Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li.

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC50 ) values of the embryos and larvae were 1.71 and 0.22 mg l-1 for copper and 2.57 and 2.72 mg l-1 for zinc, respectively, whereas the MB LC50 after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l-1 , respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l-1 for embryos and 0.03, 0.03 and 1.78 mg l-1 for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l-1 , respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l-1 led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.

Vibrio metschnikovii, a Potential Pathogen in Freshwater-Cultured Hybrid Sturgeon.

In July 2021, a disease with a high mortality rate broke out in freshwater cultured hybrid sturgeon in Zhengzhou, Henan Province. A dominant strain, H-701, was isolated from diseased fish; physiological changes in diseased fish were investigated and molecular identification, biochemical characterization, and pathogenicity and drug sensitivity tests of H-701 were performed. The 16S rRNA gene sequence of H-701 was 99.86% homologous with that of Vibrio metschnikovii in GenBank. The 50% lethal dose of H-701 was 3.72 ± 0.929 × 104 CFU/g fish weight. The proportion of monocytes, neutrophils, and eosinophils in the blood of diseased sturgeon increased significantly, whereas the proportion of lymphocytes decreased. In diseased fish, the serum levels of total protein, albumin, globulin, and alkaline phosphatase decreased significantly, and those of aspartate aminotransferase, alanine aminotransferase, and complement C3 increased significantly. There were obvious pathological changes in several tissues of the diseased fish. H-701 was sensitive to antibiotics such as florfenicol, enrofloxacin, and doxycycline. This study not only demonstrated that V. metschnikovii was the cause of death of a large number of hybrid sturgeon but also revealed its potential risk in hybrid sturgeon aquaculture. The results provide a basis for the diagnosis and prevention of this disease.

Evaluation of the potential probiotic Bacillus subtilis isolated from two ancient sturgeons on growth performance, serum immunity and disease resistance of Acipenser dabryanus.

In the present study, we aimed to screen the potential probiotic Bacillus subtilis isolated from the gut of healthy fish using in vitro assays and to evaluate its effect on Dabry's sturgeon (Acipenser dabryanus) using in vivo feeding experiments. Among the isolates, B. subtilis BSth-5 and BSth-19 exhibited antimicrobial effect against four sturgeon-pathogenic bacteria, including Aeromonas hydrophila, A. veronii, A. media, and Streptococcus iniae. The cell number of B. subtilis BSth-5 and BSth-19 changed little after 2 h of exposure to pH 3.0 or fresh Dabry's sturgeon bile at 2.5% and 5.0%. Meanwhile, B. subtilis BSth-5 and BSth-19 produced extracellular protease, cellulose, and lipase. And it was proved that B. subtilis BSth-5 and BSth-19 were harmless after injection of Dabry's sturgeon. One group of Dabry's sturgeon was fed a control diet and two groups were fed experimental diets containing 2.0 × 108 CFU/g BSth-5 (T1 group) or BSth-19 (T2 group) for 8 weeks. No significant differences in final weight, weight gain rate, and special growth rate were observed in the T1 and T2 groups compared to the control group (P > 0.05), but a significant improvement in survival rate was detected after 4 and 8 weeks of feeding (P < 0.05). After 8 weeks, serum total antioxidant capacity, total superoxide dismutase activity, and IgM levels were significantly higher in the T1 and T2 groups compared to the control group (P < 0.05). Moreover, serum lysozyme activity was significantly higher in the T1 group relative to the control group during the whole experiment period (P < 0.05); however, the differences were not significant between the T2 and control groups (P > 0.05). Serum malondialdehyde levels in the T1 and T2 groups were significantly lower than those in the control group after 4 weeks (P < 0.05). Sturgeons in the T1 and T2 groups showed a higher survival rate after Aeromonas hydrophila infection. To summarize, dietary supplementation with BSth-5 and BSth-19 could enhance the survival rate, antioxidant activity, serum immunity, and disease resistance in A. dabryanus.

Population differentiation and genetic diversity of endangered Brachymystax tsinlingensis Li between Yangtze River and Yellow River in China based on mtDNA.

Brachymystax tsinlingensis Li, distributed scatteredly in Qinling Mountains of China, is an ideal material for studies of conservation and phylogeography. In the present study, the genetic variations and phylogeographical patterns of B. tsinlingensis collecting from the Yangtze River (n = 30) and the Yellow River (n = 70) were compared based on 1082-1163 bp control region with partial tRNA and 912 bp cyt b partial sequence of mtDNA. Analysis of variance indicated that a high proportion of the total genetic variance (96.03%) was distributed among populations, supporting strong geographic structuring of mtDNA polymorphism. Ten haplotypes were assigned to two clades that were related to geographic regions. No haplotype shared between geographic populations from the Yangtze River and the Yellow River, and two clades related to geographic regions can be clearly found from phylogenetic analysis by Bayesian approach, one from individuals in the Yangtze River and the other from individuals in the Yellow River, which probably indicated artificial carrying not happened historically. The mitochondrial results revealed an obvious differentiation of genetic structure of B. tsinlingensis between populations of Yellow River and Yangtze River, and the estimated diverge time around 0.20 MY. It was suggested that each of the two evolutionarily distinct groups of B. tsinlingensis should be protected and crossing individuals between two river systems should be avoided for conservation purpose.