Carassius Auratus Red Var Research Articles

Fish IL-26 collaborates with IL-10R2 and IL-20R1 to enhance gut mucosal barrier during the antibacterial innate immunity

IL-26 is a cytokine that is crucial for the maintenance and function of the gut mucosal barrier. IL-26 signaling pathway relies on a heterodimeric receptor complex, which is composed of two distinct subunits, IL-10R2 and IL-20R1. However, there are no reports on the antibacterial immunity of IL-26 and its receptors in fish. For this purpose, in this study we identified IL-26 and its receptors IL-10R2 and IL-20R1 in Carassius cuvieri × Carassius auratus red var. (named WR-IL-26, WR-IL10R2 and WR-IL20R1, respectively). Phylogenetic analysis confirmed the conservation of these genes, with shared structural motifs similar to those found in higher vertebrates. Upon exposure to Aeromonas hydrophila, a common fish pathogen, there was a significant upregulation of WR-IL-26, WR-IL10R2 and WR-IL20R1 in the gut, indicating a potential role in the immune response to infection. A co-immunoprecipitation assay revealed that WR-IL-26 formed complexes with WR-IL10R2 and WR-IL20R1. In vivo experiments demonstrated that administration of WR-IL-26 activated the JAK1-STAT3 signaling pathway and protected the gut mucosa barrier from A. hydrophila infection. Conversely, silencing WR-IL10R2 and WR-IL20R1 via RNA interference significantly attenuated the activation of WR-IL-26-mediated JAK1-STAT3 pathway. These results provided new insights into the role of IL-26 and its receptors in the gut mucosa barrier and could offer novel therapeutic strategies for managing bacterial infections in aquaculture.

Variation and Interaction of Distinct Subgenomes Contribute to Growth Diversity in Intergeneric Hybrid Fish.

Intergeneric hybridization greatly reshapes regulatory interactions among allelic and non-allelic genes. However, their effects on growth diversity remain poorly understood in animals. In this study, we conducted whole-genome sequencing and RNA sequencing (RNA-seq) analyses in diverse hybrid varieties resulting from the intergeneric hybridization of goldfish (Carassius auratus red var.) and common carp (Cyprinus carpio). These hybrid individuals were characterized by distinct mitochondrial genomes and copy number variations. Through a weighted gene correlation network analysis, we identified 3693 genes as candidate growth-regulated genes. Among them, the expression of 3672 genes in subgenome R (originating from goldfish) displayed negative correlations with growth rate, whereas 20 genes in subgenome C (originating from common carp) exhibited positive correlations. Notably, we observed intriguing patterns in the expression of slc2a12 in subgenome C, showing opposite correlations with body weight that changed with water temperatures, suggesting differential interactions between feeding activity and weight gain in response to seasonal changes for hybrid animals. In 40.31% of alleles, we observed dominant trans-regulatory effects in the regulatory interaction between distinct alleles from subgenomes R and C. Integrating analyses of allelic-specific expression and DNA methylation data revealed that the influence of DNA methylation on both subgenomes shapes the relative contribution of allelic expression to the growth rate. These findings provide novel insights into the interaction of distinct subgenomes that underlie heterosis in growth traits and contribute to a better understanding of multiple allele traits in animals.

Comparative study of hypoxic tolerance between the hybrids of white crucian carp (♀) × red crucian carp (♂) and its parents

The hybrid of female white crucian carp (Carassius auratus cuvieri, WCC) × male red crucian carp (Carassius auratus red var. RCC) (WR), combines desirable characteristics of both parents, including tender meat, rapid growth rate, and strong resistance. To test the hypoxia tolerance of WR, an experiment of exposure to hypoxic conditions was conducted on WR, RCC, and WCC. The results showed that WR and RCC exhibited stronger hypoxia tolerance capacity compared to WCC. The strong resistance to hypoxia of WR and RCC was manifested by the absence of damage to the hepatopancreas after hypoxia, and the fish can adapt to the hypoxic environment by enhancing the hematopoietic function of hematopoietic tissue, remodeling gill morphology, and activating the antioxidant system. By contrast, after hypoxia, the number of red blood cells and the concentration of hemoglobin in the blood of WCC decreased, and the hepatopancreas was seriously damaged, indicating that the WCC is not as tolerant to low oxygen conditions as RCC and WR.

Gckr depletion leads to growth retardation and diet-dependent visceral obesity in red crucian carp (Carassius auratus red var.)

There exists a notable contrast in the carbohydrate utilization capacity between fish and mammals. In mammals, the glucokinase regulatory protein (GCKR) is known to exert inhibitory effects on glycolysis by binding to glucokinase (GCK), but its role in fish remains unexplored. In this study, the function of gckr in red crucian carp (Carassius auratus red var.), hereafter referred to as RCC, was investigated through its knockout. Under normal dietary conditions, the growth rate of gckr knockout RCC was significantly lower compared to wild-type (WT) RCC reared in the same environment. Subsequent analysis found that gckr knockout RCC exhibited significantly higher serum glucose levels at 1 h post-feeding or glucose injection (hpi), while the difference was abolished at 3 h. Therefore, the metabolic characteristics at 1 and 3 hpi were evaluated between WT and gckr knockout RCC. The results revealed that gckr knockout led to impaired insulin signaling and glycolysis, as evidenced by a reduction in serum insulin level, hepatic insulin receptor a and pyruvate kinase expression, GCK contents, and pyruvate levels at 1 hpi. Additionally, gckr knockout resulted in compromised gluconeogenesis, as indicated by a significant decrease in hepatic expression of fructose-1,6-bisphosphatase 1 and glucose-6-phosphatase at 1 hpi, while it did not affect glycogen accumulation after glucose injection. Regarding lipid metabolism, gckr knockout caused a transient decrease in triglyceride level and reduced expression of fatty acid synthase after glucose injection. Moreover, decreased hepatic peroxisome proliferator activated receptor alpha (ppara) transcripts and proteins were observed in gckr knockout RCC at 1 dpi, indicating a reduced capacity for β-oxidation due to Gckr deficiency. Interestingly, when fed a high-lipid diet, gckr knockout resulted in a significant increase in visceral mass with higher triglyceride level, accompanied by attenuated PPARα signaling. Taken together, this study provides evidence that Gckr-mediated maintenance of Gck contributes to the facilitation of postprandial glycolysis, gluconeogenesis, lipogenesis, and fatty acid β-oxidation in RCC. This study further suggests that enhancing glycolysis may promote growth and liver health in fish under high lipid dietary conditions.

Mechanistic Insights into Nonylphenol Stress on BMP2 and BMP4 Gene Expression in Red Crucian Carp (Carassius auratus Red var.)

Nonylphenol (NP) is a known endocrine-disrupting chemical (EDC) that has been shown to affect bone development in mammals. However, the detrimental impacts of NP on the skeletal growth and development of aquatic species, especially bony fish, remain poorly understood. Bone morphogenic proteins (BMPs), essential for bone formation and osteoblast differentiation, act through the BMP-Smad signaling pathway. In this study, two BMP genes, BMP2 and BMP4, were cloned and characterized in the red crucian carp (Carassius auratus red var.). The full-length cDNAs of BMP2 and BMP4 were 2029 bp and 2095 bp, respectively, encoding polypeptides of 411 and 433 amino acids, and share a typical TGF-β domain with other BMPs. The tissue expression patterns of both genes were identified, showing ubiquitous expression across all studied tissues. Additionally, the exposure of embryos or adult fish to NP stress resulted in a downregulation of BMP2, BMP4, and other genes associated with the BMP-Smad signaling pathway. Moreover, the combined treatment of adult fish with NP and the specific BMP receptor inhibitor significantly reduced these genes’ expression. These findings elucidate the mechanism of NP stress on BMP2 and BMP4, suggesting a role for the BMP-Smad signaling pathway in the response to endocrine-disrupting chemicals in fish.

Tumor necrosis factor α1 decreases mucosal immune and antioxidant response in the midgut of hybrid fish (white crucian carp ♀ × red crucian carp ♂).

Tumor necrosis factor α1 (TNFα) is a pleiotropic cytokine involved in immune regulation and cellular homeostasis, but the crucial role of TNFα in fish gut remained unclear. The current study aimed to evaluate the immunoregulatory function of TNFα1 on gut barrier in a novel hybrid fish (WR), which was produced by crossing white crucian carp (Carassius cuvieri, ♀) with red crucian carp (Carassius auratus red var, ♂). In this study, WR-tnfα1 sequence was identified, and a high-level expression was detected in the intestine. Elevated levels of WR-tnfα1 expressions were detected in immune-related tissues and cultured fish cells on stimulation. The appearance of vacuolization and submucosal rupture was observed in TNFα1-treated midgut of WR, along with elevated levels of goblet cell atrophy, whereas no significant changes were detected in most expressions of tight-junction genes and mucin genes. In contrast, WR receiving gut perfusion with WR-TNFα1 showed a remarkable decrease in antioxidant status in midgut, whereas the expression levels of apoptotic genes and redox responsive genes increased sharply. These results suggested that TNFα1 could exhibit a detrimental effect on antioxidant defense and immune regulation in the midgut of WR.

Cloning, distribution, and effects of growth regulation of MC3R and MC4R in red crucian carp (Carassius auratus red var.).

Melanocortin-3 and -4 receptors (MC3R and MC4R), G protein-coupled receptors, play vital roles in the regulation of energy homeostasis. To understand the functions of mc3r and mc4r in the energy homeostasis of red crucian carp (Carassius auratus red var., RCC), we cloned mc3r and mc4r, analyzed the tissue expression and localization of the genes, and investigated the effects of knockout of mc3r (mc3r +/-) and mc4r (mc4r +/-) in RCC. The full-length cDNAs of RCC mc3r and mc4r were 1459 base pairs (bp) and 1894 bp, respectively. qRT-PCR indicated that mc3r and mc4r were profusely expressed in the brain, but lower expressed in the periphery tissues. ISH revealed that mc3r and mc4r were located in NPP, NPO, NAPv, NSC, NAT, NRL, NLTl, and NLTp of the brain, suggesting that mc3r and mc4r might regulate many physiological and behavioral aspects in RCC. To further verify the roles of mc3r and mc4r in energy homeostasis, the mc3r+/- and mc4r+/- fish were obtained by the CRISPR/Cas9 system. The average body weights, total lengths, body depths, and food intake of mc4r+/- fish were significantly higher than those of mc3r+/- and the normal wild-type (WT) fish, but there was no difference between the mc3r+/- and WT fish, indicating that the RCC phenotype and food intake were mainly influenced by mc4r but not mc3r. Interestingly, mc4r+/- fish displayed more visceral fat mass than mc3r+/- and WT fish, and mc3r+/- fish also exhibited slightly more visceral fat mass compared to WT. RNA-seq of the liver and muscle revealed that a large number of differentially expressed genes (DEGs) differed in WT vs. mc3r+/-, WT vs. mc4r+/-, and mc3r+/- vs. mc4r+/-, mainly related to lipid, glucose, and energy metabolism. The KEGG enrichment analysis revealed that DEGs were mainly enriched in pathways such as steroid biosynthesis, fatty acid metabolism, fatty acid biosynthesis, glycolysis/gluconeogenesis, wnt signaling pathway, PPAR signaling pathway, and MAPK signaling pathway, thereby affecting lipid accumulation and growth. In conclusion, these results will assist in the further investigation of the molecular mechanisms in which MC3R and MC4R were involved in the regulation of energy homeostasis in fish.

Evolutionarily conserved IL-22 participates in gut mucosal barrier through its receptors IL-22BP, IL-10R2 and IL-22RA1 during bacterial infection in teleost

IL-22 is a critical cytokine of epithelial mucosal barrier. In humans, IL-22 signals through a heteroduplex receptor consisting of IL-22R and IL-10Rβ. In fish, IL-22 and its receptors homologues have been cloned in a number of species, however, no studies have been reported how the receptors are involved in IL-22 transduction. For this purpose, in this study we identified IL-22 and its soluble receptor IL-22BP and transmembrane receptors IL-22RA1 and IL-10R2 in Carassius cuvieri × Carassius auratus red var. (named WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1, respectively). WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1 were relatively conserved in the evolutionary process, sharing the same conserved domains as their higher vertebrate homologues. When the fish were infected with the Aeromonas hydrophila, the expression of WR-IL-22, WR-IL-22BP, WR-IL10R2 and WR-IL22RA1 were significantly induced in the gut. The co-IP assay showed that WR-IL-22 not only interacted with WR-IL-22BP, but also with WR-IL10R2 and WR-IL22RA1. When introduced in vivo, WR-IL-22 activated the JAK1-STAT3 axis and protected the gut mucosa from A. hydrophila infection. However, overexpression of WR-IL-22BP or knockdown of transmembrane receptors WR-IL10R2 and WR-IL22RA1 significantly inhibited the activation of WR-IL-22-mediated JAK1-STAT3 axis and promoted bacterial colonization in the gut. These results provided new insights into the role of IL-22 and its receptors in the gut mucosa barrier and immune response in teleost.

Tumor necrosis factor α1 (TNFα1) administration can disrupt barrier function and attenuate redox defense in midgut of red crucian carp (Carassius auratus red var)

TNFα belongs to superfamily of tumor necrosis factor that can exert the pleiotropic effort in a large quantity of biological processes, including immune homeostasis, intracellular modulation and etiopathogenesis, but its regulatory role in mucosal immune regulation of fish is unclear. Currently, this study aimed to evaluate the immunoregulatory function of TNFα1 on gut barrier in teleost fish. In this study, TNFα1 sequences were identified from red crucian carp (RCC, Carassius auratus red var). The high-level expression of RCC-TNFα1 mRNA was detected in gill among all the isolated samples. Then, RCC-TNFα1 expression increased dramatically in immune-related tissues after A. hydrophila infection and in cultured fish cells after lipopolysaccharide (LPS) treatment. RCC-TNFα1 fusion protein was generated and purified in vitro. RCC receiving RCC-TNFα1 perfusion showed an increased levels of villi fusion and edema in injured midgut with the fuzzy appearance. In addition, The mRNA expressions of tight junction (TJ) genes, mucin genes and redox sensitive genes decreased sharply in TNFα1-treated midgut in comparison with those of the control (P < 0.05), whereas the expression levels of apoptotic genes involved in caspase signals and unfolded protein response (UPR) attained the dramatic increase. These results demonstrated that RCC-TNFα1 stimulation could impair midgut structural integrity and immune function by induction of antioxidant collapse and apoptotic activation.

WR-PTXF, a novel short pentraxin, protects gut mucosal barrier and enhances the antibacterial activity in Carassius cuvieri × Carassius auratus red var.

The pentraxin family is an evolutionarily conserved group that plays an important role in innate immunity. C-reactive protein (CRP) and serum amyloid P component (SAP) are classical members of the short pentraxins and are known to be the major acute phase proteins. In this work, we have cloned a novel pentraxin fusion protein, WR-PTXF, from Carassius cuvieri × Carassius auratus red var. In fish, the biological function of PTXF is essentially unknown. For this purpose, we report the identification and analysis of WR-PTXF and elucidate its role in the antibacterial innate immunity. WR-PTXF contains 224 amino acids and shares 79.8% and 23.0% sequence identities with crucian carp CRP and SAP, respectively. Blast analysis shows that WR-PTXF and goldfish PTXF had the highest similarity (97.3%). WR-PTXF is expressed in multiple tissues and is upregulated by Aeromonas hydrophila infection. WR-PTXF contains a short pentraxin domain and recombinant WR-PTXF protein (rWR-PTXF) can bind the A. hydrophila in a concentration-dependent manner. Further, rWR-PTXF displays apparent bacteriostatic activity against A. hydrophila in vitro by enhancing the uptake of the bound bacteria by host phagocytes. When introduced in vivo, rWR-PTXF not only protects the gut mucosa but also limits the colonization of A. hydrophila in systemic immune organs. Consistently, knockdown of WR-PTXF significantly promotes bacterial dissemination in the tissues of host. These results indicate that WR-PTXF is a classic pattern recognition molecule that exerts a protective effect against bacterial infection.

Manganese-superoxide dismutase (MnSOD) rescues redox balance and mucosal barrier function in midgut of hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var ♂) infected with Aeromonas hydrophila and Edwardsiella tarda

MnSOD is a ubiquitous metalloenzyme that constitutes the first line of antioxidant defense against oxidative stress. In this study, full length sequence of MnSOD was identified from hybrid crucian carp (WR, Carassius cuvieri ♀ × Carassius auratus red var ♂). Tissue-specific analysis revealed that the highest expression of WR-MnSOD was detected in kidney. Aeromonas hydrophila challenge could dramatically enhance WR-MnSOD mRNA expression in tissues. In vivo administration of purified WR-MnSOD peptide could maintain gut mucosal barrier function, rescue redox balance as well as decrease apoptotic cell death in midgut upon bacterial infection, suggesting that WR-MnSOD is playing a crucial role in gut mucosal barrier function and could be used as feed additive to improve gut immunity in fish.

Effects of dietary glutathione on growth performance, muscle quality and lipid metabolism of hybrid crucian carp (Carassius auratus cuvieri ♀ × Carassius auratus red var. ♂) fed a high-fat diet

A 60-day feeding trial was conducted to investigate the influence of glutathione (GSH) on growth performance, lipid metabolism and muscle quality of hybrid crucian carp (initial body weight: 15.03 ± 0.11 g) fed a high-fat diet (HFD). Five experimental diets were prepared in this study: the control diet (6.17% fat), HFD (12.37% fat), and HFD with 200 mg/kg, 400 mg/kg and 600 mg/kg GSH supplementation, respectively. The results indicated that feeding HFD to hybrid crucian carp remarkably reduced weight gain rate (WGR), specific growth rate (SGR), total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) activities of serum and liver, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) activities of liver, but significantly increased malondialdehyde (MDA) content of liver compared to the control group. After adding 200–600 mg/kg GSH to HFD, the WGR, SGR, as well as serum and liver T-AOC activity of hybrid crucian carp increased significantly. Moreover, the supplementation of 400 mg/kg GSH significantly increased the activities of T-SOD, catalase (CAT) of serum and liver, as well as the GSH-Px, glutathione-S-transferase (GST) and GR of liver, but decreased MDA content of serum and liver compared to the HFD. The HFD significantly increased muscle crude lipid, polyunsaturated fatty acids (PUFA) contents and fiber diameter, but decreased chewiness, gumminess, hardness and fiber density compared to the control group. The addition of 400–600 mg/kg GSH significantly increased muscle chewiness, gumminess, hardness, and decreased muscle fiber diameter of HFD-fed hybrid crucian carp. Meanwhile, the addition of 200–600 mg/kg GSH significantly reduced the mono-unsaturated fatty acids (MUFA) content and increased the PUFA content of muscle compared to the HFD. The HFD significantly up-regulated the expression of lipid synthesis genes (dgat2 and srebp1) and down-regulated the expression of lipolysis genes (lpl, hsl and acox1) of liver compared to the control group. The addition of 400–600 mg/kg GSH significantly decreased the expression of lipid synthesis genes (dgat2 and srebp1) and increased the expression of lipolysis genes (hsl, lpl, acox1 and pparα) of HFD-fed hybrid crucian carp. This study demonstrated that GSH effectively ameliorated the decline of growth performance and muscle quality of hybrid crucian carp caused by HFD, and reduced the risk of liver lipid deposition of hybrid crucian carp by regulating the expression of genes related to liver lipid metabolism.

Triploidization modulates intestinal microbiota and promotes growth in Carassius auratus

Intestinal microbiota plays a key role in modulating host growth. However, the effect of triploidization on intestinal microflora and fast growth has not been fully explored. Here, autotriploid Carassius auratus (3nRR, 3n = 150) derived from Carassius auratus red var. (♀, RCC, 2n = 100) × autotetraploid fish (♂, 4nRR, 4n = 200) was established. Intestinal microbial community analysis of RCC and 3nRR was performed by 16S rRNA gene sequencing. Gut characteristics, growth rate, and digestive enzyme activity of the two groups of fish were explored. Significant changes in gut assemblages were observed in 3nRR fish. Differences in intestinal characteristics (structure, relative gut density, relative gut length, relative gut mass, and Zihler's index) were observed between RCC and 3nRR groups. Associations between microbial assemblages and intestinal characteristics were evaluated. Growth rate and activities of intestinal digestive enzymes (cellulase, amylase and trypsin) were higher in 3nRR compared with that in RCC. Spearman's correlation analysis showed a positive correlation between bacteria orders (Clostridiales, Micrococcales and Lactobacillales) and fast growth in 3nRR. In summary, the findings indicated that triploidization caused changes in gut microbiota and characteristics, and digestive enzyme activity. The relative abundance of several bacteria species was positively correlated with fast growth in 3nRR. The present study provided new insights into the growth mechanism of 3nRR and its interaction with intestinal microorganisms.

Transfer of CeO2 nanoparticles between freshwater omnivorous organisms: Effect of feces and necrophagy

Transfer of CeO2 engineered nanoparticles (NPs) through feces was investigated between two omnivorous organisms, red crucian carp (Carassius auratus red var.) and crayfish (Procambarus clarkii). Upon water exposure (5 mg/L, 7 days), the highest bioaccumulation was observed in carp gills (5.95 μg Ce/g D.W.) and crayfish hepatopancreas (648 μg Ce/g D.W.), with the bioconcentration factors (BCFs) at 0.45 and 3.61, respectively. In addition, 97.4% and 73.0% of ingested Ce were excreted by carp and crayfish, respectively. The feces of carp and crayfish were collected and fed to crayfish and carp, respectively. After feces exposure, bioconcentration was observed in both carp (BCF, 3.00) and crayfish (BCF, 4.56). After feeding crayfish with carp bodies (1.85 μg Ce/g D.W.), CeO2 NPs were not biomagnified (biomagnification factor, 0.28). Upon water exposure, CeO2 NPs were transformed into Ce(III) in the feces of both carp (24.6%) and crayfish (13.6%), and the transformation was stronger after subsequent feces exposure (100% and 73.7%, respectively). Feces exposure lowered histopathological damage, oxidative stress, and nutritional quality (e.g., crude proteins, microelements, amino acids) to carp and crayfish in comparison with water exposure. This research highlights the importance of feces exposure on the transfer and fate of NPs in aquatic ecosystems.

Gut-liver immune and redox response in hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var. ♂) after gut infection with Aeromonas hydrophila

Aeromonas hydrophila can pose a great threat to fish survival. In this study, we investigated the differential immune and redox response in gut-liver axis of hybrid fish (WR) undergoing gut infection. WR anally intubated with A. hydrophila showed severe midgut injury with decreased length-to-width ratios of villi along with GC hyperplasia and enhanced antioxidant activities, but expression profiles of cytokines, chemokines, antibacterial molecules, redox sensors and tight junction proteins decreased dramatically. In contrast, immune-related gene expressions and antioxidant activities increased significantly in liver of WR following gut infection with A. hydrophila. These results highlighted the differential immune regulation and redox balance in gut-liver axis response to bacterial infection.

Integrated analysis of gene expressions and metabolite features unravel immunometabolic interplay in hybrid fish (Carassius cuvieri ♀ × Carassius auratus red var ♂) infected with Aeromonas hydrophila

Aeromonas hydrophila is one of important pathogenic bacteria that can imperil fish survival. Current study suggested that hybrid crucian carp (WR) receiving acute A. hydrophila challenge exerted a molecular regulation linking peroxisomal fatty acid β-oxidation to immune response. A. hydrophila infection may alleviate antioxidant status along with the significant alternation of immune-, apoptosis- and metabolic-related gene expressions in kidney, whereas most portions of differential metabolites (DMs) were enriched in steroid derivatives and phospholipids. In addition, mevalonate (MVA) pathway and phospholipid hydrolysis were identified as crucial cellular processes in WR after A. hydrophila challenge. These results may provide an insight into immunometabolic strategy against A. hydrophila infection.

Exogenous paternal mitochondria rescue hybrid incompatibility and the destiny of exogenous mitochondria

The mitochondria of most organisms follow strict maternal inheritance, and the mechanism of elimination of paternal mitochondria is unclear. Our previous studies showed that the paternal mtDNA presented in the embryo of hybrid Megalobrama Amblycephala (BSB,♀) ​× ​Carassius auratus red var(RCC,♂) (BR) and its reciprocal hybrid (RB), but its expression was quiescent. However, the microinjected mitochondria persisted in the embryo and its mtDNA expressed throughout embryonic development. In addition, the chromosome number of RCC (2n ​= ​100) is much larger than that of BSB (2n ​= ​48). All BR embryos were severely abnormal and ultimately died, while the RB embryos could survive and grow up into adult. It implied that the nuclear-cytoplasm incompatibility may be an important cause of BR abnormality. In this study, exogenous parental mitochondria were microinjected into BR embryos, and it was found that paternal mitochondrial DNA persisted and expressed throughout embryonic development. Meanwhile, the study also found that microinjection of isolated paternal (RCC) mitochondria into BR embryos significantly improved the degree of early embryonic abnormality with some fry swimming normally, comparing to the control embryos. However, injection of maternal (BSB) mitochondria did not improve the development of BR embryos. We safely concluded exogenous mitochondria escape the mechanism of elimination and its DNA persist and express throughout embryonic development. In addition, the expression of paternal mitochondrial genes largely reduces the cyto-nuclear conflict, so that the early BR embryos exhibit less degree of abnormality and enhanced activity.

Immunoglobulin heavy-chain loci in ancient allotetraploid goldfish

As an ancient allotetraploid species, goldfish (Carassius auratus) have two sets of subgenomes. In this study, immunoglobulin heavy-chain (IGH) genes were cloned from the red crucian carp (Carassius auratus red var.), and the corresponding loci were identified in the gynogenetic diploid red crucian carp (GRCC) genome as well as the genomes of three other goldfish strains (Wakin, G-12, and CaTCV-1). Examination showed that each goldfish strain possessed two sets of parallel IGH loci: a complete IGHA locus and a degenerated IGHB locus that was nearly 40 × smaller. In the IGHA locus, multiple τ chain loci were arranged in tandem between the μ&δ chain locus and the variable genes, but no τ-like genes were found in the IGHB locus.

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.

First report of cyprinid herpesvirus 2 isolated from the golden crucian carp in China

In this study, a virus strain designated GCC2021 was isolated from the golden crucian carp (Carassius auratus red var. ♂ × Cyprinus carpio L. mirror ♀) in Jiangsu Province, China. GCC2021 was propagated in the Gibel carp brain (GiCB) cell line with a typical cytopathic effect. Healthy golden crucian carp was injected with GCC2021 virus solution intraperitoneally, and the symptoms of the artificially infected fish were consistent with those of the naturally diseased golden crucian carps. The mortality was 6.67 ± 3.33% (kidney homogenate group) and 4.44 ± 1.93% (cell culture virus group). The histopathological results indicated systemic inflammation and changes in multiple organs. Electron microscopy observation using ultra-thin sections of the diseased fish kidney revealed a number of spherical particles with capsid that were approximately 110–120 nm in diameter, and the mature virus particle was about 170–220 nm in diameter. The helicase (Hel) gene of GCC2021 was amplified using PCR and sequenced. PCR amplification showed positive results for GCC2021 in the diseased fish, artificially infected fish, and infected GiCB cells. Sequence analysis of the amplification products showed 99.68% identity with the helicase gene of CyHV-2 SY-C1 strain. Phylogenetic analysis of the CyHV-2 ORF25B non-coding region revealed that the isolate clustered with CyHV-2 1301 strain, SY strain, and SY-C1 strain. CyHV-2 copies in different tissues were determined using digital PCR. Viral loads were detected in the liver, spleen, kidney, brain, intestine, and gill, with the highest in the kidney and lowest in the brain. In this study, it was provided solid evidence that CyHV-2 could infect golden crucian carp.