Wei Liu

Pseudomonas putida is a conditional pathogenic bacterium and affects a variety of aquatic animals, including fish, amphibians and reptiles. However, studies focusing on the immune responses post-P. putida infection of sturgeon remain limited. In this study, a Pseudomonas putida-like strain (YFI-327) was isolated and identified from diseased hybrid sturgeon (Huso dauricus × Acipenser schrenckii). Firstly, the morphological, physicochemical characteristics of the bacterium, as well as the sequences of the 16s rRNA and gyrB genes, were analyzed. Then, the histopathological analysis showed that the diseased hybrid sturgeon exhibited extensive necrosis in the liver, spleen, gut and gill. Transcriptomic analysis of the spleen samples from hybrid sturgeon infected with the P. putida-like strain revealed 3337 DEGs, including 2161 upregulated genes and 1176 downregulated genes. Differentially expressed genes analysis indicated that 84 key components of the antigen processing and presentation pathway, hematopoietic cell lineage pathway and NOD-like receptor signaling pathway were upregulated, whereas 20 key components of the complement and coagulation cascades pathway were downregulated, which suggests a potential involvement of these pathways in the responses to P. putida-like infection. Moreover, the intestinal microbiota results showed a trend toward increased richness and diversity after infection, together with an apparent shift in dominant microorganisms from Firmicutes to Proteobacteria (phylum level) and Candidatus arthromitus to Xylophilus (genus level). The results also revealed that the serum biochemical parameters were altered after P. putida-like infection. These results provide evidence that the P. putida-like strain was a potential pathogen of hybrid sturgeon, and the infection might pose a serious threat to sturgeon aquaculture.

Grass carp (Ctenopharyngodon idella), a key species in China’s freshwater aquaculture, faces hindered industrial development due to overwintering mortality disease outbreaks. A Gram-negative bacterial strain, designated Hbh25210, was isolated from overwintering mortality-diseased grass carp in a Hubei Province farm. It was identified as Flavobacterium psychrophilum by 16S rRNA gene sequencing, showing 100% homology to reference strains in GenBank. The strain can be successfully cultured at 15 °C within 3 to 5 days. Multilocus sequence typing (MLST) classified it as ST-51 (CC-ST56), while serotyping assigned it to the 0 type, indicating phylogenetic affinity to Japanese cyprinid isolates. Artificial infection experiments confirmed its pathogenicity: muscle injection resulted in 15–75% mortality in grass carp, with clinical symptoms consistent with natural infections. Histopathological changes included hepatic necrosis, splenic congestion, glomerular swelling, and muscle fiber dissolution; intestinal lesions were particularly prominent in infected fish. 16S rRNA gene sequencing of diseased tissues showed Flavobacterium psychrophilum constituted over 60% of the microbial community in most ulcerative lesions samples and reached as high as 98.8% in certain cases. Strain Hbh25210 exhibited high-level resistance to multiple antibiotics, including ciprofloxacin, cefadroxil, neomycin, florfenicol, sulfamethoxazole, doxycycline, trimethoprim–sulfamethoxazole, and flumequine. Conversely, it was relatively sensitive to enrofloxacin, gentamicin, and amikacin. Quantitative PCR showed the bacterial load in muscle remained the highest, followed by the intestine and spleen, which peaked at 96 h post-injection. In conclusion, this study indicates that Flavobacterium psychrophilum is an important pathogen associated with overwintering mortality syndrome in grass carp. Our research findings can provide a basis for the effective prevention and control of overwintering mortality syndrome in major freshwater fish species.

High-carbohydrate diets can induce metabolic disorders in carnivorous fish, compromising their health. Glucose oxidase (GOx) catalyzes glucose oxidation into gluconic acid and hydrogen peroxide, which may reduce carbohydrate absorption and alleviate the physiological burden associated with high-carbohydrate diets, thereby potentially improving fish health. Thus, this study investigated the effects of varying levels of GOx in high carbohydrate diets on the growth, health, and metabolism of the largemouth bass (Micropterus salmoides), a typical carnivorous fish widely cultured in China. The six experimental diets were formulated as follows: a low-carbohydrate diet (9.5% carbohydrate, P0), a high-carbohydrate basal diet (14.5% carbohydrate, HCG0), and four high-carbohydrate diets (14.5% carbohydrate) supplemented with glucose oxidase (GOx) at 0.2, 0.4, 0.8, or 1.6 U/g, designated as HCG02, HCG04, HCG08, and HCG16, respectively. A total of 360 juvenile fish (8.07 ± 0.04 g) were randomly distributed into 18 circular tanks (300 L each) with three replicates per diet. The feeding trial lasted 8 weeks. Results showed that supplementation with 0.2–1.6 U/g GOx did not significantly affect growth performance or feed utilization. Compared with the P0 group, the HCG0 group exhibited significantly increased whole-body moisture and muscle glycogen content, while crude fat content decreased. GOx supplementation reduced whole-body moisture and liver glycogen content, increased muscle crude fat content. High-carbohydrate diets suppressed hepatic amylase activity, which was significantly increased by GOx supplementation in the HCG02 and HCG04 groups. Similarly, stomach and intestinal lipase activities initially decreased then rose with GOx addition. Protease activity followed the same pattern, being suppressed at low GOx levels but elevated at higher levels. Dietary GOx also significantly enhanced serum catalase activity and reduced malondialdehyde levels. Regarding liver and intestinal health, appropriate GOx levels (0.2–0.8 U/g) significantly lowered serum transaminase activities, improved liver color and histopathology, increased intestinal villus width, and reduced intestinal inflammation. In conclusion, GOx supplementation can protect largemouth bass by modulating glycolipid metabolism, enhancing antioxidant capacity, and improving intestinal health. The inclusion of GOx at ≤1.6 U/g is recommended to support healthy growth.

The infectious diseases have become more frequent with the production of farmed Chinese soft-shelled turtles (Pelodiscus sinensis) increasing. In this study, bacterial strain was isolated, identified, and characterized from the liver of diseased Chinese soft-shelled turtles exhibiting body surface hemorrhages, claws and tail tips necrosis, and bleeding spots in visceral tissues. Based on the analysis results of morphology, biochemistry, and 16S ribosomal RNA (16S rRNA) sequencing, one bacterium Providencia rettgeri (P. rettgeri) was identified. The histopathological examination results revealed varying degrees of tissue damage in the spleen, liver, kidneys, and intestines of individuals with P. rettgeri. Challenge experiments results confirmed that the P. rettgeri caused morbidity and mortality in healthy Chinese soft-shelled turtles, reproducing similar clinical symptoms of naturally infected individuals. And, its mortality rate was up to 91% in the highest concentration group. Screening of eight virulence-associated genes results revealed that this P. rettgeri strain carried virulence factors including invasion protein gene, alpha-hemolysin, and others which were considered to contribute to pathogenicity. Antibiotic susceptibility testing showed that the bacterium was sensitive to amikacin and ciprofloxacin, which may be effective therapeutic options. In conclusion, this bacterium P. rettgeri was the pathogen that caused this disease in Pelodiscus sinensis. These results provide valuable research basis for the disease prevention and control of Pelodiscus sinensis farming.

The rapid increase in drug resistance in recent years has become a significant global public health concern. Escherichia coli are ubiquitous bacteria, widely distributed in various environments. This study isolated a bacterial strain (HD-593) from diseased Chinese soft-shelled turtles (Pelodiscus sinensis). The bacterium was identified based on morphology, biochemical tests, and 16S rRNA sequencing, confirming it as E. coli. Drug susceptibility tests revealed that the HD-593 strain was highly resistant to ceftriaxone, enrofloxacin, doxycycline, sulfadiazine, gentamicin, neomycin, florfenicol, carbenicillin, cefradine, erythromycin, penicillin, ampicillin, midecamycin, and streptomycin. Resistance gene analysis confirmed the presence of quinolone resistance genes (oqxA and oqxB), aminoglycoside resistance genes (aac(3)-II and aphA1), a β-lactam resistance gene (blaTEM), and an acylaminol resistance gene (floR) in HD-593. The median lethal dose (LD50) of HD-593 for P. sinensis was 6.53 × 10⁵ CFU/g. Biochemical analysis of serum revealed that HD-593 infection caused a significant reduction in total protein, albumin, and globulin levels, while markedly increasing the levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. Histopathological analysis revealed severe intestinal damage characterized by villi detachment and muscle cell necrosis. Additionally, extensive splenocyte necrosis with nuclear marginalization, glomerular swelling, and pronounced hepatic steatosis accompanied by distended sinusoids were observed. This study identified a multidrug-resistant E. coli strain from deceased P. sinensis, suggesting that drug resistance genes may circulate in aquaculture ecosystems, posing potential risks to aquaculture.

D-glucuronolactone (DGL), a hepatoprotective compound widely used in clinical and energy products, was evaluated for its effects on Chinese soft-shelled turtles (Pelodiscus sinensis) through an 8-week feeding trial with dietary supplementation (0, 200, and 400 mg kg⁻¹). DGL did not alter survival or feed intake, but induced dose-dependent growth improvements, including increased final body weight, weight gain rate, specific growth rate, and muscle/liver glycogen, alongside reduced feed conversion ratio and muscle and liver fat. Serum analysis showed decreased activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and reduced low-density lipoprotein cholesterol, total cholesterol, and triacylglycerols. Antioxidant indices revealed elevated catalase and superoxide dismutase (SOD) activities in serum and intestine, coupled with reduced malondialdehyde, though hepatic SOD activity declined. Histologically, 400 mg kg⁻¹ DGL alleviated liver lesions without impacting intestinal morphology. Molecular analyses demonstrated upregulated muscle mTOR signaling genes (mTOR, IGF1, S6K1) but downregulated hepatic/intestinal mTOR and IGF1 expression. DGL also suppressed inflammatory cytokines (TNF-α, IL-1β, IL-10) in liver and intestine. Challenge tests with Aeromonas hydrophila confirmed the enhanced disease resistance in DGL-supplemented turtles. These findings highlight DGL’s potential as a nutritional strategy to enhance growth, antioxidant capacity, and health in intensive turtle farming.

Currently, the effects of dietary levels of n-3 highly unsaturated fatty acids (HUFAs) on the growth performance, antioxidant capacity, immunity, and serum oxylipin profiles of female F₂-generation Yangtze sturgeon remain unknown. A total of 75 Yangtze sturgeons, an endangered freshwater fish species, with an average body weight of 3.60 ± 0.83 kg, were randomly allocated to 15 concrete pools, with each dietary group represented by 5 fish per pool. The fish were fed five different experimental diets containing various levels of n-3 HUFAs (0.5%, 1.0%, 1.5%, 2.0%, and 2.4%). After a feeding period of 5 months, no significant differences in the growth performances of the fish were observed among the five dietary groups (p > 0.05). However, we did note that the serum levels of low-density lipoprotein cholesterol (LDL-C), triglycerides (TGs), and total cholesterol (TCHO) exhibited a marked increase in the fish that consumed higher dietary n-3 HUFA levels (p < 0.05). Conversely, alkaline phosphatase (ALP) activities showed a notable decrease as dietary n-3 HUFA levels increased (p < 0.05). Serum antioxidant indices, such as the activity levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were significantly higher in the 2.4% HUFA group compared to the 0.5% HUFA group. Additionally, muscle antioxidant indices, including total antioxidant capacity (T-AOC), catalase (CAT), and SOD activity, exhibited notable increases as dietary n-3 HUFA levels increased (p < 0.05). Furthermore, there was a decrease in malondialdehyde (MDA) levels as dietary n-3 HUFA levels increased (p < 0.05). In relation to immune indices, only serum immunoglobulin M (IgM) and muscle complement 3 (C3) were found to be influenced by dietary n-3 HUFA levels (p < 0.05). A total of 80 oxylipins were quantified, and our subsequent K-means cluster analysis resulted in the classification of 62 oxylipins into 10 subclasses. Among the different n-3 HUFA diets, a total of 14 differential oxylipins were identified in the sera. These findings demonstrate that dietary supplementation with n-3 HUFAs exceeding a 1.0% level can enhance antioxidant capacity and regulate serum lipid metabolism, potentially through modulation of oxylipins derived from ARA, DHA, and EPA. These insights provide novel perspectives on the mechanisms underlying these observations.

In recent years, the potent influence of tocotrienol (T3) on diminishing blood glucose and lipid concentrations in both Mus musculus (rats) and Homo sapiens (humans) has been established. However, the comprehensive exploration of tocotrienol’s hypolipidemic impact and the corresponding mechanisms in aquatic species remains inadequate. In this study, we established a zebrafish model of a type 2 diabetes mellitus (T2DM) model through high-fat diet administration to zebrafish. In the T2DM zebrafish, the thickness of ocular vascular walls significantly increased compared to the control group, which was mitigated after treatment with T3. Additionally, our findings demonstrate the regulatory effect of T3 on lipid metabolism, leading to the reduced synthesis and storage of adipose tissue in zebrafish. We validated the expression patterns of genes relevant to these processes using RT-qPCR. In the T2DM model, there was an almost two-fold upregulation in pparγ and cyp7a1 mRNA levels, coupled with a significant downregulation in cpt1a mRNA (p < 0.01) compared to the control group. The ELISA revealed that the protein expression levels of Pparγ and Rxrα exhibited a two-fold elevation in the T2DM group relative to the control. In the T3-treated group, Pparγ and Rxrα protein expression levels consistently exhibited a two-fold decrease compared to the model group. Lipid metabolomics showed that T3 could affect the metabolic pathways of zebrafish lipid regulation, including lipid synthesis and decomposition. We provided experimental evidence that T3 could mitigate lipid accumulation in our zebrafish T2DM model. Elucidating the lipid-lowering effects of T3 could help to minimize the detrimental impacts of overfeeding in aquaculture.

We investigated the effects of dietary tannic acid (TA) supplementation of a high‐carbohydrate diet on growth, feed utilization, whole‐body proximate composition, serum biochemical indicators, antioxidant capacity, digestive enzyme activity, and liver and intestinal health of juvenile largemouth bass, Micropterus salmoides (initial mean weight: 8.08 ± 0.08 g). Five diets were prepared, including a positive control (dietary carbohydrate level, 16%, LC0), a negative control (dietary carbohydrate level, 21%, HC0), and three TA‐supplementation diets based on the negative control diet with TA addition at 200, 400, and 800 mg/kg, respectively. After 8 weeks of feeding, the results showed that compared with the LC0 diet, 400–800 mg/kg dietary TA significantly improved the survival rate of largemouth bass (P < 0.05) while significantly reducing its weight‐gain rate and specific growth rate (P < 0.05). Compared with the HC0 diet, 400 mg/kg dietary TA significantly increased serum catalase activity (P < 0.05), and significantly decreased serum malondialdehyde, liver glycogen, lightness (L ^∗), and yellowness (b ^∗) (P < 0.05). Moreover, compared with the HC0 diet, 200–400 mg/kg dietary TA effectively improved the vacuolation of hepatocytes caused by the high‐carbohydrate diet and reduced the occurrence of intestinal epithelial cell vacuolation and necrosis. In turn, 800 mg/kg dietary TA significantly inhibited protease activity in the pyloric caecum and intestine (P < 0.05). In conclusion, dietary supplementation with TA inhibited protease activity, which resulted in decreased growth performance in largemouth bass. However, it was also found that 200–400 mg/kg TA enhanced the antioxidant capacity of largemouth bass in the case of the high‐carbohydrate diet, reduced liver glycogen levels, and improved liver and intestinal health. Finally, it should be noted that, when the dietary TA level exceeded 800 mg/kg, TA appeared to play a pro‐oxidation role in the liver, which may cause oxidative stress in the liver.

Long-term or excessive oxidative stress can cause serious damage to fish. Squalene can be added to feed as an antioxidant to improve the body constitution of fish. In this study, the antioxidant activity was detected by 2,2-diphenyl-1-acrylhydrazyl (DPPH) test and fluorescent probe (dichloro-dihydro-fluorescein diacetate). Transgenic Tg (lyz: DsRed2) zebrafish were used to evaluate the effect of squalene on CuSO₄-induced inflammatory response. Quantitative real-time reverse transcription polymerase chain reaction was used to examine the expression of immune-related genes. The DPPH assay demonstrated that the highest free radical scavenging exerted by squalene was 32%. The fluorescence intensity of reactive oxygen species (ROS) decreased significantly after 0.7% or 1% squalene treatment, and squalene could exert an antioxidative effect in vivo. The number of migratory neutrophils in vivo was significantly reduced after treatment with different doses of squalene. Moreover, compared with CuSO₄ treatment alone, treatment with 1% squalene upregulated the expression of sod by 2.5-foldand gpx4b by 1.3-fold to protect zebrafish larvae against CuSO₄-induced oxidative damage. Moreover, treatment with 1% squalene significantly downregulated the expression of tnfa and cox2. This study showed that squalene has potential as an aquafeed additive to provide both anti-inflammatory and antioxidative properties.