Effects Of Transport Stress On Survival Rate And Physiological Indicators Of Procambarus Clarkii

Procambarus clarkii is an important aquatic product of aquaculture in China.The scale of crayfish farming increases with the increase in the demand for its aquatic products,while the survival rate of crayfish is often too low for further culture after long-term transport,especially in summer.In the process of aquatic products transport,there are many factors that affected by the external environment.These environmental stress factors can cause different degrees of damage to the body.The deepen of the degree of stress will consume too much energy of aquatic animals,which will lead to suppression of its immune response.As different individuals have different stress intensity endurance,some individuals with weak constitutions cause metabolic imbalance,physiological state disorder,and eventually death when the stress intensity exceeds the regulated threshold of the body itself.In addition,one（or some）opportunistic pathogen（s）may also be related to its death after transport.This study explored the reasons for the low survival rate of P.clarkii in aquaculture after transport by detecting immune stress response and pathogens carried before and after transport,and the survival rate after transport.It is expected to provide suggestions and a basis for improving the death phenomenon of P.clarkii caused by transport and a reference for the transport and breeding production of crayfish,and established a method for genotyping SNP loci associated with WSSV（white spot syndrome virus）resistant trait using high resolution melting analysis（HRM）.The main research contents and results are as follows:1.Effects of transport stress on the physiological of P.clarkii.The two transport methods commonly used in aquaculture production,including ice-cold and exposed-to-air transport,were used to transport for about 3 hours.The statistical results of the cumulative survival rate within two weeks after transport showed that the ice-cold transport group was 63.75%,and the exposed-to-air transport group was54.5%.In contrast,the survival rate after ice-cold transport was higher.Days 3-9 of culture were the peak mortality of P.clarkii after transport.The changes of serum glucose,cortisol content and HSP70（heat shock protein 70）gene expression levels in the heart of crayfish before and after transport stress detection results showed that transport stress caused the stress response of crayfish.The results of changes in superoxide dismutase（SOD）and catalase（CAT）activity in serum and hepatopancreas indicated the activation of the oxidation system occurred in response to transport stress.Acid phosphatase（ACP）and lysozyme（LZM）activities decreased within 1 day after transport stress,indicating that transport stress inhibited the immune response of crayfish,and the changes in prophenoloxidase（pro PO）and c-type lectin（ctl）gene expression were consistent with those of immune-related enzymes.A large number of crayfish deaths occurred on the 5^thand 7^thday after transport stress.The physiological status of the dying and vibrant crayfish on the 5th and 7th days after the transport stress was detected.The results showed that the serum cortisol and glucose activities of the dying crayfish were higher than the vibrant ones,indicating that the dying crayfish were in a state of oxidative stress,and the expression level of HSP70 in the heart was significantly lower than that of the vibrant crayfish.The expression of apoptosis gene caspase-3 in the heart of dying crayfish was significantly higher than that in vibrant.Hepatopancreas antioxidant enzyme activities（superoxide dismutase,catalase）,immune-related enzyme activities（ACP,LZM）and immune-related gene expression were significantly higher than those in vibrant crayfish.The histological examination results of hepatopancreas,intestines and gills showed that the hepatopancreas,intestines and gills of dying crayfish were damaged with varying degrees,among which the damage to the hepatopancreas and intestines was more serious.2.Transport stress and pathogen.The detection of pathogen including virus（decapod iridescend virus 1,DIV1;WSSV;infectious hypodermal and hematopoietic necrosis virus,IHHNV;and so on）,fungus（eg.crayfish plague）,parasite（eg.enterocytozoon hepatopenaei）was performed as well as the isolation and purification of bacteria of the hepatopancreas of crayfish before transport and the dying crayfish on the 5 and 7 days of culture after transport.The virus,fungus and parasite detection results showed only WSSV was carried.The detection results of WSSV copy number in the hepatopancreas of crayfish before and after transportation stress showed that the copy number of WSSV significantly increased on the 3rd to 7th day of culture compared with that before transport,which was the same time period as the death peak of crayfish,moreover,the WSSV copy number of dying crayfish was significantly high than that of vibrant on the 5 and 7 days of culture after transport,which all indicated that the death of P.clarkii was closely related to the change of WSSV copy number after transport stress.In addition to bacillus,the dominant bacteria isolated and purified from hepatopancreas was a strain with yellow bulge,irregular edge of the round shape.It was confirmed as Exiguobacterium profundum by colony morphology,physiological and biochemical identification and 16sr RNA sequencing analysis.The drug susceptibility of this strain was determined by disc diffusion method,and the results showed that it was sensitive to all 30 kinds of drugs tested.After the regression infection experiment,it was inferred that it may be involved in the pathogenic process of P.clarkii.3.Establishment of detecting genotypes of SNP site associated with WSSV-tolerant traitsBased on the early discovery of three SNP sites probably related to WSSV resistance in the intron of pro PO gene,the linkage disequilibrium and haplotype were analyzed for the SNPs,and it was found that there was a strong linkage disequilibrium relationship among them.Through the analysis on association between the haplotypes and genotype of each SNP site with the WSSV-resistant traits,the detection of the SNP＿7081 genotype was considered as the most convenient and efficient way for WSSV-resistant group selection.Furtherly,the high-resolution melting curve（HRM）,which is a rapid and economic genotyping method,was chosen to establish for SNP＿7081 site genotyping in this study.The 68 bp target fragment with 27.94%GC content was amplified and melting curve analysis were performed.However,the appearance of false negatives which led to unable automatically grouped although the melting curves of genotypes CC,C>T and T>C were obviously different,and could be treated as standard to manually genotype the samples with an accuracy rate of 97.61%.The low GC content which correlated with the Tm value,was confirmed as the reason for the false negatives by the assay about the recombinant plasmid p MD18-T-SNP＿7081 constructed with 45.24%GC content.Eventually,the adaptor primers were used to increase the GC content of the target fragment,and a modified HRM method for genotyping SNP＿7081 site that could group automatically was established,which provided a foundation for WSSV-tolerant selective breeding of P.clarkii.In conclusion,from the results of this study,it can be inferred that transport stress and changes of the culture environment after transport could trigger oxidative stress in crayfish,the production of reactive oxygen species（ROS）,and the antioxidant system was activated to scavenge ROS.After transport stress,the immune response was inhibited in the early stage of culture,and the immune-related gene expression level increased due to the increase in the copy number of WSSV in the later stage of culture.After transport stress,due to the differences among individuals,some crayfish could reach a balance through their own regulation,while some individuals with weaker constitution or more severely affected by stress exceed the threshold that could be adjusted by themselves,the production and elimination of ROS were unbalanced,which could damage DNA,proteins and tissue,activate proapoptotic genes,induce cell apoptosis,the concentration of WSSV was increased,and ultimately lead to the death.Furthermore,a modified HRM method for genotyping SNP＿7081 site which associated with WSSV-resistant traits was established to improve the problem of low survival rate of P.clarkii caused by transport stress through WSSV resistance breeding.