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.

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.

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.

Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

Physiological and Transcriptome Analysis Reveal the Regulation Mechanism Underlying the Muscle Quality Effect of Dietary Schisandra chinensis in Triploid Crucian Carp (Carassius auratus).

Schisandra chinensis (sc) is generally demonstrated to improve antioxidant and immune functions in mammal. The present study through physiological and transcriptome analysis revealed alterations in muscle metabolisms of triploid crucian carp (Carassius auratus) cultured at different concentrations of S. chinensis diets (sc0, sc0.125%, sc0.25%, sc0.5%, sc1%, sc2%) after 8 weeks. The serum antioxidant enzyme activities analysis showed that dietary S. chinensis could reduce oxidative stress and increase organismic antioxidant capacity. Meanwhile, the detected results of muscle components presented that the amino acids and two flavor nucleotides of GMP and IMP significantly elevated while muscle crude lipid significantly reduced in S. chinensis feeding groups. In addition, springiness, chewiness, and fiber density in S. chinensis feeding groups muscle were significantly upregulated while muscle fiber diameter and area showed an opposite trend. By comparative transcriptome analysis of the muscles, functional enrichments of differentially expressed genes showed that multiple terms were related to purine metabolism, glycerolipid metabolism, regulation of actin cytoskeleton, and peroxisome. Finally, some key hub genes such as egln, gst, ggct, su1b, pi3kr4, myh9, lpl, gcdh, mylk, and col4a were identified by weighted gene co-expression network analysis. Taken together, our findings facilitate the understanding of the molecular basis underlying the muscle quality effect of dietary S. chinensis in triploid crucian carp, which provides valuable insights into the nutritional strategies of the aquaculture industry.

A telencephalon cell type atlas for goldfish reveals diversity in the evolution of spatial structure and cell types.

Teleost fish form the largest group of vertebrates and show a tremendous variety of adaptive behaviors, making them critically important for the study of brain evolution and cognition. The neural basis mediating these behaviors remains elusive. We performed a systematic comparative survey of the goldfish telencephalon. We mapped cell types using single-cell RNA sequencing and spatial transcriptomics, resulting in de novo molecular neuroanatomy parcellation. Glial cells were highly conserved across 450 million years of evolution separating mouse and goldfish, while neurons showed diversity and modularity in gene expression. Specifically, somatostatin interneurons, famously interspersed in the mammalian isocortex for local inhibitory input, were curiously aggregated in a single goldfish telencephalon nucleus but molecularly conserved. Cerebral nuclei including the striatum, a hub for motivated behavior in amniotes, had molecularly conserved goldfish homologs. We suggest elements of a hippocampal formation across the goldfish pallium. Last, aiding study of the teleostan everted telencephalon, we describe substantial molecular similarities between goldfish and zebrafish neuronal taxonomies.

Identification and functional characterization of Caspase-9 in goldfish (Carassius auratus L.) in response to Aeromonas hydrophila infection.

Apoptosis plays a pivotal role in the immune response to combat pathogen infections. In mammals, caspase-9, abbreviated as Casp9, plays an irreplaceable role in the initiation phase of the apoptotic cascade. To investigate the role of Casp9 in teleosts, we conducted a functional characterization of Casp9 in goldfish (Carassius auratus L.). The open reading frame of GfCasp9 spans 1296 base pairs (bp), encoding a protein composed of 431 amino acids. GfCasp9 was ubiquitously expressed in various tissues, with the spleen and brain showing the highest levels of expression. Subcellular localization analysis revealed that GfCasp9 is distributed in both the cytoplasm and nucleus. Overexpressing of GfCasp9 in HEK293 cells elicits a robust apoptotic response. Additionally, infection with Aeromonas hydrophila significantly increases the mRNA and protein expression of GfCasp9. These findings underscore the critical importance of GfCasp9 in immune responses and apoptosis against bacterial infections.

Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model.

Adiponectin (AdipoQ) is an adipokine involved in glucose homeostasis and lipid metabolism. In mammals, its role in appetite control is highly controversial. To shed light on the comparative aspects of AdipoQ in lower vertebrates, goldfish was used as a model to study feeding regulation by AdipoQ in fish species. As a first step, goldfish AdipoQ was cloned and found to be ubiquitously expressed at the tissue level. Using sequence alignment, protein modeling, phylogenetic analysis and comparative synteny, goldfish AdipoQ was shown to be evolutionarily related to its fish counterparts and structurally comparable with AdipoQ in higher vertebrates. In our study, recombinant goldfish AdipoQ was expressed in E. coli, purified by IMAC, and confirmed to be bioactive via activation of AdipoQ receptors expressed in HepG2 cells. Feeding in goldfish revealed that plasma levels of AdipoQ and its transcript expression in the liver and brain areas involved in appetite control including the telencephalon, optic tectum, and hypothalamus could be elevated by food intake. In parallel studies, IP and ICV injection of recombinant goldfish AdipoQ in goldfish was effective in reducing foraging behaviors and food consumption. Meanwhile, transcript expression of orexigenic factors (NPY, AgRP, orexin, and apelin) was suppressed with parallel rises in anorexigenic factors (POMC, CART, CCK, and MCH) in the telencephalon, optic tectum and/or hypothalamus. In these brain areas, transcript signals for leptin receptor were upregulated with concurrent drops in the NPY receptor and ghrelin receptors. In the experiment with IP injection of AdipoQ, transcript expression of leptin was also elevated with a parallel drop in ghrelin mRNA in the liver. These findings suggest that AdipoQ can act as a novel satiety factor in goldfish. In this case, AdipoQ signals (both central and peripheral) can be induced by feeding and act within the brain to inhibit feeding behaviors and food intake via differential regulation of orexigenic/anorexigenic factors and their receptors. The feeding inhibition observed may also involve the hepatic action of AdipoQ by modulation of feeding regulators expressed in the liver.

Transcriptome analysis reveals the mechanism of the effect of perfluorocaproic acid exposure on brain injury in Carassius auratus.

Perfluorocaproic acid (PFHxA) has received much attention as an emerging pollutant linked to neurological problems in humans and fish. However, the potential mechanism remains unknown. In this study, the pathological damage to tissue sections demonstrated that perfluorocaproic acid caused brain tissue damage, and the increased antioxidant index malondialdehyde (MDA) and decrease in superoxide Dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP), glutathione peroxidase (GSH-Px), Catalase (CAT), and Lysozyme (LZM) that perfluorocaproic acid activated antioxidant stress and caused brain damage. Transcriptome sequencing discovered 1,532 divergent genes, 931 upregulated, and 601 down-regulated. Furthermore, according to GO enrichment analysis, the differently expressed genes were shown to be involved in biological processes, cellular components, and molecular functions. The MAPK, calcium, and Neuroactive ligand-receptor interaction were considerably enriched in the KEGG enrichment analysis. We then analyzed qRT-PCR and chose ten essential differentially expressed genes for validation. The qRT-PCR results followed the same pattern as the RNA-Seq results. In conclusion, our study shows that perfluorocaproic acid exposure causes oxidative stress in the brain. It establishes a theoretical foundation for future research into genes linked to perfluorocaproic acid toxicity.

The first detection of Myxobolus lentisuturalis Dyková, Fiala et Nie, 2002, a highly pathogenic muscle-infecting parasite of gibel carp (Carassius auratus gibelio Berg, 1932) in Hungary.

Myxobolus lentisuturalis is a myxosporean parasite infecting the musculature both of goldfish (Carassius auratus auratus) and gibel carp (Carassius auratus gibelio). The species was originally described in China from gibel carp that is a common fish for sport fishing in Hungary meanwhile is one of the most popular farmed fish in China due to its high demand. Eighteen gibel carp with distortions were collected from a barrage pond in southern Hungary. All fish had large humps in the dorsolateral region due to infection of the muscle between the head and the dorsal fin. The swollen degenerated tissue was filled with myxozoan spores, which were collected for morphological and molecular studies. By size and morphology, the spores were consistent with morphological description of M. lentisuturalis. Histopathological examination showed that the formation of plasmodia containing myxospores leads to severe destruction of muscle tissue. The 18S ribosomal DNA and 28S ribosomal DNA data of the samples presented matched with previous sequences of M. lentisuturalis in GenBank. Phylogenetic analyses confirmed that our sequences belong to a monophyletic group with them supported by a high bootstrap. This study highlights the occurrence of a highly pathogenic myxozoan, M. lentisuturalis in Hungary as a new geographical location.

Effects of microfiber and bead microplastic exposure in the goldfish Carassius auratus: Bioaccumulation, antioxidant responses, and cell damage.

We confirmed antioxidant-related gene expression, bioaccumulation, and cell damage following exposure to various microplastics in vivo and in vitro in the goldfish Carassius auratus. Exposure of C. auratus to a 500 µm fiber-type microplastic environment (MF; 10 and 100 fibers/L) and two sizes (0.2 and 1.0 µm) of beads (MB; 10 and 100 beads/L) for 120 h increased superoxide dismutase (SOD) mRNA expression in the liver until 24 h followed by a decrease. Whereas, catalase (CAT) mRNA expression increased from 12 h to the end of the in vivo experiment. In vitro experiments were conducted with diluted microfibers (1 and 5 fibers/L) and microbeads (1 and 5 beads/L) using cultured liver cells. The results of SOD and CAT mRNA expression analysis conducted in vitro showed a tendency similar to those of experiments conducted in vivo. The H2O2 level increased in the high-concentration experimental groups compared with that in the low-concentration groups of 0.2-µm beads. In addition, the H2O2 level increased in both MF and MB groups from 12 h of exposure. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma were used as indicators of liver damage in fish. The ALT and AST levels increased up to 120 h after exposure. Caspase-3 (casp-3) mRNA expression was higher in the MB group than in the MF group. We visually confirmed liver casp-3 mRNA signals using in situ hybridization. The degree of DNA damage in the MF and MB high-concentration groups increased with the exposure time. The tail length and percent of DNA in the tail of the MB group were significantly higher than those of the MF group, confirming that DNA damage was greater in the MB group. Both fiber- and bead-type microplastics induced oxidative stress in C. auratus, but the bead-type induced greater stress than the fiber-type.

The oral protective efficacy of magnolol against Aeromonas hydrophila and A. veronii infection via enhancing anti-inflammatory ability in goldfish (Carassius auratus).

Aeromonas hydrophila and A. veronii are widespread and important critical pathogenic bacteria in the aquaculture industry and cause severe economic damage. At present, magnolol has been proved to be a broad-spectrum antibacterial activity, such as A. hydrophila, Staphylococcus aureus and Streptococcus mutans. In order to explore the cause of in vivo disease resistance of magnolol and promote its safe application in aquaculture, the pathological detection and changes in immune indicators of fish after feeding with magnolol were conducted in this paper. Results showed that the diets supplemented with magnolol (3 g magnolol/kg commercial feed) significantly increase the expression level of anti-inflammatory cytokines (IL-10, TGF-ß and IL-4) in the liver of goldfish (p < .05). Additionally, the expression levels of proinflammatory cytokines (IL-1ß, IL-8 and IFN-γ) did not increase significantly. Subsequently, this study investigated the resistance of goldfish to A. hydrophila and A. veronii infection after feeding with magnolol. The results showed that the survival rates of treatment groups fed 3 g magnolol/kg commercial feed daily increased by 23.1% and 38.5% after 10 days post A. hydrophila and A. veronii (p = .0351) infection, respectively. Meanwhile, growth performance (body weight and length), major internal organs (liver, spleen, kidney and intestine) and the serum biochemistry indicators (ATL and AST) all exhibited no significant adverse effects after the goldfish fed with magnolol for 30 days. TP showed an increasing concentration in the treatment group (p < .05). Results of the mRNA expression of stress response indicated that the expression level of cyp1a and hsp70 was significantly down-regulated after a 30-day treatment (p < .05), and the two genes recovered to the similar level as the control group after a commercial feed diet. In brief, the diets supplemented with magnolol protected the host from the excessive immune response caused by A. hydrophila and A. veronii via enhancing its anti-inflammatory capacity and had no adverse effects with feeding.

Differential regulation of hepatic SH3 domain binding kinase 1 (SBK1) expression in mouse and goldfish.

SH3 domain binding kinase 1 (SBK1) is a serine/threonine kinase that belongs to the new kinase family (NFK) with limited information on its function. Previous studies reported that SBK1 plays a role in memory formation, lipid metabolism, and cancer cell progression. Nevertheless, the regulatory mechanism of Sbk1 expression in various tissues remains unknown. We report here that Sbk1 expression in mouse hepatocytes was downregulated by glucocorticoid, whereas saturated and unsaturated fatty acids were stimulators of Sbk1 expression. The regulatory role of glucocorticoid and fatty acid was further confirmed by the Sbk1 promoter assay, which aligned with the presence of several glucocorticoid-response elements (GRE) and peroxisome proliferator responsive elements (PPRE) in the mouse Sbk1 promoter. The inhibitory effect of glucocorticoids on hepatic Sbk1 expression and protein content could also be demonstrated in vivo after prednisolone injection. Moreover, the expression of SBK1 in goldfish (gfSBK1) was also sensitive to glucocorticoid suppression as their mouse orthologues. In contrast, insulin had a differential action on SBK1 expression that it promoted the expression of all SBK1 isoforms in the goldfish hepatocytes but inhibited Sbk1 expression in the mouse hepatocytes. Together, our findings indicate that SBK1 expression is hormone- and nutrient-sensitive with a species-specific response.

Molecular characterization, gene expression and functional analysis of goldfish (Carassius auratus L.) macrophage colony stimulating factor 2.

Background: Macrophage colony-stimulating factor 2 (MCSF-2) is an important cytokine that controls how cells of the monocyte/macrophage lineage proliferate, differentiate, and survive in vertebrates. Two isoforms of MCSF have been identified in fish, each exhibiting distinct gene organization and expression patterns. In this study, we investigated a goldfish MCSF-2 gene in terms of its immunomodulatory and functional properties. Methods: In this study, goldfish were acclimated for 3 weeks and sedated with TMS prior to handling. Two groups of fish were used for infection experiments, and tissues from healthy goldfish were collected for RNA isolation. cDNA synthesis was performed, and primers were designed based on transcriptome database sequences. Analysis of gfMCSF-2 sequences, including nucleotide and amino acid analysis, molecular mass prediction, and signal peptide prediction, was conducted. Real-time quantitative PCR (qPCR) was used to analyze gene expression levels, while goldfish head kidney leukocytes (HKLs) were isolated using standard protocols. The expression of gfMCSF-2 in activated HKLs was investigated, and recombinant goldfish MCSF-2 was expressed and purified. Western blot analysis, cell proliferation assays, and flow cytometric analysis of HKLs were performed. Gene expression analysis of transcription factors and pro-inflammatory cytokines in goldfish head kidney leukocytes exposed to rgMCSF-2 was conducted. Statistical analysis using one-way ANOVA and Dunnett's post hoc test was applied. Results: We performed a comparative analysis of MCSF-1 and MCSF-2 at the protein and nucleotide levels using the Needleman-Wunsch algorithm. The results revealed significant differences between the two sequences, supporting the notion that they represent distinct genes rather than isoforms of the same gene. Sequence alignment demonstrated high sequence identity with MCSF-2 homologs from fish species, particularly C. carpio, which was supported by phylogenetic analysis. Expression analysis in various goldfish tissues demonstrated differential expression levels, with the spleen exhibiting the highest expression. In goldfish head kidney leukocytes, gfMCSF-2 expression was modulated by chemical stimuli and bacterial infection, with upregulation observed in response to lipopolysaccharide (LPS) and live Aeromonas hydrophila. Recombinant gfMCSF-2 (rgMCSF-2) was successfully expressed and purified, showing the ability to stimulate cell proliferation in HKLs. Flow cytometric analysis revealed that rgMCSF-2 induced differentiation of sorted leukocytes at a specific concentration. Moreover, rgMCSF-2 treatment upregulated TNFα and IL-1ß mRNA levels and influenced the expression of transcription factors, such as MafB, GATA2, and cMyb, in a time-dependent manner. Conclusion: Collectively, by elucidating the effects of rgMCSF-2 on cell proliferation, differentiation, and the modulation of pro-inflammatory cytokines and transcription factors, our findings provided a comprehensive understanding of the potential mechanisms underlying gfMCSF-2-mediated immune regulation. These results contribute to the fundamental knowledge of MCSF-2 in teleosts and establish a foundation for further investigations on the role of gfMCSF-2 in fish immune responses.

Insulin Controls Clock Gene Expression in the Liver of Goldfish Probably via Pi3k/Akt Pathway.

The liver circadian clock plays a pivotal role in driving metabolic rhythms, being primarily entrained by the feeding schedule, although the underlying mechanisms remain elusive. This study aimed to investigate the potential role of insulin as an intake signal mediating liver entrainment in fish. To achieve this, the expression of clock genes, which form the molecular basis of endogenous oscillators, was analyzed in goldfish liver explants treated with insulin. The presence of insulin directly increased the abundance of per1a and per2 transcripts in the liver. The dependency of protein translation for such insulin effects was evaluated using cycloheximide, which revealed that intermediate protein translation is seemingly unnecessary for the observed insulin actions. Furthermore, the putative interaction between insulin and glucocorticoid signaling in the liver was examined, with the results suggesting that both hormones exert their effects by independent mechanisms. Finally, to investigate the specific pathways involved in the insulin effects, inhibitors targeting PI3K/AKT and MEK/ERK were employed. Notably, inhibition of PI3K/AKT pathway prevented the induction of per genes by insulin, supporting its involvement in this process. Together, these findings suggest a role of insulin in fish as a key element of the multifactorial system that entrains the liver clock to the feeding schedule.