Effects Of Euryale Ferox Extract On Immunity Of Procambarus Clarkii

Red swamp crayfish(Procambarus clarkii),as a kind of freshwater crayfish,is one of the most important aquaculture species in the world.However,due to lack of scientific management experience and negligence in disease control,diseases frequently occur in the process of culture.Antibiotics are often used to prevent and treat aquatic animal diseases,but excessive use of antibiotics is potentially dangerous,which may lead to antibiotic-resistant bacteria and endanger the health of humans or animals.Therefore,looking for a safe and efficient antibiotic substitute has become a research hotspot in the aquaculture industry.Studies have shown that the crude extract of Euryale Ferox(EF)has an anti-oxidant effect,and has an obvious antibacterial effect on Aeromonas hydrophila,Aeromonas sobria,and other pathogenic bacteria.In order to find new prevention for controlling the diseases in crayfish,EF was used as the raw material in this study.The ethanol extract of EF was divided in different concentrations and added in fodder(0g·kg-1,5g·kg-1,10g·kg-1 and 20g·kg-1,corresponding to the Control,0.5%-EF group,1%-EF group and 2%-EF group).After 28 continuous days of feeding with the ethanol extract of EF in fodder under the simulated breeding environment,the enzyme activity of serum SOD,PO,ACP,AKP and hepatopancreas CAT,SOD,ACP,AKP in crayfish were measured.Meanwhile,the mRNA expression of immune-related genes,like CypA,GPxs,and Astacidin in the hepatopancreas,were detected.And then,we recorded the changes in growth parameters and the survival rate of crayfish after Aeromonas hydrophila challenge.We also explored the intestinal tissue structure and intestinal microbiota structure and diversity for evaluating the immune effect and application value of EF.Finally,to explore the palatability of EF-added fodder,we recorded the selection time and frequency of food selection of crayfish with different fodder sizes,colors,shapes,and concentrations of EF using behavioral observation experiments,and then evaluated the influence on the food choice of crayfish.Our results were as shows:1.After 28 days of feeding,the survival rates of crayfish from 0.5%-EF group,0.5%-EF group,and 0.5%-EF group were higher than those of the control group.The growth rate of the control group and 0.5%-EF group was higher,The survival rate of 0.5%-EF group was significantly improved,but there is no significant difference in the final quality,weight gain rate and specific growth rate.After Aeromonas hydrophila challenged,there was no statistical difference in the survival rate between the experimental groups though,the survival rates of 0.5%-EF group and 2%-EF group were higher than those of the control group,The highest inhibition rate is 20%of 0.5%-EF group.The results indicated that a proper concentration of EF crude extract could improve the survival rate of crayfish.2.Compared with the control group,the ACP and AKP activities in the serum of crayfish from 0.5%-EF group significantly increased,meanwhile,the SOD activity in the serum of 2%-EF group were increased.ACP activity in 1%-EF group and 2%-EF group was significantly decreased,but the PO activity did not change.The ACP activity in the hepatopancreas of crayfish from 0.5%-EF group was significantly increased,but the activities of SOD,CAT,and AKP were not changed.Compared with the control group,CypA and GPxs mRNA expression levels of crayfish in 2%-EF group significantly increased,however,there was no significant difference in the relative expression levels of Astacidin mRNA among the groups.These results indicated that EF can be used as an antioxidant to improve the antioxidant capacity of crayfish,and an appropriate concentration of EF added in fodder can also enhance its non-specific immunity.3.It is found that the compactness of the connective tissue layer of crayfish from treated groups was better than that of the control group.The muscle bundles of crayfish in 0.5%-EF group and 2%-EF group were more complete than the control group.We also found that the intestinal mucosal fold structures were increased in treated groups.The results indicated that a proper concentration of EF crude extract can improve the structure of the intestinal tract in crayfish.The analysis of intestinal microbes showed that the intestinal microbiota structure of crayfish from treated groups was not changed,and the abundance of the intestinal microbiota at the phylum level did not change,compared with the control group.At the genus level,we observed that the intestinal microflora of crayfish from the four groups is significantly different.Among them,2%-EF group had more beneficial bacteria,like Sporocytophaga,which may help promote digestion and strengthen immunity.4.The fodder concentrations,sizes,colors and shapes of EF crude extract have no significant effect on the food selection time and food selection frequency of crayfish.It shows that the fodder concentrations,sizes,colors and shapes of EF added in fodder added in fodder will not affect the food choice of crayfish.In summary,adding a proper concentration of EF crude extract in fodder do not affect the growth of crayfish,it can increase the antioxidant capacity and immunity of crayfish,meanwhile,it can also improve the intestinal tissue structure and do not change microbiota structure.Besides,the fodder concentrations,sizes,colors and shapes of the EF crude extract added in fodder will not affect the food selection of crayfish.Therefore,the EF crude extract can be used as an immunostimulant and added in fodder for crayfish.