Occurrence of viral pathogens in Penaeus monodon post-larvae from aquaculture hatcheries.

Viral pathogens appear to exert the most significant constraints on the growth and survival of crustaceans under culture conditions. The prevalence of viral pathogens White Spot Syndrome Virus (WSSV), Hepatopancreatic Parvo Virus (HPV), Monodon Baculo Virus (MBV) and Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) in Penaeus monodon post-larvae was studied. Samples collected from different hatcheries and also samples submitted by farmers from Kerala were analyzed. Out of 104 samples collected, WSSV was detected in 12.5% of the post-larvae samples. Prevalence of concurrent infections by HPV, MBV and WSSV (either dual or triple infection) was present in 60.6% of the total post-larvae tested. Out of the 51 double positives, 98% showed either HPV or IHHNV infection. HPV or IHHNV was detected in 11 post-larval samples showing triple viral infection. This is the first report of IHHNV from India. Result of this study reveals the lack of efficient screening strategies to eradicate viruses in hatchery reared post-larvae.

White spot syndrome virus infection: Threat to crustacean biodiversity in Vembanad Lake, India.

The Vembanad Lake located on the south-west coast of India, an ecological hotspot is the nursing ground of many economically important crustaceans. The prevalence of white spot syndrome virus (WSSV) among crustaceans from farmed, estuarine and marine environments surrounding the Vembanad Lake, India was detected using PCR. A total of 308 samples from aquaculture ponds consisting of six species of crustaceans collected from five different farms were tested for the presence of WSSV. Of these, 67% were found to carry the virus. A total of 258 samples of crustaceans from the Cochin backwater system that forms a part of the Vembanad lake viz., Metapenaeus dobsoni, Metapenaeus monoceros, Penaeus monodon and Penaeus indicus were found to contain WSSV in 62% of the samples. Fifteen species of crustaceans caught from the seas off Cochin were also screened for the presence of WSSV. Out of these, twelve species had WSSV incidence levels ranging from 6-23%. WSSV was not detected from three species of deep sea crustaceans tested. The black tiger shrimp, Penaeus monodon had the highest incidence of WSSV among the species screened in farmed, estuarine and marine environments.

Differential gene expression profile of the hepatopancreas of white spot syndrome virus infected Fenneropenaeus indicus by suppression subtractive hybridization.

Suppression Subtractive Hybridization was employed in order to identify the differentially expressed genes in the hepatopancreas of white spot syndrome virus infected Fenneropenaeus indicus. A forward subtracted cDNA library generated 356 clones following a white spot syndrome virus infection. A total of 345 clones with more than 100 nucleotides were selected for further analysis using bioinformatics tools after vector screening. Twenty-three contigs and 111 singletons were generated from a total of 134 consensuses. The consensuses, on a sequence homology search using BLASTX (NCBI), revealed that 74 (55%) of them had no significant match to reported sequences in the database, suggesting that they were found for the first time and are probably associated with shrimp immune function. Out of the remaining 60 (45%) consensuses, 43 had significant homology to known protein sequences in the database while 17 consensuses are homologous to unknown proteins in the database which are considered novel. The most abundant genes in the subtracted library were antimicrobial peptides accounting for 56 clones; among which one is a member of SNF2 family of proteins and another belonged to PfP1 family of proteins on analysis using Antimicrobial peptide predictor software. The other predicted genes in the subtracted library include signal transduction molecules (GTPase, Serine threonine kinase, Armadillo repeats etc), antioxidant enzymes (Cytochrome oxidase, Monomeric sarcosine oxidase and Catalase), active transporters (Nuclear Localization Signal [NLS], calcium ATPase, sodium glutamate symporter, Store-Operated Calcium Entry [SOCE] and ribonucleoprotein [RNP]) contributing to 19, 14 and 5 clones respectively. Three clones are homologous to reverse transcriptase; a first time report in shrimp and one each belong to cell adhesion molecule and Proteinase. InterProScan at EMBL, when used for an integrated search at PROSITE predicted; signal sequences and transmembrane regions for 13 clones. This is the first report on the differential gene expression in WSSV-infected F. indicus. The high expression of immune related genes in response to virus infection in shrimp will provide a new insight into the crustacean innate immunity. Further work on the functionality of the unknown genes in shrimps will give an overview on the role of the differentially expressed genes during viral infection and increase our understanding for developing antiviral measures by making use of the shrimp defense mechanism.

Functional characterization of trehalose biosynthesis genes from E. coli: an osmolyte involved in stress tolerance.

Trehalose (1-alpha-D-glucopyranosyl-1-alpha-D-glucopyranoside), a non-reducing disaccharide is a major compatible solute, which maintains fluidity of membranes and protects the biological structure of organisms under stress. In this study, trehalose-6-phosphate synthase (otsA) and trehalose-6-phosphate phosphatase (otsB) genes encoding for trehalose biosynthesis from Escherichia coli was cloned as an operon and expressed in E. coli M15(pREP4). The recombinant E. coli strain showed a threefold increase in the activity of otsBA pathway enzymes, compared to the control strain. The transgenic E. coli accumulated up to 0.86 mg/l of trehalose. The sequence of otsA and otsB genes reported in this study contains several base substitutions with that of reported sequences in GenBank, resulting in the altered amino acid sequences of the translated proteins.

Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2.

The nucleotide sequence of a smallest cryptic plasmid pRK10 of Serratia marcescens ACE2 was determined. When compared to the all other plasmids reported so far from S. marcescens in sizes of over 70kb, pRK10 is only 4241bp long with 53% G+C content and has five coding sequences representing a coding percentage of 65.41. This small plasmid consists of one Tdh gene, four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. Analysis of the five open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization containing sequences homologous to the bom region and mobCABD genes of ColE1 and Tdh from Acinetobacter baumannii str. AYE. Results also indicate that pRK10 does not encode any gene for antibiotic/heavy metal resistance. Copy number and incompatibility of the plasmid with plasmids of ColE1 origin of replication was determined and it is quite stable in its natural host as well as in Escherichia coli DH5alpha. This relatively small plasmid will be useful for construction of shuttle vectors to facilitate the genetic analysis.

Characterization and phylogenetic analysis of ectoine biosynthesis genes from Bacillus halodurans.

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was confirmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36-73% for ectA gene, 55-81% for ectB gene and 55-80% for ectC gene indicating that the enzymes are evolutionarily well conserved.

Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli.

The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.