Fish Cell Research Articles

Cytotoxicity and apoptosis induced by enrofloxacin in loach fin cells in vitro

The cytotoxic effect and cell death were studied in loach fin cells in vitro after enrofloxacin (ENR) exposure. The semi-lethal concentration of ENR for loach cells was calculated as 1296.2 ± 3.11 mol/L (about 512.5 mg/L). Loss of cell viability, increase in vacuoles, disappearance of microvilli, and apoptotic bodies were evident in cells exposed to 400, 800, and 1200 μmol/L ENR. Besides, dose-dependent inhibitory effects on SOD, CAT, Na+-K+-ATPase, and Ca2+-ATPase activities were also observed in loach cells exposed to ENR. Quantitative gene expression results showed that ENR induced caspase-3- and caspase-8-mediated apoptosis as well as caspase-activated DNase in loach cells. The findings also indicated a role of JNK pathway in ENR-induced apoptosis in loach cells. Transcriptome sequencing results showed 10,016 differentially expressed genes in ENR vs. control groups, which were all enriched in “Molecular Function” process in GO term. Furthermore, 6763 genes were enriched in 291 KEEG pathways, with most of them belonging to immune and material metabolic pathways. The large number of transcriptome data and pathways determined in this study provide a database foundation for the toxicity analysis of ENR in loach cells, which must be thoroughly examined to further investigate the cytotoxic mechanism of antibiotics in fish cells.

Kinetics of the invasion of a non-phagocytic fish cell line, RTG-2 by Yersinia ruckeri serotype O1 biotype 1.

Yersiniosis, caused by the fish pathogen Yersinia ruckeri, is a serious bacterial septicaemia affecting mainly salmonids worldwide. The acute infection may result in high mortality without apparent external disease signs, while the chronic one causes moderate to considerable mortality. Survivors of yersiniosis outbreaks become carriers. Y. ruckeri is able to adhere to, and to invade, phagocytic and non-phagocytic fish cells by using unknown molecular mechanisms. The aim of this study was to describe the kinetics of cell invasion by Y. ruckeri serotype O1 biotype 1 in a fish cell line (RTG-2) originating from rainbow trout gonads. The efficiency of invasion by Y. ruckeri was found to be temperature dependent, having a maximum at 20 °C. The bacterium was able to survive up to 96 h postinfection. The incubation of the cells at 4 °C and the pre-incubation of the bacteria with sugars or heat-inactivated antiserum significantly decreased the efficiency of invasion or even completely prevented the invasion of RTG-2 cells. These findings indicate that Y. ruckeri is capable of adhering to, entering and surviving within non-phagocytic cells, and that the intracellular environment may constitute a suitable niche for this pathogen that can favour the spread of infection and/or the maintenance of a carrier state of fish.

Mitochondrial changes in fish cells in vitro in response to serum deprivation.

Mitochondria are critical to cellular activity that implicated in expansive networks to maintain organismal homeostasis under external stimuli of nutrient variability, a common and severe stress to fish performance during the intensive culture conditions. In the present study, zebrafish embryonic fibroblast cells were used to investigate the fish mitochondrial changes upon serum deprivation. Results showed that mitochondrial content and membrane potential were significantly reduced with increased intracellular ROS level in the serum deprivation treated fish cells. And the impaired mitochondria were characterized by rough and fracted outer membrane, and more fused mitochondria were frequently observed with the upregulated mRNA expressions of mitochondrial fusion genes (mfn1b, mfn2, and opa1). Besides, the mitochondrial DNA (mtDNA) copy numbers of mtatp6, mtcox1, mtcytb, mtnd4, and mtnd6 were overall showing the highly significant reduction, together with the mRNA expressions of these genes significantly increased, exhibiting the compensatory effects in mitochondria. Furthermore, the methyl-cytosine of whole mtDNA was compared and the methyl-reads numbers were distinctly increased in the treatment group, reflecting the instability of fish mtDNA with mitochondrial dysfunction under nutrient fluctuations. Collectively, current findings could facilitate the integrated research between fish mitochondrial response and external variables that indicates the potentially profound and durative deficits in fish health during the aquaculture processes.

Vibrio cholerae: a pathogen shared by human and aquatic animals

Vibrio cholerae: a pathogen shared by human and aquatic animals

Oxidative stress, autophagy, and apoptosis induced by doxycycline in loach fin cells in vitro

Cytotoxicity, molecular function disorder, mitophagy, and apoptosis were studied in loach fin cells in vitro after exposure to doxycycline (DOX). The semi-lethal concentration of DOX in loach cells was calculated as 668.96 ± 2.83 mol/L. Loss of cell viability and increases in vacuoles and autolysosomes were evident in cells exposed to DOX at 200 and 400 μmol/L, and apoptotic bodies occurred at 600 μmol/L. In addition, Superoxide Dismutase (SOD), catalase (CAT), Na+-K+-ATPase, and Ca2+-ATPase activities increased significantly in cells exposed to 200 μmol/L DOX, and dose-dependent inhibitory effects on activities were observed in cells exposed to 400 and 600 μmol/L DOX. Quantitative gene expression showed that 400 and 600 μmol/L DOX could induce caspase-3- and caspase-8-mediated apoptosis as well as caspase-activated DNase in loach cells. Transcriptome sequencing in DOX vs. control groups found 16,288 differentially expressed genes, among which protein binding (2633, 31.91%) was the most significant in Gene Ontology terms. Furthermore, 11,930 genes were enriched in 298 Kyoto Encyclopedia of Genes and Genomes (KEGG)pathways. The top three upregulated pathways included “lysosome”, “protein processing in endoplasmic reticulum”, and “proteasome”. FPKM analysis indicated that most genes associated with autophagy and in “protein processing in the endoplasmic reticulum”, “TNF signaling pathway”, and “NF-kappa B signaling pathway” were upregulated. This suggests that at lower concentrations, DOX induces reactive oxidative species (ROS) in loach fin cells to reduce cell proliferation. ROS in turn stimulate oxidant stress, ion excretion capability and mitophagy to maintain cell homeostasis. Apoptosis was induced in cells subjected to higher concentrations of DOX. The transcriptome data and pathways determined in this study will provide a foundation for the analysis of DOX toxicity in loach cells, which must be examined thoroughly to further understand the cytotoxic mechanism of antibiotics in fish cells.

Edwardsiella piscicida YccA: A novel virulence factor essential to membrane integrity, mobility, host infection, and host immune response

YccA is a hydrophobic protein with seven transmembrane domains. The function of YccA is largely unknown in pathogenic bacteria. Edwardsiella piscicide (formerly known as E. tarda) is an aquatic pathogen that can infect various economically important fish, including flounder (Paralichthys olivaceus) and tilapia (Oreochromis niloticus). In this study, we investigated the role of YccA in E. piscicida by the construction of a markerless yccA in-frame mutant strain, TX01ΔyccA. We found that (i) in comparison to the wild type TX01, TX01ΔyccA exhibited markedly compromised tolerance to high temperature and tobramycin; (ii) deletion of yccA significantly impaired the integrity of the cell membrane and retarded bacterial biofilm formation and mobility; (iii) deficiency of yccA reduced bacterial adhesion and invasion of fish cells and immune tissues, while the introduction of a trans-expressed yccA gene restored the lost virulence of TX01ΔyccA; and (iv) host immune responses induced by TX01 and TX01ΔyccA were different in terms of reactive oxygen species (ROS) levels and expression levels of cytokines. Taken together, the results of our study indicate that YccA is a novel virulence factor of E. piscicida, and YccA is essential for bacterial pathogenicity through evasion of the host's innate immune functions.

Comparative analysis on the immunoregulatory roles of ferritin M in hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var ♂) and its parental species after bacterial infection

Ferritin M is involved in the regulation of fish immunity. In this study, open reading frame (ORF) sequences of ferritin M from hybrid fish and its parental species were 534 bp. Tissue-specific analysis indicated that the highest level of ferritin M from red crucian carp was observed in kidney, while peaked expressions of ferritin M from white crucian carp and hybrid carp were observed in gill. Elevated levels of ferritin M from hybrid carp and its parental species were detected in immune-related tissues following Aeromonas hydrophila infection or in cultured fish cell lines after lipopolysaccharide (LPS) challenge. Ferritin M overexpression could attenuate NF-κB and TNFα promoter activity in their respective fish cells. Purified ferritin M fusion proteins elicited in vitro binding activity to A. hydrophila and Edwardsiella tarda, lowered bacterial dissemination to tissues and alleviated inflammatory response. Furthermore, treatment with ferritin M fusion proteins could mitigate bacteria-induced liver damage and rescue antioxidant activity. These results suggested that ferritin M in hybrid fish showed a similar immune defense against bacteria infection in comparison with those of its parental species.

Orphan G-Protein Coupled Receptor GPRC5B Is Critical for Lymphatic Development.

Numerous studies have focused on the molecular signaling pathways that govern the development and growth of lymphatics in the hopes of elucidating promising druggable targets. G protein-coupled receptors (GPCRs) are currently the largest family of membrane receptors targeted by FDA-approved drugs, but there remain many unexplored receptors, including orphan GPCRs with no known biological ligand or physiological function. Thus, we sought to illuminate the cadre of GPCRs expressed at high levels in lymphatic endothelial cells and identified four orphan receptors: GPRC5B, AGDRF5/GPR116, FZD8 and GPR61. Compared to blood endothelial cells, GPRC5B is the most abundant GPCR expressed in cultured human lymphatic endothelial cells (LECs), and in situ RNAscope shows high mRNA levels in lymphatics of mice. Using genetic engineering approaches in both zebrafish and mice, we characterized the function of GPRC5B in lymphatic development. Morphant gprc5b zebrafish exhibited failure of thoracic duct formation, and Gprc5b−/− mice suffered from embryonic hydrops fetalis and hemorrhage associated with subcutaneous edema and blood-filled lymphatic vessels. Compared to Gprc5+/+ littermate controls, Gprc5b−/− embryos exhibited attenuated developmental lymphangiogenesis. During the postnatal period, ~30% of Gprc5b−/− mice were growth-restricted or died prior to weaning, with associated attenuation of postnatal cardiac lymphatic growth. In cultured human primary LECs, expression of GPRC5B is required to maintain cell proliferation and viability. Collectively, we identify a novel role for the lymphatic-enriched orphan GPRC5B receptor in lymphangiogenesis of fish, mice and human cells. Elucidating the roles of orphan GPCRs in lymphatics provides new avenues for discovery of druggable targets to treat lymphatic-related conditions such as lymphedema and cancer.

Isolation and characterization of Aeromonas hydrophila-specific lytic bacteriophages

Aeromonas hydrophila can infect a broad range of fish living in freshwater and estuaries. Because the majority of A. hydrophila is resistant to antimicrobials, alternative treatment strategies should be developed. Bacteriophage may be an alternate approach to control infectious diseases. For this reason, we isolated AP-T65, AP-T5, AP-Y28, and AP-AT phages and assessed their lytic activity against A. hydrophila strains, described them phenotypically, kinetically, and genetically, and evaluated their cytotoxicity in vitro and in vivo assays. After sequencing the major capsid protein (MCP) gene of the phages, it was found that all phages belonged to the Myroviridae family. Phages demonstrated lytic activity against Aeromonas sp., A. salmonicida, A. veronii, and A. media. Phages had a short latent period of roughly 10–15 min and the burst sizes of all Aeromonas phages were found to be between 10 and 20 plaque forming units (PFU)/infected cell. The AP-T65, AP-T5, AP-Y28, and AP-AT phages survived a wide range of pH values ranging from 3 to 10, as well as temperatures ranging from 4 to 37 °C. Aeromonas phages showed no impact on fish or EPC cell lines. Therefore, Aeromonas phages are safe and they can be used as a cocktail to treat A. hydrophila infection. Because of their short latent period and greater burst size, phages isolated in the present study would be ideal candidates for phage therapy, and they might be used as a cocktail for improved control effects. The application of a cocktail of these phages may also prevent the formation of phage-resistant bacteria.

TLR23, a fish-specific TLR, recruits MyD88 and TRIF to activate expression of a range of effectors in melanomacrophages in Nile tilapia (Oreochromis niloticus)

Nile tilapia (Oreochromis niloticus) is an important food fish species that is mainly cultivated in tropical and subtropical countries. However, microbial diseases have created various difficulties for this industry. The fundamental prerequisite for tackling disease outbreak prevention and disease resistance is to know how hosts’ immune responses against invading microbes are initiated. Toll-like receptors (TLRs) are vital pattern recognition receptors and play pivotal roles in the cellular innate immunity defense that is able to recognize pathogen-associated molecular patterns (PAMPs). In this study, Oreochromis niloticus TLR23 (OnTLR23) was cloned and bioinformatic analyses revealed that OnTLR23 is not an ortholog of mammalian TLR13 as previously suggested. The basal transcript level of OnTLR23 was found to be higher in the immune-related organs and was upregulated in the spleen and/or head kidney following Aeromonas hydrophila, Streptococcus agalactiae or poly I:C injections, and increased in the melanomacrophage-like tilapia head kidney (THK) cell line after LPS and zymosan stimulation. Furthermore, we demonstrated for the first time that OnTLR23 locates mainly in the intracellular region in fish cells and the constitutively active form of OnTLR23 promotes the expression of molecules related to antigen presentation, proinflammatory cytokines, antimicrobial peptides and type I interferon in THK cells. A co-immunoprecipitation assay revealed that OnTLR23 can interact with both OnMyD88 and OnTRIF, but not with OnTIRAP. A luciferase assay showed that the NF-κB activity was not elevated in the OnTLR23 overexpressed THK cells after treatment with ligand for TLR13 as well as other known purified bacterial-derived ligands of TLRs. Taken together, OnTLR23 is likely to recruit OnMyD88 and OnTRIF as adaptors to induce the expression of various effectors in melanomacrophages, but its corresponding ligand is an issue awaiting further investigation.

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

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

Microglia, the Sentinel of Brain in the Evolution of Nervous System from Invertebrate to Vertebrate: A Short Review

The presence of microglial cells as resident macrophage population in the Central Nervous System (CNS) is well documented from the study of repairing of lesions in CNS that varies widely throughout the animal kingdom. The existence of neuroglia cells similar to vertebrate microglia and small mobile phagocytes and hemocytes were documented from ganglia of some invertebrate animal models like leech (H. medicinalis), insects (P. americana and D. melanogaster) and mollusca (M. edulis). Neuronal replacement and migration of immunocompetent cells (macrophage, microglia, ependymal cells etc.) after surgical lesions in CNS of non-mammals (fishes, reptiles and aves) are much restricted to specific neurogenic niches associated to the neural regeneration and migration of cells in invertebrates. Microglial presence is largely restricted in the optic tract of fish and amphibian ganglionic cells because they have a surprising capacity to regenerate their neurons after lesions. Hence the CNS of both invertebrates and vertebrates contain microglia like mononuclear phagocytes, ensheathing glia and reticular glia, which indicate about the evolutionary conserved innate immune response to maintain CNS development and health. But the presence and gradual changes in the structure and function of microglia and neuron-microglia relationship in the CNS along the phylogeny need to be focused thoroughly.

Identification and functional characterization of an immune adapter molecular TRIF in Northeast Chinese lamprey (Lethenteron morii)

The TIR domain-containing adaptor inducing IFN-β (TRIF) is an adaptor molecule that plays a critical role in the Toll-like receptors (TLRs)-mediated innate immune signaling pathway. Lamprey, as the most primitive jawless vertebrate, rely mainly on innate immunity to defend against various pathogens infection. The function of TRIF in lamprey remains unknown. In this study, a homologous adaptor molecule TRIF, named LmTRIF, was identified in Northeast Chinese lamprey (Lethenteron morii). The LmTRIF coding sequence (cds) is 1242 bp in length and encodes 413 amino acids (aa). Domain analysis showed that LmTRIF is characterized with the classical TIR domain and a lack of TRAF6 binding motif. The results of evolutionary tree indicated that the relationship between LmTRIF and other homologous proteins was consistent with the position of lamprey in the species evolutionary history. The relative expression of LmTRIF was highest in the liver of larvae and in the gill of adults, respectively. Cellular immunofluorescence assays showed that LmTRIF was expressed in the cytoplasma in both mammalian cell line HEK 293T and the fish cell line EPC. The double luciferase reporter gene assay showed that the overexpression of LmTRIF promoted the activity of NF-κB, an immune transcription factor downstream of the classical TLR signaling pathway. In this study, we identified the TLR adaptor molecule TRIF from L. morii, a vertebrate more primitive than fish. Our results suggested an important role of LmTRIF in the innate immune signal transduction process of L. morii and is the basis for the origin and evolution of the TLR signaling pathway in the innate immune system in vertebrates.

Production and characterization of monoclonal antibodies against grass carp CD4-1 and CD4-2

CD4 T helper cells are an important group of cells in the immune system of vertebrates and express CD4 receptor on the cell surface. In mammals, the CD4 receptor is encoded by a single copy gene, whilst in fish, two copies of cd4 genes, namely cd4-1 and cd4-2, are found. In this study, the ectodomains of grass carp (Ctenopharyngodon idella, Ci) CD4-1 and CD4-2 were expressed in the E. coli cells and used to generate monoclonal antibodies in mice. Western blotting, confocal microscopy and flow cytometry were performed to characterize the monoclonal antibodies. It has been shown that the CiCD4-1 and CiCD4-2 monoclonal antibodies had good specificity to react with the recombinant ectodomains of CiCD4-1 and CiCD4-2 expressed in the CHO–S cells and the native CD4 molecules of grass carp. The CiCD4-1 monoclonal antibody did not recognize CiCD4-2 and verse versa. In addition, the CD4-1 and CD4-2 monoclonal antibodies specifically recognized the CD4-1 and CD4-2 receptors expressed in the HEK293 cells and native molecules of fish cells. Further, the percentages of lymphocytes in immune tissues of healthy fish were analyzed by flow cytometry. It was found that 17.6% of lymphocytes were CD4-1+ cells and 22.5% were CD4-2+ in the head kidney. In the spleen, 13.1% of lymphocytes were CD4-1+ and 18.6% were CD4-2+ while 7.3% of blood lymphocytes were CD4-1+ cells and 8.8% were CD4-2+ cells. The availability of the CD4-1 and CD4-2 monoclonal antibodies provides antibody-based tools for further elucidation of the functions of CD4 T cells in grass carp.

Lack of a p16INK4a/ARF locus in fish genome may underlie senescence resistance in the fish cell line, EPC

Unlike most mammalian cell lines, fish cell lines are immortal and resistant to cellular senescence. Elevated expression of H-Ras contributes to the induction of senescence in a fish cell line, EPC, but is not sufficient to induce full senescence. Here, we focused on the absence of a p16INK4a/ARF locus in the fish genome, and investigated whether this might be a critical determinant of the resistance of EPC cells to full senescence. We found that transfected EPC cells constitutively overexpressing p16INK4a exhibited large size and flat morphology characteristic of prematurely senescent cells; the cells also showed p53-independent senescence-like growth arrest and senescence-associated β-galactosidase (SA-β-gal) activity. Furthermore, the mRNA levels of proinflammatory senescence-associated secretory phenotype (SASP) factors increased in EPC cells constitutively overexpressing p16INK4a. These results suggest that the lack of p16INK4a in the fish genome may be a critical determinant of senescence resistance in fish cell lines.

Assessment of sublethal toxicity using proliferation markers in fish cell line-ICG exposed to agrochemicals

Nanocomposites based on bacterial cellulose in combination with osteogenic growth peptide for bone repair: cytotoxic, genotoxic and mutagenic evaluationsRaquel Mantuaneli Scarel-Caminagaa, Sybele Saskab, Leonardo Pereira Franchic, Raquel A. Santose, Ana Maria Minarelli Gaspara, Ticiana S.O. Capotea, Sidney José Lima Ribeirob, Younés Messaddeqb, Reinaldo Marchetto b, Catarina S. Takahashic d

Hazard profiling of a combinatorial library of zinc oxide nanoparticles: Ameliorating light and dark toxicity through surface passivation

Zinc oxide (ZnO) is one of the high-volume production nanoparticles (NPs) currently used in a wide range of consumer and industrial goods. The inevitable seepage into environmental matrices and the photoactive nature of ZnO NPs warrants hazard profiling under environmentally related conditions. In this paper, the influence of simulated solar light (SSL) on dissolution behaviour and phototoxicity of ZnO NPs was studied using a combinatorial library of ZnO NPs with different sizes, surface coatings, dopant chemistry, and aspect ratios in a fish cell line (BF2) and zebrafish embryos. Generally, the cytotoxicity and embryo mortality increased when exposed concomitantly to SSL and ZnO NPs. The increase in toxic potential of ZnO NPs during SSL exposure concurred with release of Zn ions and ROS generation. Surface modification of NPs with poly(methacrylic acid) (PMAA), silica or serum coating decreased toxicity and ZnO with serum coating was the only NP that had no significant effect on any of the cytotoxicity parameters when tested under both dark and SSL conditions. Results from our study show that exposure to light could increase the toxic potential of ZnO NPs to environmental lifeforms and mitigation of ZnO NP toxicity is possible through modifying the surface chemistry.

Analysis of sialylated N-linked glycans on fish cell lines permissive to nervous necrosis virus for predicting cellular receptors of the virus

Nervous necrosis virus (NNV), one of the simplest spherical RNA-viruses, is the causative agent of severe diseases affecting many fish species. It appears that NNV can bind to sialic acid moieties of N-glycans on host cell surface. To predict cellular receptors for NNV, we analyzed sialylated N-glycans on six fish cell lines with differential permissiveness to NNV. SSN-1, SeGF and BF-2(H) cells were permissive to both red-spotted grouper NNV (RGNNV) and striped jack NNV (SJNNV), while BF-2(A) and RoBE-4 cells were permissive only to RGNNV. FHM cells were not permissive to RGNNV or SJNNV. Matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) analysis revealed that 12–35% of total N-glycans were complex type sialylated N-glycans and that almost all sialic acids at the non-reducing end were N-acetylneuraminic acid (Neu5Ac). Major sialylated N-glycans were mono-, bi- or tri-antennary glycans. A common motif containing Neu5Ac-N-acetyllactosamine (LacNAc) was always present on at least one of the antennae. Both RGNNV and SJNNV could bind to Neu5Ac-LacNAc regardless whether Neu5Ac was α2,3- or α2,6-linked, although RGNNV preferred the former whereas SJNNV preferred the latter. Therefore, N-glycan antennae with terminal Neu5Ac-LacNAc moieties are likely to function as cellular receptors for NNVs.

Exploring Zebrafish Larvae as a COVID-19 Model: Probable Abortive SARS-CoV-2 Replication in the Swim Bladder.

Animal models are essential to understanding COVID-19 pathophysiology and for preclinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap, and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, or bloodstream, resulted in a rapid decrease of SARS-CoV-2 RNA in wild-type larvae. However, when the virus was inoculated in the swim bladder, viral RNA stabilized after 24 h. By immunohistochemistry, epithelial cells containing SARS-CoV-2 nucleoprotein were observed in the swim bladder wall. Our data suggest an abortive infection of the swim bladder. In some animals, several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. A mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro. In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung.

Are the Main Methionine Sources Equivalent? A Focus on DL-Methionine and DL-Methionine Hydroxy Analog Reveals Differences on Rainbow Trout Hepatic Cell Lines Functions.

The replacement of fishmeal by plant proteins in aquafeeds imposes the use of synthetic methionine (MET) sources to balance the amino acid composition of alternative diets and so to meet the metabolic needs of fish of agronomic interest such as rainbow trout (RT-Oncorhynchus mykiss). Nonetheless, debates still exist to determine if one MET source is more efficiently used than another by fish. To address this question, the use of fish cell lines appeared a convenient strategy, since it allowed to perfectly control cell growing conditions notably by fully depleting MET from the media and studying which MET source is capable to restore cell growth/proliferation and metabolism when supplemented back. Thus, results of cell proliferation assays, Western blots, RT-qPCR and liquid chromatography analyses from two RT liver-derived cell lines revealed a better absorption and metabolization of DL-MET than DL-Methionine Hydroxy Analog (MHA) with the activation of the mechanistic Target Of Rapamycin (mTOR) pathway for DL-MET and the activation of integrated stress response (ISR) pathway for MHA. Altogether, the results clearly allow to conclude that both synthetic MET sources are not biologically equivalent, suggesting similar in vivo effects in RT liver and, therefore, questioning the MHA efficiencies in other RT tissues.