Establishment and characterization of a piscean PCF cell line for toxicity and gene expression studies as in vitro model.

A new piscean fibroblastic cell line termed as PCF derived from the caudal fin tissue of dark mahseer, Puntius (Tor) chelynoides was established and characterized in the present study which was found to be suitable for toxicity and gene expression studies as in vitro model. The cell line grew well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS). The cells were able to grow at a temperature ranging from 20 to 28 °C with an optimal growth at 24 °C and the cell line have been expanded in culture for more than 70 passages. Authentication of the cell line was carried out using mitochondrial DNA markers (Cytochrome Oxidase subunit I and 16S ribosomal RNA). Presence of vimentin in the cells confirmed the fibroblastic origin of cell line. Significant cytopathic effects were observed upon exposure of PCF cell line to bacterial extracellular products and the study also validated the suitability of cell line in transgenic applications as well as in genotoxicity assessment as an in vitro model.

A SRCF cell line from snowtrout, Schizothorax richardsonii: development and characterization.

Schizothorax richardsonii, commonly called snowtrout, is an important indigenous coldwater fish of the Himalayas, India with high commercial values as food and game fish. A cell line named as SRCF was developed from the caudal fin tissue of S. richardsonii. The cell line has been maintained in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS) at 24°C. The cells showed fibroblastic morphology, high plating efficiency and cell doubling time of 48h. Chromosomal analysis of SRCF cells revealed a diploid count of 98 chromosomes. The origin of the cell line was confirmed by the amplification of 655 and 578bp of cytochrome oxidase subunit I (COI) and 16S rRNA of mitochondrial DNA (mt-DNA) genes, respectively. Transfection of SRCF cells with pEGFP-C1 plasmid resulted in bright fluorescent signals, suggesting the application of cell line in transgenic and genetic improvement programme. In addition, genotoxicity assessment illustrated the utility of the cell line as an in vitro model for aquatic toxicological studies.

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland).

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies.

Cytochrome b gene sequence divergence of seven sisorid species of catfish genus Glyptothorax (siluriformes, sisoridae) from India.

Samples of seven Sisorid catfish species of the genus Glyptothorax (G. garhwali, G. dakpathari, G. brevipinnis, G. ngapang, G. granulus, G. ventrolineatus, and G. davissinghi) were collected from the Himalayan region and the Western Ghats of India. They were analyzed for the mitochondrial cytochrome b gene (Cyt b). Out of 1152 nucleotide positions analyzed, 269 (23.3%) were found to be variable and 235 (20.3%) were parsimoniously informative. The sequences showed 111 (9.6%) fourfold degenerate sites. The overall transition/transversion bias was R = 3.457. The average proportion of base substitutions measured as P-distance for all sequences of seven Glyptothorax species and other five species comprising four comparison groups was intraspecies, P = 0.17 ± 0.05%, intragenus, P = 10.75 ± 0.48%, intrafamily, P = 20.07 ± 1.43%, and intraorder, P = 21.10 ± 0.45%. Within the Glyptothorax genus maximum divergence was obtained among G. brevipinnis sequences, whereas the least divergence was obtained within G. davissinghi. The phylogenetic trees for 193 and 47 sequences of Sisorid catfishes together were developed using the Cyt b gene and four different analytical approaches: Bayesian (BA), neighbour-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). The analysis revealed a monophyletic origin for the all investigated representatives of the genus Glyptothorax (99, 100, 99, and 73% support level in our BA, NJ, MP, and ML analyses respectively) and with some reservations for Sisoridae, which is the principal family investigated. The monophyletic origin of the two subfamilies of Sisorid catfish defined in the literature was partly also supported by molecular phylogenetic data.

Gross morphology of the alimentary canal and seasonal variation in feeding of Noemacheilus montanus (McClelland).

Noemacheilus montanus (McClelland) is a bottom feeder Omnivorous fish of Garhwal hillstreams. The alimentary canal is short and the stomach consists of a corpus and a pylorus portion. As it consumes more animal food than the vegetable food, it may be better called Carni-omnivore. The study of GSI for different months revealed that the feeding intensity attains 2 peaks during a year, first in December and second in June. The change in feeding intensity may be correlated with the availability of food items in nature.