Marine Fish Cell Line Research Articles

Establishment of cell lines from a tropical grouper, Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses.

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.

Cytotoxicity and genotoxicity of polyethylenimine and nickel chloride in red sea bream (Pagrosomus major) fin cell line RSBF

A continuous marine fish cell line RSBF (i. c. Red Sea Bream Fin) was utilized to screen the cytotoxicity and genotoxicity of polyethylenimine (PEI) and nickel chloride (NiCl2) in this study on the deleterious effects of aquatic genotoxins on fish. At the 0.01 to 1 μg/ml concentration tested, PEI had acute toxicity to the treated RSBF cells (IC50=1.12, 0.92, 0.88 and 0.64 μg/ml PEI for time 0 h, 24 h, 48 h and 72 h after treatment, respectively) and markedly inhibited their proliferation in a dose-dependent manner. At the 0.001 to 5 μmol/L concentration tested, NiCl2 posed no acute toxicity but significantly stimulated their growth (107%–214% of control). Random amplified polymorphic DNA (RAPD) technique was used to detect the genotoxic effects of PEI and NiCl2 by comparing the RAPD banding patterns of the control and treated cells. RAPD analysis indicated that at the concentrations tested, PEI was more genotoxic than NiCl2 to RSBF cells; that there was a slight dose-dependent response in the genotoxic effect of PEI but not NiCl2; and that RAPD technique might provide a sensitive, non-specific genotoxic endpoint. And the potent cytotoxicity and genotoxicity of PEI on fish cells showed that we should be cautious in utilizing it as gene, vector in fish gene transfer and human gene therapy.

Characterization of lactate dehydrogenase isozyme pattern and morphology of three marine fish cell lines

Three continuous marine fish cell lines of FG (i.e., Flounder Gill) from flounder (Paralichthys olivaceus) gill, SPH (i.e., Sea Perch Heart) from sea perch (Lateolabrax japonicus) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream (Pagrosomus major) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these three cell lines and their corresponding tissues of origin were investigated and compared. The results showed: (1) No difference was found in the LDH isozyme patterns of FG and flounder gill tissue. However, the LDH isozyme patterns of SPH and RSBF were significantly different from their corresponding tissues of origin; (2) LDH isozyme patterns of FG, SPH and RSBF were markedly different from each other and could serve as genetic markers for species identification and detection of cross contamination. Morphological change analysis of these three cell lines in comparison to their original tissues indicated that FG cells still appeared epithelioid without morphological transformation. However, morphological changes were found in SPH and RSBF compared to their original tissues. Therefore, the cellular morphology was still plastic in the relatively stable culture conditions, and it was possible that change of LDH patterns was related to morphological changes of fish cellsin vitro.

In vitro cytotoxicity of the organophosphorus insecticide methylparathion to FG-9307, the gill cell line of flounder (Paralichthys olivaceus).

FG-9307, a cell line derived from the gill of flounder Paralichthys olivaceus, was used to determine the acute cytotoxic effects of the organophosphorus insecticide methylparathion. The cytotoxic effects of methylparathion were initially measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay, and cell protein assay. Results indicated that concentrations of methylparathion ranging from 5 microg/ml to 60 microg/ml were toxic, and there was no significant difference in cytotoxic effects between the three test systems. Thus, the FG-9307 cell line is one of several choices for evaluating the acute toxicities of organophosphorus insecticides such as methylparathion. The ultrastructure of the cells was also studied. It was found that the ultrastructure of the cells was markedly altered by methylparathion, as evidenced by dilation of mitochondria, breakdown of rough endoplasmic reticulum, nuclear necrosis, and production of numerous lysosomes and lipid vacuoles. This appears to be the first report that a marine fish cell line can be used for acute in vitro cytotoxicity evaluation of methylparathion.

In vitro cytotoxicity of the organophosphorus pesticide parathion to FG-9307 cells

FG-9307, a cell line derived from a gill of the flounder, Paralichthys olivaceus, was used to determine the cytotoxic effects of the organophosphorus (OP) pesticide parathion. Cytotoxicity was measured by three endpoint systems: neutral red (NR) uptake assay, tetrazolium (MTT) assay and cell protein assay. The lowest concentration of parathion tested (1 μg/ml) was toxic and there was no significant difference in cytotoxic effects among the three assays. The FG-9307 cell line is a suitable bioindicator for the screening of the acute toxicities of parathion. The fine structures of the cells were also studied. Ultrastructures were markedly altered by parathion, as evidenced by dilation of nuclear membranes and mitochondrial cristae and by the presence of lysosomes with engulfed particles. With the increase of the parathion concentration, the damage degree of the cellular structures was more serious. At the highest concentration tested (15 μg/ml), there were few visible organelles, although such changes in cell morphology were not observed under a light microscope. Apparently, this is the unnoted report of marine fish cell line used for the evaluation of the acute in vitro cytotoxicity of parathion.

Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper, Epinephelus tauvina

The morphogenesis and the ultrastructure of a marine fish iridovirus isolated from diseased grouper, Epinephelus tauvina were studied by electron microscopy. The virus was grown on a marine fish cell line (GP) at 25 °C. After appearance of advanced cytopathic effect (CPE), various morphogenetic stages of virus amplification, maturation and assembly were detected in the cytoplasm of virus-infected cells. The matured nucleocapsids were probably formed by insertion of electron-dense core material into a partly forming empty capsid just before completely sealed. The nucleocapsids were located at the assembly sites as pseudocrystalline arrays or scattered individually. In the late phase of infection, the nucleocapsids were enveloped and released by budding from the plasma membrane. The budding virus particles could directly enter neighbouring cells by endocytosis to start the next round infection. Ultrastructure of the grouper iridovirus was studied using the methods of enzymatic digestions and detergent degradations. The purified iridovirus particles showed a three-layered membrane including an external lipoprotein envelope, an inner periodic protein capsid and a lipid-containing membrane. The regular array of surface capsid subunits was observed after degradation with detergent.

In vitro study on cytotoxic effects of the organopho-sphorous pesticide profenofos on the gill cell line, FG-9307, of the flounder (Paralichihys olivaceus)

The FG-9007 cell line derived from the gill of flounderParalichthys olivaceus was used in the present study to determine the acute cytotoxic effects of the organophosphorous pesticide, profenofos. It was found that the cell growth rate was markedly reduced by profenofos at the concentrations of 2.5, 5 and 7.5 mg/L tested; and that the fine structures of the cells were also altered by profenofos, as evidenced by dilation of nuclear membranes and mitochondria cristae, and presence of enlarged lysosomes with engulfed organelles and numerous vacuoles in the cytoplasm. Probably, mitochondria, the cell energy-generating sites, are the most prominent sites of profenofos cytotoxity in the cells. This seems to be the first report of the use of marine fish cell line for evaluation of the acute in vitro cytotoxicity of organophosphorus pesticide.

Analysis of three marine fish cell lines by rapd assay.

We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines.

Development of a tropical marine fish cell line from Asian seabass ( Lates calcarifer) for virus isolation

A tropical marine fish cell line (SF) was established from Asian seabass ( Lates calcarifer) fry. The cell line was maintained in Earle's minimum essential medium (EMEM) supplemented with foetal calf serum and incubated at 25°C. The susceptibility of the cell-line at the 16th, 35th and 46th subculture to several iridoviruses, birnaviruses, reoviruses, a rhabdovirus, and a nodavirus was studied. Cytopathic effect (CPE) was observed daily after virus inoculation and viral replication efficiency was determined for six viruses. SF cell cultures infected with an iridovirus, a nodavirus and a reovirus were further elucidated by electron microscopy. All the viruses tested were shown to induce CPE on SF cells. This suggests that the SF cell line has good potential for the isolation of various fish viruses. The SF cell line consists predominantly of epithelial-like cells and has been subcultured 85 times over a period of 24 months.

C-Banding of Chromosomes from Three Established Marine Fish Cell Lines

C-Banding of Chromosomes from Three Established Marine Fish Cell Lines

Analysis of a marine fish cell line from a male sheepshead.

Chromosomes from consecutive culture passages of a developing cell line from fin fibroblasts of a male sheepshead (Archosargus probatocephalus) were analyzed. It was demonstrated that the modal chromosome number is 48. The chromosome types found in these cells included 8 submetacentrics, 8-12 acrocentrics, and 28-32 telocentrics. Evidence suggests that the primary type of chromosomal alteration that occurred in cultured SHF-1 cells is aneuploidy; a large metacentric chromosome of undetermined origin was observed.

Some properties of an established fish cell line from the marine fish, Caranx mate (Omaka).

A monolayer culture of fibroblast-like cells was initiated and established from a trypsin digest of Omaka fish larvae (Caranx mate). The cell culture has been subcultured 105 times over a period of 30 mo and appears to be contact-inhibited. The Omaka cells grew optimally at 27 degrees in Eagle's basal medium plus 10% fetal calf serum but failed to grow at 16 and 37 degrees. At the lower temperature the cells remained viable and began to divide when the temperature was raised to 27 degrees. Unlike certain marine fish cell lines the Omaka cell did not require a higher osmolarity medium for optimal growth. Karyological analysis at the 43rd passage revealed a chromosomal modal number of 50 consisting of 7 submetacentric pairs and 18 acrocentric pairs of chromosomes. Virological studies indicated that Omaka cells supported the replication of poikilothermic viruses such as, FV-3 and IPN viruses, but not the mammalian viruses examined. Attempts to induce interferon by the synthetic polynucleotide poly I:C were unsuccessful.