, 2000). The crustacean farming sector has received criticism for excessive use of formulated feed containing high protein, of which around 50% gets accumulated at the pond bottom as unconsumed (Avnimelech, 1999; Hari et al., 2004, 2006). The wasted feeds undergo the process of degradation and results in the release of toxic metabolites to the culture system. Reduction of protein in the feed, manipulation and utilisation of natural food in the culture system are the remedy for the above problems. But before reducing the feed protein, it should be confirmed that the feed with reduced protein is not affecting the growth and health of the cultured animal. In the present study, biofloc technology is identified as one of the innovative technologies for ensuring the ecological and environmental Sustainability and examines the compatibility of BFT for the sustainable aquaculture of giant prawn, M. rosenbergii. This thesis starts with a general introduction (Chapter-1), a brief review of the most relevant literature (Chapter-2), results of various experiments (Chapter-3-6), summary (Chapter-7) and recommendations and future research perspectives in the field of biofloc based aquaculture (Chapter – 8). The major objectives of this thesis are, to improve the ecological and economical sustainability of prawn farming by the applicationof BFT and to improve the nutrient utilisation in aquaculture systems.

Larval biology of the spiny lobsters of the genus panulirus

Lobsters are renowned the world over as tasty delicacies and are in great demand for Epicurian gourests. As a result these animals found in different parts of the world from countries like India they are mostly frozen and exported thus earning considerable foreign exchange for the country. Importance of this crustacean is thus well known and whatever harvested from nature are utilized to the maximum extent. The demand being much more than what is being produced the need for artificial culture becomes evident. In this context a complete knowledge of the lifecycle ,larval biology and their culture is very essential. Thus with the object of studying the life history of the commercially important lobsters fished from Indian waters the larval biology of panulirus homarus has been taken up as the project for the present study

Investigations on the stress effects of petroleum hydrocarbons on Metapenaeus dobsoni (Miers)

The present investigation has addressed the eflects of PHC contaminated culture medium on the morphology, physiology and behaviour of shrimps] The shrimp Metapenaeus dobsoni is an important member of the crustacean animal community abounding the oil contaminated benthic regions of Cochin backwater system. Since it is known that true pollutants can disrupt the sustainability of ecosystems by its eflect on species, populations and communities, a representative species was used for the study. The results discussed in this work is bound to help in understanding the ecotoxicant resistance that the animal may display under toxic conditions compared to aynamic steaay-state systems in nature

Mesozooplankton of the Arabian Sea and the Bay of Bengal with special reference to planktonic ostracods

The Arabian Sea and the Bay of Bengal are both highly dynamic ecosystems, due to the seasonally reversing monsoon winds, but the processes affecting the mesozooplankton community remain poorly understood. These are important basins exhibiting enhanced biological production as a result of upwelling, winter cooling and other episodic events such as eddies and gyres. Zooplankters are primarily the prey for almost all fish larvae. Seasonal changes in the biogeochemical processes can strongly affect zooplankton density and distribution, which in turn, strongly affect the larval growth, and consequently, the pelagic fish recruitment. It is clear that plankton biomass and biogeochemical fluxes are not in steady state. Acoustic data on mesozooplankton abundance suggests that they also exist in the mesopelagic zone. Earlier studies were confined only to the upper 200 m and hence the structure of mesozooplankton community in the deeper layers was not well known. Copepods are the dominant mesoplankton group, and therefore the majority of the studies were focused on them. The planktonic ostracods are the second major crustacean group and at times, their swarms can outnumber all other planktonic groups. The understanding of the community structure of the ostracods is essential to establish their role in the marine food web. Mesozooplankton is responsible for the vertical flux of organic matter produced by phytoplankton and is assumed to be equivalent to new production (Eppley & Peterson, 1979). Since the fate of newly produced organic matter depends upon their consumers, the zooplankton biomass must be estimated in size fractions or taxonomic components to understand the vertical flux of organic carbon. It is thus important to update our knowledge on different groups of zooplankton on the basis of seasonal and temporal distribution. The distribution in space and time is essential for modeling the carbon cycling that structure the marine ecosystems

studies on Prawn Gill Nets of The Kerala Coast

In the present study. extensive investigations were carried out on various factors affecting the selectivity of prawn gill nets with reference to material, mesh size, coefficient of hanging secolouration. Effect of tidal current on fishing height of prawn gill net and seasonal variation of catch during the course of these investigations were also studied.

Studies on sphaeroma terebrans bate of the port of Cochi

Sphaeroma terebrans Bate, one of the most destructive wood-boring isopod crustacean occurring in the Cochin harbour region was taken up for investigations on its habitat, breeding, attack on timber, salinity tolerance and respiration. The respiration of the animal under different stresses was noted using an instrument fabricated in the laboratory. Of the various hydrographic conditions of the Cochin harbour region studied, salinity is found to be the most fluctuating factor and the variations are largely influenced by the seasonal rain fall from one side and the tidal currents on the other.In spite of the great fluctuations in salinity, Sphaeroma terebrans occurs throughout the year in the Cochin harbour region, though their number varies in different months. Exposure tests show that Sphaeroma terebrans can remain for about 20 to 24 hours outside water in a damp atmosphere.