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Background: Butter catfish (Ompok bimaculatus) is a preferred species in South East Asia, with huge aquaculture potential. However, there is limited information about genetic stock composition due to insufficient markers. The goal of this study was to develop de novo microsatellite markers., Methods and Results: For sequencing, genomic SMRT bell libraries (1.5 Kbp size) were prepared for O. bimaculatus. A total of 114 SSR containing sequences were used for primer designing. Polymorphic loci were validated by genotyping 83 individuals from four distant riverine populations, viz., Brahmaputra, Bichiya, Gomti and Kaveri. A total of 30 microsatellite loci were polymorphic, of which five were found to be associated with functional genes and eight (four positive and four negative) loci were found to be under selection pressure. A total of 115 alleles were detected in all loci and PIC ranged from 0.539 to 0.927 and pair-wise F ST values from 0.1267 to 0.26002 (p < 0.001), with an overall F ST value of 0.17047, indicating the presence of population sub-structure. Cross-species transferability of 29 loci (96.67%) was successful in congener species, Ompok pabda., Conclusion: The novel SSR markers developed in this study would facilitate stock characterization of natural populations, to be used in future selection breeding programs and planning conservation strategies in these species. Identified non-neutral markers will give insights into the effect of local adaptation on genetic differentiation in the natural population of this species., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

A new species of the genus Pangasius, is described based on 17 specimens collected from the Cauvery River, India. It can be distinguished from its sister species from South and Southeast Asia, by its widely placed, small and rounded vomerine and palatine tooth plates, longer maxillary and mandibular barbels, greater vertebrae count 50 (vs. 44-48), and smaller caudal peduncle depth (6.5-8.2% SL vs. 9.89-13.09% SL). The tooth plates of the new species closely resembles that of Pangasius macronema but can be clearly distinguished from the latter by having lesser gill rakers (16-19 vs. 36-45); a smaller eye (2.4-4.4% SL vs. 5.2-9.6% SL); and larger adipose-fin base (1.5-2.9% SL vs. 0.1-1.2% SL). The mitochondrial cytochrome c oxidase (COI) gene sequence of the new species shows the genetic divergence of 3.5% and 5.1% from P. pangasius and P. silasi respectively, the two sister species found in South Asia and India. The species delimitation approaches, Poisson Tree Processes (PTP) and assemble species by automatic partitioning (ASAP) clearly resolved that the P . icaria is distinct from its sister species. Phylogenetic position of the species with its sister species was evaluated using maximum likelihood and Bayesian analysis. The discovery of this previously unknown species of genus Pangasius from the Cauvery River of peninsular India indicates important biogeographical insight that this genus migrated till the southern division of Western Ghats., Competing Interests: The authors declare there are no competing interests., (©2022 Ayyathurai et al.)

Silonia silondia is a commercially important fish distributed in Asian countries, which is under threat due to overexploitation. This study focuses on the morphological analysis and genetic variation of S. silondia individuals, through truss network and sequencing of two mitochondrial regions, respectively, from six wild populations of the Ganga and Mahanadi river systems in India. A total of 38 haplotypes was observed by analysing combined mitochondrial genes (cytochrome b + ATPase 6/8) in 247 individuals of S. silondia collected from six populations. Average haplotype and nucleotide diversities were 0.8508 and 0.00231, respectively. Genetic structure analysis showed the predominant cause of genetic variation to be within populations. The two clades were observed among the haplotypes and time of divergence from their most probable ancestor was estimated to be around 0.3949 mya. Analysis of combined mitochondrial genes in six populations of S. silondia resulted into three management units or genetic stocks. The truss network analysis was carried out by interconnecting 12 landmarks from digital images of specimens to identify phenotypic stocks. Sixty-five truss morphometric variables were analysed for geometric shape variation which revealed morphological divergence in River Son specimens. The present study presents molecular markers and genetic diversity data which can be critical input for conservation and management of differentiated populations and future monitoring of the genetic bottleneck. The morphological shape analysis clearly shows that variation in the insertion of adipose fin is an important parameter influencing the morphological discrimination., (© 2020 Fisheries Society of the British Isles.)

Germ cell (GC) transplantation (GCT) is a proven powerful reproductive technique to enhance the production efficiency of domesticated animals and aid to the recovery of endangered germ lines. In mammals, several methods have been adopted for the eradication of GCs such as treatment with cytotoxic drugs, irradiation, cold ischaemia and hyperthermic treatment. Some of these methods have also been tried in fishes, and conditions for sterilization of gonads have been established. Here, we report the production of GC-depleted male striped catfish Pangasianodon hypophthalmus in 12 weeks by the combination of heat and chemical treatments. The cytotoxic drug busulphan (40 mg/kg) was intraperitoneally injected into the animals at 2-week intervals (six doses in total) and maintained in water at 38°C between weeks 1 and 12. The effectiveness of the treatment was assessed using gonadal index and histology. At the end of 12 weeks, very severe gonadal degeneration was observed in fish treated with the heat-chemical combination, and 100% of sampled fish (n = 5) were found devoid of endogenous GCs. On contrary, high temperature alone caused minor gonadal degeneration. Results obtained in this study suggest that endogenous GCs of large-bodied fish such as P. hypophthalmus can also be sterilized by heat and chemical treatments within a considerably short period., (© 2019 Blackwell Verlag GmbH.)

The Indian catfish, Clarias magur (previous name C. batrachus) is an air breathing fish, inhabitant of aquatic bodies characterized by low dissolved oxygen levels. It is exposed to hypoxic conditions in its natural habitat. Thus, it can be useful model to study the mechanism of hypoxia stress tolerance. In C. magur, molecular processes facilitating its adaptation to hypoxia stress remain largely unexplored, in part due to unavailability of genomic resources. The suppression subtractive hybridization technique (SSH) was employed to compare the differential expression of transcripts under experimental hypoxic conditions, to that of normoxic conditions. Twelve subtracted cDNA libraries (six each forward and reverse) were constructed from brain, heart, liver, muscle, spleen and head kidney tissues. A total of 2020 clones were screened and sequenced, resulting into 1805 high quality expressed sequence tags (ESTs). Annotation of these differentially expressed ESTs resulted into the identification of genes involved in vast majority of pathways/processes affecting metabolism, cellular processes, signal transduction and/or immune functions. Additionally, 18 potential novel genes expressed in hypoxia stress exposed fish were also identified. The study had catalogued the differentially expressed genes from hypoxia stress induced C. magur, where most of them are reported for the first time in a hypoxia-tolerant fish species. The results not only provided insights for the hypoxia stress altered cellular functions in C. magur, but also generated a valuable functional genomics resource to assist targeted studies on functional genomics and future genome projects., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity-capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6-24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall FST = 0.075; P < 0.0001) with maximum variation (92.5%) within populations. Cross-priming assessment revealed successful amplification (35-38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.

Silonia silondia is a commercially important food fish. Samples collected through commercial catches from four rivers in India are described by sequence analysis of two molecular markers. Cytochrome b (1140 bp) and ATPase 6/8 (842 bp) genes were analyzed, which represented high level of genetic differentiation within populations of S. silondia. The sequence alignments of cytochrome b and ATPase 6/8 genes revealed 13 and 11 different haplotypes, respectively. The sequences of both the mitochondrial regions revealed high haplotype and low nucleotide diversities. The patterns of genetic diversity and haplotype networks clearly indicated two distinct mitochondrial lineages, however, haplotypes from both the lineages were not specifically assigned to any population. The results confirm the utility of molecular markers generating baseline information, useful for planning effective strategies for conservation, management and sustainability of Silond catfish fishery.

Heat shock proteins (Hsps) are typically associated with stress response and tolerance. The Indian catfish, Clarias batrachus, is a freshwater air-breathing hypoxia tolerant teleost and is potentially important catfish species for aquaculture and for its economic value as food. The present study aimed at determining the transcriptional response of three Hsps, CbHsc71, CbHsp90α and CbHsp10 in hypoxia tolerant Indian catfish, C. batrachus, under experimental and natural hypoxia. The expression profile of above three genes were studied under different periods of hypoxia, through qRT-PCR. Primers were designed from ESTs obtained through SSH libraries constructed from hypoxia treated fishes. The Hsp10 ESTs and deduced protein was in silico characterized for its ORF and for its physical and chemical properties, respectively, using GeneScan, blastp, scanprosite, superfamily and other softwares. A phylogenetic tree was constructed based on deduced amino acid sequences of Hsc71, Hsp90α, Hsp90β of Homo sapiens and other fishes along with CbHsp10 protein in MEGA4. The deduced protein sequences of CbHsp10 was found to have characteristic Hsp10 family signatures, and it is proposed for inclusion of methionine in the consensus sequences of Hsp10 family signature, after the "proline" residue. At transcription level, these genes were found to be differentially regulated under hypoxia stress, in different tissues of C. batrachus. The CbHsc71 and CbHsp90α were up-regulated after short and long-term hypoxia, whereas CbHsp10 was significantly down-regulated after short-term hypoxia. The differential expression of these Hsps may play a role in protection and survival under hypoxia induced oxidative stress in C. batrachus.

Pangasius pangasius, an endangered freshwater fish species, is an important component of capture fishery from Indian rivers. Samples collected through commercial catches from three riverine populations were analyzed with cytb (307 bp) and ATPase6&8 (842 bp) regions for population variation and differentiation. The sequences of the both the mitochondrial regions revealed high haplotype and low nucleotide diversity. Shallow genetic diversity based on ATPase6&8 was observed, however its haplotypes network clearly indicated two distinct mitochondrial lineages. Mismatch distribution suggested population bottlenecks followed by expansion in Mahanadi population. The present study indicated the ATPase6&8 to be a potential mitochondrial marker for studying the population sub-structuring in the wild population of P. pangasius.

Complete mitogenome sequences for Clarias batrachus (magur) and Pangasius pangasius (family Claridae and Pangasiidae, respectively) were generated, which were 16,511 and 16,476 bp, respectively. Both the mitogenomes contained 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs and 1 non-coding (control) region. The order of the genes was identical to that observed in most other vertebrates. The genome resource of complete mitogenome sequence of Indian catfish species generated during this study would be useful in phylogenetic studies.

Several physiologically important genes were found to be regulated by hypoxia at the transcriptional level. The Pleckstrin homology-like domain, family A, member 2 (PHLDA2) gene was previously identified as an imprinted gene. The present study was aimed to determine the structure of complete cDNA and the deduced protein of PHLDA2 along with analysing the changes in its mRNA expression in Clarias batrachus tissues under hypoxic conditions. The complete cDNA of CbPHLDA2 gene consisted of 1009 nucleotides with an open reading frame of 417 nucleotides. The deduced CbPHLDA2 protein of 139 amino acids shared high homology with PHLD2A of other fishes as well as that of vertebrates. Importantly, a single amino acid (asparagine/lysine) insertion was identified in the PH domain of CbPHLDA2 and other fishes, which was absent in other vertebrates studied. Furthermore, under normoxic conditions, CbPHLDA2 was constitutively expressed with varying levels in analysed tissues. Short- and long-term hypoxia exposure resulted in significant changes in the expression of CbPHLDA2 in liver, spleen, head kidney, brain and muscle in a time-dependent manner. The results suggested that CbPHLDA2 might play an important role for adaptive significance under hypoxia.

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 Leibovitz's 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.

Hypoxia is an important environmental stressor that leads to rapid adaptive changes in metabolic organization. However, the molecular mechanisms of hypoxia tolerance in fish remain largely unknown. The present work was focused on understanding the molecular mechanisms and signaling pathways that may lead to tolerance of Clarias batrachus to hypoxic stress. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) is a new hypoxia-inducible factor (HIF) targeted gene and is regulated by HIF-1 under hypoxic conditions. Overexpression of PPP1R3C increases glycogen accumulation through activation of several enzymes and processes. In this study, for the first time, full length cDNA of PPP1R3C from C. batrachus was characterized and its expression pattern in the brain, liver, muscle and spleen under short (progressive hypoxia; PH, 1h, 6h and 12h) and long-term (natural) hypoxic conditions was investigated. The complete cDNA of PPP1R3C was of 1499 bp, encoding 285 amino acid residues. The identified protein had a protein phosphatase 1 binding motif and a carbohydrate binding domain, thought to be involved in the regulation of glycogen metabolism. Short-term hypoxia exposure caused significant increase in PPP1R3C transcripts in the liver (6h; 6.96 fold and 12h; 3.91 fold) and muscle (progressive hypoxia; 3.46 fold), while, after long-term hypoxia exposure, significant up-regulation in the liver (7.77 fold) and spleen (6.59 fold) tissues was observed. No significant differences were observed in the brain for any time periods. Thus PPP1R3C may play an important role in the tolerance of C. batrachus to hypoxia., (© 2013 Elsevier B.V. All rights reserved.)

The present study aimed at characterization of three HIF-α subunits, HIF-1α -2α and -3α from hypoxia-tolerant Clarias batrachus, as well as to elucidate their expression pattern under short and long-term hypoxic conditions and identification of biomarker candidate. The complete cDNAs of HIF-1α, -2α and -3α were 2,833, 4,270 and 3,256 bp in length, encoding 774, 818 and 628 amino acid residues, respectively. In C. batrachus, HIF-α subunits were structurally similar in DNA binding, dimerization, degradation and transcriptional activation domains, but differed in their oxygen-dependent degradation domains. Presence of c-Jun N-terminal kinase binding domain in HIF-α subunits was reported here for the first time in fish. In adult C. batrachus, three HIF-α mRNAs were detected in different tissues under normoxic conditions, however HIF-1α was highly expressed in all the tissues studied, in comparison to HIF-2α and -3α. Short-term hypoxia exposure caused significant increase in three HIF-α transcripts in brain, liver and head kidney, while after long-term hypoxia exposure, significant up-regulation of HIF-1α in spleen and -2α in muscle was observed and HIF-3α significantly down-regulated in head kidney. These observations suggest that the differential expression of HIF-α subunits in C. batrachus was hypoxic time period dependent and may play specialized roles in adaptive response to hypoxia. HIF-2α, with its highly elevated expression in muscle tissues, can be a robust biomarker candidate for exposure to hypoxic environment.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

The comparative assessment of genetic diversity using allozymes, random amplified polymorphic DNA (RAPD), and microsatellite markers was conducted in endemic and endangered yellow catfish (Horabagrus brachysoma) sampled from three locations in Western Ghats river systems of India. Among the three markers, microsatellites show more polymorphism, having 100% polymorphic loci, whereas allozymes show the least (56%). In RAPD, 60.5% of fragments were polymorphic. Observed heterozygosity and F(ST) values were very high in microsatellites, compared with the other markers. Microsatellite and RAPD markers reported a higher degree of genetic differentiation than allozymes among the populations depicted by pairwise F(ST)/G(ST), AMOVA, Nei's genetic distance, and UPGMA dendrogram. The three classes of markers demonstrated striking genetic differentiation between pairs of H. brachysoma populations. The data emphasize the need for fishery management, conservation, and rehabilitation of this species.

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.

The allozyme variation and population genetic structure of Horabagrus brachysoma in three natural populations from the southern part of the Western Ghats region, India, were investigated by polyacrylamide gel electrophoresis. Variations at 14 loci from 14 enzyme systems were analyzed. The allozyme analysis revealed a high level of genetic variation in this species, with an average of observed alleles per locus of 2.357 and observed heterozygosity of 0.178. The positive value of the fixation index (FIS=0.507) implied a significant deficiency of heterozygosity at the population level. The highly significant probability (P<0.0001) for the overall loci suggested that the three sample sets were not part of the same gene pool.