The Tolerance And Physiological Response Of Procambarus Clarkii To High-temperature Stress

Procambarus clarkii,commonly known as crayfish,is the largest economic freshwater crayfish in China.It is favored by the aquaculture market and consumers because of its wide adaptability,strong fecundity,easy feeding,delicious taste and high protein content.In recent years,with the increasing demand for P.clarkii in the domestic consumer market,its breeding area and output are also expanding rapidly.The high temperature in summer is one of the key factors restricting the development of aquaculture in China.The rise of global temperature and high-density aquaculture model in the future are also a major threat to aggravate the high-temperature stress of P.clarkii.Therefore,the research on the tolerance and stress response of P.clarkii to high temperature stress can provide the necessary theoretical basis for improving the culture technology of P.clarkii,promoting the healthy development of P.clarkii culture industry and coping with the frequent occurrence of extreme high temperature in summer.In this study,the effects of long-term and short-term high temperature stress on the survival,growth,antioxidant defense system,immunity,HSP70 gene expression in different target organs and intestinal flora response of P.clarkii were studied.The main research contents and results are as follows:1.Effects of continuous high temperature stress on growth,digestive enzyme activity and immune indexes of P.clarkiiIn order to explore the effects of continuous high temperature stress on the growth and health of P.clarkii,the crayfish [body weight(7.19 ± 0.29)g] was cultured for 30 days at the optimum temperature(24 ℃,control group)and significantly deviated from the suitable growth range temperature(29 ℃,32 ℃ and 35 ℃,high temperature group).The results showed that the survival rate(SR)decreased significantly with the increase of stress temperature(P<0.05).The growth performance indexes such as final body weight(FBW),final body length(FBL),weight gain rate(WGR),specific growth rate(SGR),digestive enzyme activity,pepsin,amylase and lipase increased first and then decreased with the increase of temperature;All indexes reached the peak at 29℃ group and were significantly the highest(P<0.05);Compared with 24℃ group,35℃ group significantly inhibited its growth(P<0.05).The activities of immune related enzymes acid phosphatase(ACP)and alkaline phosphatase(AKP)decreased significantly with the increase of temperature(P<0.05).The activities of total antioxidant capacity(TAOC)and total superoxide dismutase(T-SOD)were significantly lowest at 29℃ group(P<0.05),while the activities of aspartate aminotransferase(AST),alanine aminotransferase(ALT)and the content of malondialdehyde(MDA)increased significantly with the increase of temperature(P<0.05);The relative expression of heat stress protein gene(HSP70)increased significantly at 29℃ group(P<0.05)and was the highest at 35℃ group(P<0.01).The results showed that the continuous high temperature in the 29℃ group could significantly promote the growth and digestion of P.clarkii,but had a significant adverse effect on its immune indexes,while the continuous high temperature stress in the 32℃ group and above seriously affected its survival,growth,digestion,immunity and antioxidant functions.2.Oxidative stress and immune response mechanism of P.clarkii under short-term non-hyperthermalP.clarkii is generally subjected to heat stress in rice-crayfish integrated systems.In order to explore the adaptability of the crayfish to non-hyperthermal stress in a certain period of time,a non-hyperthermal stress test was carried out on the crayfish[body weight(9.01 ± 0.31)g] at 7 time points before(24 ℃,control)and after(30 ℃,6,12,24,48,72 and 96 h)non-hyperthermal stress for 96 h.The results showed that the content of malondialdehyde(MDA)in hepatopancreas increased significantly from6 h to 72 h after thermal stress(P<0.05),the activities of total antioxidant capacity(TAOC)and total superoxide dismutase(T-SOD)changed significantly in the early stage after thermal stress,from 48 h to 96 h,it basically recovered to the pre stress level(P>0.05),and the activity of catalase(CAT)reached the peak at 12 h and decreased to the pre stress level at 96 h(P>0.05);The activities of alkaline phosphatase(AKP),acid phosphatase(ACP)and lysozyme(LZM)in hepatopancreas reached the peak at 12 h,and increased significantly from 48 h to 72h(P<0.05),while the relative expression of tumor necrosis factor alpha-inducing protein 8(TNFAIP8),tumor necrosis factor(TNF)and Toll gene reached the peak at 48 h,and increased significantly from 72 h to 96 h(P<0.05);The relative expression of heat shock protein 70 gene(HSP70)in target organs hepatopancreas,gill and intestine increased sharply and reached the peak at 6 h,and basically returned to the pre stress level at the later stage of stress(P>0.05).The results showed that the oxidative damage,antioxidant response and HSP70 gene expression of P.clarkii could be gradually restored under short-term non-hyperthermal;And maintaining a high level of immune enzyme activity and gene expression is conducive to adapt to short-term non-hyperthermal stress.3.Response mechanism of intestinal microbiota of P.clarkii under short-term nonhyperthermalThe 24℃ breeding water temperature was set as the control group(NG),after the water temperature was raised from 24°C to 30°C at a temperature of 1.0°C/h,96 h of high temperature stress was taken as the stress group(AG),the two groups of intestinal tissue samples were collected for intestinal microbiota study.High-throughput sequencing of 16 S ribosomal DNA revealed thermal stress altered the diversity,and composition of intestinal microbiota.Specifically,abundance of beneficial bacteria Bacteroides and Anaerorhabdus decreased,while abundance of pathogenic bacteria Proteobacteriar increased.In addition,according to the predicted results of Meta Cyc metabolic pathway,the relative abundance of many metabolic pathways,such as Biosynthesis,Degradation/Utilization/Assimilation,Detoxification,Generation of Precursor Metabolite and Energy,in the intestinal tract significantly increased after thermal stress(P<0.05).