DNA barcoding Indian freshwater fishes.

DNA barcoding is a promising technique for species identification using a short mitochondrial DNA sequence of cytochrome c oxidase I (COI) gene. In the present study, DNA barcodes were generated from 72 species of freshwater fish covering the Orders Cypriniformes, Siluriformes, Perciformes, Synbranchiformes, and Osteoglossiformes representing 50 genera and 19 families. All the samples were collected from diverse sites except the species endemic to a particular location. Species were represented by multiple specimens in the great majority of the barcoded species. A total of 284 COI sequences were generated. After amplification and sequencing of 700 base pair fragment of COI, primers were trimmed which invariably generated a 655 base pair barcode sequence. The average Kimura two-parameter (K2P) distances within-species, genera, families, and orders were 0.40%, 9.60%, 13.10%, and 17.16%, respectively. DNA barcode discriminated congeneric species without any confusion. The study strongly validated the efficiency of COI as an ideal marker for DNA barcoding of Indian freshwater fishes.

Development and characterization of a new cell line CF from caudal fin of knifefish, Chitala chitala (Hamilton-Buchanan).

A new cell line was successfully obtained from caudal fin tissue of the economically important freshwater fish Chitala chitala. The cell line was optimally maintained at 28°C in Leibovitz's L-15 supplemented with 20% fetal bovine serum (FBS). The effects of temperature and concentration of FBS on the growth of CF cells were examined. The CF cell line consisted predominantly of fibroblastic-like cells. Moderately low plating efficiencies 8%, 11%, and 17% were observed, with CF cell line in L-15 Medium with 20% FBS. Chromosomal analysis of the cell line revealed a diploid number of 42 chromosomes in C. chitala. Molecular characterization of mitochondrial 16S rRNA and cytochrome oxidase subunit I confirmed the origin of the cell line. The cells were successfully cryopreserved in liquid nitrogen (-196°C) for 6 mo, and more than 85-90% of CF cells were revived.

Population structure of Tor tor inferred from mitochondrial gene cytochrome b.

Tor tor, commonly called as Tor mahseer, is a high-valued food and game fish endemic to trans-Himalayan region. Mitochondrial cytochrome b (cyt b) gene region of 967 bp was used to estimate the population structure of T. tor. Three populations of T. tor were collected from Narmada (Hosangabad), Ken (Madla), and Parbati river (Sheopur) in Madhya Pradesh, India. The sequence analysis revealed that the nucleotide diversity (π) was low, ranging from 0.000 to 0.0150. Haplotype diversity (h) ranged from 0.000 to 1.000. The analysis of molecular variance analysis indicated significant genetic divergence among the three populations of T. tor. Neighboring-joining tree also showed that all individuals from three populations clustered into three distinct clades. The data generated by cyt b marker revealed interesting insight about population structure of T. tor, which would serve as baseline data for conservation and management of mahseer fishery.

Genetic characterization of two hill stream fish species Barilius bendelisis (Ham.1807) and Barilius barna (Ham.1822) using RAPD markers.

Genetic structure of four wild populations of two hill stream fishes Barilius bendelisis (Ham.1807) and B. barna (Ham. 1822) from Uttarakhand, India, was studied using RAPD markers. Eight selective primers provided distinct and consistent RAPD profiles in both the species, producing a total of 47 and 35 scorable bands in B. bendelisis and B. barna respectively. The bands in the range 666-4,830 bp were scored for consistent results. The RAPD profiles generated by all the eight primers revealed varying degrees of polymorphism (25.00-50.00 %). The average genetic diversity (h) was estimated as 0.1661 and 0.1606 among the four populations of B. bendelisis and B. barna respectively.

Water temperature and pH influence olfactory sensitivity to pre-ovulatory and post-ovulatory ovarian pheromones in male Barilius bendelisis.

The attractive response and sexual activity elicited by pre-ovulatory steroid sulphate and post-ovulatory 15K-PGF pheromones are greater in wild caught tubercular males and immature males which express breeding tubercles on the snout (at 12-13 days post androgen implant) than in non-tubercular and non-androgen implanted males of freshwater fish Barilius bendelisis. This shows that circulatory androgens exert an activational effect on olfactory receptors of male fish. Wild caught tubercular males and androgen implanted juvenile males exhibit a high responsiveness to steroid sulphate at the water temperature and pH which fish experience during the pre-spawning phase. The male's sensitivity to 15K-PGF is almost equally high at the water temperature and pH which they experience in wild during the both pre-spawning and spawning periods. This suggests that the differential olfactory sensitivity to the two classes of pheromones in androgen implanted males is due to the varied temperature and pH of water, and that during the breeding season the male's olfactory sensitivity to PGF pheromone is more widespread than to the steroidal pheromone. An increased and decreased olfactory sensitivity in mature males to sex pheromones and L-alanine respectively during the breeding phase is in agreement with the hypothesis that pheromonal stimuli dominate over feeding stimuli to promote spawning success.

Ovarian steroid sulphate functions as priming pheromone in male Barilius bendelisis (Ham.).

The study reveals that pre-ovulatory females of the fish Barilius bendelisis (Ham.) release sex steroids and their conjugates into the water and that a steroid sulphate of these compounds functions as a potent sex pheromone which stimulates milt production in conspecific males prior to spawning. Since males exposed to the purified subfraction III of the steroid sulphate fraction have increased milt volume and more spermatozoa with greater motility, the function of this priming pheromone appears to be to enhance male spawning success. High turbulence and faster water currents render the hillstream ecosystem extremely challenging for chemical communication. Therefore, ovulatory female fish secrete highly water soluble steroid sulphates for rapid pheromonal action in males. Inhibited milt volume in olfactory tract lesioned (OTL) males exposed to the steroid sulphate fraction and 17alpha,20 beta-dihydroxy-4-pregnen-3-one supports the concept that the pheromonally induced priming effect in male fish is mediated through olfactory pathways.

Neurodepressive action of a piscicidal glycoside of plant, Aesculus indica (Colebr.) in fish.

Sublethal concentration (2.6 mg/l) of a triterpene based piscicidal glycoside of A. indica damaged the neurons, fibre tracts and central correlation sites for gustatory, tactile and visceral sensory impulses in medulla oblongata of fish after prolonged poisoning. The fear, sinking to bottom, lack of schooling and non discriminatory pattern in treated fish were because of neurodepression. The jerky movement, ventilatory inefficiency, swallowing air bubbles and light pinkness of gills were due to the degradation of respiratory centres in the vagal lobes of toxified fish.

Neurolytic manifestation of piscicidal flavanoid of plant, Engelhardtia colebrookiana (Lindle) in fish.

The sublethal concentration 6 mg.l-1 of a piscicidal flavanoid, 'Quercetin glycoside', obtained from E. colebrookiana affected neuro-architecture in medulla oblongata of freshwater fish, Barilius bendelisis (Ham.), at 32 days exposure. Since medulla of fish is primary centre for the reception of several incoming stimuli, the breakdown of neurons and their neuraxes in facial and vagal lobes impaired the transmission of sensory informations to cerebellum. Consequently fish became unconscious and could be caught easily with hands. The symptoms like lack of schooling, lack of response to food and external stimuli, sluggishness, incapability of maintaining body posture and uncoordinated functioning are accounted for by the neurolysis in hind brain of fish.