Fish Cell Research Articles

Fish cell lines as screening tools to predict acute toxicity to fish of biocidal active substances and their relevant environmental metabolites

Biocidal substances and their environmental relevant metabolites are highly toxic for fish. However, an important scarcity of toxicity data for metabolites is recognised. This article provides new data about the toxicity to fish of these compounds and evaluates the potential use of fish cell lines as screening tools to assess the acute toxicity of these compounds in fish. To this aim, acute toxicity of 7 substances was tested in Oncorhynchus mykiss (OECD TG203) and cytotoxicity of 16 substances was assessed in fish cell lines from two species; Poeciliopsis lucida (PLHC-1) and O. mykiss (RTH-149, RTG-2 and RTgill-W1) performing three cytotoxicity tests: Alamar-Blue, 5-carboxyfluorescein diacetate, acetoxymethyl ester and Neutral Red Uptake. Additionally, in vitro and in vivo data from the LIFE-COMBASE database were included in a dataset finally comprising 33 biocides and 14 metabolites. Hazard data were categorized into 4 toxicity groups, according to the intervals established in Regulation (EC) 1272/2008. Finally, the Spearman correlation test was performed and coincidences between in vitro-in vivo data established. In vitro and in vivo results revealed a high positive correlation, with a complete coincidence for 56.5% of the substances, a 2% of false positives (non-toxic in vivo) and a 13% of false negatives (toxic in vivo) for the 4 toxicity categories. However, when results were grouped in toxic or non-toxic coincidence was obtained for 85% of the substances. In conclusion, although fish denote a greater sensitivity, the use of at least two fish cell lines and three cytotoxicity endpoints appear to be valid approaches for fish acute toxicity screening of biocides and their metabolites.

Capsid protein from red-spotted grouper nervous necrosis virus induces incomplete autophagy by inactivating the HSP90ab1-AKT-MTOR pathway.

As a highly important fish virus, nervous necrosis virus (NNV) has caused severe economic losses to the aquaculture industry worldwide. Autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of several viruses. Although NNV can induce autophagy to facilitate infection in grouper fish spleen cells, how it initiates and mediates autophagy pathways during the initial stage of infection is still unclear. Here, we found that red-spotted grouper NNV (RGNNV) induced autophagosome formation in two fish cell lines at 1.5 and 3 h post infection, indicating that autophagy is activated upon entry of RGNNV. Moreover, autophagic detection showed that RGNNV entry induced incomplete autophagy by impairing the fusion of autophagosomes with lysosomes. Further investigation revealed that binding of the RGNNV capsid protein (CP) to the Lateolabrax japonicus heat shock protein HSP90ab1 (LjHSP90ab1), a cell surface receptor of RGNNV, contributed to RGNNV invasion-induced autophagy. Finally, we found that CP blocked the interaction of L. japonicus protein kinase B (AKT) with LjHSP90ab1 by competitively binding the NM domain of LjHSP90ab1 to inhibit the AKT-mechanistic target of the rapamycin (MTOR) pathway. This study provides novel insight into the relationship between NNV receptors and autophagy, which may help clarify the pathogenesis of NNV.

A novel ferritin L (FerL) in hybrid crucian carp could participate in host defense against Aeromonas hydrophila infection and diminish inflammatory signals

FerL, a multifunctional iron-storage polypeptide, not only exhibited a regulatory role in iron metabolism, but also participated in the regulation of fish immunity. In this study, ORF sequence of WR-FerL was 522 bp, encoding 173 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-FerL was detected in spleen. A. hydrophila challenge and LPS stimulation could sharply enhance WR-FerL mRNA expression in tissues and fish cells, respectively. Purified WR-FerL fusion peptide exhibited in vitro binding activity to A. hydrophila and endotoxin, limited bacterial dissemination to tissues as well as attenuated A. hydrophila-induced production of pro-inflammatory cytokines. Moreover, WR-FerL overexpression could abrogate NF-κB and TNFα promoter activity in fish cells. These results indicated that WR-FerL could play an important role in host defense against A. hydrophila infection.

Interactive effects of temperature and salinity on the growth and cytotoxicity of the fish-killing microalgal species Heterosigma akashiwo and Pseudochattonella verruculosa

Fish-killing blooms of Heterosigma akashiwo and Pseudochattonella verruculosa have been devastating for the farmed salmon industry, but in Southern Chile the conditions that promote the growth and toxicity of these microalgae are poorly understood. This study examined the effects of different combinations of temperature (12, 15, 18 °C) and salinity (10, 20, 30 psu) on the growth of Chilean strains of these two species. The results showed that the optimal growth conditions for H. akashiwo and P. verruculosa differed, with a maximum rate of 0.99 day−1 obtained at 15 °C and a salinity of 20 psu for H. akashiwo, and a maximum rate of 1.06 day−1 obtained at 18 °C and a salinity of 30 psu for P. verruculosa. Cytotoxic assays (2 × 101 – 2 × 105 cell mL−1; cells, filtrates, and cell lysates) performed at salinities of 20 and 30 psu showed a 100% reduction in the viability of embryonic fish cells exposed to intact cells of H. akashiwo and a 39% reduction following exposure to culture filtrates of P. verruculosa. Differences in the fish-killing mechanisms (direct cell contact vs. extracellular substances) and physiological traits of H. akashiwo and P. verruculosa explain the recent occurrence of very large blooms under contrasting (cold-brackish vs. hot-salty) extreme climate conditions in Chile.

Antiviral Activity of Ribavirin against Tilapia tilapinevirus in Fish Cells.

The outbreak of the novel Tilapia tilapinevirus or Tilapia lake virus (TiLV) is having a severe economic impact on global tilapia aquaculture. Effective treatments and vaccines for TiLV are lacking. In this study, we demonstrated the antiviral activity of ribavirin against TiLV in E-11 cells. Our findings revealed that at concentrations above 100 μg/mL, ribavirin efficiently attenuates the cytopathic effect of the TiLV infection in fish cells. When administered in a dose-dependent manner, ribavirin significantly improved cell survival compared to the untreated control cells. Further investigation revealed that the cells exposed to ribavirin and TiLV had a lower viral load (p < 0.05) than the untreated cells. However, at concentrations above 1000 μg/mL, ribavirin led to cell toxicity. Taken together, our results demonstrate the efficacy of this antiviral drug against TiLV and could be a useful tool for future research on the pathogenesis and replication mechanism of TiLV as well as other piscine viruses.

Proliferative response avoids mutagenic effects of titanium dioxide (TiO2) nanoparticles in a zebrafish hepatocyte cell line

Titanium dioxide nanoparticles (TiO2NP) have been widely applied in numerous industrial products, and their discharge into wastewater continues to increase, representing a risk hazard for aquatic biota. In the aquatic environment, NP enter fish cells, accumulate in vesicles, and interact with organelles and DNA. This study evaluated the acute (24 h) toxic potential of TiO2NP in the emergent Neotropical model zebrafish liver (ZFL) cell lines at concentrations of 0, 250, 500, 750, and 1000 ng mL−1. The main aim was to determine if TiO2NP causes ROS production and affects cell function due to membrane damage, mitochondrial activity impairment, lysosome malfunction, and DNA damage, which may lead to cell death. High concentrations (750 and 1000 ng mL−1) of TiO2NP increased ROS production and caused cell membrane damage, cell proliferation, lysosome proliferation or swelling, DNA strand breaks, and a low frequency of micronucleus formation. Cytotoxicity and genotoxicity were likely related to ROS production and NP uptake, which impair cell function and integrity and activate necrosis and apoptosis. This study contributes to the understanding of TiO2NP cytotoxicity in fish cells and provides new insights into proliferative hepatocyte responses that allow ZFL cells to lower TiO2NP toxicity and avoid its mutagenic potential.

Violacein from an Antarctic Iodobacter sp. 7MAnt and its function as immunomodulator of the defence mechanism of innate immunity in fish cells.

Violacein from an Antarctic Iodobacter sp. 7MAnt and its function as immunomodulator of the defence mechanism of innate immunity in fish cells.

Differential Nervous Necrosis Virus (NNV) Replication in Five Putative Susceptible Cell Lines.

Viral encephalopathy and retinopathy caused by nervous necrosis virus (NNV), is one of the most threatening viral diseases affecting marine fish worldwide. In vitro propagation of NNV strains is essential for the design of effective control measures. In the present study we analysed both the susceptibility and the permissiveness of five fish cell lines (E-11, GF-1, SAF-1, DLB-1, and SaB-1) to three NNV strains (one RGNNV, one SJNNV, and one reassortant RGNNV/SJNNV). E-11 and DLB-1 were demonstrated to be highly susceptible to NNV strains, with average adsorption efficiency (AE) values higher than 90%. SAF-1 also showed high susceptibility (AE 88%), whereas GF-1 can be regarded as moderately susceptible (AE around 50%). On the contrary, SaB-1 can be considered a poorly susceptible cell line (AE values below 20%). E-11 and GF-1 cell lines provided the highest production rates for RGNNV and RG/SJ (around 103) and both cell lines can be regarded as fully permissive for these viral types. However, the SJNNV production rate in GF-1 was only 17.8 and therefore this cell line should be considered semi-permissive for this genotype. In SAF-1 cells, moderate viral replication was recorded but differences in intracellular and extracellular production suggest that viral progeny was not efficiently released. In DLB-1 and SaB-1 the final viral titres obtained in E-11 were lower than those of the inoculum. However, RNA1 synthesis values seem to indicate that RGNNV replication in DLB-1 and SAF-1 could have been underestimated, probably due to a poor adaptation of the virus grown in these cell lines to E-11. Based on all these results, E-11 seems to be the most appropriate cell for in vitro culture of RGNNV, SJNNV, and reassortant strains.

Sea Bass Primary Cultures versus RTgill-W1 Cell Line: Influence of Cell Model on the Sensitivity to Nanoparticles.

Determination of acute toxicity to vertebrates in aquatic environments is mainly performed following OECD test guideline 203, requiring the use of a large number of fish and with mortality as endpoint. This test is also used to determine toxicity of nanomaterials in aquatic environments. Since a replacement method for animal testing in nanotoxicity studies is desirable, the feasibility of fish primary cultures or cell lines as a model for nanotoxicity screenings is investigated here. Dicentrarchus labrax primary cultures and RTgill-W1 cell line were exposed to several concentrations (0.1 to 200 ug/mL) of different nanoparticles (TiO2, polystyrene and silver), and cytotoxicity, metabolic activity and reactive oxygen species formation were investigated after 24 and 48 h of exposure. Protein corona as amount of protein bound, as well as the influence of surface modification (-COOH, -NH2), exposure media (Leibovitz’s L15 or seawater), weathering and cell type were the experimental variables included to test their influence on the results of the assays. Data from all scenarios was split based on the significance each experimental variable had in the result of the cytotoxicity tests, in an exploratory approach that allows for better understanding of the determining factors affecting toxicity. Data shows that more variables significantly influenced the outcome of toxicity tests when the primary cultures were exposed to the different nanoparticles. Toxicity tests performed in RTgill-W1 were influenced only by exposure time and nanoparticle concentration. The whole data set was integrated in a biological response index to show the overall impact of nanoparticle exposures.

Insights into the functional role of grass carp IL-8 in head kidney leukocytes: pro-inflammatory effects and signalling mechanisms.

Interleukin-8 (IL-8) is a critical chemokine regulating immune cells' chemotaxis as well as their physiological or pathological activations. In fish cells, recombinant IL-8 proteins induced transcriptions of pro-inflammatory cytokines. Nonetheless, the exact mechanisms underlying the function of fish IL-8 as a pro-inflammatory cytokine are still unclear. In this paper, the authors first prepared recombinant grass carp IL-8 (rgcIL-8) using an Escherichia coli expression system, and later confirmed rgcIL-8 increased gene expression of il8, il1β and tumour necrosis factor alpha (tnfα) in grass carp head kidney leukocytes (HKLs). Using signalling pathway inhibitors, the authors showed that rgcIL-8 regulated transcriptions of pro-inflammatory cytokines via MAPK and/or NF-κB signalling pathways. They cloned gcIL-8-specific receptor CXCR1 and subsequently discovered that gcIL-8 could increase the activity of NF-κB and the transcription of IL-1β via CXCR1. Simultaneously, antibody neutralization assay showed that endogenous IL-8 is partially relevant to the self-regulation of IL-1β. Moreover, rgcIL-8 led to the expression of inducible nitric oxide synthase gene, causing an accumulation of nitric oxide in the culture medium of HKLs, suggesting the potential of gcIL-8 to mediate inflammatory response. This study not only enriched the function of IL-8 in teleost but also revealed it as a potential target for the inflammatory control in grass carp.

Fish-specific TLR18 in Nile tilapia (Oreochromis niloticus) recruits MyD88 and TRIF to induce expression of effectors in NF-κB and IFN pathways in melanomacrophages

Toll-like receptors (TLRs) are evolutionarily conserved proteins of pattern recognition receptors (PRRs) and play a crucial role in innate immune systems recognition of conserved pathogen-related molecular samples (PAMPs). We identified and characterized TLR18 from Nile tilapia (Oreochromis niloticus), OnTLR18, to elucidate its role in tissue expression patterns, modulation of gene expression after microbial challenge and TLR ligands, subcellular localization in fish and human cells, and the possible effectors TLR18 induces in a melanomacrophage-like cell line (tilapia head kidney (THK) cells). OnTLR18 expression was detected in all tissues examined, with the highest levels in the intestine and the lowest in the liver. OnTLR18 transcript was up-regulated in immune-related organs after bacterial and polyinosinic-polycytidylic acid (poly I:C) challenges and in the THK cells after lipopolysaccharide (LPS) stimulation. In transfected THK and human embryonic kidney (HEK) 293 cells, OnTLR18 localizes in the intracellular compartment. OnMyD88 and OnTRIF, but not OnTIRAP, were co-immunoprecipitated with OnTLR18, suggesting that the former two molecules are recruited by OnTLR18 as adaptors. The constitutively active form of OnTLR18 induced the production of pro-inflammatory cytokines, type I interferon (IFN), and antimicrobial peptides such as tumor necrosis factor α, interferon (IFN) d2.13, tilapia piscidin (TP)2, TP3, TP4, and hepcidin in THK cells. Our results suggest that OnTLR18 plays an important role in innate immunity through initiating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IFN signaling pathways via OnMyD88 and OnTRIF and induces the production of various effectors in melanomacrophages.

In Vitro Fish Models for the Analysis of Ecotoxins and Temperature Increase in the Context of Global Warming.

Rising temperatures can affect fish survival, especially from shallower waters, as temperatures increase faster and more intensively in these areas; thus, species-specific temperature tolerance can be exceeded. Additionally, the amounts of anthropogenic pollutants are higher in coastal waters. Although increasing metabolic activity at higher temperatures could lead to stronger effects of toxins, there are hardly any studies on this topic. Subsequently, the aim was to investigate the response of fish cells upon exposure to industrial solvents (ethanol, isopropanol, dimethyl sulfoxide (DMSO)) in relation to a temperature increase (20 °C and 25 °C). Concerning the 3Rs (the replacement, reduction and refinement of animal experiments), in vitro tests were used for two threatened, vulnerable fish species: maraena whitefish (Coregonus maraena) and Atlantic sturgeon (Acipenser oxyrinchus). Both cell lines exhibited higher proliferation at 25 °C. However, ecotoxicological results indicated significant differences regarding the cell line, toxin, temperature and exposure time. The evolutionarily older fish lineage, Atlantic sturgeon, demonstrated lower mortality rates in the presence of isopropanol and recovered better during long-term ethanol exposure than the maraena whitefish. Atlantic sturgeon cells have higher adaptation potential for these alcohols. In summary, fish species respond very specifically to toxins and changes in temperature, and new ecotoxicological questions arise with increasing water temperatures.

Nanopharmaceuticals (Au-NPs) after use: Experiences with a complex higher tier test design simulating environmental fate and effect

The current environmental hazard assessment is based on the testing of the pristine substance. However, it cannot be excluded that (nano)pharmaceuticals are excreted into sewage during the use phase followed by entry into wastewater treatment plants (WWTPs). Sorption to sewage sludge or release via effluent can result in modified ecotoxicological effects which possibly can only be detected with a modified test approach. The objective of our study was to investigate a realistic exposure scenario for metallic nanoparticles (NPs) in pharmaceutical products, excreted into effluent, and released into the environment after treatment in WWTPs. The test approach was illustrated by using gold (Au) NPs. Effluent from model WWTPs were investigated in aquatic tests (Daphnia magna, fish cell lines). Sewage sludge was used as a sole food source (Eisenia fetida) or mixed with soil and used as test medium (soil microorganisms, Folsomia candida, Enchytraeus crypticus). To cover the aspect of regulation, the test systems described in OECD-test guidelines (OECD TG 201, 211, 220, 232, 249, 317) were applied. Modifications and additional test approaches were included to meet the needs arising out of the testing of nanomaterials and of the exposure scenarios. The results were assessed regarding the suitability of the test design and the toxicity of Au-NPs. Except for activated sludge as a sole food source for E.fetida, the selected test approach is suitable for the testing of nanomaterials. Additional information can be gained when compared to the common testing of the pristine nanomaterials in the standardized test systems. Effects of Au-NPs were observed in concentrations exceeding the modeled environmental.

Engagement of gcFKBP5/TRAF2 by spring viremia of carp virus to promote host cell apoptosis for supporting viral replication in grass carp

Spring viremia of carp virus (SVCV) causes severe morbidity and mortality in grass carp (Ctenopharyngodon idellus) in Europe, America and several Asian countries. We found that FKBP5 (FK506-binding protein 5) is an SVCV infection response factor; however, its role in the innate immune mechanism caused by SVCV infection remains unknown. This study cloned gcFKBP5 (grass carp FKBP5) and made its mimic protein structure for function discussion. We found that gcFKBP5 expression in the primary innate immune organs of grass carp, including intestine, liver and spleen, was highly upregulated by SVCV in 24 h, with a similar result in fish cells by poly(I:C) treatment. gcFKBP overexpression aggravates viral damage to cells and increases viral replication. Furthermore, SVCV engages gcFKBP5 interacting with TRAF2 (tumour necrosis factor receptor-associated factor 2) to promote host cell apoptosis for supporting viral replication. The enhanced viral replication seems not to be due to the repression of IFN and other antiviral factors as expected. For the first time, these data show the pivotal role of gcFKBP5 in the innate immune response of grass carp to SVCV infection.

Evidence of Coordinated and Adjustable Osmolytes Movements Following Hyposmotic Swelling in Rainbow Trout Red Blood Cells

BACKGROUND/AIMS: The osmolytes involved in the volume regulation of hyposmotically-swollen fish cells are well identified. However, if a coordination and adjustments of their fluxes are obvious, few studies have clearly illustrated these aspects. METHODS: Trout red blood cells volume variations were estimated from water contents obtained by a gravimetric method. Intracellular K+ and Na+ contents, and Cl- content of haemolysed cells were determined by photometry and colorimetry, respectively. The taurine contribution to cell volume regulation was calculated from the net changes of water, K+, Cl- and Na+ contents. The intracellular pH was calculated from the chloride distribution across the cells membranes according to the Donnan equilibrium. RESULTS: Cells responses to a rapid change (from 296 to 176 mOsm.kg-1) of the saline osmolality were examined in three conditions designed to not impact (Hypo. I) or to reduce the K+ (Hypo. II) and Cl- (Hypo. III) contributions to the volume regulation. Hypo. I condition caused an immediate increase in water content, followed by a 90 min. full regulation, concomitant with gradual lowering of K+ and Cl- contents and a surprising increase in Na+ content. Hypo. II and III conditions showed a partial and complete volume regulation, respectively. This was made possible by an increase in the taurine involvement. These experiments allowed to confirm that K+ and Cl- were released via KCl cotransport and by separate channels. The comparison of Hypo. I and III conditions led to the observation that the partially amiloride-sensitive Na+ influx is proportional to the taurine efflux; the latter being sustained mainly by a Na+/taurine cotransport. The Hypo. II condition was suitable for the (Na+/K+)ATPase activity inhibition. This effect could explain the observed lack of Na+ uptake, the consecutive depletion of intracellular taurine stock and the incomplete volume regulation. Finally, the results support the importance of taurine in pH control under Hypo. I (physiologic) condition. The alkalosis observed in Hypo. II and III conditions were the consequences of changes in the salines compositions, not of physiologic adjustments. CONCLUSION: The regulatory volume decrease process of trout RBCs is complex and adjustable through coordinated osmolytes movements. The obliged decrease in K+ and/or Cl- contributions stimulates taurine and Na+ pathways. This study highlights the importance of taurine as a compensatory variable in cell volume regulation and explains for the first time the significance of the Na+ uptake during this process

Mechanisms of Silver Nanoparticle Uptake by Embryonic Zebrafish Cells.

Evaluation of the uptake pathways in cells during exposure to nanoparticles (NPs) is key for risk assessment and the development of safer nanomaterials, as the internalisation and fate of NPs is linked to their toxicity and mode of action. Here, we determined the uptake mechanisms activated during the internalisation of 10, 30, and 100 nm AgNPs by embryonic zebrafish cells (ZF4). The uptake results demonstrated an NP size- and time-dependent uptake, showing the highest total silver uptake for the smallest AgNP (10 nm) at the lowest exposure concentration (2.5 μg/mL) after 2 h, while after 24 h, the highest exposure concentration (10 μg/mL) of the 10 nm AgNPs revealed the highest cellular load at 8 pg/cell. Inhibition of the caveolae, clathrin, and macropinocytosis endocytic pathways by pharmaceutical inhibitors (genistein, chlorpromazine, and wortmannin respectively) revealed that uptake was mainly via macropinocytosis for the 10 nm AgNPs and via the caveolae-mediated pathway for the 30 and 100 nm AgNPs. The induction of autophagy was also strongly related to the NP size, showing the highest percentage of induction for the 10 nm (around 3%) compared to naive cells, suggesting that autophagy can be activated along with endocytosis to deal with exposure to NPs. TEM imaging revealed the distribution of NPs across the cytoplasm inside intracellular vesicles. An increase in Early Endosome formation (EE) was observed for the 30 and 100 nm sizes, whereas the 10 nm AgNPs disrupted the activity of EE. The data supports the establishment of adverse outcome pathways by increasing knowledge on the link between a molecular initiating event such as receptor-mediated endocytosis and an adverse outcome, as well as supporting the reduction of animal testing by using alternative testing models, such as fish cell lines.

Establishment, characterization, and transfection potential of a new continuous fish cell line (CAM) derived from the muscle tissue of grass goldfish (Carassius auratus).

A new continuous fish cell line (CAM) has been successfully derived from the muscle tissues of grass goldfish, Carassius auratus. The primary cell cultures were initiated by incomplete trypsinization first and then explant culture in a Leibovitz-15 medium supplemented with 15% fetal bovine serum and 10% fish muscle extract. It was found that the CAM cells were very sensitive to trypsinization and needed to be sub-cultured at a low trypsin concentration of 0.0625% to be able to go through the crisis of spontaneous immortalization transformation, and afterward a total of five derivative cell strains were isolated from the original CAM cell line. This spontaneous immortalization transformation event was recorded successively at passages 44-47, beginning with a large-scale apoptosis and senescence and followed by mitosis arrest and re-activation, thus designated as cell strain CAM-44A, 44B, 45A, 44B, and 47A. Now both the CAM cell line and strains had been sub-cultured for more than 89 times and could be well cryopreserved in the growth medium containing 5% dimethylsulfoxide. Chromosome analysis and COI gene analysis had confirmed the grass goldfish origin of these CAM cells. Transfection potential analysis indicated that Lipofectamine LTX and Xfect were two suitable transfection reagents to be used in the gene delivery of CAM cells with a transfection efficiencies up to 11±6% and 8±3% in the CAM cell lines, respectively. Among the five cell strains, CAM-47A showed the highest transfection potential with a transfection efficiency up to 28 ± 5%. This work will provide a useful cell source for works on the cell-based artificial fish meat production and functional studies of fish myogenesis-related genes.

An investigation into the pathogenesis of blue catfish alloherpesvirus in ictalurid catfish

AbstractIntensive aquaculture production systems often encounter infectious viral disease outbreaks causing substantial fish mortalities and associated economic losses. Blue catfish alloherpesvirus (BCAHV) is an Ictalurid herpesvirus with limited information on pathogenesis. To determine the host‐specificity and potential host range of BCAHV, the virus was inoculated onto various fish cell lines belonging to families Ictaluridae, Cyprinidae, Centrarchidae, and Clariidae. Viral replication and exhibition of cytopathic effects (CPEs) were restricted to cell lines from the family Ictaluridae indicating the host preference of BCAHV. Subsequently, fish challenges were conducted to evaluate intrinsic and extrinsic factors influencing BCAHV infection. Mortality because of BCAHV infection was significantly higher in blue catfish and least in channel catfish further specifying the host preference of the virus. Histopathological assessment of the BCAHV infected fish revealed splenitis with severe erythrophagia. Host susceptibility to BCAHV differed with age with fish at 60 days post hatch being more susceptible. Temperature had a significant role in the activation and pathogenesis of BCAHV. Fish exposed to BCAHV at a sustained high temperature had significantly low mortality suggesting the likelihood of virus inactivation. Virus exposure in tanks with high stocking density resulted in significantly high fish mortalities indicating horizontal transmission is a key element in BCAHV pathogenesis. The heightened pathogenicity of BCAHV in blue and hybrid catfish as observed in this study validates its potential to be a significant pathogen in catfish production.

Mathematical modeling and analysis of the heat shock protein response during thermal stress in fish and HeLa cells

The climate change has the potential to dramatically affect species’ thermal physiology and may change the ecology and evolution of species’ lineages. In this work, we investigated the transition of dynamics in the heat shock response when the thermal stress approaches the upper thermal limits of species to understand how the climate change may affect the heat shock responses in ectotherms and endotherms. The heat shock protein 70, HSP70, prevents protein denaturation or misfolding under thermal stresses. When thermal stress increases, the number of misfolded proteins increases, which leads to high levels of HSP70 protein. However, when temperatures approach limits of thermal tolerance (i.e., the critical thermal maximum, CTmax, for ectotherms and the superior critical temperature, SCT, for endotherms), levels of HSP70 protein synthesis start to decrease. Thus, we hypothesized that the temperature at the first reduction of HSP abundance indicates the thermal limits of the species. In this work, we provide a mathematical model to investigate the behavior of the heat shock responses related to HSP70 protein. This model captures the dynamics of HSP70 protein and Hsp70 mRNA, in HeLa cells (i.e., representative for endotherms) and multiple species of fishes (i.e., representative for ectotherms) with different acclimation histories. Based on our hypothesis of the relationship between the HSP70 protein level and CTmax/SCT, our model provides three methods to predict the CTmax of fishes with varying acclimation temperature and the SCT of HeLa cells. The CTmax increases as the acclimation temperature increases in fishes, however the CTmax plateaus when the acclimation temperature is higher than 20°C in brook trout, a representative cool water salmonid. Our model also captures the situation that the heat shock reaction in fish is more sensitive to the heat shock temperature than HeLa cells, when the heat shock temperature is lower than the upper thermal tolerance. However, both fish and HeLa cells are sensitive to the heat shock temperature when the temperature reaches the thermal tolerance limits. Additionally, our sensitive analysis result indicates that the status of some components in the heat shock reaction changes when the temperature reaches the thermal tolerance resulting in failure in protein refolding in fish and speeding up the refolding process in HeLa cells. Mathematical analysis is also applied on a simplified mathematical model to investigate the detailed dynamics of the model, such as the steady states of the substrate, Hsp70 mRNA, and HSP70 protein, at different thermal stresses. The comparison between the original model and simplified model shows that the inhibition on HSP70 protein transcription by thermal stresses leads to the reduction in HSP70 protein at extreme temperatures.

Establishment of a new fish cell line from the brain of humpback grouper (Cromileptes altivelis) and its application in toxicology and bacterial susceptibility.

Cromileptes altivelis, humpback grouper, belongs to the family Epinephelidae and is one popular farmed fish species because of its high economic value and ornamental value. However, more and more diseases outbreaks have been reported with C. altivelis aquaculture. Today, a new brain cell line of C. altivelis (named CAB) was established and characterized. Our results showed that CAB cells were suitable for growth at 26°C in L-15 medium supplemented with 15% fetal bovine serum (FBS). The results of 18S rRNA gene sequencing confirmed that CAB cell line was derived from C. altivelis. Moreover, chromosomal aneuploidy was observed in CAB cells, and the modal chromosome number of CAB cells was 48 by chromosome analysis. In addition, CAB cells could transfect pEGFP-N3 plasmid with high transfection efficiency, indicating that CAB cell line has the potential to investigate the function of exogenous genes in vitro. Furthermore, the bacterial susceptibility results suggested that CAB cells were susceptive to Vibrio harveyi and Edwardsiella tarda. And, heavy metals (Hg, Cd, and Cu) were toxic to the CAB cells, and the toxic effect was dose-dependent. In summary, the CAB cell line could be a powerful tool in vitro to study functional genes and has the potential application in bacterial susceptibility and toxicology.