Effects Of Bacillus Subtilis On Growth,Immunity And Intestinal Flora Of Cherax Quadricarinatus

Red claw crayfish(Cherax quadricarinatus)is an emerging economically important crayfish in China.It is an invertebrate and relies only on its body shell and hepatopancreas to resist foreign pathogenic bacteria and viruses.Studies have shown that the addition of Bacillus subtilis to feed can stimulate the innate immunity of aquatic animals and thus improve disease resistance.In this study,the effects of Bacillus subtilis on growth and non-specific immune performance and intestinal flora of C.quadricarinatus were comparatively studied by feeding feeds containing different concentrations(1.5×10~6 CFU/g,1.5×10~7 CFU/g,1.5×10~8 CFU/g)of Bacillus subtilis,to be able to obtain systematic experimental data for solving the problem of microecological agents in crayfish culture in China.The experimental data are intended to provide a reference and basis for addressing the use of micro-ecological agents in crayfish culture in China and to increase the basic research related to the healthy culture of C.quadricarinatus while realizing the scientific and accurate use of Bacillus subtilis in feed.In the culture experiment,C.quadricarinatus weighing 0.45±0.05 g were randomly divided into four treatment groups,with three parallel replicates for each treatment group.The crayfish were fed with diets containing PBS(control group),1.5×10~6CFU/g,1.5×10~7CFU/g,and 1.5×10~8CFU/g Bacillus subtilis for 45 d.The body weight of each crayfish was measured on the 15th,30th and 45th days of the culture experiment.The results showed that with the increase of culture time,the final weight and specific growth rate of C.quadricarinatus showed a trend of slowly increasing and then significantly increasing,in which the final weight and specific growth rate of 1.5×10~6CFU/g test group were the highest,it can be seen that the addition of 1.5×10~6CFU/g is more beneficial to promote the growth and development of C.quadricarinatus.On the 45th day of the experiment,nine juvenile crayfish were randomly taken from each of the four treatment groups,and hepatopancreas tissues were collected to determine the activity ofα-Amylase(α-AL),trypsin,glutamic transaminase(GPT),glutamic oxaloacetic transaminase(GOT),and superoxide dismutase(SOD)in the hepatopancreas.The results of the analysis showed that the vitality ofα-Amylase and trypsin in the hepatopancreas of juvenile crayfish increased significantly after the addition of Bacillus subtilis,with the highest in the test group at 1.5×10~6CFU/g;the vitality of GPT and GOT did not change significantly;the vitality of superoxide dismutase was significantly higher in the test group fed with Bacillus subtilis than in the control group,with the highest in the test group at 1.5×10~8 CFU/g.g was the highest in the test group.The changes in the expression of immune-related genes in hepatopancreas tissues were detected,and the results of fluorescence quantitative analysis showed that the expression of ALF and SOD genes were highly significantly up-regulated in both 1.5×10~7CFU/g and 1.5×10~8CFU/g test groups(P<0.01).On days 15,30 and 45 of the culture experiment,six young crayfish were randomly selected from the four treatment groups,and the intestinal contents were collected for macroeconomic analysis of the intestinal flora,which showed that Amoebacteria were the most abundant colonizing bacteria in the intestine of crayfish,and the Rhodobacteraceae belonging to the Alphaproteobacteria had the potential to become an indicator family for Bacillus subtilis to influence the intestinal microorganisms of crayfish.The abundance of Bacteroides,Firmicutes and Actinobacteriota was increased in different periods of culture,and the genus Aeromonas was inhibited in the first period of culture,but the effect was not significant.The Chao1,ACE,Shannon,and Simpson indices of four treatment groups in three periods were statistically analyzed,and the diversity and abundance of the intestinal flora of C.quadricarinatus at different periods and treatment concentrations were assessed;based on the functional PICRUSt prediction,it was found that the intestinal microflora of C.quadricarinatus was rich in metabolic functions,of which 45.28%were metabolic among the metabolic pathways,the main ones include carbohydrate metabolism(22.69%),amino acid metabolism(20.38%)and energy metabolism(9.71%),among which the main Level 2functional pathways are amino acid metabolism,energy metabolism,carbohydrate metabolism,and disease-like pathways.At the end of the culture trial,each group was fed a basal diet for one more week,and 24h after weaning,the remaining crayfish in each group were tested for artificial infection with A.veronii in the laboratory.The hepatopancreas tissues were extracted and the activities of alkaline phosphatase(AKP),acid phosphatase(ACP),and superoxide dismutase(SOD)in the hepatopancreas were measured.The results showed that the activity of superoxide dismutase in the hepatopancreas of juvenile crayfish was significantly higher in the test group than in the control group after 24 h of attack(P<0.05),and was highest in the test group with 1.5×10~7CFU/g;the activity of alkaline phosphatase and acid phosphatase was significantly higher in the test group than in the control group after 24 h of attack with 1.5×10~7CFU/g(P<0.05).Analysis of the differences in the expression of immune-related genes using fluorescence quantitative PCR showed that the expression of ALF gene was highly significantly upregulated in the 1.5×10~6CFU/g,1.5×10~7 CFU/g and 1.5×10~8CFU/g test groups(P<0.01),the expression of SOD gene was significantly upregulated in the 1.5×10~6CFU/g test group(P<0.05),and the expression of SOD gene was significantly upregulated in the 1.5×10~6CFU/g test group(P<0.05),and SOD gene expression was highly significantly up-regulated in the 1.5×10~7 CFU/g test group(P<0.01).The test results showed that the addition of Bacillus subtilis to the basal feed not only significantly promoted the growth and development of red claw crayfish and improved the richness and diversity of intestinal flora,but also had a significant effect on disease resistance and immunity,and significantly improved the resistance of C.quadricarinatus to A.veronii.In the breeding process,taking into account the cost and other factors,it is recommended that a concentration of 1.5×10~6CFU/g is appropriate.