| Procambarus clarkii plays an important role in the comprehensive rice-fish farm strategy of China.Its characteristis determine the fact that combining propbiotics and oral Chinese herbs is more suitable than traditional bacteriostat in the fight against bacteriosis,which meets the industrial development and food safety requirements.This study mainly includes two parts:1.The evaluation of probiotic characteristics and safety of commercially available BacillusTo investigate the current situation of Bacillus probiotics commercially available for rice and crayfish,the author isolated 19 Bacillus from existing products.Their potential as probiotics was evaluated by vitro tests of bacteriostat,cellulose dissolution,growth rate at low temperature and deficient oxygen,spores against high temperature,low p H,hyperbile salt tolerance,bacteria self-aggregation and hydrophobicity,hemolytic activity,K-B drug sensitivity and etc.And the probiotic characteristics and safety of the candidate strains were further evaluated by in vivo experiments such as mouse challenge test and Procambarus clarkii breeding test.The main findings can be summarized as follows:10 out of the 19 isolated strains of bacillus were Bacillus subtilis and five were Bacillus amyloliquefaciens.14 strains of Bacillus in this study had various inhibition on 1 to 6 of the selected 8 pathogen strains(i.e.1 strain of Staphylococcus aureus,1 strain of Aeromonas hydrophila,1 strain of Aeromonas veronii,1 strain of Anterooms salmonicida and 4 strains of Escherichiacoli)and the bacteriostatic index was between 1.30 and 5.09.14 strains of Bacillus was capable of dissolving cellulose,and the cellulose-dissolving index was between 1.22 and 1.54.The growth rate of the isolated samples dropped with lower temperature(37 ℃,25 ℃,15 ℃),and the Bacillus strain with highest growth rate was different according to temperature conditions.The fastest growing strains were B18(37 ℃),B26(25 ℃),B14(15 ℃),respectively.The growth rate of the isolated samples declined with less environmental oxygen content.The isolated Bacillus had relatively strong self-aggregation,all over 70% while their hydrophobicity was poor,the highest was27.78%.Spores of most isolated Bacillus had a strog tolerance for high temperature,low p H and hyperbile salt.8 strains of isolates(including B22 and B40)were resistant to some antibiotics.8 strains of isolates(including B14 and B40)had β-hemolytic activity on rabbit erythrocytes.Only B14,B22 and B40 could grow in 15 ℃ and deficient-oxygen environmet with bacteriostasis and cellulose dissolution.To investigate whether Bacillus amyloliquefaciens strains B14 and B40 with hemolytic activity can orally taken,they were selected as strains for subsequent in vivo tests.No death and pathological changes were found in the acute challenge test by intraperitoneal injection(109-1011 CFU / mouse)and the chronic challenge test(108 CFU /mouse)of B14 and B40 strains on mice(20 ± 2 g,5-weeks old).The 20-day feeding experiment on Procambarus clarkii(whose initial body length was 2to 4 cm)showed that B14 and B40 had growth promoting effect,while it disappeared gradually as the feeding time increased.B14 and B40 tended to reduce the feed coversion rate.They appeared to cause enterities on experimental crayfish after 20-days feeding.The analysis on intestinal flora indicated that the treatment with B14 and B40 could reduce the diversity of intestinal flora,and Aeromonas abundance is significantly higher than that of control group;it is further found that the type and concentration of Aeromonas-specific virulence genes in treatment group of B14 and B40 were higher than those of the control group.In conclusion,the probiotic effects of Bacillus amylolyticus B14 and B40 on Procambarus clarkii is not obvious in this study,and they might cause enteritis on experimental crayfish instead.The enteritis pathogenesis may involve a decrease in the diversity of intestinal flora,an increase in the abundance of Aeromonas,and an increase in the type and concentration of Aeromonas-specific virulence genes.Therefore,it is necessary to enhance the management of the effectiveness and safety of commercially available Bacillus probiotics.2.The influence of compound Chinese medicine on the growth and anti-stress of Procambarus clarkiiThis study evaluated the growth index,antioxidant index and hepatopancreas transcriptome analysis results in order to explore the influence of compound Chinese medicine on the growth and anti-stress of Procambarus clarkii(whose initial body length is 2-4 cm)at low temperature(18-22 ℃).The main findings are as follows:At a water temperature of 18-22 ℃,Chinese medicine feeding for 20 days was likely to increase the weight gain rate,specific growth rate and feed conversion rate of Procambarus clarkii along with crayfishs’ increased resistance to stress-induced enteritis.The average values of glutathione peroxidase,catalase,and superoxide dismutase activities in experimental group with Chinese medicine were higher than those in control group,and the value of catalase activity of experimental group was significantly higher than that of control group(p <0.05).Compared with the control group,there were 122 hepatopancreas differentially expressed genes in the experimental group with Chinese medicine,most of which were related to metabolism and non-specific immune function.In KEGG pathway enrichment,the pathways of steroid biosynthesis,ether lipid metabolism,endocytosis,phagosome,and lysosomal went up,and the glycerophospholipid metabolism pathway went down.The transcriptional regulation results of 6 genes verified by fluorescence quantitative PCR verification was consistent with the result of transcriptome sequencing.As discussed above,the compound Chinese medicine in this study may achieve growth promotion and anti-stress effects through upregulating the expression of non-specific immune-related genes in the hepatopancreas,enhancing antioxidant enzyme activity,improving lipid metabolism and reducing protein breakdown. |
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