The Physiological Responses Of Procambarus Clarkii To Environmental Stresses

Procambarus clarkii,commonly known as crayfish,is a freshwater economic crustacean native to the southern United States and northern Mexico.Because of its delicious taste and rich nutrition,crayfish is very popular in the aquatic product market and consumers,and has become one of the important varieties of freshwater culture in China.However,they often produce stress response due to environmental factors in the process of artificial culture.In this study,P.clarkii was used as experimental material to study the changes of related physiological and biochemical indexes,gene expression levels and intestinal flora of crayfish under three stress conditions,including low temperature aerial exposure,high temperature stress and high temperature adaptation.The physiological and ecological response of P.clarkii under environmental stress were preliminarily discussed.The main research results were as follows:1.Desiccation is a commonly stressful situation experienced by decapods during live transportation.This study investigated the effects of low-temperature aerial exposures(LTAEs)(dry exposure(DL)and moist exposure(ML)under 6 °C)and re-immersion on the antioxidant and immune responses and the hepatopancreatic histopathology of P.clarkii.Compared with the control(normally feeding in 24.0 °C of water temperature),the crayfish under LTAEs overall showed severe oxidative damage to hepatopancreas by significant increase of malondialdehyde(MDA)contents and significant decrease of total antioxidant capacity(T-AOC),and the oxidant damage could not be fully recovered at 12 h of re-immersion;the expression of hsp70 was significantly increased during 24-48 h of stress and re-immersion.The acid phosphatase(ACP)activities in hemolymph were significantly increased during 24-48 h of the stress and at 12 h of re-immersion;the activities of aspartic aminotransferase(AST)and alanine aminotransferase(ALT)were significantly increased during the whole experiment;the gene expression of pro PO or TLR was significantly increased during 12-48 h of the stress.Severe histopathological changes(lumen expansion,epithelial vacuolization and cell number reduction)were observed in hepatopancreas at 48 h of stress and at 12 h of re-immersion.The above results indicated that 48 h of low-temperature aerial exposure stress stimulated the non-specific immunity but adversely affected the antioxidation and hepatopancreatic histomorphology of P.clarkii,and 12 h of re-immersion was not sufficient for the crayfish to restore to normal states from the stress.2.Hyperthermy stress is a common environmental condition during the cultivation of P.clarkii.Based on our previous studies on the intestinal immune response of P.clarkii to short-term hyperthermy stress,this study studied the survival rate,hepatopancreas histology and intestinal microflora responses to explore the adaptation mechanism of P.clarkii to hyperthermy stress(35 ℃)in 25 d.The results indicated that under the hyperthermy stress the survival rate significantly reduced,the hepatopancreas tissue structure was severely damaged,leading to hepatocyte vacuolization and enlarged lumen,and which caused the disorder of intestinal flora.High-throughput 16 S ribosomal DNA(r DNA)sequencing revealed that hyperthermy stress significantly reduced intestinal microbial diversity and altered microbial composition.Specifically,Proteobacteria and Firmicutes were significantly decreased,Bacteroidota and Fusobacteriota were significantly increased.The abundance of the beneficial bacterium Citrobacter decreased while that of the pathogenic bacterium Shewanella increased.PICRUSt functional prediction analysis showed that continuous high temperature stress reduced the pathways related to Membrane Transport,Replication and Repair,Carbohydrate Metabolism and Amino Acid Metabolism.In conclusion,25 days of continuous hyperthermy stress(35 ℃)had adverse effects on P.clarkii.The result was reduced survival,severe structural damage to the hepatopancreas and disruption of the intestinal flora of P.clarkii.This study can provide theoretical basis for better understanding of P.clarkii healthy culture in high temperature seasons.3.In Chapter 3,it was found that sustained hyperthermy stress will have adverse effects on P.clarkii.Based on this,in order to explore the tolerance of P.Clarkii to high temperature stress and whether the body can recover when it recovers to the appropriate temperature after stress,we designed this experiment that after different high temperature stress(32 ℃-6 d,32 ℃-12 d and 32 ℃-18 d)the normal temperature(26 ℃)was restored,and we tested the immune index,hepatopancreas tissue structure and intestinal flora of crayfish [body weight(7.19 ± 0.29)g].The results showed that sustained high temperature stress had a direct negative effect.After 6 days of high temperature stress,P.clarkii could not return to normal state.However,the results of digestive enzymes(Trypsin;Amylase AMS;Lipase LPS),hepatopancreas histological structure and immune indices(malondialdehyde,MDA;Total superoxide dismutase,T-SOD;Catalase,CAT;Glutathione peroxidase,GSH-Px;Acid phosphatase,ACP;Alkaline phosphatase,AKP;Lysozyme,LZM)could prove that the organism can return to normal after 18 d of high temperature stress and about 18 d of temperature recovery.High-throughput 16 S sequencing of intestinal microorganisms showed that Sobs and Shannon index increased after the recovery of sustained high temperature stress,while Simpson index decreased,indicating that intestinal microbial α diversity increased in P.clarkii.At the phylum level,Proteobacteria,Firmicutes,Bacteroidota and Actinobacteria were predominant.At the genus level,as the duration of sustained high temperature stress was prolonged,after the water temperature was restored,the abundance of Bacteroides in the intestinal microbial community of P.clarkii increased,while that of Citrobacter decreased.KEGG function prediction was mainly concentrated in Metabolism,Environmental information processing and Genetic information processing pathways.We conclude that P.clarkii can recover by establishing an adaptive mechanism after being exposed to a long period of stable high temperature for 18 days,and the physiological response was reversible.