Fish cell line: depositories, web resources and future applications.

Cell lines are important bioresources to study the key biological processes in the areas like virology, pathology, immunology, toxicology, biotechnology, endocrinology and developmental biology. Cell lines developed from fish organs are utilized as a model in vitro system in disease surveillance programs, pharmacology, drug screening and resolving cases of metabolic abnormalities. During last decade, there were consistent efforts made globally to develop new fish cell lines from different organs like brain, eye muscles, fin, gill, heart, kidney, liver, skin, spleen, swim bladder, testes, vertebra etc. This increased use and development of cell lines necessitated the establishment of cell line depositories to store/preserve them and assure their availability to the researchers. These depositories are a source of authenticated and characterized cell lines with set protocols for material transfer agreements, maintenance and shipping as well as logistics enabling cellular research. Hence, it is important to cryopreserve and maintain cell lines in depositories and make them available to the research community. The present article reviews the current status of the fish cell lines available in different depositories across the world, along with the prominent role of cell lines in conservation of life on land or below water. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-023-00601-2.

A cell line derived from heart of rainbow trout is refractory to Tilapia lake virus.

Cell lines are important in vitro models to answer biological mechanisms with less genetic variations. The present study was attempted to develop a cell line from rainbow trout, where we obtained a cell line from the heart, named "RBT-H." The cell line was authenticated using karyotyping and cytochrome c oxidase subunit I (COI) gene sequencing. The karyotype demonstrated diploid chromosome number (2n) as 62 and the sequence of partial COI gene was 99.84% similar to rainbow trout COI data set, both suggesting the origin of RBT-H from the rainbow trout. The heart cell line was mycoplasma-free and found to be refractory to infection with the Tilapia lake virus. The RBT-H cell line is deposited in the National Repository of Fish Cell Line (NRFC) at ICAR-NBFGR, Lucknow, India, with Accession no. NRFC0075 for maintenance and distribution to researchers on request for R&D.

Genome size estimation and its associations with body length, chromosome number and evolution in teleost fishes.

Precise estimation of genome size (GS) is vital for various genomic studies, such as deciding genome sequencing depth, genome assembly, biodiversity documentation, evolution, genetic disorders studies, duplication events etc. Animal Genome Size Database provides GS of over 2050 fish species, which ranges from 0.35 pg in pufferfish (Tetraodon nigroviridis) to 132.83 pg in marbled lungfish (Protopterus aethiopicus). The GS of majority of the fishes inhabiting waters of Indian subcontinent are still missing. In present study, we estimated GS of 51 freshwater teleost (31 commercially important, 7 vulnerable and 13 ornamental species) that ranged from 0.58 pg in banded gourami (Trichogaster fasciata) to 1.92 pg in scribbled goby (Awaous grammepomus). Substantial variation in GS was observed within the same fish orders (0.64-1.45 pg in cypriniformes, 0.70-1.41 pg in siluriformes and 0.58-1.92 pg in perciformes). We examined the relationship between the GS, chromosome number and body length across all the fishes. Body length was found to be associated with GS, whereas no relationship was noticed between the GS and the chromosome number. The analysis using ancestral information revealed haploid chromosome number 25, 27 and 24 for the most recent common ancestor of cypriniformes, siluriformes and perciformes, respectively. The study led to generation of new records on GS of 43 fish species and revalidated records for 8 species. The finding is valuable resource for further research in the areas of fish genomics, molecular ecology and evolutionary conservation genetics.

Protein-protein networks analysis of differentially expressed genes unveils the key phenomenon of biological process with respect to reproduction in endangered catfish, C. Magur.

Clarias magur (magur) is an important freshwater catfish with high potential in the aquaculture sector in its geographical ranges of distribution. One of the impediments to realise its full aquaculture potential is the lack of understanding key genes involved in its reproduction pathways. Nonetheless, very limited information is available on brain and gonads, with respect to reproduction related issues of magur at molecular level. The present study was aimed at understanding the interaction of the brain-gonad system by analysing differentially expressed genes (DEG) in brains and gonads of male and female magur using a protein-protein network interaction study. In brief, 641, 541, 225 and 245 DEGs, respectively, in ovary, testis and female brain and male-brain of magur were used as input in String database 11.0 and Cytoscape v 3.8.0 plug-in Network Analyzer for PPI network construction followed by network superimposition, network merging and analysis. A total of 13 key genes in female brain & ovary and 12 key genes in male brain & testis were obtained based on the network topological parameter betweenness centrality and nodes degree. Among them, cyp19a1b and amh genes in male brain-testis and Tp53 and exo1 genes in female brain-ovary were identified as hub genes having a high level of interaction and expression with other key genes in the network. Further, functional annotation study of these genes revealed their active involvement in important pathways related to reproduction. This is the first report exploring the interaction of brain and gonads in the regulation of magur reproduction through a protein-protein interaction network. The 25 key genes identified in the combined network are involved in various pathways, like neuropeptide signalling pathway, oxytocin receptor-mediated signalling pathway, corticotrophin-releasing factor receptor signalling pathway and reproduction process, which could lead to a better understanding of the magur reproductive system.

Transcriptome Analysis Revealed Osmoregulation Related Regulatory Networks and Hub Genes in the Gills of Hilsa shad, Tenualosa ilisha, during the Migratory Osmotic Stress.

Tenualosa ilisha (Hilsa shad), an anadromous fish, usually inhabits coastal and estuarine waters, and migrates to freshwater for spawning. In this study, large-scale gill transcriptome analyses from three salinity regions, i.e., fresh, brackish and marine water, revealed 3277 differentially expressed genes (DEGs), out of which 232 were found to be common between marine vs freshwater and brackish vs freshwater. These genes were mapped into 54 KEGG Pathways, and the most significant of these were focal adhesion, adherens junction, tight junction, and PI3K-Akt signaling pathways. A total of 24 osmoregulatory genes were found to be differentially expressed in different habitats. The gene members of slc16 and slc2 families showed a dissimilar pattern of expressions, while two claudin genes (cldn11 & cldn10), transmembrane tm56b, and voltage-gated potassium channel gene kcna10 were downregulated in freshwater samples, as compared to that of brackish and marine environment. Protein-protein interaction (PPI) network analysis of 232 DEGs showed 101 genes to be involved in PPI, while fn1 gene was found to be interacting with the highest number of genes (36). Twenty-five hub genes belonged to 12 functional groups, with muscle structure development with seven genes, forming the major group. These results provided valuable information about the genes, potentially involved in the molecular mechanisms regulating water homeostasis in gills, during migration for spawning and low-salinity adaptation in Hilsa shad. These genes may form the basis for the bio-marker development for adaptation to the stress levied by major environmental changes, due to hatchery/culture conditions.

BAC-FISH Based Physical Map of Endangered Catfish Clarias magur for Chromosome Cataloguing and Gene Isolation through Positional Cloning.

Construction of a physical chromosome map of a species is important for positional cloning, targeted marker development, fine mapping of genes, selection of candidate genes for molecular breeding, as well as understanding the genome organization. The genomic libraries in the form of bacterial artificial chromosome (BAC) clones are also a very useful resource for physical mapping and identification and isolation of full-length genes and the related cis acting elements. Some BAC-FISH based studies reported in the past were gene based physical chromosome maps of Clarias magur (magur) to understand the genome organization of the species and to establish the relationships with other species in respect to genes' organization and evolution in the past. In the present study, we generated end sequences of the BAC clones and analyzed those end sequences within the scaffolds of the draft genome of magur to identify and map the genes bioinformatically for each clone. A total of 36 clones mostly possessing genes were identified and used in probe construction and their subsequent hybridization on the metaphase chromosomes of magur. This study successfully mapped all 36 specific clones on 16 chromosome pairs, out of 25 pairs of magur chromosomes. These clones are now recognized as chromosome-specific makers, which are an aid in individual chromosome identification and fine assembly of the genome sequence, and will ultimately help in developing anchored genes' map on the chromosomes of C. magur for understanding their organization, inheritance of important fishery traits and evolution of magur with respect to channel catfish, zebrafish and other species.

Establishment and characterization of a continuous cell line from caudal fin of Labeo calbasu (Hamilton, 1822).

Labeo calbasu is an important food fish and candidate species for diversification of carp aquaculture. In the present study, we have established a continuous cell line, designated as L. calbasu fin (LCF), from caudal fin of L. calbasu using explant method. The cell line has been subcultured for over 73 passages and the LCF cells show optimal growth in Leibovitz's L-15 medium supplemented with 20% fetal bovine serum at a temperature of 28°C. In karyotype analysis, the modal chromosome number of LCF cells at 35th passage was found to be 50. The amplification and sequencing of partial fragments of mitochondrial genes, namely 16S rRNA and COI from LCF cells confirmed the origin of cell line from L. calbasu. The LCF cells could be successfully transfected with GFP reporter gene, indicating suitability of these cells for expression of foreign genes. Further, following inoculation with supernatant from Tilapia lake virus (TiLV) infected cell line, no cytopathic effects were observed in the LCF cells and cell pellet was negative for TiLV in RT-PCR, indicating that LCF cells were not susceptible to TiLV. The developed cell line has been submitted to National Repository of Fish Cell Lines being maintained at ICAR-National Bureau of Fish Genetic Resources, Lucknow (accession no. NRFC063). The newly developed LCF cell line would be helpful in investigating diseases affecting this candidate species particularly the ones suspected to be of viral etiology, and for cytotoxicity and transgenic studies.

Thermal stress induces expression of Nuclear protein and Parkin genes in endangered catfish, Clarias magur.

Fluctuation in water temperature can create thermal stress, which may impact many aspects of fish life, such as survival, growth, reproduction, disease occurrence etc. The endangered catfish, Clarias magur, has been reported to survive at higher thermal stress, even though the exact mechanism is unknown. The genes coding for Nuclear protein 1 (Nupr1) and Parkin E3 ubiquitin protein ligase (Park2) have been reported to protect cells from stress-induced damage and death. In this study, we characterized both the genes and assessed their quantitative expression in C. magur. Structural features of both the genes were found similar to a related catfish, Ictalurus punctatus, and model fish zebrafish. The genes were fairly conserved in fishes as observed through phylogenetic analysis. The real time expression of the two stress-associated genes were also assessed in brain, kidney, liver and muscle tissues of C. magur exposed to warm (34 °C) and cold (15 °C) water. RT-PCR analysis revealed up-regulation in the relative expression levels of Nupr1 and Park2 genes at both temperatures with maximum positive fold change during stress to cold water, even though the posteriori Dunnett's test after ANOVA revealed that there were significant differences between the control and challenged groups. The study indicated that Nupr1 gene plays role in muscle tissue at both high and low thermal stress, but at high thermal stress in liver, while Park2 plays role in muscle, brain and kidney at low temperature and in liver at high temperature stress in C. magur. The study has generated first-hand information under warm- and cold water, which pave the way to understand the expression response of these genes to thermal vacillations and to establish evolutionary significance in catfishes and other species.

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.

Evol2Circos: A web-based tool for genome synteny and collinearity analysis and its visualization in fishes.

The advent of high throughput next generation sequencing technologies and improved assembly algorithms have ensued in accumulation of voluminous genomic data in public domains. It has opened up entries for large scale comparative genome studies, especially the identification of conserved syntenic blocks among the species, facilitating the evolutionary importance of the conservation and variation in genomic organization. Synteny construction and visualization requires computational and bioinformatics skills to prepare input file for the synteny analysis pipeline. The syntenic information in fishes is still in juvenile stage and are scattered in different research domains. Here, we present a web-based tool 'Evol2Circos' to provide a user-friendly GUI- and web-based tool to analyse user specific data for synteny construction and visualization, and to facilitate the browsing of syntenic information of different fishes using the circos, bar, dual and dot plots. The information generated from the tool can also be used for further downstream analyses. Evol2Circos software tool is tested under Ubuntu Linux. The web-browser, source code, documentation, user manual, example dataset and scripts are available online at: 203.190.147.148/evole2circos/.

Improved protocols for BAC insert DNA isolation, BAC end sequencing and FISH for construction of BAC based physical map of genes on the chromosomes.

Bacterial artificial chromosome (BAC) library is an important genomic resource useful in targeted marker development, positional cloning, physical mapping and a substrate for genome sequencing for better understanding the genome organization of a species. The present manuscript elucidates the improvement in protocols for economical and efficient BAC insert DNA isolation, BAC end sequencing and FISH for physical localization on the metaphase chromosome complements. BAC clones of Clarias magur, maintained in 384-well plate format in our laboratory, were used in this study. The protocols gave consistent and efficient results. We use routinely these protocols for BAC insert DNA extraction, generating end sequence data of the clone and constructing DNA probes to hybridize on the metaphase spreads of C. magur using FISH for physical their localization.

Isolation and characterization of hypoxia inducible heme oxygenase 1 (HMOX1) gene in Labeo rohita.

The HMOX1 gene plays role in several biological processes and is also responsive to hypoxia stress. Freshwater carp fish, Labeo rohita, is reported as hypoxia sensitive, but the information of annotated hypoxia genes in public domain is very scanty for this species. Here, an attempt was made to isolate and characterize HMOX1 gene in L. rohita using information from zebrafish. HMOX1 gene was obtained by mapping HMOX1 protein of zebrafish over assembled genome of L. rohita. Aligned region was used for designing primers for HMOX1 amplification. Eight overlapping sets of primers were designed for amplifying ~540 bp long successive overlapping fragments. Splicing of overlapping amplicons generated 3715 bp fragment that was confirmed as HMOX1 gene having full coding region with 6 exons between 184 and 2156 bp positions. HMOX1 characterization is an initiative for L. rohita genes annotation to support the characterization of new genes in the important species.

Evolutionary analysis of genus Channa based on karyological and 16S rRNA sequence data.

A wide range of diploid number of chromosomes and the body size of Channa congeners are useful combination of characters for studying the factors controlling the body size. In this study, the karyological information was superimposed on the evolutionary tree generated by 16S rRNA mitochondrial gene sequences. Here, the metaphase chromosome complements stained with Giemsa, AgNO3 and CMA3 were prepared from six snakehead murrel fish species collected from northeast India. The diploid chromosome numbers and the fundamental arms of C. aurantimaculata (2n = 52, NF = 98), C. gachua (2n = 56, NF = 84), C. marulius (2n = 44, NF = 58), C. orientalis (2n = 52, NF = 74), C. punctata (2n = 32, NF = 60) and C. striata (2n = 40, NF = 48) were calculated by the analysis of metaphase chromosome complements. Both methods of nucleolar organizer region (NOR) localization, silver nitrate and chromomycin A3, revealed NOR pairs of 1, 2, 3, 1, 4 and 3 in C. aurantimaculata, C. gachua, C. marulius, C. orientalis, C. punctata and C. striata, respectively. The subject species showed primitive type of asymmetrical chromosomes, except the C. punctata. The variation in 2n for C. orientalis (2n = 52, 78) and C. gachua (2n = 52, 78, 104) of a complete haploid set indicates the possibility of either ploidy change in C. orientalis and C. gachua, if we consider 2n = 52 or the Robertsonian rearrangements in different populations of these two species. The chromosome evolution tree was constructed on 16S rRNA ML-phylogenetic tree using ChromEvol 1.3. The analysis of chromosome evolution explained the loss or gain of chromosome, duplications or semiduplications mechanism. For time scaling the chromosomeevolution, the node age of available 16S rRNA gene of Channa species were estimated, which was also used for estimating the time when chromosomal changes occurred in context of geological time-scale.

Genome-Wide Comparative Analysis of HIF Binding Sites in Cyprinus Carpio for In Silico Identification of Functional Hypoxia Response Elements.

Cyprinus carpio is world's most widely distributed freshwater species highly used in aquaculture. It is a hypoxia-tolerant species as it lives in oxygen-deficient environment for a long period. The tolerance potential of an animal against hypoxia relates it to induced gene expression, where a hypoxia-inducible factor (HIF) binds to a transcriptionally active site, hypoxia response element (HRE), a 5-base short motif that lies within the promoter/enhancer region of a certain gene, for inducing gene expression and preventing/minimizing hypoxia effects. HRE is functionally active when it contains another motif, the hypoxia ancillary sequence (HAS), which is typically adjacent to downstream of HRE within 7- to 15-nt space. Here, an attempt was made for mining HRE and identifying functional HIF binding sites (HBS) in a genome-wide analysis of C. carpio. For this, gene information along with the 5,000-nt upstream (-4,900 to +100) sequences of 31,466 protein coding genes was downloaded from "Gene" and "RefSeq" databases. Analysis was performed after filtration of the impracticable genes. A total of 116,148 HRE consensus sequences were mined from 29,545 genes in different promoter regions. HRE with HAS consensus motifs were found in the promoter region of 9,589 genes. Further, the already reported genes for hypoxia response in humans and zebrafish were reanalyzed for detecting HRE sites in their promoters and used for comparative analysis with gene promoters of C. carpio for providing support to identify functional HBS in the gene promoter of C. carpio. An interactive user interface HREExplorer was developed for presenting the results on the World Wide Web and visualizing possible HBS in protein coding genes in C. carpio and displaying the comparative results along with the reported hypoxia-responsive genes of zebrafish and reported hypoxia-inducible genes in humans. In this study, a set of Perl program was written for the compilation and analysis of information that might be used for a similar study in other species. This novel work may provide a workbench for analyzing the promoter regions of hypoxia-responsive genes.

Isolation and characterization of hypoxia inducible gene connective tissue growth factor (CTGF) in Labeo rohita.

The connective tissue growth factor gene plays important role in several biological processes and also responsive to hypoxia stress in fishes. The freshwater fish, Labeo rohita, highly cultured in Indian subcontinent for food, is reported as hypoxia sensitive but annotation and sequences of nuclear genes were not available for this species so far in the public domain, except some transcripts. In this study, an attempt was made for isolation and annotation of the CTGF gene in L. rohita using information of zebrafish from the same family. The CTGF gene sequence was obtained by aligning assembled genome of L. rohita, (NCBI BioProject ID: PRJNA437789), with the CTGF protein of zebrafish. Eight overlapping sets of forward and reverse primers from aligned region were designed for amplification of around 600 bp long successive overlapping fragments of CTGF gene in L. rohita. Assembly and annotation of overlapping fragments confirmed a complete 2421 bp long CTGF gene sequence with a full coding region that comprised of five exons between 308 and 1921 positions. This annotated CTGF gene sequence was submitted to GenBank (Acc. No. KY940466). Characterization of CTGF will be an initiative in identification of hypoxia response genes in L. rohita which may further help in understanding the mechanism of hypoxia tolerability in this species.

FisOmics: A portal of fish genomic resources.

An online portal, accessible at URL: http://mail.nbfgr.res.in/FisOmics/, was developed that features different genomic databases and tools. The portal, named as FisOmics, acts as a platform for sharing fish genomic sequences and related information in addition to facilitating the access of high-performance computational resources for genome and proteome data analyses. It provides the ability for quarrying, analysing and visualizing genomic sequences and related information. The featured databases in FisOmics are in the World Wide Web domain already. The aim to develop portal was to provide a nodal point to access the featured databases and work conveniently. Presently, FisOmics includes databases on barcode sequences, microsatellite markers, mitogenome sequences, hypoxia-responsive genes and karyology of fishes. Besides, it has a link to other molecular resources and reports on the on-going activities and research achievements.

WGSSAT: A High-Throughput Computational Pipeline for Mining and Annotation of SSR Markers From Whole Genomes.

Mining and characterization of Simple Sequence Repeat (SSR) markers from whole genomes provide valuable information about biological significance of SSR distribution and also facilitate development of markers for genetic analysis. Whole genome sequencing (WGS)-SSR Annotation Tool (WGSSAT) is a graphical user interface pipeline developed using Java Netbeans and Perl scripts which facilitates in simplifying the process of SSR mining and characterization. WGSSAT takes input in FASTA format and automates the prediction of genes, noncoding RNA (ncRNA), core genes, repeats and SSRs from whole genomes followed by mapping of the predicted SSRs onto a genome (classified according to genes, ncRNA, repeats, exonic, intronic, and core gene region) along with primer identification and mining of cross-species markers. The program also generates a detailed statistical report along with visualization of mapped SSRs, genes, core genes, and RNAs. The features of WGSSAT were demonstrated using Takifugu rubripes data. This yielded a total of 139 057 SSR, out of which 113 703 SSR primer pairs were uniquely amplified in silico onto a T. rubripes (fugu) genome. Out of 113 703 mined SSRs, 81 463 were from coding region (including 4286 exonic and 77 177 intronic), 7 from RNA, 267 from core genes of fugu, whereas 105 641 SSR and 601 SSR primer pairs were uniquely mapped onto the medaka genome. WGSSAT is tested under Ubuntu Linux. The source code, documentation, user manual, example dataset and scripts are available online at https://sourceforge.net/projects/wgssat-nbfgr.

Heat and chemical treatments in adult Cyprinus carpio (Pisces cypriniformes) rapidly produce sterile gonads.

Several options have been proposed for eradication of germ cells (GCs) in mammals such as treatment with cytotoxic drugs, irradiation, cold ischemia and hyperthermic treatment. Some of these methods have been also tried in fish but conditions for complete sterilisation of gonads have not been established. Here, we report the production of sterile adult common carp Cyprinus carpio in 10 weeks by the heat and chemical treatments. The cytotoxic drug busulfan (40mg/kg) was intraperitoneally injected into the animals at 2-week intervals (5 doses in total), and they were maintained in water at 38°C between Weeks 1 and 10. The effectiveness of the treatments was assessed using gonadal index, histology, and vasa gene expression. At the end of Week 10, very severe gonadal degeneration was observed in fish treated with the heat-chemical combination, and 100% of male and female fish were devoid of endogenous GCs. The average levels of vasa transcript were 0.01±0.005 and 0.02±0.016 for males and females, respectively. By contrast, high temperature alone caused minor gonadal degeneration and the gene transcript were 0.59±0.131 for male and 0.62±0.13 for female. In Week 20, after the recovery period of 10 weeks at 25°C, the gonadal germ cell did not recover from the sterile condition in any of the sampled individuals. The change in colouration of gonads was an additional useful index of the degree of gonadal sterility.

HRGFish: A database of hypoxia responsive genes in fishes.

Several studies have highlighted the changes in the gene expression due to the hypoxia response in fishes, but the systematic organization of the information and the analytical platform for such genes are lacking. In the present study, an attempt was made to develop a database of hypoxia responsive genes in fishes (HRGFish), integrated with analytical tools, using LAMPP technology. Genes reported in hypoxia response for fishes were compiled through literature survey and the database presently covers 818 gene sequences and 35 gene types from 38 fishes. The upstream fragments (3,000 bp), covered in this database, enables to compute CG dinucleotides frequencies, motif finding of the hypoxia response element, identification of CpG island and mapping with the reference promoter of zebrafish. The database also includes functional annotation of genes and provides tools for analyzing sequences and designing primers for selected gene fragments. This may be the first database on the hypoxia response genes in fishes that provides a workbench to the scientific community involved in studying the evolution and ecological adaptation of the fish species in relation to hypoxia.