Transcriptome generation and analysis from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758) through normalized cDNA library.

Catfishes are commercially important fish for both the fisheries and aquaculture industry. Clarias batrachus, an Indian catfish species is economically important owing to its high demand. A normalized cDNA library was constructed from spleen of the Indian catfish to identify genes associated with immune function. One thousand nine hundred thirty seven ESTs were submitted to the GenBank with an average read length of approximately 700 bp. Clustering analysis of ESTs yielded 1,698 unique sequences, including 184 contigs and 1,514 singletons. Significant homology to known genes was found by homology searches against data in GenBank in 576 (34 %) ESTs, including similarity to functionally annotated unigenes for 158 ESTs. Additionally, 433 ESTs revealed similarity to unigenes and ESTs in the dbEST but the remaining 658 EST sequences (39 %) did not match any sequence in GenBank. Of a total of 1,698 ESTs generated, 65 ESTs were found to be associated with immune functions. Gene Ontology and KEGG pathway analyses of C. batrachus ESTs collectively revealed a preponderance of immune relevant pathways apart from the presence of pathways involved in protein processing, localization, folding and protein degradation. This study constitutes first EST analysis of lymphoid organ in aquaculturally important Indian catfish species and could pave the way for further research of immune-related genes and functional genomics in this catfish.

Role of nitric oxide in motility and fertilizing ability of sperm of Heteropneustes fossilis (Bloch.).

The presence of neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS) and nitric oxide (NO) in the testis of seasonally breeding catfish has previously been demonstrated. The present study was aimed to investigate the presence of NO in fish gametes and its role in sperm physiology and strengthen the previous findings. NO, a biological signalling molecule is synthesized during the conversion of l-arginine to l-citrulline by nitric oxide synthases (NOS) in the presence of NADPH and oxygen. In the present study, a considerable amount of NO content was detected in the fresh sperm suspension of fish. A drastic reduction in NO content of the sperm suspension was observed after cryopreservation. Ovarian fluid collected from freshly stripped eggs also contained a substantial amount of NO. Further, effects of a NO donor, sodium nitroprusside (SNP), and NOS inhibitor, N-(G)-nitro-l-arginine-methyl ester (l-NAME) were evaluated on motility and fertilizing ability of fresh and cryopreserved sperm. The percentage of motile fresh sperm (66.2±3.7%) was enhanced (P<0.05) by SNP at the dose of 10(-3)M, whereas the greater and lesser concentrations of SNP did not influence the percentage of motility as compared to the control. However, a significant reduction in the percentage of motile fresh sperm was observed at the tested doses of 10(-3)M and 10(-4)M of l-NAME. The maximum reduction (23.0±3.4%) was evoked at the greatest concentration (10(-3)M); while at the smallest dose of 10(-5)M, reduction was insignificant. Ovarian fluid also enhanced sperm motility. Altogether, findings of the present study suggest that NO plays an important role in sperm performance of fish.

Development of EST derived SSRs and SNPs as a genomic resource in Indian catfish, Clarias batrachus.

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.

Evaluation of cytochrome b mtDNA sequences in genetic diversity studies of Channa marulius (Channidae: Perciformes).

Channa marulius (Hamilton, 1822) is a commercially important freshwater fish and a potential candidate species for aquaculture. The present study evaluated partial Cytochrome b gene sequence of mtDNA for determining the genetic variation in wild populations of C. marulius. Genomic DNA extracted from C. marulius samples (n = 23) belonging to 3 distant rivers; Mahanadi, Teesta and Yamuna was analyzed. Sequencing of 307 bp Cytochrome b mtDNA fragment revealed the presence of 5 haplotypes with haplotype diversity value of 0.763 and nucleotide diversity value of 0.0128. Single population specific haplotype was observed in Mahanadi and Yamuna samples and 3 haplotypes in Teesta samples. The analysis of data demonstrated the suitability of partial Cytochrome b sequence in determining the genetic diversity in C. marulius population.