Exploring the origin of a unique mutant allele in twin-tail goldfish using CRISPR/Cas9 mutants.

Artificial selection has been widely applied to genetically fix rare phenotypic features in ornamental domesticated animals. For many of these animals, the mutated loci and alleles underlying rare phenotypes are known. However, few studies have explored whether these rare genetic mutations might have been fixed due to competition among related mutated alleles or if the fixation occurred due to contingent stochastic events. Here, we performed genetic crossing with twin-tail ornamental goldfish and CRISPR/Cas9-mutated goldfish to investigate why only a single mutated allele-chdS with a E127X stop codon (also called chdAE127X)-gives rise to the twin-tail phenotype in the modern domesticated goldfish population. Two closely related chdS mutants were generated with CRISPR/Cas9 and compared with the E127X allele in F2 and F3 generations. Both of the CRISPR/Cas9-generated alleles were equivalent to the E127X allele in terms of penetrance/expressivity of the twin-tail phenotype and viability of carriers. These findings indicate that multiple truncating mutations could have produced viable twin-tail goldfish. Therefore, the absence of polymorphic alleles for the twin-tail phenotype in modern goldfish likely stems from stochastic elimination or a lack of competing alleles in the common ancestor. Our study is the first experimental comparison of a singular domestication-derived allele with CRISPR/Cas9-generated alleles to understand how genetic fixation of a unique genotype and phenotype may have occurred. Thus, our work may provide a conceptual framework for future investigations of rare evolutionary events in domesticated animals.

Effects of supplementing coated methionine in a high plant-protein diet on growth, antioxidant capacity, digestive enzymes activity and expression of TOR signaling pathway associated genes in gibel carp, Carassius auratus gibelio.

This study explored the impacts of supplementation of different levels of coated methionine (Met) in a high-plant protein diet on growth, blood biochemistry, antioxidant capacity, digestive enzymes activity and expression of genes related to TOR signaling pathway in gibel carp (Carassius auratus gibeilo). A high-plant protein diet was formulated and used as a basal diet and supplemented with five different levels of coated Met at 0.15, 0.30, 0.45, 0.60 and 0.75%, corresponding to final analyzed Met levels of 0.34, 0.49, 0.64, 0.76, 0.92 and 1.06%. Three replicate groups of fish (initial mean weight, 11.37 ± 0.02 g) (20 fish per replicate) were fed the test diets over a 10-week feeding period. The results indicated that with the increase of coated Met level, the final weight, weight gain (WG) and specific growth rate initially boosted and then suppressed, peaking at 0.76% Met level (P< 0.05). Increasing dietary Met level led to significantly increased muscle crude protein content (P< 0.05) and reduced serum alanine aminotransferase activity (P< 0.05). Using appropriate dietary Met level led to reduced malondialdehyde concentration in hepatopancreas (P< 0.05), improved superoxide dismutase activity (P< 0.05), and enhanced intestinal amylase and protease activities (P< 0.05). The expression levels of genes associated with muscle protein synthesis such as insulin-like growth factor-1, protein kinase B, target of rapamycin and eukaryotic initiation factor 4E binding protein-1 mRNA were significantly regulated, peaking at Met level of 0.76% (P< 0.05). In conclusion, supplementing optimal level of coated Met improved on fish growth, antioxidant capacity, and the expression of TOR pathway related genes in muscle. The optimal dietary Met level was determined to be 0.71% of the diet based on quadratic regression analysis of WG.

Effect of microplastics on oxytetracycline trophic transfer: Immune, gut microbiota and antibiotic resistance gene responses.

Microplastics and antibiotics are prevalent and emerging pollutants in aquatic ecosystems, but their interactions in aquatic food chains remain largely unexplored. This study investigated the impact of polypropylene microplastics (PP-MPs) on oxytetracycline (OTC) trophic transfer from the shrimp (Neocaridina denticulate) to crucian carp (Carassius auratus) by metagenomic sequencing. The carrier effects of PP-MPs promoted OTC bioaccumulation and trophic transfer, which exacerbated enterocyte vacuolation and hepatocyte eosinophilic necrosis. PP-MPs enhanced the inhibitory effect of OTC on intestinal lysozyme activities and complement C3 levels in shrimp and fish, and hepatic immunoglobulin M levels in fish (p < 0.05). Co-exposure of MPs and OTC markedly increased the abundance of Actinobacteria in shrimp and Firmicutes in fish, which caused disturbances in carbohydrate, amino acid, and energy metabolism. Moreover, OTC exacerbated the enrichment of antibiotic resistance genes (ARGs) in aquatic animals, and PP-MPs significantly increased the diversity and abundance of ARGs and facilitated the trophic transfer of teta and tetm. Our findings disclosed the impacts of PP-MPs on the mechanism of antibiotic toxicity in aquatic food chains and emphasized the importance of gut microbiota for ARGs trophic transfer, which contributed to a deeper understanding of potential risks posed by complex pollutants on aquatic ecosystems.

Effects of Water Temperature on the Body Color and Expression of the Genes Related to Body Color Regulation in the Goldfish.

Melanin-concentrating hormone (MCH), melanocyte-stimulating hormone (MSH), and somatolactin (SL) in the hypothalamus-pituitary axis are associated with body color regulation in teleost fish. Although these hormones' production and secretion respond well to light environments, such as background color, little is known about the effects of different water temperatures. We investigated the effects of water temperature, 10°C, 20°C, and 30°C, on body color and the expression of these genes and corresponding receptor genes in goldfish. The body color in white background (WBG) becomes paler at the higher water temperature, although no difference was observed in black background (BBG). Brain mRNA contents of proMCH genes (pmch1 and pmch2) increased at 30°C and 20°C compared to 10°C in WBG, respectively. Apparent effects of background color and temperature on the pituitary mRNA contents of a POMC gene (pomc) were not observed. The pituitary mRNA contents of the SLα gene were almost double those on a WBG at any temperature, while those of the SLß gene (slb) at 30°C tended to be higher than those at 10°C and 20°C on WBG and BBG. The scale mRNA contents of the MCH receptor gene (mchr2) in WBG were higher than those in BBG at 30°C. The highest scale mRNA contents of MSH receptor (mc1r and mc5r) on BBG were observed at 20°C, while the lowest respective mRNA levels were observed at 30°C on WBG. These results highlight the importance of temperature for the endocrinological regulation of body color, and darker background color may stabilize those endocrine functions.

Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Visual shape discrimination in goldfish, modelled with the neural circuitry of optic tectum and torus longitudinalis.

There is no satisfactory neurally-based theory as to how vertebrates that lack a neocortex discriminate even simple geometric shapes. In fishes, an intact optic tectum is necessary for such discriminations, but physiological studies of it have found nothing like the hierarchically arranged feature detecting neurons of mammalian visual cortex. Here, a neural model attempts a solution by basing shape discrimination upon the responses of only those elementary detectors (e.g. of size) that are within a focus of attention, formed by a winner-take-all arrangement of retinotopically mapped units representing tectal pyramidal cells. While this relatively primitive mechanism could recognize an object irrespective of position in space, it fails to distinguish patterns that differ only in their features' spatial relationships. The model's solution - imitating goldfish that naturally attend to the top of shapes - is to shift attention to the edges of a shape by spatially offsetting inputs to the pyramidal neurons, effected by the torus longitudinalis and its prolific synapses on pyramidal dendrites. The model's shape discrimination was compared to an extensive behavioral study using shapes with points and projections. In one test series fish were sensitive to the relative number of points on the tops of shapes. In another, fish were trained to discriminate points on the sides. By using different offset connections and only one elementary feature detector for small dark spots, the model successfully emulated the two sets of goldfish data, as judged by significant correlations between model response and fish discrimination.

Identification and effective regulation of scarb1 gene involved in pigmentation change in autotetraploid Carassius auratus.

The autotetraploid Carassius auratus (4nRR, 4 n=200, RRRR) is derived from whole-genome duplication of Carassius auratus red var. (RCC, 2 n=100, RR). In the current study, we demonstrated that chromatophores and pigment changes directly caused the coloration and variation of 4nRR skin (red in RCC, brownish-yellow in 4nRR). To further explore the molecular mechanisms underlying coloration formation and variation in 4nRR, we performed transcriptome profiling and molecular functional verification in RCC and 4nRR. Results revealed that scarb1, associated with carotenoid metabolism, underwent significant down-regulation in 4nRR. Efficient editing of this candidate pigment gene provided clear evidence of its significant role in RCC coloration. Subsequently, we identified four divergent scarb1 homeologs in 4nRR: two original scarb1 homeologs from RCC and two duplicated ones. Notably, three of these homeologs possessed two highly conserved alleles, exhibiting biased and allele-specific expression in the skin. Remarkably, after precise editing of both the original and duplicated scarb1 homeologs and/or alleles, 4nRR individuals, whether singly or multiply mutated, displayed a transition from brownish-yellow skin to a cyan-gray phenotype. Concurrently, the proportional areas of the cyan-gray regions displayed a gene-dose correlation. These findings illustrate the subfunctionalization of duplicated scarb1, with all scarb1 genes synergistically and equally contributing to the pigmentation of 4nRR. This is the first report concerning the functional differentiation of duplicated homeologs in an autopolyploid fish, substantially enriching our understanding of coloration formation and change within this group of organisms.

Multi-omics analysis of miRNA-mediated intestinal microflora changes in crucian carp Carassius auratus infected with Rahnella aquatilis.

Infection by an emerging bacterial pathogen Rahnella aquatilis caused enteritis and septicemia in fish. However, the molecular pathogenesis of enteritis induced by R. aquatilis infection and its interacting mechanism of the intestinal microflora associated with microRNA (miRNA) immune regulation in crucian carp Carassius auratus are still unclear. In this study, C. auratus intraperitoneally injected with R. aquatilis KCL-5 was used as an experimental animal model, and the intestinal pathological changes, microflora, and differentially expressed miRNAs (DEMs) were investigated by multi-omics analysis. The significant changes in histopathological features, apoptotic cells, and enzyme activities (e.g., lysozyme (LYS), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate transaminase (AST), and glutathione peroxidase (GSH-Px)) in the intestine were examined after infection. Diversity and composition analysis of the intestinal microflora clearly demonstrated four dominant bacteria: Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes. A total of 87 DEMs were significantly screened, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes were mainly involved in the regulation of lipid, glutathione, cytosine, and purine metabolism, which participated in the local immune response through the intestinal immune network for IgA production, lysosome, and Toll-like receptor (TLR) pathways. Moreover, the expression levels of 11 target genes (e.g., TLR3, MyD88, NF-κB, TGF-ß, TNF-α, MHC II, IL-22, LysC, F2, F5, and C3) related to inflammation and immunity were verified by qRT-PCR detection. The correlation analysis indicated that the abundance of intestinal Firmicutes and Proteobacteria was significantly associated with the high local expression of miR-203/NF-κB, miR-129/TNF-α, and miR-205/TGF-ß. These findings will help to elucidate the molecular regulation mechanism of the intestinal microflora, inflammation, and immune response-mediated miRNA-target gene axis in cyprinid fish.

Alfaxalone does not have long-term effects on goldfish pyramidal neuron action potential properties or GABAA receptor currents.

Anesthetics have varying physiological effects, but most notably alter ion channel kinetics. Alfaxalone is a rapid induction and washout neuroactive anesthetic, which potentiates γ-aminobutyric acid (GABA)-activated GABAA receptor (GABAA-R) currents. This study aims to identify any long-term effects of alfaxalone sedation on pyramidal neuron action potential and GABAA-R properties, to determine if its impact on neuronal function can be reversed in a sufficiently short timeframe to allow for same-day electrophysiological studies in goldfish brain. The goldfish (Carassius auratus) is an anoxia-tolerant vertebrate and is a useful model to study anoxia tolerance mechanisms. The results show that alfaxalone sedation did not significantly impact action potential properties. Additionally, the acute application of alfaxalone onto naive brain slices caused the potentiation of whole-cell GABAA-R current decay time and area under the curve. Following whole-animal sedation with alfaxalone, a 3-h wash of brain slices in alfaxalone-free saline, with saline exchanged every 30 min, was required to remove any potentiating impact of alfaxalone on GABAA-R whole-cell currents. These results demonstrate that alfaxalone is an effective anesthetic for same-day electrophysiological experiments with goldfish brain slices.

Ginger polysaccharide alleviates the effects of acute exposure to carbonate in crucian carp (Carassius auratus) by regulating immunity, intestinal microbiota, and intestinal metabolism.

Alkaline stress poses a significant challenge to the healthy growth of fish. Ginger polysaccharide (GP) is one of the main active substances in ginger and has pharmacological effects, such as anti-oxidation and immune regulation. However, the physiological regulatory mechanism of GP addition to diet on alkalinity stress in crucian carp remains unclear. This study aimed to investigate the potential protective effects of dietary GP on antioxidant capacity, gene expression levels, intestinal microbiome, and metabolomics of crucian carp exposed to carbonate (NaHCO3). The CK group (no GP supplementation) and COG group (NaHCO3 stress and no GP supplementation) were set up. The GPCS group (NaHCO3 stress and 0.4% GP supplementation) was stressed for seven days. Based on these data, GP significantly increased the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), acid phosphatase (ACP), and alkaline phosphatase (AKP) in carp under alkalinity stress (p < 0.05) and decreased the activity of malon dialdehyde (MDA) (p < 0.05). GP restored the activity of GSH-PX, ACP, and AKP to CK levels. The expression levels of tumor necrosis factor ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin 8 (IL-8) genes were decreased, and the expression levels of determination factor kappa-B (NF-κB) and interleukin 10 (IL-10) genes were increased (p < 0.05). Based on 16 S rRNA high-throughput sequencing, GP improved the changes in the intestinal microbial diversity and structural composition of crucian carp caused by NaHCO3 exposure. In particular, GP increased the relative abundance of Proteobacteria and Bacteroidetes and decreased the relative abundance of Actinobacteria. The metabolic response of GP to NaHCO3 exposed crucian carp guts was studied using LC/MS. Compared to the COG group, the GPCS group had 64 different metabolites and enriched 10 metabolic pathways, including lipid metabolism, nucleotide metabolism, and carbohydrate metabolism. The addition of GP to feed can promote galactose metabolism and provide an energy supply to crucian carp, thus alleviating the damage induced by alkalinity stress. In conclusion, GP can mitigate the effects of NaHCO3 alkalinity stress by regulating immune function, intestinal flora, and intestinal metabolism in crucian carp. These findings provide a novel idea for studying the mechanism of salt-alkali tolerance in crucian carp by adding GP to feed.

Extreme olfactory sensitivity of silver and bighead carp to overlapping suites of 21-carbon steroids suggests that these species, and likely all other Cyprinoidei, employ them as pheromones.

Although well established that several fishes including goldfish in the suborder Cypinoidei within the family Cypriniformes use the maturation-inducing steroid 17,20ß-dihydroxy-pregn-4-ene-3-one (17,20ßP) and its metabolites as a priming pheromone which they detect with sensitivity and specificity, it is unclear whether and how other Cypriniformes might have evolved to do so. This study examined this question in the family Xenocyprididae. Using electro-olfactogram recording we tested the olfactory sensitivity of silver (Hypophthalmichthys molitrix) and bighead carp (H. nobilis) to a range of 213 steroids in 21 mixtures at 10-9M. While silver carp detected 6 of 21 mixtures, bighead carp detected 5 (p< 0.05). Silver carp were sensitive to 13 21-carbon steroids in these mixtures including 17,20ßP while bighead carp detected 9, including 8 detected by silver carp. This assortment of steroids overlapped that detected by goldfish (family Cyprinidae) but no non-Cyprinoid, suggesting common evolutionary origin and function with differences characteristic of species-specificity.

Immune response and apoptosis of gibel carp (Carassius auratus gibelio) gills to Chilodonella hexasticha infection: Insights from histopathological and multi-omics analyses.

Gibel carp (Carassius auratus gibelio) is an important economically farmed fish in China. Chilodonella hexasticha parasitizes on the gills and fins of host fish, causing disruption to their normal respiration and movement, ultimately resulting in death of the fish. In this study, a combination of histopathological, immunohistochemical, transferase dUTP nick end labeling (TUNEL), multi-omics, and molecular approaches were employed to identify the immune reaction and cell apoptosis in gill tissue in response to C. hexasticha infection. Significant lamellae fusion, hyperplasia, hyperemia, necrosis, and desquamation of infected gibel carp gills were observed. In total, the expression of 3619 genes was higher, and 3143 lower, for gills in the infected group compared to the control group. Furthermore, 76 metabolites were significantly increased and 105 were significantly decreased in the infected group compared with the control group. From the qRT-PCR analysis results, immune system-related genes encoding IL-8, CXCL8a, and CXC11 were significantly up-regulated in infected gibel carp, while ZAP70 was significantly down-regulated. Immunohistochemical results also showed the down-regulated ZAP70 in the infected group. Apoptosis-related genes encoding CASP3 and Mcl-1b were up-regulated in response to C. hexasticha infection. These genes indicate the activation of CASP family-related apoptosis and Bim-mediated mitochondrial apoptotic pathways. TUNEL assays also revealed severe apoptosis in the infected group. Based on this study's results, it can be concluded that C. hexasticha infection leads to histopathological changes in the gills of infected fish, and induces both a significant immune response and apoptosis.

Expression and localization of HPG axis-related genes in Carassius auratus with different ploidy.

Introduction: In the Dongting water system, the Carassius auratus (Crucian carp) complex is characterized by the coexistence of diploid forms (2n=100, 2nCC) and polyploidy forms. The diploid (2nCC) and triploid C.auratus (3n=150, 3nCC) had the same fertility levels, reaching sexual maturity at one year. Methods: The nucleotide sequence, gene expression, methylation, and immunofluorescence of the gonadotropin releasing hormone 2(Gnrh2), Gonadotropin hormone beta(Gthß), and Gonadotropin-releasing hormone receptor(Gthr) genes pivotal genes of the hypothalamic-pituitary-gonadal (HPG) axis were analyzed. Results: The analysis results indicated that Gnrh2, follicle-stimulating hormone receptor(Fshr), and Lethal hybrid rescue(Lhr) genes increased the copy number and distinct structural differentiation in 3nCC compared to that in 2nCC. The transcript levels of HPG axis genes in 3nCC were higher than 2nCC (P<0.05), which could promote the production and secretion of sex steroid hormones conducive to the gonadal development of 3nCC. Meanwhile, the DNA methylation levels in the promoter regions of the HPG axis genes were lower in 3nCC than in 2nCC. These results suggested that methylation of the promoter region had a potential regulatory effect on gene expression after triploidization. Immunofluorescence showed that the localization of the Fshß, Lhß, and Fshr genes between 3nCC and 2nCC remained unchanged, ensuring the normal expression of these genes at the corresponding sites after triploidization. Discussion: Relevant research results provide cell and molecular biology evidence for normal reproductive activities such as gonad development and gamete maturation in triploid C. auratus, and contribute to further understanding of the genetic basis for fertility restoration in triploid C. auratus.

Streptomyces enissocaesilis L-82 has broad-spectrum antibacterial activity and promotes growth for Carassius auratus.

Aeromonas is the main pathogen causing bacterial diseases in fish. The disadvantages of chemical drugs to control fish diseases have been highlighted, and it is urgent to find an eco-friendly control method. In this study, an actinomycete strain with antibacterial activity against fish pathogenic bacteria was screened from soil samples. Combined with morphological characteristics, physiological and biochemical characteristics, and gyrB gene and whole genome comparison analysis, it was identified as a new strain of Streptomyces enissocaesilis, named Streptomyces enissocaesilis L-82. The strain has broad-spectrum antibacterial activity against fish pathogens. A substance with a mass-to-charge ratio of 227.20 [M + H] + was isolated and purified by high-performance liquid chromatography and mass spectrometry. It was presumed to be a derivative of 5-dimethylallylindole-3-acetonitrile. The strain is safe and non-toxic to crucian carp, and can stably colonize crucian carp and inhibit the proliferation of A. hydrophila. After feeding the feed containing 1 × 108 CFU/mL strain concentration, the weight growth rate and specific growth rate of crucian carp increased, the activity of ACP and SOD in serum increased, and the survival rate of crucian carp increased after challenge. Genome-wide analysis showed that the strain had strong ability to metabolize and tolerate extreme environments. And has a strong potential for disease resistance. Therefore, the strain is expected to be developed as a feed additive for fish farming. KEY POINTS: • The new Streptomyces enissocaesilis L-82 has a broad spectrum and stable antibacterial activity and meets the safety standards of feed additives. • Strain L-82 can colonize crucian carp, improve the growth, antioxidant, and immune performance of the host, and improve the survival rate after being infected with A. hydrophila. • Genome-wide analysis suggests that the strain has great disease resistance potential and is expected to be developed as a feed additive for fish culture.

Controlled synthesis of α-Fe2O3 nanocubes for gas-sensing applications: Feasibility of assessing crucian carp (Carassius auratus) freshness via trimethylamine levels.

Trimethylamine (TMA) is an organic amine with strong pungent smell which is an indicator gas for evaluating fish freshness according to the international standard. In this work, as-synthesize α-Fe2O3 solid nanocubes (NCs), α-Fe2O3 nucleoshell NCs and α-Fe2O3 hollow NCs were used as sensing material to develop an outstanding TMA gas sensor. The response of the α-Fe2O3 hollow NCs sensors towards 20 ppm TMA at 230 ℃ was 6.3. Meanwhile, these sensors showed exceptional response/recovery time, low limit of detection, great selectivity, and outstanding linear relationship. Furthermore, the analysis of gases released during the decomposition of Carassius auratus (0-10 days) was conducted, which demonstrated the assessment of TMA by α-Fe2O3 hollow NCs sensor can evaluate the freshness of Carassius auratus. Such a novel sensor signifies the outstanding application potential in efficient gas-sensing properties of TMA, which will make the tremendous contribution for Carassius auratus product evaluation in the future.

Effects of environmental hypoxia on the goldfish skeletal muscle: Focus on oxidative status and mitochondrial dynamics.

The skeletal muscle is a highly plastic tissue. Its ability to respond to external stimuli and challenges allows it to face the functional needs of the organism. In the goldfish Carassius auratus, a model of hypoxia resistance, exposure to reduced oxygen is accompanied by an improvement of the swimming performance, relying on a sustained contractile behavior of the skeletal muscle. At the moment, limited information is available on the mechanisms underlying these responses. We here evaluated the effects of short- (4 days) and long- (20 days) term exposure to moderate water hypoxia on the goldfish white skeletal muscle, focusing on oxidative status and mitochondrial dynamics. No differences in lipid peroxidation, measured as 2-thiobarbituric acid-reacting substances (TBARS), and oxidatively modified proteins (OMP) were detected in animals exposed to hypoxia with respect to their normoxic counterparts. Exposure to short-term hypoxia was characterized by an enhanced SOD activity and expression, paralleled by increased levels of Nrf2, a regulator of the antioxidant cell response, and HSP70, a chaperone also acting as a redox sensor. The expression of markers of mitochondrial biogenesis (TFAM) and abundance (VDAC) and of the mtDNA/nDNA ratio was similar under normoxia and under both short- and long-term hypoxia, thus excluding a rearrangement of the mitochondrial apparatus. Only an increase of PGC1α (a transcription factor involved in mitochondrial dynamics) was detected after 20 days of hypoxia. Our results revealed novel aspects of the molecular mechanisms that in the goldfish skeletal muscle may sustain the response to hypoxia, thus contributing to adequate tissue function to organism requirements.

Functional, structural, and molecular remodelling of the goldfish (Carassius auratus) heart under moderate hypoxia.

The goldfish (Carassius auratus) is known for its physiologic ability to survive even long periods of oxygen limitation (hypoxia), adapting the cardiac performance to the requirements of peripheral tissue perfusion. We here investigated the effects of short-term moderate hypoxia on the heart, focusing on ventricular adaptation, in terms of hemodynamics and structural traits. Functional evaluations revealed that animals exposed to 4 days of environmental hypoxia increased the hemodynamic performance evaluated on ex vivo cardiac preparations. This was associated with a thicker and more vascularized ventricular compact layer and a reduced luminal lacunary space. Compared to normoxic animals, ventricular cardiomyocytes of goldfish exposed to hypoxia showed an extended mitochondrial compartment and a modulation of proteins involved in mitochondria dynamics. The enhanced expression of the pro-fission markers DRP1 and OMA1, and the modulation of the short and long forms of OPA1, suggested a hypoxia-related mitochondria fission. Our data propose that under hypoxia, the goldfish heart undergoes a structural remodelling associated with a potentiated cardiac activity. The energy demand for the highly performant myocardium is supported by an increased number of mitochondria, likely occurring through fission events.

CyHV-2 infection triggers mitochondrial-mediated apoptosis in GiCF cells by upregulating the pro-apoptotic gene ccBAX.

Apoptosis is a physiological cell death phenomenon, representing one of the fundamental physiological mechanisms for maintaining homeostasis in living organisms. Previous studies have observed typical apoptotic features in Carassius auratus gibelio caudal fin cell (GiCF) infected with Cyprinid herpesvirus 2 (CyHV-2), and found a significant up-regulation of ccBAX expression in these infected cells. However, the specific apoptotic mechanism involved remains unclear. In this study, we utilized the GiCF cell line to investigate the apoptotic mechanism during CyHV-2 infection. Immunofluorescence staining revealed translocation of ccBAX into mitochondria upon CyHV-2 infection. Flow cytometry analysis demonstrated that overexpression of ccBAX expedited virus-induced apoptosis, characterized by heightened mitochondrial depolarization, increased transcriptional levels of Cytochrome c (Cyto c) in both the cytoplasm and mitochondria, and augmented Caspase 3/7 enzyme activity. Bax inhibitor peptide V5 (BIP-V5), an inhibitor interfering with the function of Bax proteins, inhibited Bax-mediated apoptotic events through the mitochondrial pathway and attenuated apoptosis induced by CyHV-2. In this study, it was identified for the first time that CyHV-2 induces apoptosis via the mitochondrial pathway in GiCF cells, bridging an important gap in our understanding regarding cell death mechanisms induced by herpesvirus infections in fish species. These findings provide a theoretical basis for comprehending viral apoptotic regulation mechanisms and the prevention and control of cellular pathologies caused by CyHV-2 infection.

Gibberellic acid induced oxidative stress, endoplasmic reticulum stress, and apoptosis in the livers of gibel carp (Carassius auratus gibelio).

Gibberellic acid (GA3), one of the most plant growth stimulator, is widely applied in agricultural regions and in beer industry. However, GA3 residue remained in soil and water can cause toxicity to all organisms. In this study, we investigated the mechanisms of GA3-induced hepatic injury in gibel carp (Carassius auratus gibelio). We found that GA3 exposure caused oxidative stress, endoplasmic reticulum stress (ERS), and apoptosis. The gibel carp exposed to GA3 exhibited significant alteration in erythrocyte nuclei. GA3 induced liver damage, as indicated by increasing the aminopherase activities. GA3 led to oxidative stress by increasing malondialdehyde content and decreasing the activities of CAT and GPx. GA3 stimulated ERS and increased the expression of grp78, perk, eif2s1α, chop, atf4, ire1α, xbp1, and atf6. Additionally, GA3 down-regulated the level of anti-apoptotic gene Bcl-2 and up-regulated the levels of pro-apoptotic genes bax and caspase-3. Overall results demonstrated that GA3 caused hepatic injury in gibel carp by increasing oxidative stress, ERS, and apoptosis.

The goldfish primary kidney macrophage system.

Miodrag (Mike) Belosevic and collaborators profoundly influenced the development of primary kidney macrophage culturing system (PKM) to study fish immunology in various aspects of comparative immunology. Their application of using PKM model, opened a new path for studying the development of macrophages, regulation of hematopoiesis, and cell specific response against various pathogens. By measuring histopathological and immunological outcomes, the biological implications of a variety of cytokines and signal transduction molecules could be elucidated with the established PKM system. A variety of growth factors mediating hematopoiesis and cytokines regulating the immune responses were functionally characterized, which served as a fundamental basis for making goldfish an excellent model to study fish immunology. Specifically, using in vivo and PKM based in vitro assays, the Belosevic lab advanced the goldfish-M. marinum model to study the anti-mycobacteria responses in teleosts, thus paving a way for the development of novel therapeutic approaches which could be applied in aquaculture settings or utilized as a model for human disease. In this review, we will look at the contribution of Dr. Mike Belosevic to teleost macrophage development, multiple cytokine functional characterization, and host-pathogen interactions.