Grass carp (Ctenopharyngodon idella) is a major aquaculture species, yet production efficiency is often constrained by growth performance and metabolic disorders such as hepatic lipid deposition. Conventional selective breeding faces challenges in further improving these complex traits, necessitating precision genetic interventions. In this study, we combined comparative transcriptomics with CRISPR/Cas9 mediated gene disruption to investigate the regulatory role of Pik3r1. Comparative RNA-seq of fast- and slow-growing individuals together with protein-protein interaction network analysis identified Pik3r1 as a candidate negative regulator of growth. To validate its function, we generated Pik3r1 mosaic F0 mutants by targeting the SH3 domain of Pik3r1. Disruption of Pik3r1 conferred a profound growth advantage, increasing body weight by 51.8% and body length by 13.5% compared to wild-type controls. Histological analyses revealed that enhanced muscle accretion was driven by both hypertrophy with a 22.50% increase in myofiber cross-sectional area and hyperplasia characterized by an 18.90% rise in fiber density, coupled with a 67.7% reduction in hepatic lipid accumulation. At the molecular level, Pik3r1 disruption relieved the inhibitory effect on the PI3K/AKT/mTOR cascade, with upregulation of PI3K, AKT, mTOR, S6K1, Myod1, and Mybpc1 and downregulation of 4ebp1 and Pik3ca in muscle, accompanied by upregulation of PPARα and CPT1a and downregulation of FASN and ACC1 in the liver. Furthermore, a clear genotype phenotype correlation was observed: fish carrying confirmed premature termination codons exhibited substantially greater growth enhancement (77.6% increase in body weight) than those with in-frame mutations. Collectively, our results support Pik3r1 as a key regulator linking somatic growth and lipid metabolism in grass carp, and highlight its potential as a target for molecular breeding aimed at improving yield and carcass quality.

The transforming growth factor-beta (TGF-beta) signaling pathway is a central regulator of skeletal muscle growth in vertebrates, but the function of Acvr1b (activin receptor type 1B) remains poorly characterized in teleosts. Here, we used CRISPR/Cas9 to disrupt Acvr1b in grass carp (Ctenopharyngodon idella) and evaluated growth performance, muscle histology, and myogenic gene expression. Cas9 protein and sgRNAs targeting exons 2 and 3 were co-injected into fertilized eggs, and edited juveniles were identified by T7E1 assay and Sanger sequencing. Acvr1b-edited fish showed significantly improved growth relative to wild-type controls, including a 25.8% increase in body weight and a 7.39% increase in body length. qRT-PCR revealed reduced Acvr1b expression in all examined tissues. Consistent with enhanced muscle growth, mstn was downregulated, myog was upregulated, and myod was unchanged in edited fish. Histological analysis showed increased muscle fiber size and density, indicating that both hypertrophy and hyperplasia contributed to the growth advantage. Together, these findings identify Acvr1b as a promising target for genetic improvement in grass carp aquaculture.

Carassius auratus is one of the most important aquaculture species in China. The infection with C. auratus herpesvirus (CaHV) often causes 100% mortality. Tumor necrosis factor (TNF) and its receptors (TNFRs) are essential in host antiviral defense. However, viral homologs of TNFRs (vTNFRs) are encoded by viruses to carry out immune evasion. In this study, the functions of the two vTNFRs in CaHV (CaHV-vTNFRs), designated ORF144L and ORF146R, were firstly described. Molecular docking identified host TNF1 as a potential ligand for CaHV-vTNFRs, which was then experimentally validated by co-immunoprecipitation (Co-IP). Overexpression of CaHV-vtnfrs conferred a survival advantage on infected cells and promoted viral propagation in vitro, alongside the downregulation of host tnf1 and tnfr genes. The onset of apoptosis was delayed and inflammatory reactions were inhibited, as indicated by the expression of relevant genes (bax, bcl, il-1β, il-10, il-8, and tgf-β). Among these, CaHV-ORF146R exhibited a stronger immune evasion activity compared to CaHV-ORF144L. Our findings elucidate the function of vTNFRs in immune evasion and enhance our understanding of herpesvirus-host interactions. Furthermore, this work establishes a solid foundation for the development of targeted strategies to prevent and control future CaHV outbreaks.

Cyprinid herpesvirus 2 (CyHV-2) is a major pathogen causing high mortality in farmed goldfish and crucian carp, for which effective prophylactic or therapeutic treatments are currently limited. Lauric acid (LA) and glycerol monolaurate (GML), representative of a medium-chain fatty acid (MCFA) and its corresponding monoglyceride, respectively, have been reported to possess antiviral and immunomodulatory properties. This study systematically evaluated the anti-CyHV-2 effects of LA and GML through in vitro assays, transcriptomic profiling, and in vivo experiments. In vitro, both LA and GML significantly reduced viral copy numbers in gibel carp caudal fin (GiCF) cells, attenuated virus-induced cytopathic effects (CPE), and suppressed intracellular viral replication. Transcriptomic analysis revealed that LA and GML induced widespread alterations in host signalling pathways, with significant enrichment of pathways related to steroid biosynthesis, ECM-receptor interaction, protein digestion and absorption, and cell survival-associated signalling. Quantitative PCR analysis further demonstrated that the expression levels of insr, akt2, pdk1, lamtor3, and hsp90b were significantly upregulated, whereas inpp4b expression was downregulated. These results further validate that host cell metabolic processes, stress responses, and signal transduction pathways are substantially modulated following LA and GML treatment. The in vivo protective efficacy of GML was further assessed. In goldfish infected with CyHV-2, GML administration significantly increased survival rate and mitigated histopathological damage in the gills, liver, spleen, and kidney. At 3 days post-infection (dpi), expression levels of pro-inflammatory cytokines il-1β, il-6, and tnf-α were significantly lower in the GML-treated group compared to the virus-infected control group, whereas the anti-inflammatory cytokine il-10 was significantly upregulated. By 7 dpi, differences in inflammatory cytokine expression between groups had diminished, suggesting that GML not only exerts direct antiviral activity but may also modulate host immune responses during the early stage of infection. Collectively, these findings provide a theoretical basis for the practical application of LA and GML and propose a novel strategy for developing safe, effective, and environmentally friendly anti-CyHV-2 agents.

Grass carp (Ctenopharyngodon idella) is an economically important aquaculture species known for its considerable variability in growth performance. In this study, we investigated the growth phenotype by comparing fast-growing and slow-growing groups. Microstructural analyses revealed that slow-growing fish exhibited significantly larger myofibrillar gaps and lower muscle fiber density. To elucidate the underlying molecular basis, we performed transcriptome (RNA-Seq) analysis of brain and dorsal muscle tissues. 328 differentially expressed genes (DEGs) were identified in dorsal muscle tissue (33 up and 295 down-regulated) and 228 in brain tissue (17 up and 211 down-regulated). Gene Ontology and KEGG enrichment analyses indicated that the DEGs were closely associated with apoptosis and angiogenesis pathways. Among the candidate genes, gpr116 was significantly up-regulated in the brain and dorsal muscle tissue of the fast-growing group. Finally, CRISPR-Cas9-mediated knockout in a zebrafish model confirmed that gpr116 deletion significantly restricted growth, underscoring its pivotal role in the growth regulation of grass carp. These discoveries lay significant groundwork for deeper exploration of growth regulation mechanisms in grass carp and offer important clues for selective breeding of key growth marker genes in this species.

Frog virus 3-like ranaviruses (FV3-like viruses), particularly FV3 (Frog virus 3), represent typical species within the genus Ranavirus, primarily infecting amphibians and reptiles, thereby posing serious threats to aquaculture and biodiversity conservation. We designed a pair of universal primers and a probe targeting the conserved region of the major capsid protein (MCP) genes of FV3-like viruses. By integrating recombinase-aided amplification (RAA) with lateral flow dipstick (LFD) technology and real-time fluorescence (RF) modification, we established RAA-LFD and RF-RAA assays. The limit of detection (LoD) of RAA-LFD was determined to be 35.4 copies/μL under optimized amplification conditions at 35 °C for 15 min. Similarly, the RF-RAA assay exhibited high specificity with a satisfactory LoD of 3.54 × 102 copies/μL at 39 °C over a duration of 17-20 min. In diagnosing 53 clinical samples infected by an isolated strain of FV3-like viruses, both assays demonstrated consistency with results obtained from real-time fluorescence PCR assay. These experiments indicate that both methods serve as promising alternatives for point-of care testing (POST), adaptable to various scenarios. This study represents the first establishment of RAA-LFD and RF-RAA assays for FV3-likes viruses, enabling rapid and intuitive assessment of detection results while fulfilling on-site testing requirements, thus contributing positively to swift diagnosis and long-term monitoring in aquaculture.

To elucidate the molecular mechanisms by which grass carp respond to high-concentration NaHCO3 stress, this study employed RNA-sequencing technology to perform transcriptome analysis on the gill and liver tissues of the treatment group with 30 mmol/L NaHCO3 and the control group (0 mmol/L). Through sequencing of 12 libraries constructed from 6 gill samples and 6 liver samples, a total of 41.86 GB of high-quality data from gill tissues and 41.49 GB of high-quality data from liver tissues were obtained. The analysis revealed that there were 1223 and 439 significantly differentially expressed genes (DEGs) in the gill and liver tissues, respectively, under NaHCO3 stress. Functional enrichment (GO) analysis indicated that grass carp respond to alkaline stress by regulating biological processes such as protein hydrolysis, ATP-binding activity, and lipid metabolism. Pathway (KEGG) analysis further revealed that immune-related signaling pathways were significantly activated to resist external stress, and the elevation of energy metabolism levels may provide necessary support for the stress-resistance process. This study systematically revealed the molecular adaptation strategies of grass carp in a high-alkali environment, providing an important theoretical basis for the molecular breeding of saline-alkali-tolerant grass carp varieties and the research on stress-resistance mechanisms.

Cyprinid herpesvirus 2 (CyHV-2) poses a substantial global threat to goldfish (Carassius auratus) and crucian carp (Carassius carassius). Despite the development of several sensitive molecular diagnostic techniques, there is an ongoing demand for alternative visualisation platforms to streamline the workflow, enhance safety profiles, and improve accessibility for end-users. In this study, we have integrated recombinase-aided amplification (RAA) technology with the CRISPR/Cas12a system to establish a rapid diagnostic system for CyHV-2, termed one-pot RAA-CRISPR/Cas12a. This method enables the results of detection within 60 min. The RAA-CRISPR/Cas12a platform is capable of detecting as few as 10 copies of CyHV-2 per reaction cycle without exhibiting cross-reactivity with other pathogens. The positive detection rate in clinical samples exceeds that of conventional PCR approaches, underscoring its high precision. Furthermore, the method could be used in conjunction with iron flocculation for the concentration and detection of viruses within aquaculture settings. This approach minimises the dissection of aquatic organisms, thereby maximising animal welfare and bolstering detection efficiency. Collectively, our findings validate the RAA-CRISPR/Cas12a method as a robust, specific, confirmatory, user-friendly and promising approach for on-site diagnosis of CyHV-2.

The medaka spermatogonial stem cell line (SG3) is a well-established cell line that can be induced to differentiate into sperm in vitro. Ranavirus, a genus of viruses widely distributed among fish, amphibians, and reptiles, poses a significant threat to these organisms. This study aimed to investigate the morphological changes and transcriptomic responses in SG3 following infection with three ranavirus strains: Andrias davidianus ranavirus (ADRV), Rana grylio virus (RGV), and Siniperca chuatsi ranavirus (SCRaV). Microscopic observations confirmed that SG3 cells were susceptible to all three ranaviruses, resulting in cellular swelling and membrane rupture, with infection confirmed through Real-time PCR. Transcriptomic profiling identified conserved responses: downregulation of early growth response protein (Egr1) and upregulation of myelocytomatosis oncogene (MYC). Ranavirus infection elevated alternative splicing events, with skipped exon events dominated and the maximal divergence observed at the 12 h post-infection. Key cellular metabolic pathways including glycolysis were disrupted, marked by coordinated upregulation of glycolytic enzymes and suppression of gluconeogenic enzymes. These results elucidate conserved molecular strategies underlying ranavirus-host interactions and metabolic mechanisms.

As an ectothermic animal that lives in water for a lifetime, grass carp ( Ctenopharyngodon idella) has a strong dependence on environmental temperature. The objective of this study was to estimate the genetic parameters for heat tolerance and growth traits in grass carp using reconstructed pedigree and molecular relatedness. The experimental population consisted of 800 offspring at 60 days old and 55 parents (female, 32; 5, 23) from selected line F4 and 539 at 6 months old offspring from selected line F5 with 87 parents (female, 44; 5, 43). Genetic genotyping of the experimental population was conducted using 10 pairs of microsatellite markers, and all 10 markers exhibited a high degree of genetic polymorphism across all 1394 grass carp (Ho = 0.751-0.981; He = 0.783-0.942; PIC = 0.759-0.938). Parentage assignment divided F4 individuals into 203 full-sib, and the Pearson correlation coefficient between reconstructed pedigree relatedness and molecular relatedness was 0.475. For the selected line F4, the heritability for each trait based on reconstructed pedigree relatedness (PR) were 0.320 f 0.092 for body weight (BW), 0.303 f 0.093 for standard length (SL), 0.246 f 0.082 for body height (BH), 0.193 f 0.074 for body thickness (BT), and 0.131 f 0.063 for upper thermal tolerance (UTT) was 0.131 f 0.063. Similarly, the heritability for each trait based on molecular relatedness (MR) were 0.330 f 0.092 for body weight (BW), 0.348 f 0.097 for standard length (SL), 0.248 f 0.080 for body height (BH), 0.197 f 0.073 for body thickness (BT) and the heritability for upper thermal tolerance (UTT) was 0.260 f 0.037. For the F5 generation, the heritability of body weight (BW) and standard length (SL) estimated by molecular relatedness (MR) was 0.281 f 0.045 and 0.283 f 0.044. These results indicate that molecular relatedness is a viable method for estimating genetic parameters when pedigree information is inaccurate or missing. The findings of this study can provide theoretical support for the optimization of breeding programs aimed at developing productive and heat tolerant grass carp strains.

The grass carp (Ctenopharyngodon idella) is highly susceptible to infections caused by Aeromonas species, particularly A. hydrophila and A. veronii. However, the immunological mechanisms underlying co-infection by these pathogens remain largely uncharted. This study investigated the pathogenesis and host immune response in grass carp following concurrent infection with A. hydrophila and A. veronii. Mortality was observed as early as 24 h post-infection, with cumulative mortality reaching 68%. Quantitative analysis demonstrated significantly elevated bacterial loads in hepatic tissue at 3 days post-infection (dpi). Histopathological evaluation revealed severe hepatic lesions characterized by cellular necrosis, cytoplasmic vacuolization, and hemorrhagic manifestations. Comparative transcriptomic analysis of hepatic tissues between co-infected and control specimens identified 868 and 411 differentially expressed genes (DEGs) at 1 and 5 dpi, respectively. Gene ontology and KEGG pathway analyses revealed significant enrichment of immune-related genes primarily associated with Toll-like receptor signaling and TNF signaling cascades. Notably, metabolic pathways showed substantial suppression while immune responses were significantly activated after infected. These findings provide novel insights into the host–pathogen interactions during Aeromonas co-infection in grass carp, which may facilitate the development of effective prevention and control strategies.

Grass carp (Ctenopharyngodon idella), a globally important aquaculture species, exhibits protein-dependent growth plasticity, requiring genetic improvement for sustainable production. This study integrates genome-wide association analysis (GWAS) and genomic selection (GS) to unravel the genetic architecture of four growth traits, body weight, body length, body height, and body depth, in grass carp fed with diets of varying protein levels (20%, 25%, 30%, and 35%). Using a 21K liquid SNP array, we identified 62,736 high-quality SNPs across 24 chromosomes, with 90 SNPs significantly associated with growth traits. Notably, three SNPs (SLG14_24417024, SLG14_24417039, SLG24_30276273) exhibited pleiotropic effects on multiple traits. Functional annotation of 276 candidate genes near significant SNPs revealed enrichment in keratinocyte development, septin cytoskeleton organization, and heat acclimation pathways. Genomic prediction achieved accuracies up to 0.79 for body weight traits using 1533 optimal markers. This study provides the first comprehensive SNP resource for grass carp growth traits with different dietary treatments, bridging GWAS and genomic prediction to accelerate marker-assisted selection. Our findings not only advance genetic breeding strategies but also inform protein diet optimization, minimizing economic and environmental costs in aquaculture.

The long-term-cultured Opsariichthys bidens spermatogonial stem cell line (ObSSC) was established in our previous study, which could be induced to differentiate into sperm in vitro. The genus Ranavirus is a large double-stranded DNA virus with a high degree of pathogenicity, which can be widely transmitted in fish, amphibians and reptiles. It is highly representative and possesses significant research value. The present study aimed to investigate the morphological changes and transcriptome analyses of ObSSC following infection by the three strains of ranaviruses (Andrias davidianus ranavirus, ADRV; Rana grylio virus, RGV; Siniperca chuatsi ranavirus, SCRaV). Microscopic observation and polymerase chain reaction (PCR) detection techniques were employed to ascertain that ObSSC could be invaded by ranaviruses, resulting in the formation of cell shed plaques and other lesions. Transcriptome analysis revealed the high degree of similarity among different infections of ranaviruses. The genes Egr1 (early growth response protein 1) and Dusp2 (dual specificity protein phosphatase 2) were downregulated after infection. The downregulation of PI3K-AKT and MAPK signaling pathways was observed in all infected groups, which may be related to the interference of viral infection on cell growth, biological process and apoptosis. At the same time, specific responses of the host cells were observed by infections of different types of ranaviruses. The expression of phagosome was upregulated in the ADRV-infected group, whereas the Junctional Adhesion Molecule (JAM2) and the leukocyte transendothelial migration pathway was upregulated in the RGV-infected group, and the Fc gamma R-mediated phagocytosis was upregulated in the SCRaV-infected group. These findings provided important clues for further understanding of the mechanism of ranaviruses infection and host cell response.

Citrobacter amalonaticus is widely recognized as a conditionally pathogenic bacterium, primarily associated with meningitis, brain abscesses and urinary tract infections in humans. To date, no documented cases of C. amalonaticus causing disease in fish have been reported. However, our recent study identified this bacterium as a novel pathogen responsible for over 50 % morbidity in grass carp (Ctenopharyngodon idella). Through comprehensive morphological analysis, biochemical phenotype identification and 16S rRNA gene sequencing, this pathogen was confirmed as C. amalonaticus (ca). The lethal dose, 50 % (LD50) of ca in grass carp was determined to be 2.04 x 105 CFU/mL. Infected fish exhibited symptoms consistent with naturally diseased individuals, including ulcerated and hemorrhagic skin, abdominal distension, hemorrhagic and swollen cloaca, and severe ascites. Histopathological examination revealed extensive damage to the intestine, liver, spleen, and kidney, with the intestines exhibiting particularly severe lesions. The health status of the fish was assessed through various parameters, including hematological indices, intestinal enzyme activity 72 h post-infection and expression levels of inflammation-related genes at multiple time points (0, 24, 48, 72, 96 hpi). Results indicated significant changes, including increased white blood cell count, reduced digestive enzyme activity (e.g., trypsin, TRY), elevated oxidative stress enzyme activity (e.g., catalase, CAT), and upregulation of inflammatory genes (e. g., IL-8). Drug sensitivity testing demonstrated that ca was highly susceptible to ciprofloxacin, ofloxacin and vancomycin. This study confirms the pathogenicity of ca in fish and represents the first identification of C. amalonaticus infection in grass carp, causing severe intestinal inflammation. These findings provide valuable data for the prevention and treatment of this disease.

In recent years, the concentrations of cadmium (Cd) and diclofenac (DCF) in water have frequently exceeded the standard; however, the toxic effects of these two pollutants on grass carp under single and combined exposure are unknown. In this study, the concentrations of pollutants in different tissues were detected, and the toxicities of the two pollutants to grass carp under different exposure conditions were compared based on growth traits, biochemical responses, gut microbiome, and transcriptomes. Based on these findings, the brain showed the lowest levels of Cd and DCF accumulation. Oxidative stress and pathological damage were observed in the brain and intestines. Changes in the structure and abundance of the gut microflora affect the synthesis of neurotransmitters, such as GABA and steroids. Differentially expressed genes in the brain were enriched in circadian rhythm functions. The expression of PER, CLOCK,1L-1β, 1L-17, and other genes are related to the abundance of Akkermansia, which indicates that the disorder of gut microflora will affect the normal circadian rhythm of the brain. All indices in the recovery group showed an increasing trend. Overall, the toxicity of Cd and DCF showed antagonism, and a single exposure had a stronger effect on gut microorganisms and circadian rhythm, which provided a scientific basis for exploring the comprehensive effects of different pollutants.

miRNAs are increasingly recognized for their crucial role in autophagy processes. Recent research has highlighted the significant function of autophagy in modulating immune responses. Within this context, specific miRNAs have been identified as indirect mediators of immune functions through their modulation of autophagy. In this study, we verified that miR-193b-5p simultaneously targeted the grass carp autophagy-related gene deptor, thereby reducing autophagy levels in CIK cells. Moreover, we found the expression levels of miR-193b-5p and deptor responding to pathogen infections in the GCRV-infected CIK cells. Notably, the overexpression of miR-193b-5p was found to induce the GCRV replication and reduce the irf3, irf7 and IFN1 expression. These findings also demonstrated that grass carp miR-193b-5p impacted the proliferation, migration, and antiapoptotic abilities of CIK cells. All the above results indicated that miR-193b-5p was linked to grass carp autophagy and played a vital role in antiviral immunity by targeting deptor. Our study may provide important insights into autophagy-related miRNAs and their roles in defense and immune mechanisms against pathogens in teleost.

Grass carp (Ctenopharyngodon idella) is a highly productive freshwater fish species that is widely cultivated worldwide, contributing substantial economic value to the aquaculture industry annually. Recently, a breeding program for grass carp has progressed to the fourth generation (F4) after four consecutive generations of family selection. To further assess the growth performance of the genetically selected grass carp F4, a 90-day consecutive growth comparison experiment was conducted between the selected population and a wild base population. The results showed that the selected grass carp F4 had better growth performance. Whole-genome resequencing of both wild and selected populations of grass carp yielded a total of 4,354,416 high-quality single nucleotide polymorphisms (SNPs). Parameters of genetic diversity including observed heterozygosity (Ho), expected heterozygosity (He), nucleotide diversity (pi), and polymorphic information content (PIC) were slightly decreased in the selected population compared to the wild population. The fixation index (Fst) indicated a low level of genetic differentiation (Fst = 0.021). Selection signatures analysis identified a total of 173 candidate selected regions with a cumulative size of 23.75 Mb, and 912 candidate genes including igf-1r, igfbp-2, igfbp-5 and itga6. This study provides valuable insights into the growth performance and the genetic basis of selection signatures in the process of grass carp selective breeding, and offers guidance for future breeding programs.

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen that causes significant losses to the global aquaculture industry due to mass mortality in crucian carp and goldfish. This study demonstrates that the ORF55/ORF57 deletion mutants CyHV-2-Δ55-CP and CyHV-2-Δ57-CP obtained through homologous recombination replicate effectively within the caudal fin of Carassius auratus gibelio (GiCF) cells and exhibit morphologies similar to the CyHV-2 wild-type strain. Both mutants demonstrated a decrease in virulence, with CyHV-2-Δ57-CP exhibiting a more significant reduction. This serves as a reference for the subsequent development of recombinant attenuated vaccines against CyHV-2. Additionally, both mutants expressed the inserted RGNNV-CP (capsid protein of Redspotted grouper nervous necrosis virus) fusion protein gene, and inoculation with CyHV-2-Δ57-CP-infected GiCF cell lysates elicited an antibody response in the grouper. These results indicate that, while ORF55 and ORF57 genes of CyHV-2 are not required for viral replication in vitro, they do play a role in virulence in vivo. Additionally, expression of foreign protein in CyHV-2 suggests that the fully attenuated mutant of CyHV-2 could potentially function as a viral vector for developing subunit vaccines or multivalent recombinant attenuated vaccines.

In recent years, Microcystin-LR (MC-LR) has been frequently detected in aquatic environments, exerting detrimental effects on the health of aquatic organisms. The grass carp (Ctenopharyngodon idella), an economically important fish, inhabits environments prone to MC-LR contamination. To investigate the toxicological mechanisms of MC-LR on grass carp, the fish were exposed to water containing 35.8 μg/L MC-LR. The physiological, biochemical, gut microbiota, and transcriptomic responses of the grass carp were evaluated at various time points (days 0, 1, 3, 7, and 21). The results showed that total superoxide dismutase activity in the liver and intestine was significantly increased after 21 days of exposure. Additionally, pathological damage was observed, including impaired intestinal epithelial barrier and nuclear pyknosis in the liver cell. MC-LR exposure also altered the diversity and composition of the gut microbiota and reshaped the microbiota interaction network, increasing the abundance of Cetobacterium and Vibrio by 32.43% and 25.7%, respectively. The intestinal microbiota functions were enriched in carbohydrate metabolism (10.45%) and amino acid metabolism (9.89%). RNA sequencing identified 504 shared differentially expressed genes (DEGs). The expression of the immune genes ITGB1 and GART was significantly upregulated in the D21 group. The upregulated DEGs were enriched in the complement and coagulation cascade pathways. MC-LR exposure induced metabolic disorders in grass carp, with upregulated genes significantly enriched in amino acid and carbohydrate metabolism pathways, while downregulated genes were enriched in lipid metabolism. This study provides valuable insights into the effects and toxicological mechanisms of MC-LR on aquatic organisms.

This research elucidates the potential of Lycium barbarum residue (LBR), a by-product rich in bioactive substances, as a dietary supplement in aquaculture, especially for herbivorous fish like grass carp. In a detailed 120-day feeding trial, the impacts of varying LBR levels on juvenile grass carp were assessed, focusing on growth performance, survival rate, biochemical markers, and liver health. The study identified a 6% inclusion rate of LBR as optimal for enhancing survival and growth while mitigating hepatic lipid accumulation. Composition analysis of this diet revealed high concentrations of polysaccharides and flavonoids. Notably, the intake of LBR was found to enhance the antioxidant and immune-related enzymatic activities in the liver. Furthermore, it contributed to a reduction in hepatic fat deposition by decreasing the levels of triglycerides (TG) and total cholesterol (T-CHO) both in the liver and serum. Transcriptomic analysis of the liver highlighted LBR's substantial influence on lipid metabolism pathways, including the PPAR signaling pathway, primary bile acid biosynthesis, cholesterol metabolism, bile secretion, fat digestion and absorption, fatty acid degradation and fatty acid biosynthesis. Further, the expression level of genes pinpointed significant downregulation of fasn and dgat2, alongside upregulation of genes like pparda, cpt1b, cpt1ab and abca1b, in response to LBR supplementation. Overall, the findings present LBR as a promising enhancer of growth and survival in grass carp, with significant benefits in promoting fat metabolism and liver health, offering valuable insights for aquacultural nutrition strategies.