Functional characterization of Labeo rohita muscle cell line for in vitro research.

BACKGROUND: Labeo rohita represents the most dominant fish species in Indian aquaculture and the fish cell lines have been used as an excellent in vitro platform for performing various biological research. METHODS AND RESULTS: The LRM cell culture developed from the muscle tissue of L. rohita was used to study the in vitro applications. The developed muscle cells were maintained in a Leibovitz's-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng/ml bFGF at 28 oC temperature. The LRM cells showed fibroblastic-like morphology and was authenticated by sequencing mitochondrial gene 16S rRNA. The expression of myogenic regulatory factors (MRFs) was studied in different stages of LRM cells; however, the expression patterns varied at different passages. The MEF2A, Mrf-4, and Myogenin expressions were higher in passage 25, while the expression of MyoD was maximum in passage 15, and the expression of Myf-5 was highest in passage 1. The transfection efficiency of LRM cells revealed 14 % of the GFP expression with a pmaxGFP vector DNA. The LRM cells were susceptible to the extracellular products prepared from Aeromonas hydrophilla and Edwardsiella tarda. The acute cytotoxicity of six heavy metals (Hg, Cd, Zn, Cu, Pb, Ni) was assessed in LRM cells by a dose-dependent manner in comparison to IC50 values obtained from MTT and NR assays. A revival rate of 70-75% was achieved when the LRM cells were cryopreserved at - 196 °C using liquid nitrogen. CONCLUSION: The developed muscle cells serve as an functional in vitro tool for toxicological and biotechnological studies.

Identifying miRNAs in the modulation of gene regulation associated with ammonia toxicity in catfish, Clarias magur (Linnaeus, 1758).

BACKGROUND: The small non-coding microRNAs play a vital role in post-transcriptional gene regulation associated with different physiological events such as metabolism, stress, etc. The freshwater catfish, Clarias magur, can grow within hyper ammonia containing stagnant water bodies and/or muddy substratum. We intended to identify organ-specific miRNAs associated with ammonia stress management. METHODS AND RESULTS: The miRNA-libraries were generated from QC passed total RNA extracted from liver, muscle, and kidney of ammonia-treated (exposed to 25 mM NH4Cl for 14 days) and untreated catfish. The libraries were validated using High sensitivity D1000 Screen tape. The trimmed quality-filtered reads for control and treated samples of kidney were 19,406,210; 14,904,423; for liver 15,467,727; 18,582,072; and for muscle 25,081,345; 19,782,182 respectively. Total 120 known and 150 novel differentially expressed miRNAs were identified, out of which miR-200, miR-217, miR-122, miR-133, miR-145, miR-221, miR-19, miR-138, miR-34, and miR-184 were predicted to be involved in the metabolism of nitrogen. The key miRNAs targeted several genes associated with urea synthesis like Glutaminase 2, Argininosuccinate lyase, Glutamate dehydrogenase 1, Alanine aminotransferase 2-like, Aspartate aminotransferase, cytoplasmic-like, Glutamate ionotropic receptor NMDA type subunit 2A, etc. CONCLUSIONS: This is the first report of miRNAs, which serve as a vital resource for regulating nitrogen metabolism in freshwater catfish, C. magur. The data will be resourceful for further evaluating the regulatory role of miRNAs in fishes, which grow and reproduce very well in hazardous ammonia-contaminated water bodies.

Revealing liver specific microRNAs linked with carbohydrate metabolism of farmed carp, Labeo rohita (Hamilton, 1822).

The role of microRNA in gene regulation during developmental biology has been well depicted in several organisms. The present study was performed to investigate miRNAs role in the liver tissues during carbohydrate metabolism and their targets in the farmed carp rohu, Labeo rohita, which is economically important species in aquaculture. Using Illumina-HiSeq technology, a total of 22,612,316; 44,316,046 and 13,338,434 clean reads were obtained from three small-RNA libraries. We have identified 138 conserved and 161 novel miRNAs and studies revealed that miR-22, miR-122, miR-365, miR-200, and miR-146 are involved in carbohydrate metabolism. Further analysis depicted mature miRNA and their predicted target sites in genes that were involved in developmental biology, cellular activities, transportation, etc. This is the first report of the presence of miRNAs in liver tissue of rohu and their comparative profile linked with metabolism serves as a vital resource as a biomarker.

Revealing Alteration in the Hepatic Glucose Metabolism of Genetically Improved Carp, Jayanti Rohu Labeo rohita Fed a High Carbohydrate Diet Using Transcriptome Sequencing.

Although feed cost is the greatest concern in aquaculture, the inclusion of carbohydrates in the fish diet, and their assimilation, are still not well understood in aquaculture species. We identified molecular events that occur due to the inclusion of high carbohydrate levels in the diets of genetically improved 'Jayanti rohu' Labeo rohita. To reveal transcriptional changes in the liver of rohu, a feeding experiment was conducted with three doses of gelatinized starch (20% (control), 40%, and 60%). Transcriptome sequencing revealed totals of 15,232 (4464 up- and 4343 down-regulated) and 15,360 (4478 up- and 4171 down-regulated) differentially expressed genes. Up-regulated transcripts associated with glucose metabolisms, such as hexokinase, PHK, glycogen synthase and PGK, were found in fish fed diets with high starch levels. Interestingly, a de novo lipogenesis mechanism was found to be enriched in the livers of treated fish due to up-regulated transcripts such as FAS, ACCα, and PPARγ. The insulin signaling pathways with enriched PPAR and mTOR were identified by Kyoto Encyclopedia of Genes and Genome (KEGG) as a result of high carbohydrates. This work revealed for the first time the atypical regulation transcripts associated with glucose metabolism and lipogenesis in the livers of Jayanti rohu due to the inclusion of high carbohydrate levels in the diet. This study also encourages the exploration of early nutritional programming for enhancing glucose efficiency in carp species, for sustainable and cost-effective aquaculture production.

Ontogeny and tissue specific expression profiles of recombination activating genes (RAGs) during development in Nile tilapia, Oreochromisniloticus.

Recombination activating genes (RAGs) mediates the process of rearrangement and somatic recombination (V(D)J) to generate different antibody repertoire. Studies on the expression pattern of adaptive immune genes during ontogenic development are crucial for the formulation of fish immunization strategy. In the present study, Nile tilapia was taken to explore the relative expression profile of RAG genes during their developmental stages. The developmental stages of Nile tilapia, i.e., unfertilized egg, 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 days post-hatch (dph) and kidney, blood, gill, liver and spleen tissues from adult fish were collected and the cDNA synthesis was carried out. Gene specific primers for RAG-1 and RAG-2 of Nile tilapia were designed and their annealing temperature (Tm) was optimized by gradient PCR. Consequently, PCR was performed to confirm the specific amplification of RAG-1 and RAG-2 genes. Quantitative real-time PCR (qRT-PCR) gene expression of RAG-1 and RAG-2 were noticed in all the developmental stages; however, a significant increase was observed after 12 dph and peaked at 24 dph, followed by a gradual decrease until 30 dph. Tissue-specific gene expression profiling revealed that the highest expression of RAG-1 and RAG-2 was observed in the kidney, followed by spleen, gill, liver and blood. The findings of the study explored the suitable timing of lymphoid maturation that could be technically used for the adoption of strategies to improve disease resistance of fish larvae for mitigating larval mortality.

DNA Methylation Profiling in Genetically Selected Clarias magur (Hamilton, 1822) Provides Insights into the Epigenetic Regulation of Growth and Development.

DNA methylation is an epigenetic alteration that impacts gene expression without changing the DNA sequence affecting an organism's phenotype. This study utilized a reduced representation bisulfite sequencing (RRBS) approach to investigate the patterns of DNA methylation in genetically selected Clarias magur stocks. RRBS generated 249.22 million reads, with an average of 490,120 methylation sites detected in various parts of genes, including exons, introns, and intergenic regions. A total of 896 differentially methylated regions (DMRs) were identified; 356 and 540 were detected as hyper-methylated and hypo-methylated regions, respectively. The DMRs and their association with overlapping genes were explored using whole genome data of magur, which revealed 205 genes in exonic, 210 in intronic, and 480 in intergenic regions. The analysis identified the maximum number of genes enriched in biological processes such as RNA biosynthetic process, response to growth factors, nervous system development, neurogenesis, and anatomical structure morphogenesis. Differentially methylated genes (DMGs) such as myrip, mylk3, mafb, egr3, ndnf, meis2a, foxn3, bmp1a, plxna3, fgf6, sipa1l1, mcu, cnot8, trim55b, and myof were associated with growth and development. The selected DMGs were analyzed using real-time PCR, which showed altered mRNA expression levels. This work offers insights into the epigenetic mechanisms governing growth performance regulation in magur stocks. This work provides a valuable resource of epigenetic data that could be integrated into breeding programs to select high-performing individuals.

Genetic improvement and genomic resources of important cyprinid species: status and future perspectives for sustainable production.

Cyprinid species are the most cultured aquatic species around the world in terms of quantity and total value. They account for 25% of global aquaculture production and significantly contribute to fulfilling the demand for fish food. The aquaculture of these species is facing severe concerns in terms of seed quality, rising feed costs, disease outbreaks, introgression of exotic species, environmental impacts, and anthropogenic activities. Numerous researchers have explored biological issues and potential methods to enhance cyprinid aquaculture. Selective breeding is extensively employed in cyprinid species to enhance specific traits like growth and disease resistance. In this context, we have discussed the efforts made to improve important cyprinid aquaculture practices through genetic and genomic approaches. The recent advances in DNA sequencing technologies and genomic tools have revolutionized the understanding of biological research. The generation of a complete genome and other genomic resources in cyprinid species has significantly strengthened molecular-level investigations into disease resistance, growth, reproduction, and adaptation to changing environments. We conducted a comprehensive review of genomic research in important cyprinid species, encompassing genome, transcriptome, proteome, metagenome, epigenome, etc. This review reveals that considerable data has been generated for cyprinid species. However, the seamless integration of this valuable data into genetic selection programs has yet to be achieved. In the upcoming years, genomic techniques, gene transfer, genome editing tools are expected to bring a paradigm shift in sustainable cyprinid aquaculture production. The comprehensive information presented here will offer insights for the cyprinid aquaculture research community.

DNA Methylation Profiling of Ovarian Tissue of Climbing Perch (Anabas testudienus) in Response to Monocrotophos Exposure.

Epigenetic modifications like DNA methylation can alter an organism's phenotype without changing its DNA sequence. Exposure to environmental toxicants has the potential to change the resilience of aquatic species. However, little information is available on the dynamics of DNA methylation in fish gonadal tissues in response to organophosphates. In the present work, reduced-representation bisulfite sequencing was performed to identify DNA methylation patterns in the ovarian tissues of Anabas testudienus exposed to organophosphates, specifically monocrotophos (MCP). Through sequencing, an average of 41,087 methylated cytosine sites were identified and distributed in different parts of genes, i.e., in transcription start sites (TSS), promoters, exons, etc. A total of 1058 and 1329 differentially methylated regions (DMRs) were detected as hyper-methylated and hypo-methylated in ovarian tissues, respectively. Utilizing whole-genome data of the climbing perch, the DMRs, and their associated overlapping genes revealed a total of 22 genes within exons, 45 genes at transcription start sites (TSS), and 218 genes in intergenic regions. Through gene ontology analysis, a total of 16 GO terms particularly involved in ovarian follicular development, response to oxidative stress, oocyte maturation, and multicellular organismal response to stress associated with reproductive biology were identified. After functional enrichment analysis, relevant DMGs such as steroid hormone biosynthesis (Cyp19a, 11-beta-HSD, 17-beta-HSD), hormone receptors (ar, esrrga), steroid metabolism (StAR), progesterone-mediated oocyte maturation (igf1ar, pgr), associated with ovarian development in climbing perch showed significant differential methylation patterns. The differentially methylated genes (DMGs) were subjected to analysis using real-time PCR, which demonstrated altered gene expression levels. This study revealed a molecular-level alteration in genes associated with ovarian development in response to chemical exposure. This work provides evidence for understanding the relationship between DNA methylation and gene regulation in response to chemicals that affect the reproductive fitness of aquatic animals.

Muscle Transcriptome Sequencing Revealed Thermal Stress-Responsive Regulatory Genes in Farmed Rohu, Labeo rohita (Hamilton, 1822).

Rohu, Labeo rohita, is one of the most important aquaculture species in the Indian subcontinent. Understanding the molecular-level physiological responses to thermal stress or climate change is essential. In the present work, transcriptome sequencing was carried out in the muscle tissue of the rohu in response to heat stress (35 °C) in comparison with the control (28 °C). A total of 125 Gb of sequence data was generated, and the raw-reads were filtered and trimmed, which resulted in 484 million quality reads. Reference-based assembly of reads was performed using L. rohita genome, and a total of 90.17% of reads were successfully mapped. A total of 37,462 contigs were assembled with an N50 value of 1854. The differential expression analysis revealed a total of 107 differentially expressed genes (DEGs) (15 up-, 37 down-, and 55 neutrally regulated) as compared to the control group (Log2FC > 2, P < 0.05). Gene enrichment analysis of DEGs indicates that transcripts were associated with molecular, biological, and cellular activities. The randomly selected differentially expressed transcripts were validated by RT-qPCR and found consistent expression patterns in line with the RNA-seq data. Several transcripts such as SERPINE1(HSP47), HSP70, HSP90alpha, Rano class II histocompatibility A beta, PGC-1 and ERR-induced regulator, proto-oncogene c-Fos, myozenin2, alpha-crystallin B chain-like protein, angiopoietin-like protein 8, and acetyl-CoA carboxylases have been identified in muscle tissue of rohu that are associated with stress/immunity. This study identified the key biomarker SERPINE1 (HSP47), which showed significant upregulation (~ 2- to threefold) in muscle tissue of rohu exposed to high temperature. This study can pave a path for the identification of stress-responsive biomarkers linked with thermal adaptations in the farmed carps.

Aquaculture omics: An update on the current status of research and data analysis.

Aquaculture is the fast-growing agricultural sector and has the ability to meet the growing demand for protein nutritional security for future population. In future aquaculture is going to be the major source of fish proteins as capture fisheries reached at its maximum. However, several challenges need to overcome such as lack of genetically improved strains/varieties, lack of species-specific feed/functional feed, round the year availability of quality fish seed, pollution of ecosystems and increased frequencies of disease occurrence etc. In recent years, the continuous development of high throughput sequencing technology has revolutionized the biological sciences and provided necessary tools. Application of 'omics' in aquaculture research have been successfully used to resolve several productive and reproductive issues and thus ensure its sustainability and profitability. To date, high quality draft genomes of over fifty fish species have been generated and successfully used to develop large number of single nucleotide polymorphism markers (SNPs), marker panels and other genomic resources etc in several aquaculture species. Similarly, transcriptome profiling and miRNAs analysis have been used in aquaculture research to identify key transcripts and expression analysis of candidate genes/miRNAs involved in reproduction, immunity, growth, development, stress toxicology and disease. Metagenome analysis emerged as a promising scientific tool to analyze the complex genomes contained within microbial communities. Metagenomics has been successfully used in the aquaculture sector to identify novel and potential pathogens, antibiotic resistance genes, microbial roles in microcosms, microbial communities forming biofloc, probiotics etc. In the current review, we discussed application of high-throughput technologies (NGS) in the aquaculture sector.

Liver-Specific microRNA Identification in Farmed Carp, Labeo bata (Hamilton, 1822), Fed with Starch Diet Using High-Throughput Sequencing.

The liver is an important central organ, which controls carbohydrate metabolism through maintaining glucose homeostasis by a tightly regulated system of genes or enzymes. The microRNAs are small non-coding RNAs playing an important role in the regulation of genes associated with developmental biology, physiology, metabolism, etc. Thus, in this study, we have intended to detect liver-specific microRNAs in farmed carp, Labeo bata, upon being fed a diet with different levels of carbohydrates. Here, we have conducted the experiment for 45 days using fingerlings of farmed carp fed with 20% (control), 40%, and 60% gelatinized starch levels. The liver tissues were collected from each treatment and processed for RNA isolation, small RNA library preparation, and high-throughput sequencing using Illumina NexSeq500. Through sequencing, 15,779,417 reads in 20% CHO, 13,959,039 in 40% CHO, and 13,661,950 in 60% CHO reads were generated for control and treated fishes using three small RNA libraries. We have investigated 445 novel and 231 conserved microRNAs in 20%, 40%, and 60% carbohydrate (CHO), respectively, through computational analysis. The differential expression analysis of miRNAs was carried out between different treatments compared with control and this study depicted 117 known and 114 novel miRNA genes involved in carbohydrate metabolic pathways. Further, target prediction and gene ontology analysis revealed that miRNAs were involved in several pathways such as signaling pathway, G protein pathway, complement receptor-mediated pathway, dopamine receptor signaling pathway, epidermal growth factor pathway, and notch signaling pathway. The predicted miRNA sites in targeted genes were associated with cellular activities, developmental biology, DNA binding, Golgi apparatus, extracellular region, catalytic activity, MAPK cascade, etc. Overall, we have generated a vital resource of liver-specific miRNAs involved in metabolic gene regulation. These studies further will help develop miRNA inhibitors to study their role during carbohydrate metabolism in farmed carp.

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.

Analysis of consequences of non-synonymous SNP in feed conversion ratio associated TGF-ß receptor type 3 gene in chicken.

The recent advances in high throughput sequencing technology accelerate possible ways for the study of genome wide variation in several organisms and associated consequences. In the present study, mutations in TGFBR3 showing significant association with FCR trait in chicken during exome sequencing were further analyzed. Out of four SNPs, one nsSNP p.Val451Leu was found in the coding region of TGFBR3. In silico tools such as SnpSift and PANTHER predicted it as deleterious (0.04) and to be tolerated, respectively, while I-Mutant revealed that protein stability decreased. The TGFBR3 I-TASSER model has a C-score of 0.85, which was validated using PROCHECK. Based on MD simulation, mutant protein structure deviated from native with RMSD 0.08 Å due to change in the H-bonding distances of mutant residue. The docking of TGFBR3 with interacting TGFBR2 inferred that mutant required more global energy. Therefore, the present study will provide useful information about functional SNPs that have an impact on FCR traits.