Guodong Zheng

Although fibroblast growth factor 8 (FGF8) is a critical regulator of skeletal morphogenesis in vertebrates, its specific role in the formation and development of intermuscular bones (IBs) in teleost fish remains insufficiently characterized. In this study, we generated double mutants (fgf8a^+/- + fgf8b^+/-) in diploid Chongming crucian carp (Carassius auratus) using CRISPR/Cas9-mediated gene editing. Compared to wild-type fish, the double mutants exhibited significantly reduced IB number (p < 0.01), demonstrating a synergistic role of fgf8a and fgf8b in IB formation. Notably, reduced number of IBs did not compromise overall growth, muscle architecture, or reproductive performance. Integrated transcriptomic and metabolomic analyses revealed that the reduced IB phenotype was linked to modifications in relevant signaling pathways and a concomitant upregulation of metabolites beneficial for muscle quality and health. Our findings highlight the crucial role of fgf8 in regulating IB formation and development in crucian carp, providing insights into the genetic mechanisms underlying this process in teleosts.

In the present study, vertebral bone tissues derived from Chongming crucian carp (Carassius carassius), a dominant species during the summer and autumn seasons on Chongming Island in the lower Yangtze River, were used to establish and characterize a Carassius carassius osteoblast cell line (COBC). The established COBCs were assessed using chromosome analysis, osteocalcin enzyme-linked immunosorbent assay (ELISA), and osteogenesis-related gene expression analysis. Additionally, cellular responses to environmental stress were assessed. The results showed that COBC exhibited optimal proliferation in L-15 medium supplemented with 20% fetal bovine serum at 28 °C. The histochemical staining assay results were all positive, thereby confirming that the isolated cells display typical osteoblast characteristics. Quantitative PCR analysis revealed that osteogenic marker genes, including runx2a and runx2b, were expressed at significantly higher levels in COBCs than in fish tissues. Under hypoxic stress, COBCs exhibited morphological changes, an increase in cell death, significant alterations in gene expression, and variations in antioxidant enzyme activity. These responses facilitate adaptation to hypoxic stress. This study established the first osteoblast cell line of the Chongming crucian carp and characterized its biological properties and response to hypoxic stress. These findings offer a valuable in vitro cell model and technical support for research on fish bone tissue biology and the assessment of environmental stress effects.

A reliable and reproducible method for the isolation, culture, and identification of an osteoblast cell line from crucian carp (Carassius auratus) was established in this study using vertebral bone tissue from Chongming crucian carp, a locally important aquaculture strain from the lower Yangtze River region. O steoblast cells were isolated using a tissue explant culture method, and optimal in vitro culture conditions were systematically evaluated. The established osteoblast cell line, designated Chongming Carassius auratus osteoblast cells (COBC), was characterized through chromosomal karyotype analysis, osteocalcin enzyme-linked immunosorbent assay (ELISA), and osteogenesis-related gene expression analysis. Additionally, cellular responses to environmental stress were assessed. The results showed that COBC exhibited optimal proliferation in L-15 medium supplemented with 20% fetal bovine serum at 28 ℃ under 5% CO₂. Alkaline phosphatase staining, Alizarin Red staining, and von Kossa staining all yielded positive results, thereby confirming that the isolated cells possessed typical and stable osteoblastic properties, with the osteocalcin content of 36,884 ng / L. Quantitative PCR analysis revealed that osteogenic marker genes, including runx2a and runx2b, were expressed at significantly higher levels in COBC s than in muscle tissue. Under hypoxia - reoxygenation stress, COBC exhibited enhanced apoptotic responses, marked alterations in related gene expression, and modulation of antioxidant enzyme activities, suggesting a certain degree of adaptive capacity to oxygen fluctuations. This study provides the first systematic description of the establishment and biological characterization of COBC, as well as its responses to hypoxic stress. These findings offer a valuable in vitro cell model and technical support for studies on fish bone tissue biology and the assessment of environmental stress effects.

Ammonia–nitrogen is a common stress factor for aquatic organisms in their habitation environment, which is enriched in water due to high-density farming and environmental pollution. Ammonia nitrogen can enter fish body through gill, epidermis, digestive tract, and other tissues, causing fish ammonia poisoning. In the present study, juvenile blunt snout bream (average weight, 45 ± 5 g) were exposed to high concentrations of ammonia–nitrogen stress (25.0 ± 0.5 mg/L) for six different treatment times (0, 3, 6, 12, 24, 48, and 72 h); the tissue ultrastructure, mRNA levels of antioxidation system, and apoptosis patterns were studied. The antioxidant systems of malondialdehyde (MDA), catalase (CAT), acid phosphatase (ACP), and reduced glutathione (GSH) in various tissues were highly transcripted at 6 or 12 h (hpt) after treatment under high ammonia–nitrogen, which may play a role in preventing cells from being attacked by highly toxic reactive oxygen species (ROS). After 24 hpt, the antioxidant capacity threshold is breached, followed by the decline of antioxidant enzyme activity. Thus, with the prolonging of high ammonia–nitrogen processing time, ammonia–nitrogen stress caused irreversible damage of organs (gill, liver, and kidney). Furthermore, the expression of caspase-3 apoptotic pathway was highly induced in different tissues, implying the apoptotic system is activated, which causes extensive cell apoptosis in different tissues as shown using TUNEL analysis. In conclusion, we observed that, in response to acute ammonia–nitrogen stress, blunt snout bream enhances antioxidant capacity and cell apoptosis.

With the gradual decrease in freshwater resources, the available space for freshwater aquaculture is diminishing. As a result, saline-alkaline water aquaculture has emerged as a crucial method to fulfill the increasing demand. This study investigates the impact of alkaline water on the growth performance, tissues (gill, liver, and kidney), digestive enzyme activity, and intestinal microbiology in grass carp (Ctenopharyngodon idella). The aquarium conditions were set with sodium bicarbonate (18 mmol/L (LAW), 32 mmol/L (HAW)) to simulate the alkaline water environment. A freshwater group was the control (FW). The experimental fish were cultured for 60 days. The findings revealed that NaHCO₃ alkaline stress significantly reduced growth performance, caused alterations in the structural morphology of gill lamellae, liver, and kidney tissues, and led to decreased activity of intestinal trypsin and lipase amylase (P < 0.05). Analysis of 16S rRNA sequences demonstrated that alkalinity influenced the abundance of dominant bacterial phyla and genera. Proteobacteria showed a significant decrease under alkaline conditions, while Firmicutes exhibited a significant increase (P < 0.05). Furthermore, alkalinity conditions significantly reduced the abundance of bacteria involved in protein, amino acid, and carbohydrate metabolism, cell transport, cell decomposition, and environmental information processing. Conversely, the abundance of bacteria associated with lipid metabolism, energy metabolism, organic systems, and disease functional flora increased significantly under alkalinity conditions (P < 0.05). In conclusion, this comprehensive study indicates that alkalinity stress adversely affected the growth performance of juvenile grass carp, likely due to tissue damage, reduced activity of intestinal digestive enzymes, and alterations in intestinal microorganisms.

Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia environment. Hypoxia-inducible factor (HIF) is the most critical factor in the HIF pathway, which strictly regulates the hypoxia stress process of fish. In this study, we found six hifα genes in blunt snout bream that demonstrated different expressions under hypoxia conditions. In HEK293T cells, all six hifαs were detected to activate the HRE region by luciferase reporter assay. More importantly, we identified two linkage-disequilibrium SNP sites at exon 203 and 752 of the hif2αb gene in blunt snout bream. Haplotype II (A²⁰³A⁷⁵²) and its homozygous diplotype II (A²⁰³A²⁰³A⁷⁵²A⁷⁵²) appeared frequently in a selected strain of blunt snout bream with hypoxia tolerance. Diplotype II has a lower oxygen tension threshold for loss of equilibrium (LOE_crit) over a similar range of temperatures. Moreover, its erythrocyte number increased significantly (p < 0.05) than those in diplotype I and diplotype III strains at 48 h of hypoxia. The enzymes related with hypoxia tolerant traits, i.e., reduced glutathione, superoxide dismutase, and catalase, were also significantly (p < 0.05) induced in diplotype II than in diplotype I or III. In addition, the expression of epo in the liver of diplotype II was significantly (p < 0.01) higher than that in the diplotype I or III strains at 48 h of hypoxia. Taken together, our results found that the hypoxia-tolerant-related diplotype II of hif2αb has the potential to be used as a molecular marker in future genetic breeding of hypoxia-tolerant strain.