Genetic diversity and population structure of the threatened chocolate mahseer (Neolissochilus hexagonolepis McClelland 1839) based on SSR markers: implications for conservation management in Northeast India.

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.

Transcriptome profiling and expression analysis of immune responsive genes in the liver of Golden mahseer (Tor putitora) challenged with Aeromonas hydrophila.

Transcriptome profiling has been used to decipher the novel mechanisms behind immune responses of the fishes. However, the molecular mechanism underlining immune response in mahseer is not studied so far. Fishes are greatly affected by bacterial pathogens such as Aeromonas hydrophila. In this study, transcriptome response of golden mahseer (Tor putitora) infected with A. hydrophila was examined using paired end Illumina sequencing of liver tissue to understand the immune response of the fish. The de novo assembly generated 61,042 unigenes ranging from 200 to 9322 bp in length and an average length of 463 bp. The gene ontology annotations resulted a total of 131,826 term assignments to the annotated transcriptome including 60,846 (46.16%) allocations from the biological process; 21,603 (16.39%) from molecular function and 49,377 (37.46%) from cellular components. Differential gene expression analysis of the transcriptome data from challenged and control group revealed 1104 upregulated and 1304 down-regulated unigenes. The differentially expressed genes were mainly involved in the pathways including cell surface receptor signaling, TH1 and TH2 cell differentiation, pathogen recognition, and immune system process/defense response especially complement cascade. Twelve unigenes including ankyrin, serum amyloid, hsp4b, STAT3, complement factor c3 and c7 were validated using qPCR and found differentially expressed in accordance with in silico expression analysis. The results obtained in this study will provide the first and crucial information on the molecular mechanism of mahseer fishes against bacterial infection.

Transcriptional response to heat shock in liver of snow trout (Schizothorax richardsonii)--a vulnerable Himalayan Cyprinid fish.

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 impact of a moderate chronic temperature increase on spleen immune-relevant gene transcription depends on whether Atlantic cod (Gadus morhua) are stimulated with bacterial versus viral antigens.

Exposure to elevated temperature is an inherent feature of Atlantic cod (Gadus morhua) sea-cage culture in some regions (e.g., Newfoundland) and may also become an increasingly prevalent challenge for wild fish populations because of accelerated climate change. Therefore, understanding how elevated temperatures impacts the immune response of this commercially important species may help to reduce the potential negative impacts of such challenges. Previously, we investigated the impacts of moderately elevated temperature on the antiviral responses of Atlantic cod (Hori et al. 2012) and reported that elevated temperature modulated the spleen transcriptome response to polyriboinosinic polyribocytidylic acid (pIC, a viral mimic). Herein, we report a complementary microarray study that investigated the impact of the same elevated temperature regime on the Atlantic cod spleen transcriptome response to intraperitoneal (IP) injection of formalin-killed Aeromonas salmonicida (ASAL). Fish were held at two different temperatures (10 °C and 16 °C) prior to immune stimulation and sampled 6 and 24 h post-injection (HPI). In this experiment, we identified 711 and 666 nonredundant ASAL-responsive genes at 6HPI and 24HPI, respectively. These included several known antibacterial genes, including hepcidin, cathelicidin, ferritin heavy subunit, and interleukin 8. However, we only identified 15 differentially expressed genes at 6HPI and 2 at 24HPI (FDR 1%) when comparing ASAL-injected fish held at 10 °C versus 16 °C. In contrast, the same comparisons with pIC-injected fish yielded 290 and 339 differentially expressed genes (FDR 1%) at 6HPI and 24HPI, respectively. These results suggest that moderately elevated temperature has a lesser effect on the Atlantic cod spleen transcriptome response to ASAL (i.e., the antibacterial response) than to pIC (i.e., antiviral response). Thus, the impacts of high temperatures on the cod's immune response may be pathogen dependent.

Molecular cloning and expression profile of snow trout GPDH gene in response to abiotic stress.

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.

Complete mitochondrial genome of nearly threatened freshwater ornamental fish, Microphis deocata and its phylogenetic relationship within Syngnathidae.

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 OL 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.

Data on solute carrier transporter genes of a threatened Himalayan fish species - Schizothorax richardsonii.

The Snowtrout, Schizothorax richardsonii, is a vulnerable fish species found in different rivers and rivulets of the Himalayan region. The species is also a suitable poikilotherm to study the low-temperature tolerance as it dwells well at a temperature range of 5-20 °C. The solute carrier (SLC) group of membrane transport proteins play an integral role in cellular acclimation response. The present RNA sequencing was done to identify solute carrier transporter which are the major gene cascades responsible for transport of sugars, amino acids, oligonucleotides, ions, drugs, etc. to and from the cell organelles. A reference transcriptome database was created from liver tissue of Schizothorax richardsonii through RNA sequencing on Illumina HiSeq 2000 platform. The sequences were annotated and characterized under various solute carrier families in the species. So far, 113 transcripts were identified as solute carrier transporter genes categorized under 13 different families. This data will be useful for many researchers working on gene cloning and differential expression of solute carriers.

Molecular identification and genetic diversity analysis of Chocolate mahseer (Neolissochilus hexagonolepis) populations of Northeast India, using mitochondrial DNA markers.

The population genetic structure and genetic diversity of Neolissochilus hexagonolepis were studied using three mitochondrial genes (CoxI, Cytb, ATPase 6/8). A total of 120 individuals representing nine populations from different drainages of Northeast India were used for the study. Thirty-three distinct haplotypes were identified from concatenated gene analysis. The total haplotype and nucleotide diversities are 0.8880 and 0.0280, respectively. The analysis of molecular variance (AMOVA) reveals that the main variation (89.33%) was among populations. Most of the populations showed high polymorphisms, parsimony and haplotype diversity which indicate genetically healthy stocks in the wild. The genetic differentiation patterns were consistent with geographical distributions. Pairwise FST comparison of populations showed significant genetic differentiation (0.9088, p < .05). The pattern of haplotype network and phylogenetic tree revealed six major groups. Results suggested that chocolate mahseer populations in Northeast India having high haplotype diversity and genetic differentiation can be utilized in breeding programs to maintain genetic diversity in the descendant populations. The present study would be beneficial for sustainable management, stock-specific strategies for breeding and conservation of the wild population of N. hexagonolepis in future.

Solute carriers (SLCs) identified and characterized from kidney transcriptome of golden mahseer (Tor putitora) (Fam: Cyprinidae).

The solute carriers (SLC) are trans-membrane proteins, those regulate the transport of various substances (sugars, amino acids, nucleotides, inorganic cations/anions, metals, drugs etc.) across the cell membrane. There are more than 338 solute carriers (slc) reported in fishes that play crucial role in cellular influx and efflux. The study of solute carrier families may reveal many answers regarding the function of transporter genes in the species and their effect in the existing environment. Therefore, we performed RNA sequencing of kidney tissue of the golden mahseer (Tor putitora) using Illumina platform to identify the solute carrier families and characterized 24 putative functional genes under 15 solute carrier families. Out of 24 putative functional genes, 11 genes were differentially expressed in different tissues (head kidney, trunk kidney, spleen, liver, gill, muscle, intestine and brain) using qRT-PCR assay. The slc5a1, slc5a12, slc12a3, slc13a3, slc22a13 and slc26a6 were highly expressed in kidney. The slc15a2, slc25a47, slc33a1 and slc38a2 were highly expressed in brain and slc30a5 was over-expressed in gill. The unrooted phylogenetic trees of slc2, slc5, slc13 and slc33 were constructed using amino acid sequences of Homo sapiens, Salmo salar, Danio rerio, Cyprinus carpio and Tor putitora. It appears that all the putative solute carrier families are very much conserved in human and fish species including the present fish, golden mahseer. This study provides the first hand database of solute carrier families particularly transporter encoding proteins in the species.

Complete mitochondrial genome organization of Schizothorax plagiostomus (Heckel, 1838).

The complete mitochondrial genome of Schizothorax plagiostomus, an endemic coldwater fish of Himalayas, was determined for the first time. The genome is 16,576 bp in length and consists of 13 protein coding genes, 22 tRNAs, 2rRNA genes and 1 putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was A 30%, G 17.4%, C 26.8%, T 25.8%, A + T content 55.8% and the G + C content 44.2%. The control region consists of a microsatellite locus (TA)12 between 16,460 and 16,483 bp. The present study will provide the rationale for the management and conservation of S. plagiostomus.

De novo assembly and characterization of tissue-specific transcriptome in the endangered golden mahseer, Tor putitora.

The golden mahseer (Tor putitora) graces most of the Himalayan Rivers of India and neighboring South Asian countries. Despite its several importance as a research model, as food, and in sport fishing, knowledge on transcriptome database is nil. Therefore, it was targeted to develop reference transcriptome databases of the species using next-generation sequencing. In the present study, 100,540,130 high-quality paired-end reads were obtained from six cDNA libraries of spleen, liver, gill, kidney, muscle, and brain with 28.4 GB data using Illumina paired-end sequencing technology. Tissue-specific transcriptomes as well as complete transcriptome assembly were analyzed for concise representation of the study. In brief, the de novo assembly of individual tissue resulted in an average of 31,829 (18,512-46,348) contigs per sample, while combined transcriptome comprised 77,907 unique transcript fragments (unigenes) assembled from reads of six tissues. Approximately 75,407 (96.8%) unigenes could be annotated according to their homology matches in the nr, SwisseProt, GO, or KEGG databases. Comparative analysis showed that 84% of the unigenes have significant similarity to zebra fish RefSeq proteins. Tissue-specific-dominated genes were also identified to hypothesize their localization and expression in individual tissue. In addition, 2485 simple sequence repeats (SSRs) were detected from 77,907 transcripts in the combined transcriptome of the golden mahseer. This study has generated organ-specific transcriptome profiles, which will be helpful to understand the local adaptation, genome evolution, and also future functional studies on immune system of the golden mahseer.

Complete mitochondrial genome organization of Tor tor (Hamilton, 1822).

The complete mitochondrial genome of Tor tor, a threatened "Mahseer" was sequenced for the first time. The mitochondrial genome size determined to be 16,554 bp in length and consisted of 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNA genes and a control region or displacement loop (D-Loop) region, resembling the typical organizational pattern of most of the teleost. The overall base composition found was A: 31.8%, T: 25%, G: 15.7% and C: 27.4%; A + T: 56.9% and G + C: 43.1%. The phylogenetic tree constructed using 11 other cyprinids' total mtDNA datasets confirmed the location of present species among mahseers. The total sequence data could support further study in molecular systematics, species identification, evolutionary and conservation genetics.

The complete mitogenome of brown trout (Salmo trutta fario) and its phylogeny.

The complete mitochondrial genome of Salmo trutta fario, commonly known as brown trout, was sequenced using NGS technology. The mitochondrial genome size was determined to be 16 677 bp and composed of 13 protein-coding gene (PCG), 22 tRNAs, 2 rRNA genes, and 1 putative control region. The overall mitogenome composition of S. trutta fario is A: 28.13%, G: 16.44%, C: 29.47%, and T: 25.96% with A + T content of 54.09% and G + C content of 45.91%. The gene arrangement and the order are similar to other vertebrates. The phylogenetic tree constructed using 42 complete mitogenomes of Salmonidae fishes confirmed the position of the present species under the genus Salmo of subfamily Salmoninae. NGS platform was proved to be a rapid and time-saving technology to reveal complete mitogenomes.

Complete mitochondrial genome organization of Schizothorax progastus (McClelland, 1839).

The complete mitochondrial genome of Schizothorax progastus, an endemic coldwater fish of Himalayas, was determined for the first time. The genome was 16,575 bp in length and consists of 13 protein coding genes, 22 tRNAs, 2rRNA genes and one putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was--A: 29.8%, G: 17.8%, C: 27%, T: 25.4%, A+T content: 55.2% and the G+C content: 44.8%. The control region also consisted of a microsatellite locus (TA)12 between 16,466 and 16,489 bp. The present study will provide the rationale for the management and conservation of S. progastus.

Molecular cloning and characterization of mannose binding lectin homologue from snow trout (Schizothorax richardsonii).

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.

Complete mitochondrial genome organization of Schizothorax esocinus (Heckel, 1838).

The complete mitochondrial genome of Schizothorax esocinus, an endemic coldwater fish of Himalayas, was determined for the first time. The genome is 16,583 bp in length and consists of 13 protein-coding genes, 22 tRNAs, 2rRNA genes and 1 putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was; A 29.8%, G 17.8%, C 27%, T 25.4%, A + T content 55.2% and the G + C content 44.8%. The control region was also consisted of a microsatellite locus (TA) 13 between 16,463 to 16,488 bp. The present study will provide the rationale for the management and conservation of S. esocinus.

Complete mitochondrial genome organization of Schizopyge niger (Heckel, 1838).

The complete mitochondrial genome of Schizopyge niger, an endemic coldwater fish of Himalayas was determined for the first time. The genome is 16,585 bp in length and consists of 13 protein-coding genes, 22 tRNAs, 2rRNA genes and 1 putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was; A: 29.9%, G: 17.7%, C 27.1%, T 25.3%, A + T content 55.2% and the G + C content 44.8%. The control region was also consisted of a microsatellite locus (TA)13 between 16,471 to 16,496 bp. The present study will provide the rationale for the management and conservation of S. niger.

Genetic characterization of Golden mahseer (Tor putitora) populations using mitochondrial DNA markers.

Golden Mahseer (Tor putitora) is an economically important fish of India and Southeast Asia. The present study examined the genetic variations between seven geographically isolated populations of T. putitora using Cyt b (Cytochrome b) and ATPase6/8 gene sequences of mitochondrial DNA. Analysis of 133 sequences of Cyt b (1141 bp) and 130 sequences of ATPase6/8 gene (842 bp) revealed 47 and 44 haplotypes, respectively. The estimated haplotype and nucleotide diversity was high in River Jia Bhoreli (Bhalukpong) population (h = 1.00000, π = 0.007121 for Cyt b and h = 0.90441 π = 0.004867 for ATPase6/8). Results of AMOVA indicated that majority of the genetic variations in both genes were due to variation among populations (60.79% for Cyt b and 51.41% for ATPase6/8 gene). The pairwise F(ST) comparison and neighbor-joining tree revealed high genetic divergence of River Jia Bhoreli population from other populations. The understanding of genetic variations of T. putitora populations will play a key role in conservation and management of this endangered fish species.

Complete mitochondrial genome organization of Schizothorax labiatus (McClelland, 1842).

The complete mitochondrial genome of Schizothorax labiatus, an endemic coldwater fish of Himalayas was determined for the first time. The genome is 16,582 bp in length and consists of 13 protein coding genes, 22 tRNAs, 2rRNA genes and one putative control region. The gene organization and its order were similar to other vertebrates. The overall base composition was; A: 29.8%, G: 17.7%, C 27%, T 25.5%, A+T content 55.3% and the G+C content 44.7%. The control region was also consisted of a microsatellite locus (TA)12 between 16,467 and 16,490 bp. The present study will provide the rationale for the management and conservation of S. labiatus.

The complete mitochondrial genome of threatened chocolate mahseer (Neolissochilus hexagonolepis) and its phylogeny.

The chocolate mahseer (Neolissochilus hexagonolepis) is an important food and game fish of North Eastern India. To study the phylogenetic status we sequenced the complete mitochondrial genome of N. hexagonolepis. The mitogenome is 16,563 bp in length and composed of 13 protein coding genes, 22 tRNAs, 2 rRNAs and one putative control region. The overall base composition was A 31.8%, T 25.0%, G 15.8%, C 27.4% and A+T content 56.9%, G+C content 43.1%. The phylogenetic analysis using the complete mitochondrial genome revealed that the chocolate mahseer belonged to same clade of mahseer group of fishes but different from genera Barbus and Acrossocheilus. The present study will be helpful for the evolution and conservation genetic studies of N. hexagonolepis.