High Genetic Differentiation and Genetic Diversity in Endangered Mahseer Tor khudree (Sykes, 1839) as Revealed from Concatenated ATPase 6/8 and Cyt b Mitochondrial Genes.

The Deccan mahseer, Tor khudree (Sykes 1839), belonging to family Cyprinidae is an important food and a game fish distributed in peninsular India. Due to overfishing and habitat destruction, the species is declared endangered and placed on the IUCN red list. Therefore, a well-designed conservation program may be essential to get this species protected in its natural habitat. We used a total of 152 samples from four rivers of peninsular India to assess the genetic diversity and structure of the mahseer using concatenated sequences of two mitochondrial genes, ATPase 6/8 (790 bp) and Cyt b (1000 bp). High haplotypic diversity was seen with 44 haplotypes. Individual gene wise haplotypes included 10 and 21 haplotypes for ATPase6/8 and Cyt b, respectively. AMOVA revealed most of the genetic variations (71.02%) to be within the populations. Significant genetic differentiation was observed between all population pairs, with FST values ranging from 0.121 to 0.372, with minimum between Tunga and Tungabhadra population and maximum between Tunga and Periyar population. Haplotype network showed one ancestral haplotype (TKACH04). Significant negative Fu's F and unimodal mismatch distribution suggested recent demographic expansion. The results of the present study would serve as a useful resource for further research on population genetics and conservation programs of the species.

Revelation of candidate genes and molecular mechanism of reproductive seasonality in female rohu (Labeo rohita Ham.) by RNA sequencing.

BACKGROUND: Carp fish, rohu (Labeo rohita Ham.) is important freshwater aquaculture species of South-East Asia having seasonal reproductive rhythm. There is no holistic study at transcriptome level revealing key candidate genes involved in such circannual rhythm regulated by biological clock genes (BCGs). Seasonality manifestation has two contrasting phases of reproduction, i.e., post-spawning resting and initiation of gonadal activity appropriate for revealing the associated candidate genes. It can be deciphered by RNA sequencing of tissues involved in BPGL (Brain-Pituitary-Gonad-Liver) axis controlling seasonality. How far such BCGs of this fish are evolutionarily conserved across different phyla is unknown. Such study can be of further use to enhance fish productivity as seasonality restricts seed production beyond monsoon season. RESULT: A total of ~ 150 Gb of transcriptomic data of four tissues viz., BPGL were generated using Illumina TruSeq. De-novo assembled BPGL tissues revealed 75,554 differentially expressed transcripts, 115,534 SSRs, 65,584 SNPs, 514 pathways, 5379 transcription factors, 187 mature miRNA which regulates candidate genes represented by 1576 differentially expressed transcripts are available in the form of web-genomic resources. Findings were validated by qPCR. This is the first report in carp fish having 32 BCGs, found widely conserved in fish, amphibian, reptile, birds, prototheria, marsupials and placental mammals. This is due to universal mechanism of rhythmicity in response to environment and earth rotation having adaptive and reproductive significance. CONCLUSION: This study elucidates evolutionary conserved mechanism of photo-periodism sensing, neuroendocrine secretion, metabolism and yolk synthesis in liver, gonadal maturation, muscular growth with sensory and auditory perception in this fish. Study reveals fish as a good model for research on biological clock besides its relevance in reproductive efficiency enhancement.

Activin receptor type IIB in rohu (Labeo rohita): molecular characterization, tissue distribution and immunohistochemical localization during different stages of gonadal maturation.

Activin receptor type IIB (ActRIIB) is a transmembrane serine/threonine kinase receptor which plays a pivotal role in regulating the reproduction in vertebrates including teleost. Earlier studies have documented its importance in governing gonadal maturation in higher vertebrates. However, reports on the regulation of fish reproductive system by ActRIIB gene are still limited. Here, we report the identification and characterization of ActRIIB cDNA of Labeo rohita, a commercially important fish species of the Indian subcontinent. The full-length gene encoding rohu ActRIIB was cloned and found to be of 1674 bp in length. Functional similarities were evident from evolutionary analysis across vertebrates. Real-time PCR to measure the expression of ActRIIB transcript in rohu revealed significant mRNA levels in gonads followed by non-reproductive tissues, including the brain, pituitary and muscle. With respect to different gonadal maturation stages, predominant expression of ActRIIB mRNA was observed during the pre-spawning phase of both sexes. To further delineate its role in rohu reproduction, a recombinant protein of the extracellular domain of ActRIIB (rECD-ActRIIB) was produced, and polyclonal antibody is raised against the protein for its immuno-localization studies during different gonadal maturation stages. Strong immunoreactivity was noticed in the pre-vitellogenic oocytes which decreased dramatically in the fully mature oocytes. Similarly, the strong and intense immunoreactivity was found in the spermatids and spermatocytes of the immature testis, and eventually the intensity reduced with the progression of the maturation stage. These results provide the first evidence of the presence of ActRIIB in rohu gonadal tissues. Taken together, our observations lay the groundwork for further understanding and investigating on the potential role of ActRIIB in fish reproduction system in the event of gonadal maturation.

The genome of walking catfish Clarias magur (Hamilton, 1822) unveils the genetic basis that may have facilitated the development of environmental and terrestrial adaptation systems in air-breathing catfishes.

The walking catfish Clarias magur (Hamilton, 1822) (magur) is an important catfish species inhabiting the Indian subcontinent. It is considered as a highly nutritious food fish and has the capability to walk to some distance, and survive a considerable period without water. Assembly, scaffolding and several rounds of iterations resulted in 3,484 scaffolds covering ∼94% of estimated genome with 9.88 Mb largest scaffold, and N50 1.31 Mb. The genome possessed 23,748 predicted protein encoding genes with annotation of 19,279 orthologous genes. A total of 166 orthologous groups represented by 222 genes were found to be unique for this species. The Computational Analysis of gene Family Evolution (CAFE) analysis revealed expansion of 207 gene families and 100 gene families have rapidly evolved. Genes specific to important environmental and terrestrial adaptation, viz. urea cycle, vision, locomotion, olfactory and vomeronasal receptors, immune system, anti-microbial properties, mucus, thermoregulation, osmoregulation, air-breathing, detoxification, etc. were identified and critically analysed. The analysis clearly indicated that C. magur genome possessed several unique and duplicate genes similar to that of terrestrial or amphibians' counterparts in comparison to other teleostean species. The genome information will be useful in conservation genetics, not only for this species but will also be very helpful in such studies in other catfishes.

In Silico Analysis of nsSNPs of Carp TLR22 Gene Affecting its Binding Ability with Poly I:C.

Immune response mediated by toll-like receptor 22 (TLR22), only found in teleost/amphibians, is triggered by double-stranded RNA binding to its LRR (leucine-rich repeats) ecto-domain. Accumulated evidences suggested that missense mutations in TLR genes affect its function. However, information on mutation linked pathogen recognition for TLR22 was lacking. The present study was commenced for predicting the effect of non-synonymous single-nucleotide polymorphisms (nsSNPs) on the pathogen recognizable LRR domain of TLR22 of farmed carp, Labeo rohita. The sequence-based algorithms (SIFT, PROVEAN and I-Mutant2.0) indicated that three SNPs (out of 27) such as p.L159F (rs76759876) and p.L529P (rs749355507) of LRR, and p.I836M (rs750758397) of intracellular motifs could potentially disrupt protein function. The 3D structure was generated using MODELLER 9.13 and further validated by SAVEs server. The simulated molecular docking of native TLR22 and mutants with poly I:C ligand indicated that mutations positioned at p.L159F and p.L529P of the LRR region affects the binding affinity significantly. This is the first kind of study of predicting nsSNPs of teleost TLR22 with disturbed ligand binding affinity with its extra-cellular LRR domain and thereby likely hindrance in subsequent signal transduction. This study serves as a guide for in vivo evaluation of impact of mutation on immune response mediated by teleost TLR22 gene.

Gene editing tools: state-of-the-art and the road ahead for the model and non-model fishes.

Advancements in the DNA sequencing technologies and computational biology have revolutionized genome/transcriptome sequencing of non-model fishes at an affordable cost. This has led to a paradigm shift with regard to our heightened understandings of structure-functional relationships of genes at a global level, from model animals/fishes to non-model large animals/fishes. Whole genome/transcriptome sequencing technologies were supplemented with the series of discoveries in gene editing tools, which are being used to modify genes at pre-determined positions using programmable nucleases to explore their respective in vivo functions. For a long time, targeted gene disruption experiments were mostly restricted to embryonic stem cells, advances in gene editing technologies such as zinc finger nuclease, transcriptional activator-like effector nucleases and CRISPR (clustered regulatory interspaced short palindromic repeats)/CRISPR-associated nucleases have facilitated targeted genetic modifications beyond stem cells to a wide range of somatic cell lines across species from laboratory animals to farmed animals/fishes. In this review, we discuss use of different gene editing tools and the strategic implications in fish species for basic and applied biology research.

Molecular cloning and characterization of LrTLR4, analysis of its inductive expression and associated down-stream signaling molecules following lipopolysaccharide stimulation and Gram-negative bacterial infection.

Toll-like receptors (TLRs) play key roles in innate immunity from lower to higher vertebrates. Among various TLR types, TLR4 was reported to recognize LPS in higher vertebrates resulting in the activation of down-stream signaling pathway. Except in some teleosts, function of TLR4 in most fish species including rohu (Labeo rohita) a commercially important fish species in the South-East Asian countries remained unknown. To investigate it, full-length cDNA of Labeo rohita TLR4 (LrTLR4) was cloned, and it consisted of 2729 bp, with a single ORF of 2469 bp encoding a polypeptide of 822 aa with a predicted molecular mass of 94.753 kDa. Structurally, LrTLR4 consisted of 25 LRRs (leucine rich repeat regions), one TM (trans-membrane) domain and one TIR (Toll/interleukin-1 receptor) domain, and was similar to higher vertebrate's TLR4. Phylogenetically, LrTLR4 exhibited highest (85%) identity with the common carp TLR4b amino acids sequence, and formed a separate subgroup in the phylogenetic tree. LrTLR4 was widely expressed in all tested organs/tissues, and amidst the tissues highest expression was detected in blood and the lowest in eye. In response to LPS-stimulation, LrTLR4 was induced with the activation of MyD88-dependent and TRIF-dependent signaling pathway resulting in pro-inflammatory cytokines (interleukin 6 and 8) and type I IFN gene expression. Infection of rohu with a Gram-negative fish pathogen (Aeromonas hydrophila), also activated LrTLR4. Together, these findings suggest the important role of TLR4 in LPS sensing and augmentation of innate immunity against Gram-negative bacterial infection in fish.

Molecular characterization of Activin Receptor Type IIA and its expression during gonadal maturation and growth stages in rohu carp.

Activin receptor type IIA (ActRIIA), a transmembrane serine/threonine kinase receptor is an important regulator of physiological traits, viz., reproduction and body growth in vertebrates including teleosts. However, existing knowledge of its role in regulating fish physiology is limited. To address this, we have cloned and characterized the ActRIIA cDNA of Labeo rohita (rohu), an economically important fish species of the Indian subcontinent. Comparative expression profiling of the receptor gene at various reproductive and growth stages supports to its role in promoting oocyte maturation, spermatogenesis and skeletal muscle development via interaction with multiple ligands of transforming growth factor-ß (TGF-ß) family. The full-length cDNA of rohu ActRIIA was found to be of 1587bp length encoding 528 amino acids. The three-dimensional structure of the intracellular kinase domain of rohu ActRIIA has also been predicted. Phylogenetic relationship studies showed that the gene is evolutionarily conserved across the vertebrate lineage implicating that the functioning of the receptor is more or less similar in vertebrates. Taken together, these findings could be an initial step towards the use of ActRIIA as a potential candidate gene marker for understanding the complex regulatory mechanism of fish reproduction and growth.

Immunoglobulin (Ig) D in Labeo rohita is widely expressed and differentially modulated in viral, bacterial and parasitic antigenic challenges.

Immunoglobulins (Igs) play critical roles in protecting host against diverse pathogenic invasion and diseases. Among all Ig isotypes, IgD is the most recently-evolved and enigmatic molecule detected in all vertebrates species except birds. In South-East Asia, Labeo rohita (rohu) is the leading candidate fish species for freshwater aquaculture, and this article describes about IgD gene expression in rohu following viral, bacterial and parasitic antigenic challenges. The partial cDNA (761bp) of Labeo rohita-IgD (LrIgD) was cloned and submitted in the GenBank with the accession no KT883581. Phylogenetically, LrIgD was closely related to grass carp IgD. Analysis of LrIgD gene expression in juveniles by quantitative real-time PCR (qRT-PCR) assay revealed gradual increase in IgD expression with the advancement of time. In the healthy rohu fingerlings, LrIgD expression occurred predominantly in kidney followed by liver and spleen. In response to rhabdoviral antigenic stimulation, LrIgD expression was significantly enhanced in all tested tissues. In bacterial (Aeromonas hydrophila) infection, transcripts of LrIgD increased more dramatically in liver followed by kidney and gill. In parasitic (Argulus) infection, most significant expression of IgD was noted in the skin, followed by kidney, liver, spleen and gill. These results collectively suggest the key role of IgD in the immune response of rohu during viral, bacterial and parasitic infections.

B cell activating factor is induced by toll-like receptor and NOD-like receptor-ligands and plays critical role in IgM synthesis in Labeo rohita.

B-cell activating factor (BAFF), an important member of the tumor necrosis factor superfamily, plays critical roles in the modulation of B-cell functions and enhancement of immune response in the host. Like higher vertebrates, the important role of BAFF in boosting immune response against diverse pathogens was also envisaged in fishes. We therefore, studied BAFF in rohu (Labeo rohita), a freshwater food fish species of highest economic importance in the Indian subcontinent. Full-length rohu-BAFF- cDNA comprised of 804bp nucleotide long ORF, encoding 267 amino acid residues, and shared high structural similarity with human-BAFF. It was expressed in the embryonic developmental stages suggesting its key role in immune response at the early life of fish. In Aeromonas hydrophila infection and rhabdoviral antigen stimulation, BAFF-gene expression in rohu was induced across the organs/tissues. Stimulation of un-treated healthy rohu fish leukocytes, and viral or bacterial or BSA (bovine serum albumin) antigen stimulated rohu fish leukocytes with recombinant-BAFF (r-BAFF) resulted in enhanced expression of immunoglobulin (Ig)M. Both in-vitro and in-vivo treatment with toll-like receptor (TLR)- ligand (poly I:C) or nod-like receptor (NLR)- ligands (iE-DAP and MDP) resulted in TLR and NLR activation and BAFF-gene expression. This is the first report showing BAFF-expression by innate immune receptor-ligands and its critical role in enhancing adaptive immune response in fish.

Production of fertile sperm from in vitro propagating enriched spermatogonial stem cells of farmed catfish, Clarias batrachus.

Spermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9.

Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes.

Variation in susceptibility pattern of fish to Argulus siamensis: Do immune responses of host play a role?

Branchiuran ectoparasites of the genus Argulus can have extensive damaging effects on cultured fish. There exist no systematic studies that evaluate susceptibility or resistance of various carp species to Argulus sp. and the underlying mechanisms. The present study aimed at identifying the most susceptible and resistant cultured species, studying settlement and survival of parasite on these species, and finally unravelling the variations of immune response in both resistant and susceptible species. Fish from eight species (Labeo rohita, Cirrhinus mrigala, Catla catla, Hypophthalmichthys molitrix, Cyprinus carpio, Ctenopharyngodon idella, Carassius auratus, Labeo fimbriatus) were individually challenged with metanauplii of A. siamensis (100 metanauplii/fish) before rearing them in single tank in triplicate for 45 days. Based on the observed parasite load on each species, L. rohita was found to be the most susceptible and C. idella the resistant species. The settlement and survival of the parasite on L. rohita and C. idella was compared at 24, 48, 72 and 96h post experimental infection. Survival was significantly low at 72h onwards in C. idella indicating it is an unsuitable/poorly preferred host for A. siamensis. The inflammatory responses which are known to be related to susceptibility were analysed. Individuals of both the species were exposed to A. siamensis (100 parasites/fish), and after 24h and 3 d, skin samples directly from the attachment site and non-attachment sites were assessed for transcriptomic profiles of selected innate defence genes. Artificial skin abrasion permitted comparisons between abrasion associated injury and louse-associated injury. The inflammatory responses varied significantly between both species indicating their role in determining susceptibility of a host to A. siamensis. The expression of major histocompatibility class II and matrix metalloproteinase 2 was significantly higher in C. idella compared to L. rohita and therefore appeared to be involved in the early protective response against A. siamensis. It is essential to study the expression pattern of more participatory genes of the inflammation related pathways to understand species specific susceptible patterns.

Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita) Using Modeling and Molecular Dynamic Simulation Approaches.

The myostatin (MSTN) is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A) of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P) were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies.

Genetic variation in Labeo fimbriatus (Cypriniformes: Cyprinidae) populations as revealed by partial cytochrome b sequences of mitochondrial DNA.

Labeo fimbriatus, a medium sized carp is assessed as a commercially important aquaculture species in Indian subcontinent. In the present study, the genetic diversity and population structure of four Indian riverine populations of L. fimbriatus have been evaluated using partial cytochrome b sequences of mitochondrial DNA. Sequencing and analysis of this gene from 108 individuals defined 7 distinct haplotypes. Haplotype diversity (Hd) and nucleotide diversity (π) ranged from 0.067 to 0.405 and 0.00023 to 0.03231, respectively. The Mahanadi population had the highest π level. Analysis of molecular variance (AMOVA) indicated that 47.36% of genetic variation contained within population and 53.76% of genetic variation among groups. Pairwise FST analysis indicated that there was little or no genetic differentiation among populations (-0.0018 to 04572) from different geographical regions except Mahanadi population. The Mahanadi population can be considered as a separate stock from rest three riverine populations. Accordingly, the genetic information generated from this study can be implemented while taking decision in formulating base population for the sustainable selective breeding programs of this species.

The ß-actin gene promoter of rohu carp (Labeo rohita) drives reporter gene expressions in transgenic rohu and various cell lines, including spermatogonial stem cells.

We previously characterized the ß-actin gene promoter of Indian domesticated rohu carp (Labeo rohita) and made a reporter construct via fusion to green fluorescence protein (GFP) cDNA. In this study, the same construct was used to breed transgenic rohu fish. About 20% of the transgenic offspring showed ubiquitous expression of the reporter GFP gene. In a few of the transgenic fish, we documented massive epithelial and/or muscular expression with visible green color under normal light. The expression of GFP mRNA was higher in the muscle tissue of transgenic fish than in that of non-transgenic fish. A highly efficient nucleofection protocol was optimized to transfect proliferating spermatogonial cells of rohu using this reporter construct. The ß-actin promoter also drove expressions in HEK293 (derived from human embryonic kidney cells), K562 (human leukemic cells) and SF21 (insect ovarian cells) lines. These findings imply conserved regulatory mechanisms of ß-actin gene expression across eukaryotes. Furthermore, the isolated ß-actin promoter with consensus regulatory elements has the potential to be used in generating transgenic carp with genes of interest and in basic biology research.

Construction, De-Novo Assembly and Analysis of Transcriptome for Identification of Reproduction-Related Genes and Pathways from Rohu, Labeo rohita (Hamilton).

Rohu is a leading candidate species for freshwater aquaculture in South-East Asia. Unlike common carp the monsoon breeding habit of rohu restricts its seed production beyond season indicating strong genetic control over spawning. Genetic information is limited in this regard. The problem is exacerbated by the lack of genomic-resources. We identified 182 reproduction-related genes previously by Sanger-sequencing which were less to address the issue of seasonal spawning behaviour of this important carp. Therefore, the present work was taken up to generate transcriptome profile by mRNAseq. 16 GB, 72 bp paired end (PE) data was generated from the pooled-RNA of twelve-tissues from pre-spawning rohu using IlluminaGA-II-platform. There were 64.97 million high-quality reads producing 62,283 contigs and 88,612 numbers of transcripts using velvet and oases programs, respectively. Gene ontology annotation identified 940 reproduction-related genes consisting of 184 mainly associated with reproduction, 223 related to hormone-activity and receptor-binding, 178 receptor-activity and 355 embryonic-development related-proteins. The important reproduction-relevant pathways found in KEGG analysis were GnRH-signaling, oocyte-meiosis, steroid-biosynthesis, steroid-hormone biosynthesis, progesterone-mediated oocyte-maturation, retinol-metabolism, neuroactive-ligand-receptor interaction, neurotrophin-signaling and photo-transduction. Twenty nine simple sequence repeat containing sequences were also found out of which 12 repeat loci were polymorphic with mean expected-&-observed heterozygosity of 0.471 and 0.983 respectively. Quantitative RT-PCR analyses of 13-known and 6-unknown transcripts revealed differences in expression level between preparatory and post-spawning phase. These transcriptomic sequences have significantly increased the genetic-&-genomic resources for reproduction-research in Labeo rohita.

First evidence of molecular characterization of rohu carp Sox2 gene being expressed in proliferating spermatogonial cells.

Because little is known about the function of Sox2 (Sry-related box-2) in teleosts, the objective of this study was to clone and characterize Sox2 complementary DNA (cDNA) from the testis of Indian major carp, Labeo rohita (rohu). The full-length cDNA contained an open reading frame of 936 nucleotides bearing the typical structural features. Phylogenetically, Sox2 of L rohita was most closely related to freshwater counterparts than marine water. The sequence information of cDNA and genomic DNA together revealed that the Sox2 gene is encoded by an uninterrupted exon. Furthermore, comparative mRNA expression profile in various organs including proliferating spermatogonial stem cells (SSCs) suggested about the participatory role of Sox2 during fish male germ cell development and maintenance of stem cells. In support, we have also provided evidence that Sox2 protein is indeed present in rohu SSCs by Western blot analysis. The evolutionarily conserved high-mobility group box domain indicated its possible involvement in common networking pathways for stem cell maintenance and pluripotency between mammals and nonmammals. Our findings could be the first step toward the use of Sox2 as a potential biomarker for proliferating SSCs and understanding the transcriptional regulatory network involved during male germ cell development and maintenance in fish species.

Characterization and Inductive Expression Analysis of Interferon Gamma-Related Gene in the Indian Major Carp, Rohu (Labeo rohita).

Interferon gamma (IFN-γ) is one of the key cytokines that plays a major role against viral and intracellular bacterial infection. In addition to the IFN-γ gene, teleost fish possess a second copy known as IFN-γ-related (IFN-γrel) gene. This report describes structural and functional properties of IFN-γrel gene in the Indian major carp, rohu (Labeo rohita), a commercially important freshwater fish species in the Indian subcontinent. The rohu IFN-γrel gene consisted of four exons with three intervening introns and phylogenetically closely related to grass carp. The full-length IFN-γrel cDNA comprised 927 bp nucleotides with a single open reading frame of 504 bp, encoding 167 amino acids (aa) polypeptide with a signal peptide of 24 aa. The mature rohu IFN-γrel protein was 143 aa with a predicted molecular weight of 16.85 kDa. Basal expression analysis of IFN-γrel showed its wide range of expression in all examined tissues: The highest was in the skin and the lowest was in the liver. In response to LPS, poly I:C, iE-DAP, muramyl dipeptide stimulations, and bacterial infections, IFN-γrel gene expression was significantly (p<0.05) induced in treated fish tissues as compared with their control. The IFN-γrel was expressed as recombinant protein (rIFN-γrel) and confirmed through western blot. Stimulation of peripheral blood leukocytes with rIFN-γrel protein resulted in the activation of IFN-γ receptor and marked induction of inducible nitric oxide synthase gene expression. These results together may suggest the important role of IFN-γrel as an antimicrobial cytokine in fish.