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In this study, we produced a population of gynogenetic mrigal carp (Cirrhinus mrigala , GMCJ) through artificial gynogenesis using UV-inactivated white crucian carp spermatozoa. The GMCJ exhibited a faster growth rate and strong resistance to low temperatures, surviving natural winter conditions with temperatures below 10 °C. To gain a deeper insight into GMCJ's molecular response to acute cold stress (a decrease in water temperature from 26 °C to 14 °C within 1 h at a rate of 0.2 °C/min), we conducted a comparative analysis of the histology and transcriptomics of the brain-liver axis. Transmission electron microscopy of the heart tissues revealed that acute cold stress result in widened myofilament spacing, along with enlarged, deformed mitochondria that developed vacuoles and ruptured. We identified differentially expressed genes (DEGs) in the brain (2463 upregulated and 3288 downregulated genes) and liver (1210 upregulated and 1224 downregulated genes). Analysis of the DEGs' correlations indicated that major pathways involved in immunity, disease, metabolism, and growth were active and interconnected. KEGG functional analysis showed that the most enriched pathways in the brain-liver axis involved the regulation of the glucagon pathway, PI3K-Akt signaling pathway, and herpes simplex infection. Numerous genes associated with the nervous-endocrine system were activated, suggesting a coordinated response of the brain-liver axis to acute stress. These findings enhance our understanding of how farmed, particularly gynogenetic fish respond to acute cold stress. • GMCJ fish show strong resistance to low temperatures. • Immunity and metabolism genes active under acute stress. • Brain-liver axis coordinates response to acute stress. [ABSTRACT FROM AUTHOR]

Crucian carp (Carassius auratus) is one of the main freshwater cultured fish species in China, and its species degeneration and diseases outbreak for aquaculture brings great economic losses. However, the blood single cellular morphology and molecular identification still needs further investigation in fish. Here, the blood parameters, single-cell types and shapes, physicochemical indexes and phagolysosome (PL)-related gene expression analysis of C. auratus were examined. The results showed that four single-cell types of blood cells, including hematopoietic progenitor cells (HPCs), red blood cells (RBCs), proerythrocytes (PEs) and platelets (PLTs) were identified by pseudotime trajectory analysis in the whole blood and kidney, respectively. The involvement in the PL signaling pathway was significantly observed in GSEA enrichment analysis, and their expression distribution (e.g., CD63, ITGB2, CXCR4b, MMP2, MHCI uka, B2ML, CD74a, FTH1a, COX8b, COX7a2) were evaluated in various tissues. A protein-protein interaction network analysis was performed on the highly expressed cellular markers and co-expressed genes. Moreover, CaCD63 gene cloning and expression, and molecular docking interaction were presented with the integrin (ITGB2) and chemokine receptor (CXCR4b). Overall, this study firstly revealed the molecular marker features of blood cells and provided a valuable reference for further function verification in cyprinid fish. • Blood cell and physicochemical indexes analysis were examined in crucian carp C. auratus. • Four types of single cells (HPCs, RBCs, PEs, and PLTs) were described in detail. • Molecular docking interaction of CD63 with ITGB2 and CXCR4b were explored for the first time. • Expression distribution of 17 phagolysosome-related genes was evaluated in various tissues. [ABSTRACT FROM AUTHOR]

The application of cold shock to inhibit the extrusion of the second polar body in fertilized eggs is one of the main methods to induce triploid in diploid fish. In this study, we originally planned to induce hexaploid by this method in tetraploid carp. However, among the surviving offspring, we observed only 50.7% tetraploid and 49.3% diploid individuals, which could potentially result from either androgenetic diploids or gynogenetic diploids. The absence of irradiation and the high proportion of diploids attracted us. However, the lack of sex-specific markers for tetraploid carp, makes it difficult to determine their origin. Fortunately, we also carried out the cross of yellow carp (Cyprinus carpio yellow var., 2n, ♀) × tetraploid carp (♂) and red crucian carp (Carassius auratus red var., 2n, ♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis, diploid offspring have also been found, and whether they have undergone gynogenesis or androgenesis can be judged based on phenotype and genotype. In terms of phenotypes, the key phenotypes of two diploids, such as the whisker length, number of lateral scales, and presence of melanin, are consistent with tetraploid carp. In terms of genotypes, through single-copy nuclear gene cloning and fluorescence in situ hybridization (FISH), it is supported that both diploids are androgenetic offspring. In addition, 14.9% of tetraploid offspring were found in red crucian carp (♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis. By the above-mentioned means of detection, it was found that they were created by inhibiting the extrusion of the second polar body of the fertilized eggs. From this it will be seen that the application of cold shock before the extrusion of the second polar body of fertilized eggs cause not only polyploidy, but also the elimination of both second polar body and female nucleus. Previous studies have found that cold shock can induce lethal androgenetic haploids in diploid fish, but these androgenetic diploid fish produced by tetraploid carp sperm were surviving. We summarize the mechanism behind the above phenomenon, which is mainly related to meiosis occurring at the edge of the cell membrane. Cold shock may disrupt the function of the spindle, resulting in the simultaneous retention or extrusion of female nucleus and second polar body. This study will provide valuable insights into androgenesis, polyploidization, and hybridization in fish genetic breeding. • Androgenetic diploids were induced from tetraploid carp sperm by cold shock without irradiating the eggs. • New-type allotetraploids were induced in the cross between male tetraploid carp and female diploid carp by cold shock. • Nuclear gene cloning is suitable for qualitative analysis of the genetic composition of hybrids, but not for quantitative analysis. [ABSTRACT FROM AUTHOR]

The effects of different concentrations of sodium bicarbonate (0 g/L, 1 g/L, 2 g/L, 3 g/L) on Hefang crucian carp (HCC) were studied. H&E staining, oil red O staining, scanning electron microscopy, transmission electron microscopy, 16S rDNA sequencing, transcriptomic analysis and other techniques were used to observe the pathological changes of intestinal and liver tissues, and detect the changes of intestinal flora and liver gene expression. The main findings are as follows: It was found that the WGR and SGR of the three treatment groups was significantly higher than that of the CK group (p < 0.05). Intestinal H&E staining results showed that the number of goblet cells and the width of submucosa gradually increased with the increase of carbonate concentration. The results of intestinal scanning electron microscopy showed that the roughness of intestinal villi increased slightly with the increase of carbonate concentration. The analysis of populations with large differences among groups treated with sodium bicarbonate of different concentrations found that the relative abundance of Lachnospiraceae varied greatly. LPS levels in the serum, intestinal tract and liver tissue were detected. The results showed that LPS content in MCA and HCA groups was significantly higher than that in CK group (p < 0.05). The expression of claudin 12, 7, c and occludin genes in HCA group and MCA group was significantly lower than that in CK group in intestinal (p < 0.05). The ALT, AST, T-CHO, TG, HDL C, ACC, ACS, CPT-1, HL, LPL and FAS content in HCA group was significantly higher than that in CK group in liver (p < 0.05). The liver was damaged by oil red O staining and transmission electron microscopy. Compared with the control group, the enrichment of steroid biosynthesis is the highest number of DEGs in the HCA group. The iron death pathway and autophagy pathway in HCA group were significantly different from those in CK group (p < 0.05). In summary, this study elucidates that carbonate exposure alters the gut microbiota homeostasis of HCC, leading to intestinal inflammation and increased mucosal barrier permeability, promoting LPS synthesis and absorption, subsequently entering the liver through the gut-liver, increasing liver autophagy and iron death, inducing lipid metabolism disorders, and causing liver injury. In summary, carbonate exposure may cause intestinal microbial disturbance and intestinal injury, and induce liver autophagy and iron death and lipid metabolism disturbance, leading to liver injury • Carbonate stress inhibited the growth of Hefang crucian carp through enterohepatic axis pathway. • Carbonate stress can destroy intestinal flora homeostasis of Hefang crucian carp. • Carbonate stress induced lipid metabolism disorder in the liver of Hefang crucian carp. [ABSTRACT FROM AUTHOR]

Aeromonas veronii has serious implications for food safety and the aquaculture industry. The iron uptake system can effectively regulate the life activities and pathogenicity of microorganisms, and Fur is one of the key proteins. In this experiment, we constructed the Δ fur mutant strain of A. veronii TH0426 using the pRE112 suicide plasmid and the C- fur complementary strain using the pBBR1-MCS generalized host expression plasmid to research the effects of fur gene on the biological function and mechanisms of virulence regulation of TH0426. It was found that when the fur gene was deleted, the polar flagella of TH0426 was disrupted and the biofilm formation ability was significantly reduced, with consequent changes in motility, flagellation-related gene expression, drug resistance, and tolerance. Transcriptome analysis of TH0426 wild-type and Δ fur mutant showed that fur gene could directly or indirectly regulate bacterial flagella assembly, antibiotic resistance, growth and metabolism. In the pathogenic test, compared with the TH0426 wild-type, Δ fur significantly reduced the cytotoxicity to EPC cells at 30 min, 1 h and 2 h, reduced the adhesion and invasion ability to EPC cells by 2.38-fold (P < 0.001), increased LD 50 of zebrafish by 58.33-fold (P < 0.001) and decreased bacterial load at 12 h, 24 h, and 48 h. Meanwhile, the result of C- fur was higher than that of Δ fur and lower than that of the TH0426 wild-type in all experiments. In summary, the fur gene is essential for flagellar assembly and biofilm formation and is able to indirectly affect some of the biological properties and animal pathogenicity of TH0426. This research helps to explain the function of the fur gene of the iron uptake system and its role in the pathogenesis of animal. • fur gene can directly regulate the assembly of polar flagella in A. veronii TH0426. • fur gene could directly regulate the biofilm formation of A. veronii. • Deletion of fur gene could significantly reduce the pathogenicity of A. veronii to crucian carp and zebrafish. • Deletion of fur gene can affect multiple metabolic pathways in A. veronii. [ABSTRACT FROM AUTHOR]

Establishing beneficial intestinal microbiota using autochthonous probiotics is a promising approach for disease control and growth regulation during fish rearing. Therefore, it is crucial to identify indigenous probiotics with high application potential in aquaculture. In this study, we screened Bacillus coagulans BC1, a strain with strong application potential. After a comprehensive safety assessment, diets containing varying doses of BC1 were formulated and administered to crucian carp. Subsequently, a feeding experiment was conducted to evaluate non-specific immune response, intestinal structure, microbiota, and challenge protection rate. The results demonstrate that oral supplementation of BC1 can lead to tissue-dependent upregulation of various serum immune enzymes and IgM levels, while significantly enhancing the expression of IL-1β , IL-10 , TNF-α , and TGF-β in various organs of crucian carp (p < 0.05). The BC1 diet effectively promoted intestinal morphology in crucian carp and improved resistance to Aeromonas veronii TH0426 infection. Intestinal microbiota analysis revealed that the oral addition of BC1 significantly changed the intestinal microbial community structure of crucian carp but did not clearly affect intestinal microbial diversity. Oral administration of BC1 significantly improved the relative abundance of Cetobacterium in the intestines of crucian carp; reduced the abundance of opportunistic pathogens such as Bacteroides , Vibrio and Aeromonas and effectively regulating the nutrient metabolism in intestinal microbiota. These results indicate that BC1 could regulate immune function, effectively improving the intestinal morphology and intestinal flora structure and function of crucian carp, and therefore is a potential candidate strain for aquatic feed additives. • B. coagulans strain BC1 with strong application potential in aquatic feed additive was obtained by in situ screening. • Oral BC1 effectively enhanced the non-specific immunity of crucian carp juvenile. • BC1 diet could regulate the structure and function of intestinal flora of crucian carp juvenile. [ABSTRACT FROM AUTHOR]

Rahnella aquatilis is widely recognized as an opportunistic pathogen of fish, but molecular detection and diagnostic techniques for it remain to be developed. In a previous study, we isolated the pathogenic R. aquatilis strain KCL-5 from diseased crucian carp Carassius auratus and identified it as a new fish pathogen causing enteritis and septicemia. Here, we describe novel real-time quantitative PCR (RT-qPCR) and loop-mediated isothermal amplification (LAMP) assays for the rapid and sensitive detection of R. aquatilis. Specific RT-qPCR and LAMP primers were developed for the outer membrane protein (OmpA) gene. Their reaction systems were optimized using the recombinant plasmid pMD18-T containing the OmpA gene and their specificities were confirmed using various bacterial fish pathogens including Aeromonas hydrophila , A. veronii , A. allosaccharophila , Yersinia ruckeri , Vibrio vulnificus , V. alfacsensis, Providencia rettgeri , and R. aquatilis. The sensitivities of the RT-qPCR and LAMP assays were further evaluated using serial dilutions of the positive recombinant plasmid pMD18-T/ OmpA. Both the RT-qPCR and LAMP assays were able to reliably detect the presence of R. aquatilis with high accuracy and a specificity of 100%. The lowest detection limits of the RT-qPCR and LAMP assays were 2.3 copies/μL and 2.3 × 101 copies/μL, respectively. To the best of our knowledge, this is the first report of RT-qPCR and LAMP methods for R. aquatilis detection, and provides a means for its rapid detection and diagnosis in fish. • Development of qPCR assay for detection of R. aquatilis in fish. • Establishment of LAMP assay for detection of R. aquatilis with MDL of 2.3 × 101 copies/μL. • The qPCR and LAMP methods were firstly established for the rapid detection of R. aquatilis. • Analysis of bacterial loads was performed for R. aquatilis infection in fish. [ABSTRACT FROM AUTHOR]

Whilst the hybrids of F1 generations usually experience heterosis for fitness-related traits (including the resistance to parasites), post-F1 generations, due to Dobzhansky–Muller genetic incompatibilities, express numerous disadvantageous traits (including susceptibility to parasites). Genetic disruption in hybrids may also result from the broken system of cyto-nuclear coadaptation. Maternal backcrosses (having parents with the same mtDNA) and paternal backcrosses (having parents with different mtDNA) have the same nuclear genetic compositions, but differ in cytoplasmic genetic elements, affecting their viability and survival. Spring viraemia of the carp virus (SVCV), a disease with a serious economic impact in aquacultures, affects almost exclusively cyprinids, primarily common carp (Cyprinus carpio), and causes high mortality, whilst gibel carp (Carassius gibelio) is a less susceptible species. Our study was focused on the transcriptome profile analysis of head kidney to reveal differential gene expression in highly susceptible common carp, weakly susceptible gibel carp, and hybrid lines, hypothetizing that the patterns of differential gene expression will reflect hybrid heterosis in F1 generations and hybrid breakdown in backcrosses and F2 generations. We expected the differences in differential gene expression between maternal and paternal backcrosses to be in line with the hypothesis of broken cyto-nuclear coadaptation. The pattern of differential gene expression revealed from the transcriptomic analysis of head kidney was linked to the resistance or susceptibility to SVCV infections in two pure species, C. gibelio and C. carpio , and their various generations of inter-specific hybrids. We evidenced hybrid heterosis for F1 generations of hybrids, mostly reflected by similarity in the down-regulation of differentially expressed genes classified within the viral infection disease category. High hybrid breakdown was documented throughout the transcriptomic analysis for paternal backcross generations and F2 generation of hybrids, confirming the broken system of cyto-nuclear coadaptation in those post-F1 generations. This evidence may support the role of intrinsic selection acting against inter-specific hybridization in freshwater fish. • Spring viraemia of carp is a disease with a serious economic impact for aquacultured fish. • We performed transcriptome profile analysis of the head kidney to reveal differential gene expression (DEG) in highly susceptible common carp, weakly susceptible gibel carp, and hybrid lines, hypothesizing that the patterns of differential gene expression reflect heterosis in F1 hybrids and breakdown in backcrosses and F2 hybrids. • DEG was linked to the resistance or susceptibility to SVCV infections in pure species and their various generations of hybrids. • We evidenced the similarity in DEGs between F1 hybrids and weakly susceptible gibel carp supporging hybrid heterosis for F1 hybrids. • Hybrid breakdown was suggested for paternal backcross generations and F2 generation of hybrids, confirming the broken system of cyto-nuclear coadaptation. [ABSTRACT FROM AUTHOR]

TNFα is a multifunctional cytokine that can modulate immune homeostasis and promote cellular regulation in mammals, but the crucial role of TNFα in fish gut remained unclear. The current study aimed to evaluate the immunoregulatory function of TNFα2 on gut barrier breach by A. hydrophila and systemic pathogenesis in gut and liver of WR (White crucian carp ♀ × red crucian carp ♂). WR-TNFα2 encoded 232 amino acid residues and showed a high-level of WR-TNFα2 expression in spleen. After A. hydrophila and lipopolysaccharide (LPS) stimulated, elevated levels of WR-TNFα2 expressions were detected in immune-related tissues and cultured fish cells, respectively. Gut perfusion with WR-TNFα2 alone could alter antioxidant status, increase extrinsic apoptosis as well as promote inflammatory response in midgut, causing severe injury of epithelial barrier. Moreover, WR-TNFα2-treated midgut exhibited a decline of mucosal protection against invasive A. hydrophila , which could elevate bacterial burden in tissues, aggravate tissue injury as well as attenuate immune activation. Our results suggested that WR-TNFα2 could exhibit a detrimental effect on gut barrier defense against A. hydrophila infection and dysregulate immune response in midgut and liver of hybrid crucian carp. • The predicted structure and expression patterns of TNFα2 were firstly reported in WR. • WR-TNFα2 altered antioxidant status and increased extrinsic apoptosis in midgut. • WR-TNFα2 facilitated bacterial infection by impairing gut barrier and then exacerbated liver lesion. [ABSTRACT FROM AUTHOR]

Based on the formation of fertile allotetraploid fish (4nJB, 4n = 148) derived from female Carassius cuvieri (JCC, 2n = 100) × male Megalobrama amblycephala (BSB, 2n = 48), an improved Japanese white crucian carp (IJCC) was obtained by mating female 4nJB and male 4nJB, and thus far, the population of IJCC has reached the fifth generations (F 1 –F 5). The purpose of the present study was to comparatively investigate the biological characteristics of the IJCC lineage (F 1 –F 5) and their original female parent JCC. The results of DNA content and chromosome numbers showed that the IJCC lineage was diploid with 100 chromosomes. For morphological characteristics, it appears that IJCC-F 1 has smaller head and a higher body width than that of JCC, and some countable traits as well as the ratio of measurable traits were significantly different (P < 0.05) between these two fish. With the passing of generations, the appearance of the IJCC lineage has been stable. Regarding reproductive characteristics, the fecundity, sperm volume, sperm density, fertilization rate and hatching rate were not significantly different (P > 0.05) between the JCC and IJCC lineages, indicating that the reproductive capacity of the IJCC lineage was similar to that of the JCC. Furthermore, the growth performance showed that the average weight of IJCC-F 1 and IJCC-F 5 was significantly greater (P < 0.05) than that of JCC both at 6 months and 1 year of age. The good fertility of the IJCC lineage ensures the multigeneration utilization of its advantage, and the IJCC lineage has significant value in aquatic applications. • The improved IJCC lineage was established derived from JCC (♀) × BSB (♂). • The produced IJCC lineage grows faster than JCC. • The reproductive capacity of IJCC lineage was similar to that of JCC. [ABSTRACT FROM AUTHOR]

Certain bacterial strains could efficiently improve the conversion of insoluble phosphorus to active phosphorus due to their phosphate-solubilizing ability, but there is little research on its specific effects on aquaculture. In this study, a mutant strain Bacillus cereus S458-M was obtained via compound mutagenesis of UV and LiCl. We found it exhibited higher phosphate-solubilizing ability and phytase activity than the original strain S458-1 reported in our previous report. The sequence analysis of phytase gene (phyC) in S458-M revealed mutations in amino acids 81, 89 and 148. Its predicted protein contained an additional Ca2+ binding site that might be associated with higher phytase activity. To verify phosphate-solubilizing and probiotic properties of S458-M, no bacteria (CG: control group), S458-1 (LS: 1 × 105 CFU/mL; HS: 1 × 107 CFU/mL) and S458-M (LM: 1 × 105 CFU/mL; HM: 1 × 107 CFU/mL) were supplemented into aquaculture water separately. The results showed that S458-M could improve the concentration of active phosphorus in aquaculture water and enhance the antioxidant ability of crucian carp. Overall, S458-M obtained by UV-LiCl mutagenesis could support the application of phosphate-solubilizing bacteria in aquaculture water. • UV-LiCl mutagenesis was used in the genetic mutation of Bacillus cereus S458-M. • S458-M exhibited higher phosphate-solubilizing ability and phytase activity. • An additional Ca2+ binding site was observed in the predicted phytase of S458-M. • S458-M could improve the concentration of active phosphorus in aquaculture water. • S458-M could enhance the antioxidant ability of crucian carp. [ABSTRACT FROM AUTHOR]

A 70-day feeding trial was conducted to investigate the effects of dietary supplementation with prebiotics β-glucan (BG group) and probiotics Bacillus subtilis (BS group) on growth performance, fillet quality, immune response, and antioxidant status of Pengze crucian carp (Carassius auratus var. Pengze). The results showed that supplementation of B. subtilis significantly improved the growth performance than other groups whereas feed efficiency was unaffected by dietary treatments. The textures of muscle in terms of hardness, springiness, cohesiveness, gumminess, chewiness, and resilience were higher in BG and BS groups than Control group. Supplementation of β-glucan and B. subtilis acted as an hypolipidemic in terms of decreasing the total cholesterol, high-density lipoprotein, and low-density lipoprotein whereas, increased the immune responses in serum measured by acid phosphatase, alkaline phosphatase, and catalase activities. Dietary supplementation of β-glucan and B. subtilis significantly improved the fold height and microvillus height in contrast to basal diet. Moreover, β-glucan could significantly increase digestive capacity observed in terms of an increase in amylase and trypsase activities, and B. subtilis significantly increased amylase and lipase activities in intestine. β-glucan and B. subtilis supplementation enhanced the immunity and antioxidant capacity via an increase in acid phosphatase, alkaline phosphatase, glutathione peroxidase, reduce glutathione, catalase, and total superoxide dismutase activities, accompanied by a significant reduction in malondialdehyde. Overall, the results indicated that B. subtilis improved the growth performance by increasing the digestive enzymes activities and improving the intestinal morphology. β-glucan and B. subtilis supplementation significantly improved the fillet quality, immune responses and antioxidant status of Pengze crucian carp. • Dietary supplementation with Bacillus subtilis significantly improved the growth of Pengze crucian carp. • β-Glucan and B. subtilis supplementation significantly affected the fillet quality. • β-Glucan and B. subtilis supplementation significantly improved the immune responses and antioxidant status of fish. [ABSTRACT FROM AUTHOR]

Abstract Hybridization is an important technique and is widely used in fish genetic breeding; this technique prevents variety degeneration and improves variety. In this research, we obtained a hybrid lineage (WR-F 1 -F 4) by interspecific hybridization derived from female white crucian carp (Carassius cuvieri , WCC) and male red crucian carp (Carassius auratus red var., RCC), and a new type of improved fish (WR-II) was obtained by back-crossing between the female hybrid (WR-F 1) and the male WCC. We investigated the morphological traits, chromosomal numbers, DNA content, gonadal development, sperm shape and 5S rDNA in the hybrid lineage and WR-II. The results showed that all the assessed morphological traits were not significantly different between the progenies of the hybrid lineage and their original parents, but the WR-II showed smaller head and higher body compared with its original parents; the hybrid lineage and WR-II were diploid with 100 chromosomes; all the tested males and females of the hybrid lineage and WR-II had normal gonadal development and produced mature sperm and egg, respectively; the 5S rDNA of the hybrid lineage and WR-II inherited the characteristics of their parents and had some mutant bases or chimeric sequences, which revealed heredity, variability and heterozygosity in the hybrid progenies; finally, the 5S rDNA of the hybrid lineage could be highly inherited from the second generation (WR-F 2) to the next generations. This study will be beneficial for the establishment and application of hybrid lineages in genetic breeding. Highlights • A hybrid lineage (WR-F 1 -F 4) was obtained by hybridization derived from female WCC and male RCC. • A new type of improved fish (WR-II) was obtained by back-crossing between female WR-F 1 and male WCC. • WR-II showed smaller head, higher body and faster growth compared with its original parents. • This study will be beneficial for the establishment and application of hybrid lineage in genetic breeding. [ABSTRACT FROM AUTHOR]

Abstract The pharmacokinetic profiles of florfenicol (FF) and florfenicol amine (FFA) following a single oral dose of FF at 10 mg·kg−1 body weight were compared in crucian carp reared at three different temperatures of fresh water. The concentrations of FF and FFA were determined simultaneously by high-performance liquid chromatography coupled with fluorescence detection, followed by compartmental analysis using a one-compartment open model. At water temperatures of 10 ± 0.5, 20 ± 0.5, and 25 ± 0.5 °C, the maximum concentration (C max) of FF was 2.435, 2.795, and 3.555 μg·mL−1, respectively, while that of FFA was 0.810, 0.945, and 1.063 μg·mL−1, respectively. The absorption half-life (t 1/2ka) of FF was 0.72, 0.73, and 0.70 h, and the elimination half-life (t 1/2kel) was 22.86 12.29, and 9.56 h, respectively. The volume of distribution per fraction absorbed (V d /F) for FF at water temperatures of 10, 20, and 25 °C was 3.67, 2.99, and 2.29 L·kg−1, respectively. For FFA, the formation half-life (t 1/2kf) was 2.13, 3.47, and 5.11 h, respectively, whereas the t 1/2kel was 27.79, 20.33, and 15.44 h at water temperatures of 10, 20, and 25 °C, respectively. At higher water temperatures, FF was absorbed and eliminated more quickly and was more extensively distributed than at lower water temperatures; in addition, the formation and elimination of FFA were faster at higher water temperatures. From the values of time > minimum inhibitory concentration (T > MIC) calculated in the current study at 10 °C, an oral dose of FF at 10 mg·kg−1 body weight administered at 48-h intervals would be sufficient for the treatment of bacterial infection with an MIC of ≤0.5 μg·mL−1, whereas a 24-h interval would be necessary at 20 and 25 °C to treat bacterial infections with the same MIC. Highlights • The plasma concentrations of florfenicol amine were reported for the first time in crucian carp. • The pharmacokinetic profiles of florfenicol and florfenicol amine in crucian carp were compared at three water temperatures. • The dosing regimens were obtained for florfenicol in crucian carp reared at three different water temperatures. [ABSTRACT FROM AUTHOR]

Abstract In recent years, Cyprinid herpesvirus 2 (CyHV-2) has severely affected the culture of crucian carp Carassius auratus gibelio , causing massive economic losses. However, the responses of crucian carp during infection with CyHV-2 have not been explored. In this study, physiological and biochemical indicators and enzyme activities of crucian carp before and after infection with CyHV-2 were analyzed. The number of white blood cells in the diseased fish was significantly increased. Compared to control group, red blood cell content, hemoglobin content, mean corpuscular volume, mean corpuscular hemoglobin content and mean corpuscular hemoglobin concentration in the experimental group were significantly decreased. In the diseased fish group, the levels of the glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, alkaline phosphatase, lactate dehydrogenase and urea nitrogen in the blood were also significantly increased compared to the control group. In addition, the activity of serum total antioxidant capacity, acid phosphatase, glutathione-S transferase and catalase in diseased fish were significantly higher than in the control group. LC-Q/TOF-MS analysis platform was used in this study for the first time to detect changes in the serum metabolome of crucian carp infected with CyHV-2. A total of 79 differential metabolites have been identified. Significant increases in concentrations were found for several carbohydrates (glucose, mannose, ribose, etc.), fatty acids (pentadecanoic acid, arachidonic acid, palmitic acid, eicosapentaenoic acid, hydroxylic tetracarbonate alkenoic acid, linoleic acid and its derivatives) and most of the amino acids and their derivatives. However, guanine, cytosine, uridine and VB2 were significantly decreased. KEGG pathways enrichment showed that amino acid metabolism and lipid metabolism were severely influenced. The above results provide a theoretical basis for the infection mechanism of CyHV-2 Highlights • The WBC was increased in the blood of CyHV-2 infected fish while RBC, HB, MCV, MCH and MCHC were decreased. • ALT, AST, ALP, LDH, BUN, T-AOC, ACP, GST and CAT were significantly increased in the blood of CyHV-2 infected fish. • 79 differential metabolites (75 with significant differences) were identified after CyHV-2 infection. [ABSTRACT FROM AUTHOR]

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. • Triploidization could alter intestinal flora and gut characteristics in autotriploid Carassius auratus. • Growth rate and activities of intestinal digestive enzymes (cellulase, amylase and trypsin) were higher in autotriploid Carassius auratus compared with that in Carassius auratus red var.. • Spearman's correlation analysis showed a positive correlation between bacteria orders (Clostridiales, Micrococcales and Lactobacillales) and fast growth in autotriploid Carassius auratus. [ABSTRACT FROM AUTHOR]

Previously, we have reported diploid YY male fish obtained by the combination of androgenetic technology and distant hybridization and described its special reproductive characteristics producing unreduced and diploid homogametes. The above diploid sperm displayed developmental advantages, forming all-male offspring, and directly developing into individuals without chromosome doubling during androgenesis. However, the related molecular advantages of YY fish sperm have not been reported. In this study, we firstly compared the semen of two kinds of male fish (XY and YY), both obtained by androgenetic technology with the autotetraploid fish sperm. Morphological features exhibited by electron microscope revealed that YY fish sperm is normal, while partial XY fish sperm has defects with uncompacted nucleus or cell degradation. Moreover, a comparison of global transcriptomes showed that the expression of genes related to immunity and energy production was increased in YY fish semen, while genes related to meiosis and spermiogenesis were highly expressed in XY fish semen. Then, we compared the semen of YY fish and red crucian carp (RCC), in which the sperm has normal structure but the different developmental potential for different ploidy. Epigenetic analysis revealed that di/tri- methylation on histone H3 lysine 9 (H3K9me2/3) exhibited higher levels in YY fish semen than in RCC semen. Consisting with the variation of H3K9me2/3, expression of histone methylase gene setdb1b increased, while demethylase gene kdm4a decreased in YY fish semen. Taken together, this is the first time reported the molecular dominance of diploid sperm of YY fish, which will be helpful for the following practice aquaculture breeding application. • The YY sperm are normal and exhibit advantages in immunity and energy metabolism. • Genes related to spermatogenesis and maturation expressed abnormally in the XY semen. • Compared to haploid sperm, the diploid sperm contained higher levels of H3K9me2/3. [ABSTRACT FROM AUTHOR]

Elimination of intermuscular bones (IMBs) is vital to the aquaculture industry of cyprinids. In our previous study, we characterized bmp6 as essential in the development of IMBs in zebrafish. Knockout of bmp6 results in the absence of IMBs in zebrafish without affecting growth and reproduction. Therefore, we hypothesized that bmp6 could be used to generate new cyprinid strains without IMBs by gene editing. In this study, we established a gene editing strategy for knocking out the two orthologs of bmp6 in diploid crucian carp (Carassius auratus). We obtained an F 3 population with both orthologs knocked out, in which no IMBs were detected by bone staining and X-ray, indicating that the new strain without IMBs (named WUCI strain) was successfully generated. Furthermore, we extensively evaluated the performance of the new strain in growth, reproduction, nutrient components, muscle texture and structure, and metabolites in muscle. The results showed that the WUCI strain grew faster than the wild-type crucian carp at 4-month-age. The reproductive performance and flesh quality did not show significant differences between the WUCI strain and wild-type crucian carp. Moreover, the metabolomics analysis suggested that the muscle tissues of the WUCI strain significantly enriched some metabolites belonging to the Thiamine metabolism, Nicotinate and Nicotinamide Metabolism pathway, which plays beneficial effects in anti-aging, anti-oxidant, and anti-radiation damage. In conclusion, we established a strategy to eliminate IMBs in crucian carp and obtain a WUCI strain whose performance was expected compared to the wild-type crucian carp; meanwhile, the WUCI strain enriched some beneficial metabolites to human health in muscle tissue. This study is the first report that a farmed cyprinid strain without IMBs, which could be stably inherited, was obtained worldwide; it provided excellent germplasm for the cyprinids aquaculture industry and a useful molecular tool for eliminating IMBs in other cyprinids. • A gene-editing strategy was established for rapidly knocking out multiple orthologs • The first report of an inherited farmed cyprinid strain without intermuscular bones in the world • The new cyprinid strain without intermuscular bones enriched beneficial metabolites in muscle • Production performance of the new cyprinid strain without intermuscular bones is similar to wildtype [ABSTRACT FROM AUTHOR]

Aquaculture needs effective antibiotic alternatives to control outbreaks of intestinal inflammation, as well as liver inflammation and metabolic disorder caused by enteritis. Specific metabolites have been shown to have immunomodulatory effects by the field of immunometabolism. As derivatives of the Krebs cycle metabolites, the itaconate and fumarate, 4-octyl itaconate (OI) and dimethyl fumarate (DMF) have recently attracted a lot of interest as anti-inflammatory medications. To evaluate the effects of oral administration of OI and DMF on fish growth and health, an 8-week feeding experiment followed by induction of enteritis with 2,4,6-trinitrobenzene sulfonic acid (TNBS) was carried out in gibel carp. The results showed that dietary 0.1%DMF improved the growth performance, and 0.1%OI up-regulated the abundance of beneficial intestinal microbiota. Both of OI and DMF alleviated intestinal inflammation by increasing intestinal anti-inflammatory cytokines, and relieved enteritis-induced hepatopancreas injury by inhibiting glycolysis both in vivo and in vitro. The 0.1%DMF also enhanced the tight junction and altered the microbiota to maintain the intestinal barrier. Collectively, dietary supplementation with 0.1% OI and DMF might be novel strategies to prevent enteritis and intestinal inflammation-induced hepatopancreas damage in gibel carp. [Display omitted] • Dietary 0.1%DMF improved the growth performance. • Both 0.1% OI and 0.1% DMF alleviated TNBS-induced intestinal inflammation in gibel carp. • DMF enhanced the tight junction and altered the microbiota to maintain the intestinal barrier. • OI and DMF alleviated enteritis-induced liver injury by inhibiting glycolysis both in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Herpesviral hematopoietic necrosis caused by cyprinid herpesvirus-2 (CyHV-2) is highly prevalent in gibel carp (Carassius gibelio Bloch) and is associated with high mortality and great economic loss. Currently, no efficient methods of prevention and control of CyHV-2 infection are available. Increasing evidence suggests that antigen-specific egg yolk immunoglobulins (IgY) are highly effective against various pathogens in farmed fish. Therefore, the aim of this study was to develop a specific IgY against CyHV-2 infection in gibel carp. We first produced a recombinant D4ORFs protein that encodes four open reading frames of CyHV-2 and then generated specific IgY by immunizing laying hens with this protein. The eggs laid by immunized hens were processed into spray-dried egg powder containing D4ORFs IgY. The D4ORFs IgY showed high neutralizing activity in vitro against CyHV-2, as evidenced by a significant reduction in cytopathic effect in gibel carp brain cells cultured in the presence of the IgY antibodies. Whole-egg powder containing IgY specific to D4ORFs protein was incorporated into the basal diet at levels of 0, 0.1, 0.3, and 0.6% to prepare experimental diets for use in intervention studies in gibel carp. Another group received non-specific IgY. For preventive intervention, all fish were fed these experimental diets for 25 days before being immersion challenged with CyHV-2, after which the fish were switched to the basal diet for 21 days. For therapeutic intervention, fish were first challenged with CyHV-2 and then fed the diet with 0.6% D4ORFs IgY. Fish that received D4ORFs IgY prior to CyHV-2 challenge had significantly higher survival rates and significantly lower viral load in the trunk kidney compared to those that received non-specific IgY. Improved survival and reduced viral load were also observed in CyHV-2-challenged fish that received dietary supplementation of D4ORFs IgY. Hematological and serum biochemical analysis also showed improved health and enhanced antiviral defense in fish that received D4ORFs IgY. Overall, these findings demonstrated both preventive and therapeutic effects of anti-D4ORFs IgY against CyHV-2 infection. • D4ORFs encoded by four tandem ORFs of CyHV-2 showed high immunogenicity. • Anti-D4ORFs IgY can effectively neutralize CyHV-2 in vitro. • Dietary inclusion of anti-D4ORFs IgY improves survival in CyHV-2-infected gibel carp. • Anti-D4ORFs IgY is prophylactically and therapeutically effective against CyHV-2 infection. [ABSTRACT FROM AUTHOR]

Distant hybridization occurs widely in fishes and is a useful strategy to produce different ploidy offsprings. In this study, we obtained androgenetic blunt snout bream (2n = 48, ADBSB), triploid hybrids (3n = 124, 3nJB) and tetraploid hybrids (4n = 148, 4nJB) from the hybridization of two species from different subfamilies: Carassius cuvieri (♀, 2n = 100, JCC) and Megalobrama amblycephala (♂, 2n = 48, BSB). The ploidy levels of ADBSB, 3nJB and 4nJB hybrids were confirmed by counting chromosomal numbers, forming chromosomal karyotype, and measuring DNA content. In the phenotypes and reproductive traits, 3nJB and 4nJB exhibited significantly divergences from JCC and BSB, but ADBSB took after BSB. Fluorescence in situ hybridization (FISH) and 5S rDNA analyses revealed that the genetic traits of the offsprings compared with those of their parents. This is the first report on the coexistence in vertebrates of viable androgenetic, triploid and tetraploid hybrids being produced by crossing JCC and BSB. The formation of the different ploidy offspring is great significance in both evolution and fish genetic breeding and it also provides a good model for studying the genomic variation in the first generation of interspecific hybrids. [ABSTRACT FROM AUTHOR]

Intermuscular bones (IBs) are ubiquitous in most of freshwater farmed fishes, especially in cyprinid species, and the creation of IB-free mutants has become a hot issue in fish genetic breeding because of the potential beneficial for consumers and fish processing industry. Recently, significant progress in the mechanism underlying IB development has been achieved in zebrafish and a decisive player, runt-related transcription factor 2b (runx2b), has been identified. However, its role has not yet been validated in other farmed fishes. Amphitriploid gibel carp (Carassius gibelio) is an important aquaculture species in China, but the injuries threats with approximately 80 IBs have strongly restricted its industry development. In this study, we first identified two homeologs of runx2b (Cgrunx2b-A and Cgrunx2b-B) in gibel carp with about 91.8% identity, each of them possessing three alleles with 99.6% identities. Then, we traced the IB ossification process. Alizarin red S staining showed that gibel carp IB began to ossify at 14 approximately days post hatching (dph) and developed from the posterior to the anterior regions. Cgrunx2b-A and Cgrunx2b-B displayed similar expression pattern but differential expression level during IB development. Moreover, we applied an optimized protocol of CRISPR/Cas9 editing in the polyploid Carassius complex to disrupt the functions of Cg Runx2b-A and Cg Runx2b-B singly or simultaneously. IB development was unaffected by single deficiency of Cg Runx2b-A or Cg Runx2b-B, but simultaneous disruption of all alleles of both Cg Runx2b-A and Cg Runx2b-B leads to complete loss of IB. The results indicate that Cg Runx2b-A and Cg Runx2b-B cooperatively regulate IB development. Importantly, we created several gynogenetic gibel carp mutants, including IB-free Cgrunx2b-A −/−/− ; Cgrunx2b-B −/−/− mutants and IB-less Cgrunx2b-A −/−/− ; Cgrunx2b-B −/−/+ mutants. The current study not only confirms the cooperatively decisive role of two homeologs of runx2b in IB formation, but also provides a feasible gene editing-based approach to create IB-free mutants in gibel carp. • Cgrunx2b-A and Cgrunx2b-B cooperatively regulate intermuscular bone (IB) development in amphitriploid gibel carp. • Single loss of Cg Runx2b-A or Cg Runx2b-B function is unable to affect normal development of intermuscular bones. • Simultaneous disruption of all alleles of both Cg Runx2b-A and Cg Runx2b-B create IB-free gibel carp mutants. [ABSTRACT FROM AUTHOR]

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. • Acute A. hydrophila infection could induce antioxidant imbalance in WR. • Changes of immune- and metabolic-related gene expressions in kidney were observed. • A. hydrophila infection exhibit a profound effect on MVA pathway and phospholipid hydrolysis. [ABSTRACT FROM AUTHOR]

Aquaculture, as one of the largest food production sectors, is a candidate industry to solve the worldwide food shortage. Nitrogenous compounds from uneaten feed and excretion continually destroy the rearing water quality during the breeding which causes slow growth, production reduction, and even death. Thus, alleviating water contamination can improve fish welfare and prevent disease occurrence. In the present study, a stable bacteria-microalgae association was used to construct a biofortified crucian carps culture system without water exchange. During the 8-week experiments, the water quality, fish production, and intestinal health were assessed. In this biofortified system, water quality was significantly improved and the content of total ammonia-N and nitrite-N was finally in a state of dynamic equilibrium at a lower concentration compared with that in blank control. The final body weight of crucian carps in the bacteria-microalgae groups was significantly improved by 10.52%. The crucian carps intestinal health including amylase and lipase activities, villus length and width, and muscle thickness was also significantly increased by 56.33%, 63.89%, 347.25%, 98.48%, and 129.53%, respectively, in bacteria-microalgae groups. Intestinal microbiota was improved significantly by changing the microbiota composition and LEfSe analysis demonstrated that some genera frequently found in the intestines of stressed and dying animals were enriched in crucian carps intestine in blank control. The KEGG prediction proved that metabolisms including carbon metabolism, pyruvate metabolism, and fatty acid metabolism were significantly improved. In summary, the bacteria-microalgae association biofortified culture system improved the rearing water quality, and then improved the intestinal health of crucian carp, eventually increased the production of crucian carp. [Display omitted] • Bacteria-microalgae association improved the water quality and fish production. • Association promoted intestinal digestive enzyme activity and morphology. • Intestinal microbiota was improved by bacteria-microalgae association. • Better water quality means healthier intestines and more fish production. [ABSTRACT FROM AUTHOR]

Bdellovibrio are obligate predators of Gram-negative bacteria that could parasitize other bacteria and cause their lysis. It has been considered as a potential probiotic and may replace antibiotics. In order to better explore the inhibitory activity of Bdellovibrio against common aquatic pathogens and its potential use in aquaculture, the Aeromonas hydrophilia was used as the host bacteria for culturing Bdellovibrio. An absolute qPCR detection system with specific primers were also established for the amount determination of Bdellovibrio sp.. The morphological characteristics of the cultured Bdellovibrio sp. were detected by scanning electron microscope. By detecting absorbance at OD 600nm and double layer plates plaque experiments, the Bdellovibrio sp. exhibited a certain lysis effect on the selected aquatic pathogens including Aeromonas veronii , Aeromonas hydrophil ia , Vibrio fluvialis , Escherichia coli , Vibrio anguillarum , Vibrio cholerae and Citrobacter freundii. Adding the cultured Bdellovibrio sp. to water could significantly reduce the mortality rate of Carassius auratus caused by the infections with A. vironii. Meanwhile, the amount of Bdellovibrio sp. on intestines and gills of C. auratus could increase over time after treatment, indicating Bdellovibrio may colonized in fish. The present study revealed that Bdellovibrio may play important roles as an alternative to antibiotics in protecting aquatic animals from pathogenic bacteria. These results will provide a certain theoretical basis for the application of Bdellovibrio in aquaculture. • An absolute qPCR detection system was established for the amount determination of Bdellovibrio sp. • The cultured Bdellovibrio sp. exhibited a certain lysis effect on aquatic pathogenic bacteria. • Bdellovibrio sp. significantly reduce the mortality rate of C. auratus infected with A. vironii. • The amount of Bdellovibrio sp. on intestines and gills of C. auratus increase over time after treatment. [ABSTRACT FROM AUTHOR]

A 105-day feeding trial was carried out to investigate the effects of dietary curcumin (CM) supplementation on growth performance, digestive enzyme activities, and intestinal antioxidant capacity of c rucian carp ( Carassius auratus ). A total of 585 crucian carp with average initial weight of 76.3 ± 0.10 g were fed three diets supplemented with 0 (basal group), 1, or 5 g kg − 1 CM. Fish fed the diet supplemented with 5 g kg − 1 CM showed significantly higher final body weight (FW), percent weight gain (PWG), and feed efficiency (FE) compares to those fed the basal or 1 g kg − 1 CM diet ( P < 0.05). Feed intake and body composition were not significantly different among dietary groups ( P > 0.05). The hepatopancreas weight, hepatopancreas protein content, intestinal weight, intestinal somatic index, intestine protein content, trypsin and lipase activities in hepatopancreas and intestine, amylase activity in hepatopancreas, Na + , K + -ATPase (NKA), alkaline phosphatase (AKP), gamma-glutamyl transpeptidase (γ-GT), and creatine kinase (CK) activities in intestine were significantly increased with dietary CM supplementation at 5 g kg − 1 ( P < 0.05). Fish fed the 5 g kg − 1 CM diet showed significantly lower malondialdehyde and protein carbonyl contents in the intestine ( P < 0.05). Superoxide anion and hydroxy radical scavenging ability, glutathione reducase (GR), catalase (CAT), total superoxide dismutase (T-SOD), glutathione peroxidase (GPx), and glutathione- S -transferase (GST) activities, and glutathione (GSH) content in the intestine were significantly increased with increased CM. The relative mRNA expressions of trypsin, lipase, NKA, AKP, γ-GT, CK, SOD1, CAT, GPx, GST, and GR in intestine were significantly up-regulated by dietary CM supplementation at 5 g kg − 1 . These results suggested that dietary CM supplementation increased intestinal antioxidant capacity, digestive and absorptive ability, and promoted fish growth. [ABSTRACT FROM AUTHOR]

Sex hormones play an important role in gametogenesis. During the breeding season, sex hormone levels in the sterile allotriploid crucian carp (3nAT) were significantly lower than those in the fertile diploid red crucian carp (2nRCC). Sex hormone rescue was performed to study the relationship between sex hormones and 3nAT sterility. For three months before breeding season, 3nAT females and males were fed the sex hormones estradiol and testosterone, respectively. During breeding season, hormone-rescued 3nAT fish (re-3nAT) showed normal reproductive behavior, and levels of estradiol in female re-3nAT and testosterone in male re-3nAT were similar to those of the diploid control (2nRCC). The vtg gene, which is associated with the hepatic synthesis of vitellogenin in the female, and the genes spef2, tekt1, and ift27 , which are associated with the formation of the sperm flagellum, were significantly upregulated in re-3nAT as compared to 3nAT. Under artificially assisted reproduction, re-3nAT individuals produced a few eggs of different sizes and sperm with abnormal structures. However, few individuals had insemination ability. Neither zygotes produced by self-crossing re-3nAT nor those produced by crossing re-3nAT with the haploid gametes produced by 2nRCC exhibited normal embryonic development. Thus, although sex hormone treatment led to the production of abnormal gametes by re-3nAT, viable offspring did not develop, meaning that re-3nAT remained sterile. These results indicated that sex hormones affected gamete maturation but were not the determinant of triploid sterility. • The sex hormone secretion of sterile allotriploid was abnormal. • Sex hormone treatment results in abnormal sterile gametes from allotriploid. • Sex hormone was not the decisive factor of triploid sterility. [ABSTRACT FROM AUTHOR]

Qihe crucian carp (Carassius auratus) is a valuable fish species in China that has two reproduction modes, unisexual gynogenesis and sexual reproduction. However, little is known about the differences between offspring of Qihe C. auratus produced by these two reproduction modes. A thorough multi-aspects comparison of the offspring produced via the two reproduction modes would provide essential guide to aquaculturists in the artificial breeding and farming of Qihe C. auratus. Therefore, this study compared the growth performance, feed utilization, and morphological traits between homologous and heterologous juvenile Qihe C. auratus after three-month successive culture. Noticeably, homologous fish grew faster than heterologous fish in terms of the body size and final body weight; the feed intake, protein efficiency ratio, and the values of all 15 measured traits of homologous fish were significantly higher than that of heterologous fish. However, for external morphology, heterologous fish had relatively larger head size and thicker body. Additionally, the significant direct effects of the morphological traits on body weight varied between homologous and heterologous fish at different culture stages. Therefore, six independent regression equations were constructed to evaluate the relationships between different morphological traits and body weight. Further, in some traits, the homologous and heterologous fish presented discrepant allometric growth patterns. Finally, discriminant function equations (total accuracy: 92.78%) were successfully constructed based on morphological indexes to ease the identification of homologous and heterologous Qihe C. auratus. These findings suggest that homologous fish can grow faster, but heterologous fish have a thicker body, which is of higher economical value and a key parameter that determines quality and price. • Homologous fish grew faster and had a more effective feed utilization than heterologous fish. • Heterologous fish had a relatively larger head size and thicker body than homologous fish. • Homologous and heterologous fish showed discrepant allometry growth patterns in some traits. • Discriminant equation was successfully constructed to identify homologous and heterologous Qihe crucian carp. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the nutrient dynamics and balance of zero–water exchange ponds of grass carp Ctenopharyngodon idella , crucian carp Carassius auratus and bighead carp Aristichthys nobilis , and explain advantages of the zero–water exchange mode from the perspective of material flow. This study was conducted for two years. The polyculture ponds of grass carp, crucian carp and bighead carp were grouped into the treatment group using the zero–water exchange mode, while the ordinary polyculture ponds of grass carp, bighead carp, and crucian carp were grouped into the control group. The results showed that 1) the treatment group reduced water conductivity, dissolved solids and sediment organic carbon significantly (P < 0.05), and improved water transparency significantly (P < 0.05); 2) the carbon, nitrogen, and phosphorus accumulation amount and rate in the water of the treatment group were significantly lower (P < 0.05) than those in the control group, so was the sediment accumulation rate of organic carbon and nitrogen (P < 0.05); 3) the carbon, nitrogen, and phosphorus utilization efficiency of stocked fish in the treatment group were significantly higher (P < 0.05) than those in the control group, with an increase of 29.49%, 21.72%, and 20.65%, respectively. Therefore, the zero–water exchange pond was a green and efficient aquaculture mode, which could improve water quality, reduce nutrient accumulation, and improve the utilization efficiency of nutrients effectively. Considering the nutrient emissions from aquaculture in China, the zero–water exchange mode has a good value for promotion. • The zero-water exchange ponds reduced the water and sediment accumulation rate. • The zero-water exchange ponds reduced the sediment accumulation rate of organic carbon and nitrogen significantly by 48.98% and 13.97%, respectively. • Utilization efficiency of organic carbon, nitrogen and phosphorus in the zero-water exchange ponds were improved. [ABSTRACT FROM AUTHOR]

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. • A virus strain named GCC2021 was isolated from diseased golden crucian carp. • GCC2021 could cause cytopathic effect in the Gibel carp brain cell line. • GCC2021 caused pathological changes in golden crucian carp tissues. • GCC2021 has been demonstrated for cyprinid herpesvirus 2. [ABSTRACT FROM AUTHOR]

The phagocytic function of peripheral blood leukocytes (PBL) in teleost fish is the bridge between cellular-mediated and humoral immunity and plays a central role in the immune system of the teleost fish. However, the phagocytic ability of PBL from Qihe crucian carp Carassius auratus remains unclear. This study aimed to build a convenient separation method to obtain high purity PBL from Qihe crucian carp and to explore their phagocytic characteristics. The results showed that the PBL of Qihe crucian carp (QPBL) could be obtained effectively by 1.085 g/ml percoll separation liquid. Most isolated QPBL were round or elliptical, with horseshoe-shaped, reniform, or irregular nucleus. Finger-like or filamentous protrusions were present on the cell surface, and some cells had a long pseudopod. According to the results of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence microscope, fluoresbrite carboxylate YG microspheres (1 μm) could be effectively phagocytosed by QPBL, and various phagocytic stages could be observed. The phagocytic cells in QPBL have morphology similar to those of "professional" phagocytes (monocyte and granulocyte). Notably, the percentage of cells, which can engulf microspheres, increased to approximately 22% after incubation with microspheres for 90 min, and it showed no obvious changes until 180 min. Furthermore, two important phagocytosis regulatory protein genes, Rab5 and Rab7 , were seriously up-regulated during the phagocytosis in QPBL. Taken together, an efficient and convenient separation method for isolating QPBL was established, and the isolated QPBL had a high phagocytic activity towards the external particles. The results could provide the basic foundation for further study of the immune function of PBL in Qihe crucian carp and other teleost fish. • The PBL of Qihe crucian carp (QPBL) could be separated by 1.085 g/ml percoll separation liquid. • The QPBL had a high phagocytic activity towards the external particles. • The proportion of phagocytes in QPBL was about 22%. • Rab5 and Rab7 were involved in the phagocytic activity of QPBL. [ABSTRACT FROM AUTHOR]

The phagocytic function of peripheral blood leukocytes (PBL) in teleost fish is the bridge between cellular-mediated and humoral immunity and plays a central role in the immune system of the teleost fish. However, the phagocytic ability of PBL from Qihe crucian carp Carassius auratus remains unclear. This study aimed to build a convenient separation method to obtain high purity PBL from Qihe crucian carp and to explore their phagocytic characteristics. The results showed that the PBL of Qihe crucian carp (QPBL) could be obtained effectively by 1.085 g/ml percoll separation liquid. Most isolated QPBL were round or elliptical, with horseshoe-shaped, reniform, or irregular nucleus. Finger-like or filamentous protrusions were present on the cell surface, and some cells had a long pseudopod. According to the results of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence microscope, fluoresbrite carboxylate YG microspheres (1 μm) could be effectively phagocytosed by QPBL, and various phagocytic stages could be observed. The phagocytic cells in QPBL have morphology similar to those of "professional" phagocytes (monocyte and granulocyte). Notably, the percentage of cells, which can engulf microspheres, increased to approximately 22% after incubation with microspheres for 90 min, and it showed no obvious changes until 180 min. Furthermore, two important phagocytosis regulatory protein genes, Rab5 and Rab7 , were seriously up-regulated during the phagocytosis in QPBL. Taken together, an efficient and convenient separation method for isolating QPBL was established, and the isolated QPBL had a high phagocytic activity towards the external particles. The results could provide the basic foundation for further study of the immune function of PBL in Qihe crucian carp and other teleost fish. • The PBL of Qihe crucian carp (QPBL) could be separated by 1.085 g/ml percoll separation liquid. • The QPBL had a high phagocytic activity towards the external particles. • The proportion of phagocytes in QPBL was about 22%. • Rab5 and Rab7 were involved in the phagocytic activity of QPBL. [ABSTRACT FROM AUTHOR]

An acute cyprinid herpesvirus 2 (CyHV-2) infection of crucian carp (Carassius auratus gibelio) is characterized with hemorrhagic inflammation and high mortality. Berberine is a biologically active plant alkaloid with an anti-inflammation function involving the regulation of transcription factor NF-κB and its downstream inflammation-related genes in mammalian organisms. Our previous study had indicated that BBH (berberine chloride hydrate) inhibited gene expression and replication of CyHV-2, and might serve as a potential small molecular drug for controlling CyHV-2 infection in crucian carp. Here, NF-κB signaling in CyHV-2-infected crucian carp treated with BBH was investigated both in vitro and in vivo. Real-time RT-PCR and ELISA assays revealed that CyHV-2 could activate the NF-κB signaling resulting in up-regulation of its downstream pro-inflammatory gene expression including IL-6, IL-8, IL-1β and TNF-ɑ. BBH could attenuate NF-κB signaling and downregulate the expression of its downstream inflammatory cytokines in a dose-dependent manner both in vivo and in vitro. BBH not only dose-dependently inhibits viral load in CyHV-2-susceptible crucian carp cell lines in infection and inhibition assays, but also ameliorates inflammatory injury in CyHV-2-targeted organ in histopathology examinations. Thus, negative regulation of NF-κB by BBH should contribute to BBH-mediated protection of crucian carp from CyHV-2 infection via attenuating viral gene expression and host inflammatory response simultaneously. • CyHV-2 induces NF-κB signaling pathway and its downstream inflammatory factors. • BBH down-regulates NF-κB signaling pathway and downstream its inflammatory factors. • BBH inhibits CyHV-2 infection both in vitro and in vivo. • BBH protects crucian carp from CyHV-2 infection by suppressing host inflammation. [ABSTRACT FROM AUTHOR]

Previous studies have shown that emodin (EMO) has a wide range of pharmacological activities, with the potential to protect against a variety of diseases. However, it is not clear whether EMO can alleviate impairment induced by acute hypoxia in gibel carp. Thus, an acute hypoxia experiment was conducted using gibel carp fed with EMO diets for eight weeks. The results showed that EMO decreased hypoxia signaling by down-regulating protein levels of hypoxia-inducible factor (HIF-1α). This also protected the liver from oxidative damage induced by acute hypoxia, as indicated by the upregulation of mRNA levels and antioxidant enzyme activity of genes related to nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Moreover, EMO also inhibited endoplasmic reticulum (ER) stress in the liver and hypothalamus induced by acute hypoxia. EMO enhanced autophagy and inhibited apoptosis in gibel carp by mediating the AMPK/mTOR signaling pathway. Overall, EMO could alleviate impairment in gibel carp induced by acute hypoxia via the AMPK/mTOR pathway. [Display omitted] • Acute hypoxia induced oxidative stress, ER stress and apoptosis in gibel carp. • EMO decreased hypoxia signaling by down-regulating protein levels of HIF-1α. • EMO improved antioxidant defense as well as inhibited ER stress effects. • Autophagy and inhibited apoptosis was enhanced by EMO in gibel carp. • EMO exerts protective effects by regulating Nrf2/Keap1 and AMPK/mTOR pathways. [ABSTRACT FROM AUTHOR]

Viperin is increasingly thought as a key protein in antiviral response. However, the in vivo antiviral effects of fish viperin remains unresolved. Hexaploid gibel carp (Carassius gibelio) is an important aquaculture species in China and its culture industry has suffered enormous economic losses due to an epizootic disease caused by C. auratus herpesvirus (Ca HV) since 2012. In our previous studies, two duplicated viperin homeologs (Cgviperin-A and Cgviperin-B) were identified and their divergent antiviral mechanisms were revealed through in vitro overexpression in CAB (C. auratus blastulae embryonic) cell. Here, we tried to explore their function difference in vivo by using CRISPR/Cas9 editing. Firstly, we uncovered their biased expression patterns and differential promoter activities. Then we mutated them singly or simultaneously in resistant clone H of gibel carp by using CRISPR/Cas9 and finally confirmed that they cooperatively participated in the antiviral battle between gibel carp and Ca HV through virus challenge and comparative transcriptome analysis. Cg Viperin-A depletion mainly affected immune-related pathways and Cgviperin-B deficiency influenced autophagy-associated pathways. Simultaneous disruption of them resulted in complete loss of resistance ability of gibel carp against Ca HV. Significantly, the deficiency of Cgviperin-A influenced the expression of Cgviperin-B in Cgviperin-A −/−/− mutant, but Cgviperin-A transcripts kept a high level of expression in the Cgviperin-B −/−/− mutant, same as in the WT. Therefore, the current findings are not only the first time to clarify the antiviral activities of Viperin in the battle between teleost and virus in vivo, but also explain the detailed evolutionary divergence of a duplicated gene in a recurrent polyploid fish. • Two duplicated viperin homeologs were edited singly or simultaneously in hexaploid gibel carp by using CRISPR/Cas9. • It is the first time to clarify the antiviral activities of Viperin in the battle between teleost and virus in vivo. • Cg Viperin-A and Cg Viperin-B have sub-functionalized, Cg viperin-B has evolved a novel function related to autophagy. • Cg Viperin-A and Cg Viperin-B cooperatively participate in the antiviral response of gibel carp against CaHV. [ABSTRACT FROM AUTHOR]

Blunt snout bream (BSB) is an important economic freshwater fish in China. One of technical bottlenecks in practice lies in improving its hypoxia tolerance. In this study, gynogenesis BSB (GBSB-R) is generated by artificial gynogenesis with UV-inactivated sperms of red crucian carp (RCC), which is regarded as one of freshwater fish with the strongest hypoxia-tolerance. Asynchronous development of the eye pigment further provides an objective morphologic evidence of distinguishing the gynogenesis BSB progeny with the hybrids of BSB (♀) × RCC (♂). It is found that the GBSB-R is a kind of all-female population with normal fertility and its genetic homozygosity is higher than that of wildtype BSB. In view of hypoxia stress tests, physiological indexes and gene expression, the obtained experimental data from this research confirm that the GBSB-R is a new strain with improved hypoxia tolerance, especially compared with the wildtype BSB. These results indicate that artificial gynogenesis is an efficient method to enlarge capacity of the BSB hypoxia tolerance, and the conducted research in this article is valuable to the practical aquaculture. • Gynogenesis blunt snout bream (GBSB-R) generated with UV-inactivated sperms of red crucian carp (RCC) • GBSB-R is a new strain with higher genetic homozygosity and stronger hypoxia tolerance than blunt snout bream (BSB) • An objective evidence to distinguish GBSB-R progeny with hybrids of BSB x RCC by asynchronous development of eye pigments [ABSTRACT FROM AUTHOR]

Blunt snout bream (BSB) is an important economic freshwater fish in China. One of technical bottlenecks in practice lies in improving its hypoxia tolerance. In this study, gynogenesis BSB (GBSB-R) is generated by artificial gynogenesis with UV-inactivated sperms of red crucian carp (RCC), which is regarded as one of freshwater fish with the strongest hypoxia-tolerance. Asynchronous development of the eye pigment further provides an objective morphologic evidence of distinguishing the gynogenesis BSB progeny with the hybrids of BSB (♀) × RCC (♂). It is found that the GBSB-R is a kind of all-female population with normal fertility and its genetic homozygosity is higher than that of wildtype BSB. In view of hypoxia stress tests, physiological indexes and gene expression, the obtained experimental data from this research confirm that the GBSB-R is a new strain with improved hypoxia tolerance, especially compared with the wildtype BSB. These results indicate that artificial gynogenesis is an efficient method to enlarge capacity of the BSB hypoxia tolerance, and the conducted research in this article is valuable to the practical aquaculture. • Gynogenesis blunt snout bream (GBSB-R) generated with UV-inactivated sperms of red crucian carp (RCC) • GBSB-R is a new strain with higher genetic homozygosity and stronger hypoxia tolerance than blunt snout bream (BSB) • An objective evidence to distinguish GBSB-R progeny with hybrids of BSB x RCC by asynchronous development of eye pigments [ABSTRACT FROM AUTHOR]

Viperin is increasingly thought as a key protein in antiviral response. However, the in vivo antiviral effects of fish viperin remains unresolved. Hexaploid gibel carp (Carassius gibelio) is an important aquaculture species in China and its culture industry has suffered enormous economic losses due to an epizootic disease caused by C. auratus herpesvirus (Ca HV) since 2012. In our previous studies, two duplicated viperin homeologs (Cgviperin-A and Cgviperin-B) were identified and their divergent antiviral mechanisms were revealed through in vitro overexpression in CAB (C. auratus blastulae embryonic) cell. Here, we tried to explore their function difference in vivo by using CRISPR/Cas9 editing. Firstly, we uncovered their biased expression patterns and differential promoter activities. Then we mutated them singly or simultaneously in resistant clone H of gibel carp by using CRISPR/Cas9 and finally confirmed that they cooperatively participated in the antiviral battle between gibel carp and Ca HV through virus challenge and comparative transcriptome analysis. Cg Viperin-A depletion mainly affected immune-related pathways and Cgviperin-B deficiency influenced autophagy-associated pathways. Simultaneous disruption of them resulted in complete loss of resistance ability of gibel carp against Ca HV. Significantly, the deficiency of Cgviperin-A influenced the expression of Cgviperin-B in Cgviperin-A −/−/− mutant, but Cgviperin-A transcripts kept a high level of expression in the Cgviperin-B −/−/− mutant, same as in the WT. Therefore, the current findings are not only the first time to clarify the antiviral activities of Viperin in the battle between teleost and virus in vivo, but also explain the detailed evolutionary divergence of a duplicated gene in a recurrent polyploid fish. • Two duplicated viperin homeologs were edited singly or simultaneously in hexaploid gibel carp by using CRISPR/Cas9. • It is the first time to clarify the antiviral activities of Viperin in the battle between teleost and virus in vivo. • Cg Viperin-A and Cg Viperin-B have sub-functionalized, Cg viperin-B has evolved a novel function related to autophagy. • Cg Viperin-A and Cg Viperin-B cooperatively participate in the antiviral response of gibel carp against CaHV. [ABSTRACT FROM AUTHOR]

Abstract: Two experiments were conducted to evaluate the feasibility of including high levels of rapeseed meal (RSM) and peanut meal (PNM) in juvenile crucian carp (Carassius auratus gibelio♀× Cyprinus carpio♂) diets by investigating the growth performance, immunity, intestinal morphology, and microflora of these animals. Fish meal (FM)-based diets were used as controls in both experiments. In experiment 1, RSM and PNM replaced 250 (RSM25 and PNM25) and 500 (RSM50 and PNM50) gkg−1 of FM in test diets. In experiment 2, RSM and PNM substituted 500gkg−1 of FM in test diets (RSM50′ and PNM50′, respectively). The diets in experiment 2 were isonitrogenous and isocaloric, and supplemental crystalline amino acids were added to compensate for the reduction in essential amino acids. More than 100gkg−1 of FM was maintained in the test diets in both experiments to ensure palatability, and fish were fed these diets for 8weeks. No significant changes in feed intake were noticed among groups in both experiments. In experiment 1, the growth rate (GR) of fish fed test diets was significantly lower than in fish fed control diets; the feed conversion ratios (FCR) were higher in fish fed test diets than in fish fed control diets with the exception of fish fed the PNM25 diet. The lower GR observed in fish fed test diets was partly due to reduced dietary energy and protein levels. No significant differences were observed in the GR of fish in experiment 2; however, the FCR of fish fed the RSM50′ diet increased, whereas the protein efficiency ratio decreased in fish of both test groups, suggesting that growth retardation would have occurred over a longer period. No significant changes were observed in serum superoxide dismutase activity in both experiments. Compared with control groups, the lymphocyte proliferation index (LPI) decreased in fish fed the RSM50 diet but not in those fed the RSM50′ diet; the LPI increased in fish fed the PNM50 and PNM50′ diets. The secretion of mucus, which formed a peritrophic membrane-like structure, was enhanced when diets included high levels of RSM and PNM. Mild intestinal histological changes, but no significant inflammatory responses or intestinal microflora changes, were observed. From these results, we came to the general conclusion that the inclusion of high levels of RSM and PNM in the diet not only retarded growth but also produced health risks to the crucian carp. [Copyright &y& Elsevier]

Phosphatidylinositol 3-kinase (PI3K) signaling plays an indispensable role in vertebrate metabolism and energy homeostasis. Heterozygous disruption of P85α, a PI3K subunit encoded by the pik3r1 gene, is known to improve insulin sensitivity in mammals. However, there is no direct evidence on the effects of altered expression of pik3r1 on metabolism and somatic growth in teleosts. In this study, the effects of heterozygous disruption of pik3r1 on metabolism and somatic growth in Gibel carp were investigated. Enhancement of the PI3K/AKT/mTOR signaling pathway was achieved by inducing haploinsufficiency of P85α in heterozygous pik3r1 -deficient Gibel carp. Unlike in mammalian models, there was no significant changes in the plasma glucose levels and hepatic glucose uptake. However, significant reduction in the levels of plasma triglycerides (TG), hepatic TG, amino acids, and lactate was observed. Furthermore, the haploinsufficiency of P85α significantly improved somatic growth, lipid utilization, and feed conversion efficiency (FCE) in the mutant Gibel carp. Taken together, our results demonstrate that enhancement of the PI3K/AKT/mTOR signaling pathway could effectively improve somatic growth and FCE in practical aquaculture. Therefore, genetic engineering for partial activation of the PI3K signaling pathway may be a potential breeding strategy for improvement of the traits of cultured fish in the future. • Heterozygous pik3r1 targeting in gibel carp enhanced PI3K and mTOR activity. • Improved somatic growth and feed conversion efficiency (FCE) were achieved in heterouzygous pik3r1 mutant gibel carp. • In pik3r1 mutant fish, reduction in hepatic lipid content has been achieved under regular- and even over-feeding conditions. [ABSTRACT FROM AUTHOR]

Abstract: Tissue distribution and elimination of florfenicol (FF) in crucian carp were investigated after a single dose (40mg/kg) of intramuscular (i.m.) or oral gavage (per os; p.o.) administration in freshwater at 25°C. FF concentrations in tissues (muscle, liver, kidney, gill and bile) were analyzed by means of high-performance liquid chromatography using an ultraviolet detector. Compared to p.o. administration, it took a longer time to reach FF peak concentration (Cmax) in tissues (except for bile) after i.m. administration. The Cmaxs (except for bile) in these tissues were lower, as compared to p.o. administration. The elimination half-lives (t1/2β) in all the tissues (except for bile) were on an average two-fold longer when administered i.m. than administered p.o. Also, the area under the concentration time curve (AUC) and the mean residence time (MRT) was almost two times greater as well in all tissues when administered i.m. than administered p.o. These results indicate that higher levels of FF in tissues were achieved and took a longer time to eliminate the drug when administered i.m. than p.o. In order to achieve effective dosing regimens in crucian carp, the differences in pharmacokinetics should be considered in future studies. [ABSTRACT FROM AUTHOR]

Abstract: An experiment was conducted to compare the effects of selenium nanoparticle (T-1) and selenomethionine (T-2) on crucian carp, Carassius auratus gibelio. There were significant differences (P <0.05) in relative gain rate and final weight of T-1 and T-2 compared with the control. Survival rate and feed conversion ratio (FCR) were not affected by the dietary treatments. Fish fed the basal diet (control) showed lower (P <0.05) selenium content in muscle (6.10±0.78 μg g−1) compared to T-1 and T-2 (16.42±1.07 μg g−1 and 13.52±1.31 μg g−1, respectively). Furthermore, the highest value (P <0.05) of selenium content in muscle was observed in T-1. Glutathione peroxidase (GSH-Px) activities in carps plasma and liver of T-1 and T-2 were significantly different (P <0.05) with that of the control. However, no significant differences were observed in GSH-Px activities both in plasma and liver between T-1 and T-2. [Copyright &y& Elsevier]

Abstract: In the present study, viable diploid androgenetic individuals (AT-ag) (2n =100) have been developed in UV-radiated eggs of goldfish (GF) (Carassius auratus) following fertilization with diploid spermatozoa from male allotetraploid hybrid (AT) F13 (4n =200) of red crucian carp (C. auratus red var.) (♀)×common carp (Cyprinus carpio L.) (♂). In the experimental groups, eggs were inactivated by UV-rays, the range of UV-radiation dose tested varied from 240 to 600 mJ/cm2, the relative high hatching rate (4.1±0.4%) and survival from hatching to start of feeding (41.3±2.6%) were given at UV dose of 300 mJ/cm2. The androgenetic nature was confirmed by chromosome observation and DNA content measurement of somatic cells by a flow cytometer. The AT-ag were diploid (2n =100) whereas the controls (GF×AT) were triploids (3n =150). The diploid AT-ag grew slower than the triploid GF×AT. The AT-ag and GF×AT were also morphologically distinguishable. The AT-ag began to sexually mature at the age of 2 years, and their fertility further increased at the age of 3 years. The tetraploids were obtained in the offspring (F1ag×ag) produced by mating the males with females of AT-ag and in the progeny (AT-ag×AT) generated by crossing the males of AT with the females of AT-ag, indicating that diploid AT-ag could produce diploid gametes. All the sex ratios of males to females in AT-ag, F1ag×ag, AT-ag×AT were not significant (P > 0.05) from the ratio of 1:1, suggesting the XXXY sex-determining mechanism in the males of AT. The present study indicated that bisexual fertile diploid AT-ag were successfully induced from diploid spermatozoa of AT. [Copyright &y& Elsevier]

Abstract: The study was conducted to evaluate the pharmacokinetic disposition of difloxacin (DIF) with a single dose of 20mg/kg body weight after oral administration in crucian carp (Carassius auratus, 250±30g) at water temperatures of 10°C and 20°C. DIF concentration in plasma and tissues was determined using high-performance liquid chromatography (HPLC). The plasma drug concentration–time data were fitted using a single-compartment model and a two-compartment model at 10°C and 20°C, respectively. The absorption rate constant (K a) of DIF was found to be 0.85 and 1.27h−1 at 10°C and 20°C, respectively, whereas the elimination half-life (t 1/2β ) of the drug was 95.36h at 10°C and 48.93h at 20°C. The area under the serum concentration–time curve (AUC) was 1361.79μgml−1 h at 10°C and 355.19μgml−1 h at 20°C. The distribution volume (V d/F) of DIF was computed as 2.02 and 4.02lkg−1 at 10°C and 20°C, respectively. The total clearance of DIF (CLb) was estimated to be 0.015 and 0.056lh−1 kg−1 at 10°C and 20°C, respectively. The time-point of maximum plasma concentration of the drug (T p) and the maximum plasma concentration (C max) were calculated as 5.65h and 9.49μg/ml at 10°C and 2.21h and 10.28μg/ml at 20°C. After repeated oral administration at a dosage of 20mg/kg body weight, DIF was detected in the muscle, liver, kidney, and skin for 15days after the last administration. The results revealed that the depletion of DIF in the crucian carp becomes slower with a long half-life time, especially at lower water temperature. [Copyright &y& Elsevier]

Pengze crucian carp (Carassius auratus of pengze), just like silver crucian carp, is known to be a gynogenetic crucian carp. It is one of the most popular cultured fishes in China. The offspring developed from pengze crucian carp eggs activated by heterologous sperm shows obvious allogynogenetic biological effects in growth and fertilization rate, although the offspring is almost the same in morphology as the maternal pengze crucian carp. Recently, a multiple tetraploid form, which showed obvious morphological changes compared with the normal allogynogenetic offspring, was found in the offspring developed from pengze crucian carp eggs stimulated by sea carp (Cyprinus acutidorsalis Wang) sperm. In this paper, the chromosome number and karyotype of the multiple tetraploid form and its parents were analyzed, using chromosomes of kidney cell-PHA culture in vivo prepared by flame-drying technique. The paternal sea carp had 2n=100 diploid chromosomes and the karyotype of 28 metacentric, 24 submetacentric, 26 subtelocentric and 22 telocentric, NF=152. The maternal pengze crucian carp had 156 chromosomes in its karyotype, with 42 metacentric, 36 submetacentric, 39 subtelocentric, 33 telocentric, and 6 supernumerary microchromosomes. The karyotype of the multiple tetraploid allogynogenetic pengze crucian carp contained 206 chromosomes, with 56 metacentric, 48 submetacentric, 44 subtelocentric, 52 telocentric, and 6 supernumerary microchromosomes. Both the multiple tetraploid form and its maternal parent had six supernumerary microchromosomes. The results indicated that the chromosomes of the multiple tetraploid allogynogenetic pengze crucian carp consist of the whole maternal chromosome set and a haploid paternal chromosome set, which suggests that a few of the gynogenetic pengze crucian carp eggs have the gonochoristic reproductive ability to integrate with heterologous sperm nuclei. [Copyright &y& Elsevier]

Thirty-six new microsatellites were identified from common carp (Cyprinus carpio L.) by screening through genes found in GenBank, EST sequences from a testis cDNA library and a genomic DNA library enriched for CA repeats. Eleven of the twenty-eight microsatellites identified from genes and ESTs were AT repeats, suggesting their high abundance in the genome of common carp. Characterization of the 36 microsatellites on a panel of 18 unrelated common carp individuals revealed that all, except two, were polymorphic with an average allele number of 7.3/locus (range: 2–15 alleles/locus). The microsatellites located in genes and ESTs showed higher allele number than those eight which were isolated from a genomic DNA library (7.7/locus vs. 4.9/locus df=32, P<0.05). Cross-species amplification showed that 41.7% (15/34) of the primer pairs from common carp amplified specific and polymorphic PCR products in the silver crucian carp (Carassius auratus gibelio). Interestingly, the success rate of cross-species amplification was lower for microsatellites located within genes and ESTs, than for those presumably located in non-coding regions. These novel microsatellites will be very useful for genome mapping and population genetic studies in both species, as well as for studying reproductive strategies of the silver crucian cap. A set of 21 polymorphic microsatellites from our study and those of others were selected as a standardized marker set to be used in studies on genetic diversity of common carp. [Copyright &y& Elsevier]

Although Cyprinid herpesvirus 2 (CyHV-2, species Cyprinid herpesvirus 2) was identified as the causative agent of hemorrhagic disease of gills in gibel carp, opportunistic pathogens, including Aeromonas hydrophila , were found frequently in CyHV-2-infected fish. However, the pathogenicity and mechanism of viral and bacterial coinfection as well as the interaction between viral and bacterial pathogens remain unknown. In this study, epidemiological investigation and experimental infection suggested that CyHV-2 infection triggered secondary bacterial infections and that the outcome of a secondary infection was dependent on the time interval between viral and bacterial administration. Lethal synergy and antagonism of CyHV-2 and bacterial coinfection occurred in the group administrated A. hydrophila on day 3 and day 14 after CyHV-2 infection, respectively. The results of pathogen burden analysis demonstrated that the mortality of crucian carp coinfected with CyHV-2 and A. hydrophila in is dependent on pathogen burden. Evaluation of the integrity of the intestinal mucosal barrier and transcriptome analysis demonstrated the disruption of gut homeostasis after CyHV-2 infection in crucian carp. In total, these results imply that CyHV-2 infection disrupts the intestinal mucosal barrier and results in bacterial translocation, which contributes to the severe mortality of CyHV-2 infection. • CyHV-2 infection triggered secondary bacterial infections. • Synergy and antagonism of CyHV-2 and bacteria was dependent on the time interval between viral and bacterial administration. • The mortality of gibel carp coinfected with CyHV-2 and A. hydrophila is dependent on pathogen burden. [ABSTRACT FROM AUTHOR]

The regulation of the disease resistance, mTOR and NF-kB signaling pathway of Aristichthys nobilis using Rhodopseudomonas wastewater treatment, Developmental & Comparative Immunology, 2020 Pan Wu, Wentao Mo, Yanling Wang, Yuan Wu, Ying Zhang, Zhaobo Chen, Ning Li. Effluent containing Rubrivivax gelatinosus promoting the yield, digestion system, disease resistance, mTOR and NF-kB signaling pathway, intestinal microbiota and aquaculture water quality of crucian carp, Fish & Shellfish Immunology, 2019 Pan Wu, Liying Xie, Xiaozhen Wu, Yanling Wang, Yuan Wu, Ning Li, Ying Zhang, Zhaobo Chen. This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). [Extracted from the article]

Pan Wu, Yaxin Liu, Xiaoting Li, Yonghu Gu et al. "The regulation of the disease resistance, mTOR and NFkB signaling pathway of Aristichthys nobilis using Rhodopseudomonas wastewater treatment", Developmental & Comparative Immunology, 2020. "Regulation of the disease resistance and mTOR and NF-kB signaling pathway of Tilapia mossambica by Rhodopseudomonas capsulatus wastewater treatment", Fish & Shellfish Immunology, 2019. "Effluent containing Rubrivivax gelatinosus promoting the yield, digestion system, disease resistance, mTOR and NF-kB signaling pathway, intestinal microbiota and aquaculture water quality of crucian carp", Fish & Shellfish Immunology, 2019. [Extracted from the article]

Citrobacter freundii , a common pathogen of freshwater fish, severely restricts the sustainable development of the aquaculture industry. In this study, the novel C. freundii strain CFC202 was isolated from the intestines of crucian carp, which was annotated with three virulence factors related to promoting bacterial colonization and causing anemia by the whole genome analysis. We found that the strain adhered to the surface of hepatic L8824 cells, and actin aggregation occurred at the adhesion site of the strain. Then, C. freundii was fluorescently labelled and imaged the distribution of crucian carp under an In Vivo Imaging System (IVIS) during natural infection. The results showed that this strain mainly gathered in the intestines of crucian carp, and the intestinal villi were severely damaged by TEM. From the results of histopathological examination, the intestinal mucosa was obviously shed and various organs were damaged. Hence, we speculated that C. freundii could cause attaching and effacing (A/E) lesions to fish cells, which in turn caused pathological changes to various organs. Moreover, we constructed C. freundii ghost (CFG) and evaluated their immune effects by oral and injection immunization in crucian carp. Compared with the non-immunized group, non-specific immune indicators (ACP, AKP, SOD, and LZM), agglutination antibody titres of the serum, and the expression of immune-related genes (TNF-α and IL-6) of the head kidney and spleen were significantly increased in the immunized group (P < 0.01), indicating that innate and specific immune responses were induced by the CFG. In addition, the immunized fish showed higher protection than the non-immunized fish after challenge: oral control (20.00% survival); oral test (80.21% survival); injected control (6.67% survival); injected test (87.50% survival). Taken together, our research was the first to demonstrate that the constructed CFG might be developed as either oral or injected vaccines against C. freundii in aquaculture. • Citrobacter freundii CFC202 was firstly isolated from the diseased crucian carp in the Dongting Lake area of China. • Genome analysis of CFC202 showed it could mediate adhesion to cell surface through virulence factors such as hop AJ2 and fep E. • Strain CFC202 could cause attaching and effacing lesions to fish cells in vivo and in vitro. • This study was the first to construct Citrobacter freundii ghost and evaluate their immune effects in crucian carp. • Immunization with CFG vaccine in advance in crucian carp could improve disease resistance against Citrobacter freundii. [ABSTRACT FROM AUTHOR]