Role and relevance of fish cell lines in advanced in vitro research.

INTRODUCTION: Cell line derived from fish has been established as a promising tool for studying many key issues of aquaculture covering fish growth, disease, reproduction, genetics, and biotechnology. In addition, fish cell lines are very useful in vitro models for toxicological, pathological, and immunological studies. The easier maintenance of fish cell lines in flexible temperature regimes and hypoxic conditions make them preferable in vitro tools over mammalian cell lines. Great excitement has been observed in establishing and characterizing new fish cell lines representing diverse fish species and tissue types. The well-characterized and authenticated cell lines are of utmost essential as these represent cellular functions very similar to in vivo state of an organism otherwise it would affect the reproducibility of scientific research. CONCLUSION: The fish cell lines have exhibited encouraging results in several key aspects of in vitro research in aquaculture including virology, nutrition and metabolism, production of vaccines, and transgenic fish production. The review paper reports the cell lines developed from fish, their characterization, and biobanking along with their potential applications and challenges in in vitro research.

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies.

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.

De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, Silonia silondia (Hamilton, 1822) and their validation for population genetic studies.

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity-capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6-24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall FST = 0.075; P < 0.0001) with maximum variation (92.5%) within populations. Cross-priming assessment revealed successful amplification (35-38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.

Ion torrent next-generation sequencing reveals the complete mitochondrial genome of endangered mahseer Tor khudree (Sykes, 1839).

The complete mitochondrial genome of an endangered mahseer (Deccan mahseer), Tor khudree was sequenced using Ion torrent platform for the first time. The genome sequence was 16 573 bp in size, and consists of 13 protein coding genes, 22 tRNAs, 2 rRNA genes and 1 control region. The gene organization and its order were similar to other vertebrates. The overall base composition was A: 31.9%, G: 15.6%, C: 27.68%, T: 24.76%, A + T content 56.6% and the G + C content 43.32%. The phylogenetic tree constructed using a maximum likelihood model showed sister relationship between T. khudree and Tor tambroides.

Genetic characterization of Silond catfish, Silonia silondia (Hamilton, 1822) inferred from two mitochondrial markers.

Silonia silondia is a commercially important food fish. Samples collected through commercial catches from four rivers in India are described by sequence analysis of two molecular markers. Cytochrome b (1140 bp) and ATPase 6/8 (842 bp) genes were analyzed, which represented high level of genetic differentiation within populations of S. silondia. The sequence alignments of cytochrome b and ATPase 6/8 genes revealed 13 and 11 different haplotypes, respectively. The sequences of both the mitochondrial regions revealed high haplotype and low nucleotide diversities. The patterns of genetic diversity and haplotype networks clearly indicated two distinct mitochondrial lineages, however, haplotypes from both the lineages were not specifically assigned to any population. The results confirm the utility of molecular markers generating baseline information, useful for planning effective strategies for conservation, management and sustainability of Silond catfish fishery.

DNA barcoding of marine ornamental fishes from India.

India has rich marine ornamental fish diversity with 400 fish species distributed in Gulf of Munnar/Palk Bay, Gulf of Kutch, and in reefs around Andaman & Nicobar and Lakshadweep Islands. Marine ornamental fish identification at the field level is very difficult because of their high diversity and profound changes in appearance during their developmental stages and camouflage. To facilitate ornamental fish trading with ease and in compliance with the biodiversity act, DNA barcoding technique could be used to accurately identify species. In this study, DNA barcodes were generated for 31 species of commercially important marine ornamental fishes from India. The average genetic distance (K2P model) within species, genus, and family was 0.446, 13.08, and 20.09%, respectively. Intraspecific variation has increased several folds (15-20 times) after including conspecific sequences from different geographical locations. The presence of allopatric lineages/cryptic species was observed in the Indo-pacific region. The NJ tree constructed based on K2P values showed distinct clusters shared by congeneric species specific to populations.

Isolation and characterization of Pseudomonas stutzeri and its potential application in biological nitrification.

Ammonia-oxidizing bacteria (AOB) were isolated from sediment samples of fishponds with an aim to use them for application in biological nitrification of water. Isolation of AOB was done in an inorganic medium and nitrite-producing bacterial isolates were selected. These isolates were further screened by polymerase chain reaction using specific primers forAOB. Out of 119 nitrate positive isolates, only 12 showed positive amplification and yielded a PCR product of ~465 bp. Treatment of aquaculture pond and riverwaterwith one of the bacterial isolate (HC-5) resulted in lowering of soluble ammonia level from 3.50 to 0.05 mgl(-1) and 7.5 to 0.01 mgl(-1), respectively. Partial 16S rRNA gene sequencing of isolate HC-5 identified the microorganism as Pseudomonasstutzeri.

DNA barcoding of elasmobranchs from Indian coast and its reliability in delineating geographically widespread specimens.

Identification of elasmobranchs by conventional taxonomy is difficult due to similarities in morphological characters. Species-specific molecular markers are good choice for identifying species irrespective of it's life stage. Recently, mitochondrial cytochrome c oxidase subunit I (COI) gene got global recognition as a barcode gene to discriminate all animals up-to species level. In this study, mitochondrial COI partial gene was used to develop DNA barcodes for 18 species of elasmobranchs (10 species of sharks and 8 species of rays). The COI barcodes clearly distinguished all the species with high interspecific distance values than intraspecific values. The average interspecific and intraspecific distance values are 8.6% and 0.3% for sharks, respectively and 12.4% and 0.63% for rays, respectively using K2P method. The Neighbor-Joining tree showed distinct clusters shared by the species of same genera. The COI barcodes were also used to estimate allopatric divergences for selected species across broad geographical locations and found that Sphyrna lewini, Aetobatus narinari and Neotrygon kuhlii have cryptic diversity.

DNA barcoding of gobiid fishes (Perciformes, Gobioidei).

Gobiids constitute a major proportion of fish population in both tropical and temperate freshwater as well as marine ecosystem. Due to their small size, cryptic ecology and ambiguous morphological characters, gobiids diversity was not documented completely. In this study, DNA barcodes were generated for 11 species of gobiids, collected from the Ashtamudi Lake, India. The mitochondrial COI gene was amplified using universal primers and the resulted 650 bp amplicon was sequenced. The COI barcodes clearly distinguished all the species with high inter-specific genetic distance values than intra-specific values based on K2P (Kimura 2 Parameter) model. The average genetic distance (K2P model) within species, genus and family was 1.2%, 22.2% and 25.3%, respectively. In addition to barcode-based species identification system, Nucleotide Diagnostic (ND) characters specific for species were identified. The Neighbor-Joining tree revealed distinct clusters shared by the species of same genera.

Establishment of a leukocyte cell line derived from peritoneal macrophages of fish, Labeo rohita (Hamilton, 1822).

A continuous leukocyte cell line with phagocytic activity was established from peritoneal macrophages of rohu, Labeo rohita (LRPM). LRPM was initiated from adherent mononuclear leukocytes isolated from peritoneal cavity of rohu, without use of any growth factors or feeder cells. These cells exhibited maximum growth at 30 °C in L-15 medium containing 20 % foetal bovine serum, and has been subcultured for more than 60 passages till date. The cells showed 85 % viability after 6 months of storage in liquid nitrogen. The species of origin of the LRPM was confirmed by the amplification and sequencing of 655 bp fragment of cytochrome oxidase subunit I of mitochondrial DNA. Functionally, LRPM showed phagocytic activity of yeast cells and fluorescent latex beads as evaluated by phase contrast and scanning electron microscopy, respectively. Immuno-modulators such as bacterial lipopolysaccharide and phorbol myristate acetate resulted in functional activation of LRPM; and enhanced their microbicidal activity through release of reactive oxygen species and nitric oxide. Culture supernatant from activated cells also revealed lysozyme activity. Cells of LRPM were positive for alpha-naphthyl acetate esterase enzyme indicating macrophage lineage. Our results indicate that this cell line can be a useful in vitro tool to study the role of macrophages in teleost immune system and to evaluate the effects of new aquaculture drugs. The LRPM cell line represents the first reported leukocyte cell line of peritoneal origin from any freshwater species of fish.

Isolation and characterization of head kidney derived macrophages of Labeo rohita.

Macrophages play a significant role in non-specific defense mechanisms of all vertebrates against pathogens. One critical element in the area of fish immunology is the unavailability of in-vitro model of immune cells. Therefore, it is essential to develop methods for harvesting and culture of macrophages for assessing innate immune functions of rohu, Labeo rohita, an important culture fish of India. Head kidney leukocytes from were isolated by density gradient sedimentation, so as to exclude other cells. Among isolated leukocytes, only macrophages showed the unique property of sustained adherence on plastic surfaces. These cells exhibited optimum growth at 28 degrees C in L-15 containing 20% FBS. Cultured head kidney macrophages (HKM) demonstrated the property of phagocytosis as evidenced by engulfment of yeast cells. Bacterial lipopolysaccharide (20 microg/ml) resulted in functional activation of macrophages as seen by enhanced reactive oxygen and nitrite production; and lysosomal enzyme activity. These results show that in-vitro model of HKM cells can be used to study the role of macrophages in innate immune responses against various immunomodulators.

Establishment and characterization of a piscean PCF cell line for toxicity and gene expression studies as in vitro model.

A new piscean fibroblastic cell line termed as PCF derived from the caudal fin tissue of dark mahseer, Puntius (Tor) chelynoides was established and characterized in the present study which was found to be suitable for toxicity and gene expression studies as in vitro model. The cell line grew well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS). The cells were able to grow at a temperature ranging from 20 to 28 °C with an optimal growth at 24 °C and the cell line have been expanded in culture for more than 70 passages. Authentication of the cell line was carried out using mitochondrial DNA markers (Cytochrome Oxidase subunit I and 16S ribosomal RNA). Presence of vimentin in the cells confirmed the fibroblastic origin of cell line. Significant cytopathic effects were observed upon exposure of PCF cell line to bacterial extracellular products and the study also validated the suitability of cell line in transgenic applications as well as in genotoxicity assessment as an in vitro model.

Mitochondrial ATPase 6/8 genes reveal genetic divergence in the Coilia dussumieri (Valenciennes, 1848) populations of north east and northwest coasts of India.

The golden anchovy, Coilia dussumieri, though possessing discontinuous distribution along northeast and northwest coasts of India, it is being managed as unit stock for fishery assessment purposes. By considering the need for stock specific management of the species, mitochondrial ATP synthase 6 and 8 (ATPase 6/8) genes were analyzed for delineating genetic stock structure of the species. Sequence analysis revealed a total of 34 haplotypes across four populations from both the east and west coasts of India. Haplotype diversity (h) was found in the range of 0.7421-0.9368. Similarly, nucleotide diversity (π) varied from 0.0012 to 0.0025. AMOVA results indicated a high total variance of 72.66% between east and west coast populations and less (1.34%) among populations within the respective coast. Phylogenetic tree constructed using pair wise FST also indicated the genetic divergence of populations of east and west coasts of India. The findings of the present study will be helpful in developing stock specific management measures for conservation and sustainable utilization of the species.

Microsatellite markers to determine population genetic structure in the golden anchovy, Coilia dussumieri.

Coilia dussumieri (Valenciennes, 1848) commonly called as golden anchovy, constitutes a considerable fishery in the northern part of both the west and east coasts of India. Despite its clear-cut geographic isolation, the species is treated as a unit stock for fishery management purposes. We evaluated 32 microsatellite primer pairs from three closely related species (resource species) belonging to the family Engraulidae through cross-species amplification in C. dussumieri. Successful cross-priming was obtained with 10 loci, which were sequenced for confirmation of repeats. Loci were tested for delineating the genetic stock structure of four populations of C. dussumieri from both the coasts of India. The number of alleles per locus ranged from 8 to 18, with a mean of 12.3. Results of pairwise F ST indicated genetic stock structuring between the east and west coast populations of India and also validated the utilization of identified microsatellite markers in population genetic structure analysis.

Molecular phylogeny of elasmobranchs inferred from mitochondrial and nuclear markers.

The elasmobranchs (sharks, rays and skates) being the extant survivors of one of the earliest offshoots of the vertebrate evolutionary tree are good model organisms to study the primitive vertebrate conditions. They play a significant role in maintaining the ecological balance and have high economic value. Due to over-exploitation and illegal fishing worldwide, the elasmobranch stocks are being decimated at an alarming rate. Appropriate management measures are necessary for restoring depleted elasmobranch stocks. One approach for restoring stocks is implementation of conservation measures and these measures can be formulated effectively by knowing the evolutionary relationship among the elasmobranchs. In this study, a total of 30 species were chosen for molecular phylogeny studies using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA gene and nuclear Internal Transcribed Spacer 2. Among different genes, the combined dataset of COI and 12S rRNA resulted in a well resolved tree topology with significant bootstrap/posterior probabilities values. The results supported the reciprocal monophyly of sharks and batoids. Within Galeomorphii, Heterodontiformes (bullhead sharks) formed as a sister group to Lamniformes (mackerel sharks): Orectolobiformes (carpet sharks) and to Carcharhiniformes (ground sharks). Within batoids, the Myliobatiformes formed a monophyly group while Pristiformes (sawfishes) and Rhinobatiformes (guitar fishes) formed a sister group to all other batoids.

Genetic characterization of Clupisoma garua (Hamilton 1822) from six Indian populations using mtDNA cytochrome b gene.

Clupisoma garua (Hamilton, 1822) is a commercially important freshwater fish and a potential candidate species for aquaculture. This study investigates the genetic diversity and population structure of six Indian populations of C. garua using cytochrome b (cyt b) sequences of mitochondrial DNA (mtDNA). We sequenced cyt b gene of 64 individuals collected from five distant rivers: Ganga, Gomti, Betwa, Gandak and Brahmaputra. Sequencing of 1054 bp cyt b mtDNA fragment revealed the presence of 19 haplotypes with a haplotype diversity value of 1.000 and a nucleotide diversity value of 0.0258 ± 0.00164. The Gandak river fish population showed highest nucleotide diversity. The fixation index analysis indicated significant genetic divergence among populations from different geographical areas. Both the neighbor-joining tree and median-joining network analysis of the haplotype data showed distinct patterns of phylo-geographic structure. The hierarchical analysis of molecular variance revealed that intra-group variation among populations was highly significant. The results of this study suggest that C. garua populations, especially geographically isolated groups, have developed significant genetic structures within the population. In addition, tests of neutrality suggest that C. garua may have experienced a population expansion. The study results establish cyt b as polymorphic and a potential marker to determine the population structure of C. garua. Information of genetic variation and population structure generated from this study would be useful for planning effective strategies for the conservation and rehabilitation of Schilibid cat fishes.

Development and characterization of a new cell line CF from caudal fin of knifefish, Chitala chitala (Hamilton-Buchanan).

A new cell line was successfully obtained from caudal fin tissue of the economically important freshwater fish Chitala chitala. The cell line was optimally maintained at 28°C in Leibovitz's L-15 supplemented with 20% fetal bovine serum (FBS). The effects of temperature and concentration of FBS on the growth of CF cells were examined. The CF cell line consisted predominantly of fibroblastic-like cells. Moderately low plating efficiencies 8%, 11%, and 17% were observed, with CF cell line in L-15 Medium with 20% FBS. Chromosomal analysis of the cell line revealed a diploid number of 42 chromosomes in C. chitala. Molecular characterization of mitochondrial 16S rRNA and cytochrome oxidase subunit I confirmed the origin of the cell line. The cells were successfully cryopreserved in liquid nitrogen (-196°C) for 6 mo, and more than 85-90% of CF cells were revived.

DNA damage and oxidative stress modulatory effects of glyphosate-based herbicide in freshwater fish, Channa punctatus.

The present study was undertaken to evaluate the genotoxic and oxidative stress modulatory effects of commercial formulation of glyphosate-based herbicide (Roundup(®)) in freshwater fish Channa punctatus. Three sublethal test concentrations of the herbicide viz., SL-I (1/10th of LC50=∼3.25mgL(-1)), SL-II (1/8th of LC50=∼4.07mgL(-1)) and SL-III (1/5th of LC50=∼6.51mgL(-1)) were calculated using 96-LC50 value and the test specimens were exposed to these concentrations. Blood and gill cells of the exposed specimens were sampled on day 1, 7, 14, 21, 28 and 35 to examine the DNA damage using comet assay and to assess the alteration in lipid peroxidation and antioxidant enzymes activities. The highest DNA damage was observed on day 14 at all test concentrations followed by gradual non-linear decline. Induction of oxidative stress in the blood and gill cells were evidenced by increased lipid peroxidation level, while antioxidants namely superoxide dismutase, catalase and glutathione reductase responded in a concentration-dependent manner. The results supported the integrated use of comet and antioxidant assays in determining the toxicity of water pollutants which could be used as part of monitoring programs.

Molecular characterization of major and minor rDNA repeats and genetic variability assessment in different species of mahseer found in North India.

Relationship among the mahseer species (Family: Cyprinidae) has long been debated in fish systematics. Present study concentrates on the nature of the phylogenetic relationship among the five mahseer species using the sequence of major ribosomal DNA (45S rDNA). We have covered rDNA sequence of approximately 5.2 kb per individual, 26.0 kb per species and 130.0 kb as a whole. We also characterized the 45S and 5S rDNA regions with respect to their nucleotide composition. For phylogenetic analyses, nucleotide sequences were divided into four datasets. First and second datasets contained 18S rDNA and ITS1 sequence, whereas third and fourth datasets consisted of ITS2 and complete 18S-ITS1-5.8S-ITS2-28S, respectively. The NJ tree was constructed for all the datasets. The mahseer species under study formed a monophyletic group well separated from the outgroup species. Similarly, the individuals of Neolissochilus hexagonolepis form monophyletic group with Tor species, indicating Neolissochilus as a sister genus of Tor. The findings from the present study provide greater insights into taxonomic status of mahseer, and set the stage for future investigations dealing with phylo-geography, taxonomy, conservation and co-evolution within this interesting and important group of fish.

A SRCF cell line from snowtrout, Schizothorax richardsonii: development and characterization.

Schizothorax richardsonii, commonly called snowtrout, is an important indigenous coldwater fish of the Himalayas, India with high commercial values as food and game fish. A cell line named as SRCF was developed from the caudal fin tissue of S. richardsonii. The cell line has been maintained in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS) at 24°C. The cells showed fibroblastic morphology, high plating efficiency and cell doubling time of 48h. Chromosomal analysis of SRCF cells revealed a diploid count of 98 chromosomes. The origin of the cell line was confirmed by the amplification of 655 and 578bp of cytochrome oxidase subunit I (COI) and 16S rRNA of mitochondrial DNA (mt-DNA) genes, respectively. Transfection of SRCF cells with pEGFP-C1 plasmid resulted in bright fluorescent signals, suggesting the application of cell line in transgenic and genetic improvement programme. In addition, genotoxicity assessment illustrated the utility of the cell line as an in vitro model for aquatic toxicological studies.