Molecular Genetic Characterization of Endemic Yellow Catfish, Horab rus brachysoma (Gunther)

The present study Molecular genetic characterization of endemic yellow catfish ,generated an important information on the genetic variation and stock structure of the endangered yellow catfish(Horabagrus brachysoma) endemic to the western Ghats. Three genetically discrete stocks of the species have been identified for the first time using allozymes, RAPD(Random Amplified Polymorphic DNA) and microsatelite markers and it is a significant step towards realizing the goal of management of fishery and conservation of the yellow catfish populations in the rivers of the Western Ghats region. In conclusion genetic markers were found to be powerful tools to analyze the population genetic structure of the yellow catfish. Geographic isolation by land distance,inbreading as a result of over-exploitation etc are some reasons for the genetic differenciation between the pairs and deficiency of hetrozygosity revealed by the two co dominant markers, allozyme, and microsatelites.the study emphasizes the need for stock-wise, propagation assisted-rehabilitation of the natural populations yellow catfish

Production, purification, genetic characterization and application studies of tannase enzyme from marine fungus Aspergillus awamori

Marine fungi remain totally unexplored as a source of industrial enzyme and prospective applications. Further tannase production by a marine organism has so far not been established. The primary objective of this study included the evaluation of the potential of Aspergillus awamori isolated from sea water as part of an earlier study and available in the culture collection of the Microbial technology laboratory for tannase production through different fermentation methods, optimization of bioprocess variables by statistical methods, purification and characterization of the enzyme, genetic study, and assessment of its potential applications.

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.

Biochemical and molecular investigations on Salmonella serovars from seafood

The present investigation was envisaged to determine the prevalence and identify the different Salmonella serovar in seafood from Cochin area. Though, the distribution of Salmonella serovars in different seafood samples of Cochin has been well documented, the present attempt was made to identify the different Salmonella serovars and determine its prevalence in various seafoods. First pan of this investigation involved the isolation and identification of Salmonella strains with the help of different conventional culture methods. The identified isolates were used for the further investigation i.e. serotyping, this provides the information about the prevalent serovars in seafood. The prevalent Salmonella strains have been further characterized based on the utilization of different sugars and amino acids, to identify the different biovar of a serovar.A major research gap was observed in molecular characterization of Salmonella in seafood. Though, previous investigations reported the large number of Salmonella serovars from food sources in India, yet, very few work has been reported regarding genetic characterization of Salmonella serovars associated with food. Second part of this thesis deals with different molecular fingerprint profiles of the Salmonella serovars from seafood. Various molecular typing methods such as plasmid profiling, characterization of virulence genes, PFGE, PCR- ribotyping, and ERIC—PCR have been used for the genetic characterization of Salmonella serovars.The conventional culture methods are mainly used for the identification of Salmonella in seafood and most of the investigations from India and abroad showed the usage of culture method for detection of Salmonella in seafood. Hence, development of indigenous, rapid molecular method is most desirable for screening of Salmonella in large number of seafood samples at a shorter time period. Final part of this study attempted to develop alternative, rapid molecular detection method for the detection of Salmonella in seafood. Rapid eight—hour PCR assay has been developed for detection of Salmonella in seafood. The performance of three different methods viz., culture, ELISA and PCR assays were evaluated for detection of Salmonella in seafood and the results were statistically analyzed. Presence of Salmonella cells in food and enviromnental has been reported low in number, hence, more sensitive method for enumeration of Salmonella in food sample need to be developed. A quantitative realtime PCR has been developed for detection of Salmonella in seafood. This method would be useful for quantitative detection of Salmonella in seafood.

Isolation, purification and characterization of Protease Inhibitor from Moringa oleifera Lam.

Protease inhibitors are one of the most important tools of nature for regulating the proteolytic activity of their target proteases. They are synthesized in biological systems and they play a critical role in controlling a number of diverse physiological functions. The current investigation focused on the isolation, purification and characterization of a novel protease inhibitor from Moringa oleifera. The results obtained during the course of study opens new perspectives for the utilization of protease inhibitor from Moringa oleifera for various pharmaceutical, agricultural and food industries. The biological and physicochemical properties exhibited by the novel protease inhibitor from Moringa oleifera clearly testify its suitability for the development as a drug for application in pharmaceutical industries such as anticoagulant agent or biocontrol agent in agriculture and even as a food preservant. There is a scope for further research on the structure elucidation and protein engineering towards a wide range of further applications. Detailed structure/function analysis of these proteins is important to facilitate their use in genetic engineering for various applications.

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.

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.

Characterization and virulence potential of phenotypically diverse Aeromonas veronii isolates recovered from moribund freshwater ornamental fishes of Kerala, India

In the present study, we investigated the involvement of Aeromonas spp. in eliciting disease outbreaks in freshwater ornamental fishes across the state of Kerala, India. We investigated three incidences of disease, in which the moribund fishes exhibited clinical signs such as haemorrhagic septicemia (in gouramy, Trichogaster sp.), dropsy (in Oscar, Astronotus ocellatus) and tail rot/fin rot (in gold fish, Carassius carassius). Pure cultures (n = 20 from each fish; 60 in total) of Aeromonas spp. were recovered from the abdominal fluid as well as from internal organs of affected fishes, although they could not be identified to species level because of the variations in their phenotypic characters. The molecular fingerprinting of the isolates using Enterobacterial Repetitive Intergenic Consensus PCR proved the genetic diversity of the isolates from the three sites. The phylogenetic trees constructed using concatenated sequences (using 16S rRNA, gyrA, gyrB and rpoD genes) indicated that they were related to Aeromonas veronii. They exhibited marked cytotoxic and haemolytic activity, which were responsible for the pathogenic potential of the isolates. The isolates possessed multiple virulence genes such as enterotoxins (act and alt), haemolytic toxins (aerA and hlyA), genes involved in type III secretion system (ascV, aexT and ascF–ascG), glycerophospholipid-cholesterol acyltransferase (gcat) and a type IV pilus (tapA) gene, as determined by PCR. Virulence of representative isolates to goldfish was also tested, and we found LD50 values of 104.07–105.35 cfu/fish. Furthermore, the organisms could be recovered as pure cultures from the lesions as well as from the internal organs.

Integron study, molecular typing and characterization of Salmonella serotypes isolated from seafood and poultry

The primary habitat of Salmonella is the gastrointestinal tract of animals and they are discharged into the water bodies through the feces. Aquatic animals act as asymptomatic reservoirs of a wide range of Salmonella serotypes. The inevitable delay in the detection of Salmonella contamination and the low sensitivity of the conventional methods is a serious issue in the seafood industry. Due to the indiscriminate use, the antibiotics are finally accumulated in the aquatic environment which provides the required antibiotic stress for the emergence of more and more antibiotic resistant phenotypes ofSalmonella. Several genetic determinants like integrons, genomic islands etc. play their role in acquisition and reshuffling of antibiotic resistance genes. A large number of virulence determinants are required for Salmonella pathogenicity. The virulence potential of Salmonella is determined, to some extent, by the presence of phages or phage mediated genes in the bacterial genome. There is much intra-serotype polymorphism in Salmonella and epidemiological studies rely on genetic resemblance of the isolated strains. Proper identification of the strain employing the traditional and molecular techniques is a prerequisite for accurate epidemiological studies (Soto et al., 2000). In this context, a study was undertaken to determine the prevalence of different Salmonella serotypes in seafood and to characterize them

Molecular genetic profiling of functional genes of pearl oyster, Pinctada fucata

There are a number of genes involved in the regulation of functional process in marine bivalves. In the case of pearl oyster, some of these genes have major role in the immune/defence function and biomineralization process involved in the pearl formation in them. As secondary filter feeders, pearl oysters are exposed to various kinds of stressors like bacteria, viruses, pesticides, industrial wastes, toxic metals and petroleum derivatives, making susceptible to diseases. Environmental changes and ambient stress also affect non-specific immunity, making the organisms vulnerable to infections. These stressors can trigger various cellular responses in the animals in their efforts to counteract the ill effects of the stress on them. These include the expression of defence related genes which encode factors such as antioxidant genes, pattern recognition receptor proteins etc. One of the strategies to combat these problems is to get insight into the disease resistance genes, and use them for disease control and health management. Similarly, although it is known that formation of pearl in molluscs is mediated by specialized proteins which are in turn regulated by specific genes encoding them, there is a paucity of sufficient information on these genes.In view of the above facts, studies on the defence related and pearl forming genes of the pearl oyster assumes importance from the point of view of both sustainable fishery management and aquaculture. At present, there is total lack of sufficient knowledge on the functional genes and their expressions in the Indian pearl oyster Pinctada fucata. Hence this work was taken up to identify and characterize the defence related and pearl forming genes, and study their expression through molecular means, in the Indian pearl oyster Pinctada fucata which are economically important for aquaculture at the southeast coast of India. The present study has successfully carried out the molecular identification, characterization and expression analysis of defence related antioxidant enzyme genes and pattern recognition proteins genes which play vital role in the defence against biotic and abiotic stressors. Antioxidant enzyme genes viz., Cu/Zn superoxide dismutase (Cu/Zn SOD), glutathione peroxidise (GPX) and glutathione-S-transferase (GST) were studied. Concerted approaches using the various molecular tools like polymerase chain reaction (PCR), random amplification of cDNA ends (RACE), molecular cloning and sequencing have resulted in the identification and characterization of full length sequences (924 bp) of the Cu/Zn SOD, most important antioxidant enzyme gene. BLAST search in NCBI confirmed the identity of the gene as Cu/Zn SOD. The presence of the characteristic amino acid sequences such as copper/zinc binding residues, family signature sequences and signal peptides were found out. Multiple sequence alignment comparison and phylogenetic analysis of the nucleotide and amino acid sequences using bioinformatics tools like BioEdit,MEGA etc revealed that the sequences were found to contain regions of diversity as well as homogeneity. Close evolutionary relationship between P. fucata and other aquatic invertebrates was revealed from the phylogenetic tree constructed using SOD amino acid sequence of P. fucata and other invertebrates as well as vertebrates

Characterization and applications of two protease enzymes obtained by culture dependent and independent approaches from mangrove sediments

Soil community genomics or metagenomics is employed in this study to analyze the evolutionary related - ness of mangrove microbial community. The metagenomic DNA was isolated from mangrove sediment and 16SrDNA was amplified using universal primers. The amplicons were ligated into pTZ57R/T cloning vector and transformed onto E. coli JM109 host cells. The recombinant plasmids were isolated from positive clones and the insert was confirmed by its reamplification. The amplicons were subjected to Amplified Ribosomal DNA Restriction Analysis (ARDRA) using three different tetra cutter restriction enzymes namely Sau3A1, Hha1 and HpaII. The 16SrDNA insert were sequenced and their identity was determined. The sequences were submitted to NCBI database and accession numbers obtained. The phylo - genetic tree was constructed based on Neighbor-Joining technique. Clones belonged to two major phyla of the bacterial domain, namely Firmicutes and Proteobacteria, with members of Firmicutes predominating. The microbial diversity of the mangrove sediment was explored in this manner.

Characterization and Pathogenicity of Vibrio cholerae and Vibrio vulnificus from Marine environments

The genus Vibrioof the family Vibrionaceae are Gram negative, oxidasepositive, rod- or curved- rodshaped facultative anaerobes, widespread in marine and estuarine environments. Vibrio species are opportunistic human pathogens responsible for diarrhoeal disease, gastroenteritis, septicaemia and wound infections and are also pathogens of aquatic organisms, causing infections to crustaceans, bivalves and fishes. In the present study, marine environmental samples like seafood and water and sediment samples from aquafarms and mangroves were screened for the presence of Vibrio species. Of the134 isolates obtained from the various samples, 45 were segregated to the genus Vibrio on the basis of phenotypic characterization.like Gram staining, oxidase test, MoF test and salinity tolerance. Partial 16S rDNA sequence analysis was utilized for species level identification of the isolates and the strains were identified as V. cholerae(N=21), V. vulnificus(N=18), V. parahaemolyticus(N=3), V. alginolyticus (N=2) and V. azureus (N=1). The genetic relatedness and variations among the 45 Vibrio isolates were elucidated based on 16S rDNA sequences. Phenotypic characterization of the isolates was based on their response to 12 biochemical tests namely Voges-Proskauers’s (VP test), arginine dihydrolase , tolerance to 3% NaCl test, ONPG test that detects β-galactosidase activity, and tests for utilization of citrate, ornithine, mannitol, arabinose, sucrose, glucose, salicin and cellobiose. The isolates exhibited diverse biochemical patterns, some specific for the species and others indicative of their environmental source.Antibiogram for the isolates was determined subsequent to testing their susceptibility to 12 antibiotics by the disc diffusion method. Varying degrees of resistance to gentamycin (2.22%), ampicillin(62.22%), nalidixic acid (4.44%), vancomycin (86.66), cefixime (17.77%), rifampicin (20%), tetracycline (42.22%) and chloramphenicol (2.22%) was exhibited. All the isolates were susceptible to streptomycin, co-trimoxazole, trimethoprim and azithromycin. Isolates from all the three marine environments exhibited multiple antibiotic resistance, with high MAR index value. The molecular typing methods such as ERIC PCR and BOX PCR revealed intraspecies relatedness and genetic heterogeneity within the environmental isolatesof V. cholerae and V. vulnificus. The 21 strains of V. choleraewere serogroupedas non O1/ non O139 by screening for the presence O1rfb and O139 rfb marker genes by PCR. The virulence/virulence associated genes namely ctxA, ctxB, ace, VPI, hlyA, ompU, rtxA, toxR, zot, nagst, tcpA, nin and nanwere screened in V. cholerae and V. vulnificusstrains.The V. vulnificusstrains were also screened for three species specific genes viz., cps, vvhand viu. In V. cholerae strains, the virulence associated genes like VPI, hlyA, rtxA, ompU and toxR were confirmed by PCR. All the isolates, except for strain BTOS6, harbored at least one or a combination of the tested genes and V. choleraestrain BTPR5 isolated from prawn hosted the highest number of virulence associated genes. Among the V. vulnificusstrains, only 3 virulence genes, VPI, toxR and cps, were confirmed out of the 16 tested and only 7 of the isolates had these genes in one or more combinations. Strain BTPS6 from aquafarm and strain BTVE4 from mangrove samples yielded positive amplification for the three genes. The toxRgene from 9 strains of V. choleraeand 3 strains of V. vulnificus were cloned and sequenced for phylogenetic analysis based on nucleotide and the amino acid sequences. Multiple sequence alignment of the nucleotide sequences and amino acid sequences of the environmental strains of V. choleraerevealed that the toxRgene in the environmental strains are 100% homologous to themselves and to the V. choleraetoxR gene sequence available in the Genbank database. The 3 strains of V. vulnificus displayed high nucleotide and amino acid sequence similarity among themselves and to the sequences of V. cholerae and V. harveyi obtained from the GenBank database, but exhibited only 72% homology to the sequences of its close relative V. vulnificus. Structure prediction of the ToxR protein of Vibrio cholerae strain BTMA5 was by PHYRE2 software. The deduced amino acid sequence showed maximum resemblance with the structure of DNA-binding domain of response regulator2 from Escherichia coli k-12 Template based homology modelling in PHYRE2 successfully modelled the predicted protein and its secondary structure based on protein data bank (PDB) template c3zq7A. The pathogenicity studies were performed using the nematode Caenorhabditiselegansas a model system. The assessment of pathogenicity of environmental strain of V. choleraewas conducted with E. coli strain OP50 as the food source in control plates, environmental V. cholerae strain BTOS6, negative for all tested virulence genes, to check for the suitability of Vibrio sp. as a food source for the nematode;V. cholerae Co 366 ElTor, a clinical pathogenic strain and V. cholerae strain BTPR5 from seafood (Prawn) and positive for the tested virulence genes like VPI, hlyA, ompU,rtxA and toxR. It was found that V. cholerae strain BTOS6 could serve as a food source in place of E. coli strain OP50 but behavioral aberrations like sluggish movement and lawn avoidance and morphological abnormalities like pharyngeal and intestinal distensions and bagging were exhibited by the worms fed on V. cholerae Co 366 ElTor strain and environmental BTPR5 indicating their pathogenicity to the nematode. Assessment of pathogenicity of the environmental strains of V. vulnificus was performed with V. vulnificus strain BTPS6 which tested positive for 3 virulence genes, namely, cps, toxRand VPI, and V. vulnificus strain BTMM7 that did not possess any of the tested virulence genes. A reduction was observed in the life span of worms fed on environmental strain of V. vulnificusBTMM7 rather than on the ordinary laboratory food source, E. coli OP50. Behavioral abnormalities like sluggish movement, lawn avoidance and bagging were also observed in the worms fed with strain BTPS6, but the pharynx and the intestine were intact. The presence of multi drug resistant environmental Vibrio strainsthat constitute a major reservoir of diverse virulence genes are to be dealt with caution as they play a decisive role in pathogenicity and horizontal gene transfer in the marine environments.

Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.