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The snow trout ( Schizothorax richardsonii ) belonging to family Cyprinidae, is an endemic fish of the Himalayan region. The species is tagged as vulnerable species in the IUCN red list of threatened species. The fish thrives well in snowmelt water of several streams and rivers in the region but are occasionally exposed to more than 20 °C during the summer season. Therefore, we have used deep RNA sequencing to decipher the transcriptome of snow trout and characterize the genes and molecular pathways involved in heat shock response. In this study 72,601,298 and 65,428,283 raw reads for heat-shocked and control, respectively, were obtained by Illumina paired-end sequencing technology. The de novo assembled transcriptome was tested for differential gene expression across the treatment groups. The quality of assembly was high with N75 and N50 lengths of 461 and 1274 bases, respectively. A total of 65 unique transcripts were differentially expressed in liver under heat shock and control. Annotated blast matches reveal that differentially expressed transcripts correspond to critical chaperones and molecular pathways, previously shown to be important for thermal stress in other fish species. Eight randomly selected heat-stressed responsive transcripts were also observed to be upregulated during qRT-PCR analysis. This study is the preliminary step to understanding the responses during sudden environmental changes like heat shock. The reference transcriptome database would also aid further studies on biological and physiological aspects of the snow trout under abiotic stresses.

The stream loach, Schistura reticulofasciata has high demand in ornamental fish trade due to its mesmerizing colorful banding pattern and size. In recent times, its abundance has declined severely in its habitat, and presently it has been assessed as vulnerable fish. Despite its threatened status, no nucleotide sequence information from this fish is publicly available in GenBank and the BOLD System. Here we describe the first complete mitogenome of S. reticulofasciata. The resulting circular mitogenome of S. reticulofasciata is 16,581 in length, comprising 13 protein-coding genes (PCG), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA genes (rRNA), one replication origin (OL) and one control region (D-loop). The overall genome size, gene arrangement, A + T content, AT-skew, GC-skew and partiality towards A and T in mitogenome were similar to those of other Schistura species. Phylogenetic analysis confirmed the high sequence similarity and conserved synteny among the Schistura species. The primary genetic data developed in this study could be successively applied for species identification, genetic diversity studies and the formulation of effective conservation strategies for steam loaches.

Microphis deocata (deocata pipefish), belonging to family Syngnathidae, is one of the important indigenous ornamental fish species listed as near threatened in the IUCN red list. Here, we first report the complete mitochondrial genome of deocata pipefish using Illumina next-generation sequencing platform. The total length of the mitogenome is 16,526 bp. It encompasses 13 protein coding genes, 2 ribosomal rRNAs, and 22 tRNAs. The WANCY region (a cluster of five tRNA genes) contains the 50 bp O L light strand origin of replication. Phylogenetic analysis of Syngnathidae revealed M. deocata to cluster with Oostethus manadensis, forming a sister group with Doryrhamphus japonicas and Dunckerocampus dactyliophorus. The mitochondrial genome sequence data generated in the present study will play an important role in population genetic analysis and developing conservation strategies for this species.

A set of 12 microsatellites was developed for a freshwater fish golden mahseer, Tor putitora . Golden mahseer is an endangered and endemic in mid-hill regions of South-Asian countries. Constructing a partial genomic library, we obtained 44 clones containing putative microsatellite motifs and 12 microsatellite with enough flanking sequence for primer designing. The number of alleles per locus ranged from 3 to 8 (mean 5.41), with a mean expected and observed heterozygosity of 0.68 and 0.66, respectively. Most of them were in Hardy–Weinberg equilibrium except TPM20a. There was no significant evidence for linkage disequilibrium between any pair of loci. These microsatellites were highly polymorphic (mean PIC 0.68) and will be useful for genetic variability and conservation genetics studies.

A set of 8 microsatellites was developed for a freshwater fish chush snowtrout, Schizopyge niger. Chush snowtrout is an endemic fish to Kashmir valley, India. Constructing a partial genomic library, we obtained 36 clones containing putative microsatellite motifs and 12 microsatellite with enough flanking sequence for primer designing. The number of alleles per locus ranged from 3 to 7 (mean 4.75), with a mean expected and observed heterozygosity of 0.71 and 0.68, respectively. All the loci were in Hardy–Weinberg equilibrium. There was no significant evidence for linkage disequilibrium between any pair of loci. These microsatellites were highly polymorphic (mean PIC 0.63) and will be useful for genetic variability and conservation genetics studies.

Neolissochilus hexagonolepis (MacClend 1839), commonly known as chocolate mahseer, is an ecologically threatened fish species. The species is reported from Southeast Asia and in India, it is distributed across the Northeast region. The present study was carried out to develop species-specific novel microsatellite markers using next-generation sequencing and to assess the genetic diversity of wild chocolate mahseer populations distributed through Northeastern India. A total of 25 polymorphic loci (mean PIC = 0.933) were amplified in 194 individuals belonging to seven different populations. We observed high genetic diversity across the loci with mean observed (Ho) and expected heterozygosity (He) of 0.557 and 0.939 respectively. The studied loci didn’t show significant deviation from Hardy–Weinberg equilibrium. Genetic analyses indicate substantial pairwise Nei’s genetic distance and moderate to high levels of genetic differentiation among populations (mean FST = 0.23). The structure, factor and cluster analysis identified five major clusters that can be considered as different conservation units while formulating any management measures. Furthermore, the migration analysis inferred that there is no active migration among the studied populations. Results suggested that two populations i.e. Dikrong river (Arunanchal Pradesh) and Umiam river have high genetic diversity. These populations can be utilized for the breeding programme to achieve substantial genetic variations in the descendant populations. The Nongbareh populations showed very less genetic vigor and need an immediate attention for conservation. The SSR markers developed in the present study will provide a valuable resource for future population genetic assessment and implementation of effective conservation strategies for wild chocolate mahseer.

The teleost fishes depend on their innate immune system for first line of defense. Mannose binding lectin (MBL) is a member of the collectin family of proteins that plays an important role in the innate immune response in many vertebrates including fish. The present study was thus carried out to identify and characterize MBL homologue from snow trout. The cDNA of snow trout MBL homologue (galactose binding; Sch-GBL) was cloned and characterized. The obtained cDNA (778-bp) contains 762 bp long ORF (open reading frame) and a 16 bp partial 3′UTR (untranslated region); transcripts are ubiquitously expressed in all the tissues under study. Sch-GBL encodes a 253-aa protein that clusters with that of common carp, goldfish and contain several common structural motifs. In addition, the K a /K s ratios of the C-type lectin domain sequences depicts that they are under negative selection. Sch-GBL transcript level increases in response to challenge to Aeromonas hydrophila infection and decreases in response to downshift in temperature. Altogether, these findings suggest that Sch-GBL play an important role in the first line of fish defense mechanisms.

This study examined the phylogenetic relationship and identification of five Mahseer species (Tor putitora, Tor tor, Tor khudree , Tor chelynoides and Neolissochilus hexagonolopis) using partial sequencing of a Cytochrome Oxidase I (COI) DNA barcodes. The sequence analysis data showed that 134 (21.61%) sites out of 628 sites were variable without insertion or deletion. Rate of transition (70.5%) were higher than transversion (29.41%). There was a high inter-species divergence (range 4.1% to 12.2%) in Mahseer species as compared to intra-specific sequence divergence (1.7% for T. putitora, 1.2% for T. tor, 1.4% for T. khudree, 3.0% for T. chelynoides, 0.26 % for N. hexagonolopis). The phylogenetic tree, constructed by maximum parsimony, maximum likelihood and unweighted pair group average methods revealed similar results suggesting that T. putitora, T. tor and T. khudree had a close relationship to each other while maximum divergence was observed in T. chelynoides, which was also confirmed by the genetic distance data. The results indicate that COI sequencing or bar-coding is useful in unravelling phylogenetic relationship and identification of Mahseer species. [PUBLICATION ABSTRACT]

Glycerol-3-phosphate dehydrogenase (GPDH) gene possibly plays a key role for cold acclimation process in snow trout during winter months when water temperature goes down to 4–5 °C. In this study, 1,012 bp nucleotide fragment of GPDH gene was obtained from two snow trout species ( Schizothorax richardsonii and S. niger ; family: Cyprinidae), distributed in several Himalayan rivers. The gene encoded a protein of 334 amino acids. The encoded protein sequence was very similar to GPDH of Danio rerio (94.36 %) using BLASTx searches. In S. richardsonii the qRT-PCR showed highest expression in muscle tissue followed by liver and also revealed 19 fold gene expression in liver tissue under cold (5 °C) in comparison with warm (15 °C) condition. The elevated expression levels of GPDH cDNA on cold treatment furthermore suggest that GPDH plays a role in stress related responses in S. richardsonii . The phylogenetic analysis showed that the two snow trout species GPDH share the same clade with characterized GPDHs from other teleost fishes suggesting a common evolutionary origin and a similar catalytic function. In addition, the Ka/Ks ratios of these sequences suggested that they are under purifying selection. Moreover, the expression profile of GPDH gene among co generic species of genus Schizothorax showed that GPDH cDNA expression was highest in S. richardsonii and lowest in S. esocinus which gives an indication of species specific adaptation in relation to different geographical areas.

The Golden mahseer Tor putitora is one of the most important sport fish even its endangered status due to its declining population. The length-weight relationship and the condition factor for T putitora (N=150) from four different geographical regions in eastern and western Himalayas were studied. Fish sampling took place from December 2009 to March 2011. The b (slope) values obtained ranged from 2.69-3.10. However, no significant deviation was observed from the isometric value (b3) therefore, population from all four locations followed isometric growth pattern. The length-weight regression equation obtained for Bhalokpong, Arunachal Pradesh was Log W=-5.276+3.108Log L; for Anji, J&K was Log W=-4.633+2.811Log; for Basoli, J&K was Log W=-4.315+2.690Log L and for Jogindernagar, eastern Himalayas in Jia bhoreli river was in better condition in comparison to western Himalayas. Among the western Himalayan region, the population of Ravi river was in poor condition.