Fish Cell Cultures Research Articles

Xenobiotic metabolism in the fish hepatic cell lines Hepa-E1 and RTH-149, and the gill cell lines RTgill-W1 and G1B: Biomarkers of CYP450 activity and oxidative stress

The use of fish cell cultures has proven to be an effective tool in the study of environmental and aquatic toxicology. Valuable information can be obtained from comparisons between cell lines from different species and organs. In the present study, specific chemicals were used and biomarkers (e.g. 7-Ethoxyresorufin-O-deethylase (EROD) activity and reactive oxygen species (ROS)) were measured to assess the metabolic capabilities and cytotoxicity of the fish hepatic cell lines Hepa-E1 and RTH-149, and the fish gill cell lines RTgill-W1 and G1B. These cell lines were exposed to β-naphthoflavone (BNF) and benzo[a]pyrene (BaP), the pharmaceutical tamoxifen (TMX), and the organic peroxide tert-butylhydroperoxide (tBHP). Cytotoxicity in gill cell lines was significantly higher than in hepatic cells, with BNF and TMX being the most toxic compounds. CYP1-like associated activity, measured through EROD activity, was only detected in hepatic cells; Hepa-E1 cells showed the highest activity after exposure to both BNF and BaP. Significantly higher levels of CYP3A-like activity were also observed in Hepa-E1 cells exposed to TMX, while gill cell lines presented the lowest levels. Measurements of ROS and antioxidant enzymes indicated that peroxide levels were higher in gill cell lines in general. However, levels of superoxide were significantly higher in RTH-149 cells, where no distinctive increase of superoxide-related antioxidants was observed. The present study demonstrates the importance of selecting adequate cell lines in measuring specific metabolic parameters and provides strong evidence for the fish hepatocarcinoma Hepa-E1 cells to be an excellent alternative in assessing metabolism of xenobiotics, and in expanding the applicability of fish cell lines for in vitro studies.

Quantification of sterols from carp cell lines by using HPLC–MS

AbstractCholesterol is a major constituent of cell membranes and exhibits together with its oxidation products (oxysterols) several essential cellular functions. Additionally, it was recently shown to play a role during virus replication in fish. To investigate the involvement of cholesterol and oxysterols, for example, during a viral infection in fish cells, quantitative analysis of these analytes from cell cultures is necessary. A fast method based on solvent extraction followed by reversed‐phase high‐performance liquid chromatography with mass spectrometry for quantitation of cholesterol and seven oxysterols from cells and culture medium is described in this work. Detection limits were in the low ng/mL range, and the intra‐ and inter‐day precisions were above 87% for all analytes. A cholesterol content of approximately 10 μg per 2 × 106 cells, exceeding the concentration of oxysterols by at least 103‐fold, was determined for common carp brain cells. Furthermore, using the method established here, an uptake of externally supplied 25‐hydroxycholesterol by fish cells and its conversion to 7a,25‐dihydroxycholesterol could be shown. In summary, this is the first report on quantification of cholesterol and oxysterols from fish cell cultures, which can help in exploring their function during viral infections.

Use of Salmon Cardiac Primary Cultures (SCPCs) of different genotypes for comparative kinetics of mx expression, viral load and ultrastructure pathology, after infection with Salmon Pancreas Disease Virus (SPDV)

In vitro fish based models have been extensively applied in human biomedical research but, paradoxically, less frequently in the research of fish health issues. Farmed Atlantic salmon can suffer from several viral conditions affecting the heart. Therefore, species-specific, cardiac in vitro models may represent a useful tool to help further understanding and management of these diseases. The mechanisms underlying genotype based resistance are complex and usually rely on a combined effect of elements from both the innate and adaptive immune response, which are further complicated by external environmental factors. Here we propose that Salmon Cardiac Primary Cultures (SCPCs) are a useful tool to investigate these mechanisms as the basis for genotypic differences between Atlantic salmon families in susceptibility to cardiotropic viral disease.Using SCPCs produced from two different commercially available Atlantic salmon embryonated ova (Atlantic Ova IPN sensitive” (S) and “Atlantic QTL-innOva® IPN/PD” (R)), the influence of host genotype on the viral load and mx expression following Salmon Pancreas Disease Virus infection was assessed over a 15 day period. Both R and S SCPCs groups were successfully infected. A measurable difference between groups of viral nsP1 and host antiviral mx gene expression was observed (i.e. a later, but larger onset of mx expression in the R group). Mx expression peaks were followed by a decrease in viral nsP1 in both groups. Additionally, ultrastructural examination of infected SCPCs allowed the description of degenerative changes at the individual cell level. The SCPC model presents some advantages, over current fish cell culture monolayers and in vivo material, such as the presence of different cell components normally present in the target organ, as well as the removal of a layer of functional complexity (acquired immunity), making it possible to focus on tissue specific, early innate immune mechanisms. These preliminary results highlight the importance of considering genetic origin when selecting the fish source for the production of SCPCs, as well as their usefulness as screening tools for assessment of genotypic differences in disease resistance.

Two Virus-Induced MicroRNAs Known Only from Teleost Fishes Are Orthologues of MicroRNAs Involved in Cell Cycle Control in Humans.

MicroRNAs (miRNAs) are ~22 base pair-long non-coding RNAs which regulate gene expression in the cytoplasm of eukaryotic cells by binding to specific target regions in mRNAs to mediate transcriptional blocking or mRNA cleavage. Through their fundamental roles in cellular pathways, gene regulation mediated by miRNAs has been shown to be involved in almost all biological phenomena, including development, metabolism, cell cycle, tumor formation, and host-pathogen interactions. To address the latter in a primitive vertebrate host, we here used an array platform to analyze the miRNA response in rainbow trout (Oncorhynchus mykiss) following inoculation with the virulent fish rhabdovirus Viral hemorrhagic septicaemia virus. Two clustered miRNAs, miR-462 and miR-731 (herein referred to as miR-462 cluster), described only in teleost fishes, were found to be strongly upregulated, indicating their involvement in fish-virus interactions. We searched for homologues of the two teleost miRNAs in other vertebrate species and investigated whether findings related to ours have been reported for these homologues. Gene synteny analysis along with gene sequence conservation suggested that the teleost fish miR-462 and miR-731 had evolved from the ancestral miR-191 and miR-425 (herein called miR-191 cluster), respectively. Whereas the miR-462 cluster locus is found between two protein-coding genes (intergenic) in teleost fish genomes, the miR-191 cluster locus is found within an intron of a protein-coding gene (intragenic) in the human genome. Interferon (IFN)-inducible and immune-related promoter elements found upstream of the teleost miR-462 cluster locus suggested roles in immune responses to viral pathogens in fish, while in humans, the miR-191 cluster functionally associated with cell cycle regulation. Stimulation of fish cell cultures with the IFN inducer poly I:C accordingly upregulated the expression of miR-462 and miR-731, while no stimulatory effect on miR-191 and miR-425 expression was observed in human cell lines. Despite high sequence conservation, evolution has thus resulted in different regulation and presumably also different functional roles of these orthologous miRNA clusters in different vertebrate lineages.

Development of a novel in vitro method for drug development for fish; application to test efficacy of antimicrosporidian compounds

Few drugs are approved for treating diseases caused by parasites in minor species such as fish. This is due, in part, to the expense of drug development and to the...

Conditions for initiating Lake Victoria haplochromine (Oreochromis esculentus) primary cell cultures from caudal fin biopsies.

The global decline of freshwater fishes has created a need to cryopreserve biological materials from endangered species in an effort to conserve the biodiversity within this taxon. Since maternal gametes and embryos from fish are difficult to cryopreserve, somatic cells obtained from caudal fins have become an increasingly popular resource as they contain both maternal and paternal DNA ensuring valuable traits are not lost from the population. Somatic cells stored in cryobanks can be used to supplement endangered populations with genetically valuable offspring with the use of assisted reproductive technologies. However, initiating primary cell cultures from caudal fin biopsies of endangered species can be challenging as standardized protocols have not yet been developed. The objective of this study was to identify culture conditions, including antibiotic supplementation, biopsy size, and culture temperature, suitable for establishing primary cell cultures of ngege (Oreochromis esculentus), a critically endangered African cichlid. Six-millimeter caudal fin biopsies provided sufficient material to develop a primary cell culture when incubated at 25°C using standard fish cell culture medium containing 1× Primocin. Further investigation and application of these culture conditions for other endangered freshwater fishes is necessary.

Unraveling the mechanisms involved in zearalenone-mediated toxicity in permanent fish cell cultures

The world-wide occurrence of zearalenone (ZEN) as a contaminant in feed for farm animals and fish requires the evaluation of toxicity mechanisms of action of ZEN. The present study investigates possible metabolization of ZEN in fish cell lines suggesting that mainly glucuronidation takes place. It demonstrates that concentrations up to 20,000 ng ml−1 ZEN are capable of influencing cell viability in permanent fish cell cultures in a dose–response manner with different response patterns between the five tested cell lines, whereby lysosomes appeared to be the main target of ZEN. ZEN toxicity is often discussed in the context of oxidative stress. Our study shows a biphasic response of the cell lines when reactive oxygen species (ROS) production is monitored. Damage in cells was observed by measuring lipid peroxidation, DNA strand breaks, and alterations of intracellular glutathione levels. Metabolization of ZEN, especially at concentrations above 7500 ng ml−1 ZEN, does not prevent cytotoxicity. ZEN as an estrogenic compound may involve processes mediated by binding to estrogen receptors (ER). Since one cell line showed no detectable expression of ER, an ER-mediated pathway seems to be unlikely in these cells. This confirms a lysosomal pathway as a main target of ZEN in fish cells.

Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

(Eco)toxicological effects of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in zebrafish (Danio rerio) and permanent fish cell cultures

2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) is a high-production volume chemical used in paper, ink, pesticide, and adhesive industries as a wetting and anti-foaming agent. The physicochemical properties and slow biodegradation rate of TMDD indicate a low bioaccumulation potential but a high prevalence in the environment. As a consequence, TMDD has been detected in several European rivers in the nanogram per liter and lower microgram per liter range; however, its environmental risk to aquatic organisms is considered low. Recent studies almost exclusively focused on acute effects by TMDD, little is known about cytotoxic and genotoxic effects, reproduction and developmental toxicity, endocrine disruption, and any kind of long-term toxicity and carcinogenicity so far. The present study aims to provide more specific baseline information on the ecotoxicological effects of TMDD in fish. For this end, cyto- and genotoxicity assays were carried out in vitro with the permanent fish cell line RTL-W1; in addition, in vivo studies were conducted with the early life stages of zebrafish (Danio rerio) in order to fill the data gaps in developmental toxicity and endocrine disruption. TMDD showed a cytotoxic and slight genotoxic potential in fish cell lines; moreover, various sublethal and lethal effects could be detected in developing zebrafish embryos. There was no evidence of endocrine-disrupting effects by TMDD; however, mortality following prolonged exposure to TMDD during fish sexual development test was clearly higher than mortality in the fish embryo test after 96-h exposure. Our results thus confirmed previous findings of laboratory screening tests, suggesting short-term toxic effects of TMDD in the intermediate, and long-term effects in the lower milligram per liter range.

Establishment of caudal fin cell lines from tropical ornamental fishes Puntius fasciatus and Pristolepis fasciata endemic to the Western Ghats of India

Two new cell lines, PFF and CFF were established from the caudal fin of the Puntius fasciatus, and Pristolepis fasciata respectively. Since their initiation, these cell lines (PFF and CFF) have been subcultured in L-15 medium with 10% fetal bovine serum for more than 35 passages at 28°C and both the cell lines were characterized. Karyotyping analysis of PFF and CFF cells at 25th passage indicated that the modal chromosome number was 2n=50 and 2n=48 respectively. The cell line was cryopreserved in liquid nitrogen at −196°C and could be recovered from storage after six months with good cell viability. Polymerase chain reaction amplification of the fragments of two mitochondrial genes, 16S rRNA and COI confirmed that the cell lines originated from the respective species. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to PFF and CFF. Both the cells were resistant to the marine viral nervous necrosis virus (VNNV). No CPE could be found in both cell lines inoculated with the fish samples and cell culture supernatants were demonstrated free of SVC, iridovirus and KHV by molecular methods. These results indicated the absence of SVC, iridovirus and KHV in the ornamental fishes collected from the Western Ghats of India.

Fish cell cultures as in vitro models of inflammatory responses elicited by immunostimulants. Expression of regulatory genes of the innate immune response

We report the differential expression of various genes related to the regulation of the innate immune responses, including pro-inflammatory (IL-1β1, IL-8, TNF-α1, TNF-α2) and immune-suppressing (IL-10) cytokines, interferon-induced Mx-1 protein, enzymes regulating nitric oxide (inducible nitric oxide synthase, arginase-2) and eicosanoid (COX-2) production, and Toll-like pathogen pattern-recognition receptors TLR-3, TLR-5 and TLR-9, in two lympho-haematopoietic stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss), as well as in primary cultures of rainbow trout head kidney macrophages, after their exposure to the well-known immunostimulants LPS, levamisole and poly I:C.Although there were differences in the responses between the two stromal cell lines, using reverse transcription followed by real time polymerase chain reaction (RT-qPCR) we demonstrated that exposure to the immunostimulants, particularly poly I:C and LPS, resulted in significant changes in the expression of the immunoregulatory genes in the two stromal cell lines in many cases their responses resembling in fold change magnitudes and in response profiles to those observed in the primary macrophage cultures. Exposure to poly I:C and, with lower fold change values, to LPS produced upregulation of the pro- (IL-1β, IL-8, TNF-α) and anti-inflammatory (IL-10) cytokine genes, as well as of the Mx-1 gene. Furthermore, the immunostimulation elicited the upregulation of COX-2, iNOS and arginase-2 genes in the cell lines. Likewise, the TSS and TPS-2 cell lines significantly upregulated the expression of TLR-3, TLR-5 and TLR-9 genes after exposure to the immunostimulants, thus explaining the ability of the stromal cells to recognise and respond to the immunostimulants.Such results give support to an important role of lympho-haematopoietic stromal cells in the development and control of pro-inflammatory responses in fish. The upregulation of genes of pro-inflammatory cytokines and of mediators of the innate immune responses correlates well with the previously demonstrated functional capacities, including phagocytosis, microbicidal activity and NO production, exhibited by the TSS and TPS-2 stromal cell lines when exposed to the same immunostimulants. On the other hand, the expression of immunosuppressing genes (IL-10, COX-2 and arginase-2) demonstrate that the lympho-haematopoietic stromal cells are also able to contribute to the control of inflammatory responses.This study reinforce the possibility of using histotypic cell cultures, as those formed by the TSS and TPS-2 cell lines, formed by heterogeneous cell populations that partially replicates the cell–cell and cell-extracellular matrix interactions, to develop cost-effective and repetitive in vitro systems for the screening of immunostimulant candidates for aquaculture, as they are able to replicate in vitro immune regulatory networks occurring in vivo.

Infectious salmon anaemia virus infection of Atlantic salmon gill epithelial cells

Infectious salmon anaemia virus (ISAV), a member of the Orthomyxoviridae family, infects and causes disease in farmed Atlantic salmon (Salmo salar L.). Previous studies have shown Atlantic salmon endothelial cells to be the primary targets of ISAV infection. However, it is not known if cells other than endothelial cells play a role in ISAV tropism. To further assess cell tropism, we examined ISAV infection of Atlantic salmon gill epithelial cells in vivo and in vitro. We demonstrated the susceptibility of epithelial cells to ISAV infection. On comparison of primary gill epithelial cell cultures with ISAV permissive fish cell cultures, we found the virus yield in primary gill epithelial cells to be comparable with that of salmon head kidney (SHK)-1 cells, but lower than TO or Atlantic salmon kidney (ASK)-II cells. Light and transmission electron microscopy (TEM) revealed that the primary gill cells possessed characteristics consistent with epithelial cells. Virus histochemistry showed that gill epithelial cells expressed 4-O-acetylated sialic acid which is recognized as the ISAV receptor. To the best of our knowledge, this is the first demonstration of ISAV infection in Atlantic salmon primary gill epithelial cells. This study thus broadens our understanding of cell tropism and transmission of ISAV in Atlantic salmon.

Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants

We have tested the elicitation of innate defence-related responses in two stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss) after they were exposed to different concentrations of lipopolysaccharide (LPS), levamisole, or polyinosinic polycytidylic acid (poly-I:C). For comparison, cultures of rainbow trout head kidney macrophages were also included in the study, and the effect of the immunostimulants on the phagocytic activity, the intracellular and extracellular reactive oxygen species and nitric oxide production were assayed. Although the responses varied depending upon the concentration of the immunostimulants and the particular cell line, our results demonstrate that those activities were enhanced in the TSS and TPS-2 cell lines after exposure to any of the immunostimulants. These results indicate that the stromal cells of the main lympho-haemopoietic organs of O. mykiss develop innate defence responses, which are enhanced by well-known immunostimulants. In addition, such enhancement of the defence responses in the TSS and TPS-2 cell lines could be also elicited when they were exposed to conditioned supernatants from levamisole- or poly I:C-stimulated HK macrophage cultures, thus demonstrating that the haemopoietic stromal cells respond to macrophage-derived factors. Moreover, we demonstrate that the stromal cell lines constitutively expressed the Toll-like receptors TLR3, TLR5 and TLR9 genes. The results are discussed considering the role of the lympho-haemopoietic stromal cells in the innate immune responses, and the possibility of using histiotypic cell cultures of non-leucocyte cells of the haemopoietic organs to develop in vitro methods to select new immunostimulant candidates for aquaculture.

A validated quantitative polymerase chain reaction assay for the detection of ranaviruses (Family Iridoviridae) in fish tissue and cell cultures, using EHNV as a model

Viruses in the genus Ranavirus (Family Iridoviridae) are important pathogens of fish, amphibians and reptiles and likely have a global distribution. Certification of freedom from infection, prevalence surveys, surveillance and routine diagnosis are hampered by lack of a validated rapid molecular test. In this study, a quantitative PCR (qPCR) assay for the detection of the major capsid protein gene of ranaviruses was developed and validated using Epizootic Haematopoietic Necrosis Virus (EHNV) as a model ranavirus. The assay had 100-fold greater analytical sensitivity compared to virus isolation in Bluegill fry (BF-2) cells, detected all viruses in a panel of 20 ranaviruses from Australia, America, Europe and the United Kingdom, did not detect two disputed members of the genus Ranavirus (doctorfish virus and guppy virus 6) and did not detect a megalocytivirus or two cyprinid herpesviruses. When applied to the detection of EHNV in fish tissue homogenates, 94.3% of samples which caused cytopathic effect (CPE) in BF-2 cells were positive in the qPCR, together with many others which did not display CPE but that arose from fish exposed to EHNV. Similarly, 98% of cell culture supernatants from cell monolayers with CPE were positive in the qPCR, together with other cultures of tissues from EHNV-exposed fish which did not display CPE. Overall a greater number of samples from exposed fish tested positive in qPCR compared to culture in BF-2 cells. The diagnostic specificity of the qPCR assay was 100% based on tests of tissue homogenates from 100 fish which were not exposed to EHNV. The assay appears to be suitable for a range of applications including surveillance and diagnosis, and for confirming the cause of CPE in BF-2 cells.

Frequent biphasic cellular responses of permanent fish cell cultures to deoxynivalenol (DON)

Contamination of animal feed with mycotoxins is a major problem for fish feed mainly due to usage of contaminated ingredients for production and inappropriate storage of feed. The use of cereals for fish food production further increases the risk of a potential contamination. Potential contaminants include the mycotoxin deoxynivalenol (DON) which is synthesized by globally distributed fungi of the genus Fusarium. The toxicity of DON is well recognized in mammals. In this study, we confirm cytotoxic effects of DON in established permanent fish cell lines. We demonstrate that DON is capable of influencing the metabolic activity and cell viability in fish cells as determined by different assays to indicate possible cellular targets of this toxin. Evaluation of cell viability by measurement of membrane integrity, mitochondrial activity and lysosomal function after 24 h of exposure of fish cell lines to DON at a concentration range of 0–3000 ng ml −1 shows a biphasic effect on cells although differences in sensitivity occur. The cell lines derived from rainbow trout are particularly sensitive to DON. The focus of this study lies, furthermore, on the effects of DON at different concentrations on production of reactive oxygen species (ROS) in the different fish cell lines. The results show that DON mainly reduces ROS production in all cell lines that were used. Thus, our comparative investigations reveal that the fish cell lines show distinct species-related endpoint sensitivities that also depend on the type of tissue from which the cells were derived and the severity of exposure.

Development of a Fish Cell Culture Model to Investigate the Impact of Fish Oil Replacement on Lipid Peroxidation

Fish oils are rich in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), predominantly 20:5n-3 and 22:6n-3, whereas vegetable oils contain abundant C(18)-PUFA, predominantly 18:3n-3 or 18:2n-6. We hypothesized that replacement of fish oils with vegetable oils would increase the oxidative stability of fish lipids. Here we have used the long established and easily cultivated FHM cell line derived from the freshwater fish species fathead minnow (Pimephales promelas) to test this hypothesis. The FHM cells were readily able to synthesize 20:5n-3 and 24:6n-3 from 18:3n-3 but 22:6n-3 synthesis was negligible. Also, they were readily able to synthesize 20:3n-6 from 18:2n-6 but 20:4n-6 synthesis was negligible. Mitochondrial β-oxidation was greatest for 18:3n-3 and 20:5n-3 and the rates for 16:0, 18:2n-6, 22:6n-3 and 18:1n-9 were significantly lower. Fatty acid incorporation was predominantly into phospholipids (79-97%) with very little incorporation into neutral lipids. Increasing the fatty acid concentration in the growth medium substantially increased the concentrations of 18:3n-3 and 18:2n-6 in the cell phospholipids but this was not the case for 20:5n-3 or 22:6n-3. When they were subjected to oxidative stress, the FHM cells supplemented with either 20:5n-3 or 22:6n-3 (as compared with 18:3n-3 or saturated fatty acids) exhibited significantly higher levels of thiobarbituric reactive substances (TBARS) indicating higher levels of lipid peroxidation. The results are discussed in relation to the effects of fatty acid unsaturation on the oxidative stability of cellular lipids and the implications for sustainable aquaculture.

New cell line from adipopancreatic tissue of Atlantic herring Clupea harengus

AB Aquatic Biology Contact the journal Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AB 11:271-278 (2011) - DOI: https://doi.org/10.3354/ab00317 New cell line from adipopancreatic tissue of Atlantic herring Clupea harengus S. Langner*, S. Rakers, P. Ciba, A. E. Petschnik, D. H. Rapoport, C. Kruse Fraunhofer Institution for Marine Biotechnology, Fraunhofer Society, Paul-Ehrlich-Straße 1–3, 23562 Lübeck, Germany *Email: stephanie.langner@emb.fraunhofer.de ABSTRACT: We report the isolation, cultivation, and cryopreservation of cells from adipopancreatic tissue of adult Atlantic herring Clupea harengus. The cell population was cultured for >1 yr and had a mean doubling time of 2 wk. Immunocytochemical analyses revealed that >50% of cells contained glial fibrillary acidic protein and pan-cytokeratins. Vigilin, a well-conserved protein related to mRNA transport, was found in only 11% of cells. Alpha smooth-muscle actin and stage-specific embryonic antigen 1 were even less abundant (<5%). Although little is known about tissue homeostasis and regeneration in adult C. harengus, the heterogeneity and long-term proliferation of the cell population described here suggest that it may contain stem cells or progenitor cells. Before the present study, there was only one C. harengus cell line available to researchers. The newly established cell line described in the present study represents an important addition to scientific investigation as well as aquaculture industries and toxicological/virological testing of herring populations. KEY WORDS: Herring · Clupea harengus · Long-term fish cell culture · Fish cell line · Fish · Cryopreservation · Stem cells · Adipopancreatic tissue Full text in pdf format PreviousNextCite this article as: Langner S, Rakers S, Ciba P, Petschnik AE, Rapoport DH, Kruse C (2011) New cell line from adipopancreatic tissue of Atlantic herring Clupea harengus. Aquat Biol 11:271-278. https://doi.org/10.3354/ab00317 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AB Vol. 11, No. 3. Online publication date: February 10, 2011 Print ISSN: 1864-7782; Online ISSN: 1864-7790 Copyright © 2011 Inter-Research.

The versatile, changing, and advancing roles of fish in sediment toxicity assessment—a review

Sediments serve as integral and dynamic parts of our aquatic systems. Within the last 15 to 20 years, however, the scientific community has begun noticing deterioration of sediment quality at an alarming rate worldwide. Sediments are now harboring hazardous pollutants that can directly influence water quality, thereby creating very stressful conditions for aquatic life. As a consequence, global efforts were initiated in the early 1970s, to find ways to assess sediment quality. Because of their obvious ecological and economic significance, fish have remained a major taxonomic group for appraising the general quality of aquatic systems. However, for sediment risk assessment, fish have lagged behind invertebrates due to their mobility and generally, pelagic lifestyle. To our knowledge, this is the first paper that comprehensively presents and reviews the versatile role of fish in assessing the state of health of aquatic sediments. Through a literature search of the more relevant and/or more recent studies, this review attempted to trace the development of the various approaches as well as to describe the future prospects of using fish as sentinels for sediment quality assessment. Initially, the use of whole fish (juveniles or adults) bioassays contributed immensely to our understanding of sediment contamination and ecotoxicology. But due to economic as well as ethical issues linked to the use of live vertebrates for toxicity testing, the approach has shifted to using fish cell cultures and fish embryos. Much newer approaches involving receptors and gene arrays in fish cells to elucidate the mode of action of sediment-borne contaminants are very promising. The review paper also lists and explores some of the issues associated with the use of juvenile or adult fish, fish cell cultures, fish embryos, and fish gene expression profiles in sediment toxicity evaluations to stimulate further discussions, and hopefully, to serve as benchmark for future handling of similar or related aspects of fish utilization in sediment risk assessment. Overall, the present review has comprehensively explored the changing and progressing roles of fish in sediment toxicity evaluation. Indeed the usefulness of this taxon as test organisms has provided a significant contribution to the advancement of sediment toxicology. However, despite the quite optimistic and bright future for these current procedures, a number of issues and problems remain. Therefore, efforts to develop new technologies and to refine current methods and approaches continue to challenge many laboratories worldwide.

Validation of a qPCR assay for the detection of Ictalurid herpesvirus‐2 (IcHV‐2) in fish tissues and cell culture supernatants

Ictalurid herpesvirus-2 (IcHV-2) is a pathogen of cultured black bullhead, Ameiurus melas (Rafinesque), and has been shown to produce high mortality in experimental exposures of channel catfish, Ictalurus punctatus (Rafinesque). During acute infections, the virus grows readily in cell cultures but produces a cytopathic effect (CPE) similar to that of Ictalurid herpesvirus-1 (IcHV-1) and the channel catfish reovirus. We have developed a quantitative PCR assay that can be used to detect IcHV-2 in fish tissues and cell culture supernatants. The assay does not amplify other fish herpesviruses tested or host DNA. It is quantitative over a range of eight logs, and the limit of detection is <10 copies per reaction. In replicate assays carried out on different days, the coefficient of variability was 10%. The best organs for the detection of acute IcHV-2 infections by our assay are the spleen and kidney. This assay should be useful for the diagnosis of IcHV-2 disease, the identification of syncytial CPEs in cell cultures, and for the detection of latent infections in carrier fish.

SUSCEPTIBILITY OF CYPRINID AND NON-CYPRINID FISH CELL LINES TO KOI HERPESVIRUS (KHV)

Koi herpesvirus (KHV) is an emerging virus that infects koi and common carp (Cyprinus carpio) with mortality up to 95% within 7 days. The disease is rapidly spreading worldwide including to Indonesia. However, it has only been documented in koi and common carp. The aim of this research was to evaluate the susceptibility of fish cell cultures originated from cyprinid and non-cyprinid fish to KHV. Koi Fin (KF-1) and Koi Tail (KT-2) cell lines derived from koi carp and SSN-1 cells originated from fry of striped snakehead were used in this study. The cells were inoculated with tissue extract of KHV-infected koi carp (experiment 1) and virus stock of KHV (experiment 2). The cultures were incubated at 22oC and the onset and type of cytophatic effect (CPE) were observed for 21 days post inoculation. The results of experiment 1 showed that CPE was observed in KT-2 at day 6 post inoculation. In the experiment 2, however CPE was observed in KF-1 and KT-2 cells at day 4 post infection. CPE was not observed in SSN-1 of either experiment 1 or experiment 2. CPE was characterized by extensive vacuolization of the infected cells. Polymerase chain reaction (PCR) assay of cell and tissue culture supernatants confirmed that KF-1 and KT-2 showing CPE were indeed infected with KHV. The results indicated that KF-1 and KT-2 cells were susceptible and SSN-1 was resistant to KHV. The implication of these findings was also discussed in the paper.