Anindya Sundar Barman

Molecular evidence to reconcile taxonomic instability in mahseer species (Pisces: Cyprinidae) of India

The mahseers are an important group of fishes endemic to Asia with most species considered threatened. Conservation plans to save declining wild populations are hindered by unstable taxonomy, and detailed systematic review could form a solid platform for future management and conservation. D-loop and cytochrome c oxidase I (COI) mtDNA sequences were examined in nine mahseer species of Tor, Neolissochilus, and Naziritor. Pseudogenes amplified in a portion of the species limited the utility of the D-loop region. ABGD analysis, NJ, ML, and MP methods and genetic distance (TrN + I + G) using COI data revealed concordant species delimiting patterns. The three genera were monophyletic, separated as distinct clades (TrN + I + G 0.064 to 0.106), and Naziritor was flagged as a separate genus, distinct from Puntius (TrN + I + G 0.196). Out of seven nominal species known for Tor cogeners from India, only five were recovered with mtDNA data (TrN + I + G 0.000 to 0.037) and two species could not be distinguished with the molecular data set employed. Tor mosal, synonymized as Tor putitora, was rediscovered as a distinct species (TrN + I + G 0.031) based on its type locality. Tor mussulah was confirmed as a separate species (TrN + I + G 0.019 to 0.026). Two valid species, Tor macrolepis and T. mosal mahanadicus, were not distinct from T. putitora (TrN + I + G 0.00). The high divergence with mtDNA data failed to validate T. mosal mahanadicus as a subspecies of T. mosal (TrN + I + G 0.031). Morphological outliers discovered within the distribution range of Tor tor (TrN + I + G 0.022 to 0.025) shared the same lineage with T. putitora (TrN + I + G 0.002 to 0.005), indicating a new extended distribution of the Himalayan mahseer T. putitora in the rivers of the Indian central plateau. The findings indicate the need for integrating molecular and morphological tools for taxonomic revision of the Tor and Naziritor genera, so that taxa are precisely defined for accurate in situ and ex situ conservation decisions.

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.

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.

DNA barcoding and genetic diversity analyses of fishes of Kaladan River of Indo-Myanmar biodiversity hotspot

Abstract Species are considered as a fundamental unit of biodiversity. Therefore, the prerequisite for biodiversity management and conservation is to know the number of species one is dealing with. Consequently, the need of the present study was conceptualized, which dealt with the comprehensive molecular appraisal of Kaladan’s Fish fauna. A total of 291 specimens representing 49 species, 28 genera, 11 families, and 4 orders, were collected from 11 sampling stations situated along the main Kaladan River and its four major tributaries, i.e. Tiau, Tuipui, Mat, and Tuichang, flowing in Mizoram state of India (part of Indo–Myanmar biodiversity hotspot) and COI sequences of all the 291 samples were generated. All the analyses conducted in the present study, i.e. K2P genetic divergence, bPTP and Neighbour-Joining suggest that DNA Barcoding is an efficient and reliable tool for species identification and deciding the species boundary. Most of the species of Kaladan showed the clear existence of barcode gap. However, the presence of intra-specific and inter-specific genetic distance overlap in two species revealed the existence of putative sibling species or hidden taxa. This study also revealed the presence of two cryptic species and putative previously unknown species of genus Garra and Schistura. The COI barcode database of Kaladan’s fish fauna, established in the present study, may serve as a reference library for accurate identification of fishes and will help ichthyologist, researcher, students, biodiversity managers and policy makers in proper planning with regard to conservation and management of the resources.

DNA Barcoding of Freshwater Fishes of Indo-Myanmar Biodiversity Hotspot

To develop an effective conservation and management strategy, it is required to assess the biodiversity status of an ecosystem, especially when we deal with Indo-Myanmar biodiversity hotspot. Importance of this reaches to an entirely different level as the hotspot represents the area of high endemism which is under continuous threat. Therefore, the need of the present study was conceptualized, dealing with molecular assessment of the fish fauna of Indo-Myanmar region, which covers the Indian states namely, Manipur, Meghalaya, Mizoram, and Nagaland. A total of 363 specimens, representing 109 species were collected and barcoded from the different rivers and their tributaries of the region. The analyses performed in the present study, i.e. Kimura 2-Parameter genetic divergence, Neighbor-Joining, Automated Barcode Gap Discovery and Bayesian Poisson Tree Processes suggest that DNA barcoding is an efficient and reliable tool for species identification. Most of the species were clearly delineated. However, presence of intra-specific and inter-specific genetic distance overlap in few species, revealed the existence of putative cryptic species. A reliable DNA barcode reference library, established in our study provides an adequate knowledge base to the groups of non-taxonomists, researchers, biodiversity managers and policy makers in sketching effective conservation measures for this ecosystem.

Complete mitochondrial genome of near threatened fish species Osteobrama belangeri (Cypriniformes: Cyprinidae)

Abstract The complete mitochondrial genome of Osteobrama belangeri was obtained, using Illumina high-throughput NextSeq 500 with 2 × 150 bp sequencing of mitochondrial DNA. The mitochondrial genome of O. belangeri was 16,602 bp in length (GenBank Accession No. KY887473), comprised of 13 protein coding genes, 22 tRNA genes, 2 rRNA genes and a control region, i.e. D-loop. In present mitogenome, 11 short sequence repeats were identified and validated in silico. The arrangement of genes was found identical to other Cypriniformes fish mitogenomes, available in NCBI database. Phylogenetic relationship established in the present study also supported that genus Osteobrama is member of subfamily Cyprininae (tribe: smiliogastrini) not Cultrinae, which provide useful insights into taxonomic status of the genus.

Complete mitochondrial genome of near threatened butter Catfish Ompok bimaculatus (Siluriformes: Siluridae)

Abstract The complete mitochondrial genome of Ompok bimaculatus was obtained, using illumina high-throughput NextSeq 500 with 2 × 150 bp sequencing of mitochondrial DNA. The genome of O. bimaculatus was 16,482 bp in length (GenBank Accession No. KY887474) comprised of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region i.e. D-loop. In present mitogenome, 9 SSR were identified and validated in silico and secondary structures of all the 22 tRNA were predicted. The arrangement of genes was found identical to other siluriformes fish mitogenomes available in NCBI database. Phylogenetic relationship with closely related species were established which provide useful insights into taxonomic status of the species.

Derivation and Characterization of a ES-Like Cell Line from Indian Catfish Heteropneustes fossilis Blastulas

A cell line designated as HFB-ES was established from blastula stage embryos of H. fossilis (Singhi). The embryonic cells were harvested and maintained in Leibovitzs medium supplemented with 15% fetal bovine serum. The cell line had been subcultured for more than 90 passages in a period of 24 months. HFB-ES cells were able to grow at temperatures between 25 and 35°C with an optimum temperature of 28°C. The growth rate of HFB-ES was proportional to FBS concentration, with optimum growth seen at 15% FBS concentration. The originality of the cell line was confirmed by sequencing of cytochrome oxidase c subunit I (COI), cytochrome b gene, and microsatellite DNA profile. Results of chromosome complements of HFB showed normal karyo-morphology with 56 (2n) diploid number of chromosomes after 40 passages which indicated that the developed cell line is chromosomally stable. The pluripotency of HFB was demonstrated by alkaline phosphatase activity and Oct-4 gene expression. Expression of GFP reporter gene was successful in HFB-ES. These results indicated that HFB-ES could be utilized for future gene expression studies.