| The blood clam Tegillarca granosa is one of the most important mudflat shellfish in China,which belongs to the phylum Mollusca,Lamellibranchia,Taxodonta,Arcidae,and Tegillarca.It has long been loved by the residents of coastal areas.However,in recent years,outbreaks of vibriosis and large-scale mortality have often occurred during the culture proces,which has hindered the sustainable development of the clam culture industry.This is certainly related to factors such as deterioration of culture environment,increase of culture density and poor culture management,but the lack of resistant good varieties is an important reason for the above problems.Therefore,it is of great economic value to breed new strains of resistant clam by means of genetic selection and breeding.However,at present,breeding for disease resistance in aquatic animals is progressing relatively slowly compared with other selective breeding for growth(length,weight)traits,for example,and an important reason for this situation is the lack of clarity in the definition of disease resistance traits and the difficulty in their determination and evaluation.Therefore,it is urgent to mine phenotypic and genetic information at multiple levels and to construct a database integrating phenotypic,genotypic and functional markers of germplasm.In this study,we firstly analyzed the toxicity effect of Vibrio harveyi on clams by both immersion infection and injection stress.Secondly,we analyzed the changes of Vibrio load in tissues of clams under Vibrio infection.Finally,we determined the resistance-related indexes of clams based on different resistant phenotypic differences of clam families and populations.The results of the study will provide theoretical basis and data support for the analysis of disease resistance mechanism and breeding for disease resistance in clams.The main results of this study are as follows.1.The standard curve of V.harveyi concentration was established by turbidimetric method.The acute toxicity test was conducted by injection and immersion under different concentration of V.harveyi to the clam.The death of the clam was observed,and then the median lethal concentration(or dose)of V.harveyi was calculated after infection.The results of immersion infection experiment showed that the clam began death time and the maximum daily mortality time were both advanced with the increase of Vibrio in the immersed sea water,and then the death status of clam increased rapidly firstly,the cumulative mortality of clam was close to 100%after 15 days when the concentration of Vibrio reached 2×10~7 CFU/m L in sea water.The results of injection stress experiment showed that the clam mortality was positively correlated with Vibrio injection concentration(r=0.964,P<0.01).The probit analysis showed that the immersion infection experiment was stopped 7 days and the clam LC50 at 15 days was 1.35×10~7CFU/m L.The96h lethal dose(96h LD50)under Vibrio injection was 2.12×10~7 CFU/g,The results suggested that the V.harveyi was obviously toxic to T.granosa.Our results provide data referenced for the tolerance of T.granosa to V.harveyi and the breed of clam environmental requirements,and then provide a theoretical basis for the prevention and treatment of T.granosa vibriosis.2.In this study,V.harveyi immersion experiment was conducted,from which the dynamic relationship between the bacterial growth and reproduction in seawater under Vibrio challenge in 24h was described,the bacterial load of the blood clam tissues was calculated,and the relationship between the Vibrio load in hepatopancreas and the immersion different concentrations(1×10~5,1×10~6,1×10~7,5×10~7 CFU/m L)of Vibrio in seawater was specified.The results showed that the Vibrio load in the clam blood was significantly higher than in other tissues under the concentration of 1×10~7 CFU/m L.The bacterial load in the tissues was increased at first,then decreased,and finally stayed at a low level.The Vibrio load in the blood was decreased to 10~2-10~4 CFU/m L,and the Vibrio load in other tissues was decreased to 10~0-10~2 CFU/mg.Under different Vibrio concentrations,the Vibrio load in the hepatopancreas increased significantly on Day 1,which was 81.0-667.8 times of that of the control.Correlation between the bacterial load and the Vibrio dose in the seawater was significantly positive(Spearman’sρ=0.762,P<0.001).The bacterial load in the hepatopancreas was obviously decreased on day 5,which was still higher than that of the control.This study provided a reference for the immune recognition and response during pathogenesis by Vibrio infection in T.granosa.3.In this study,V.harveyi was used to infect clams by immersion,and indicators significantly associated with resistance phenotypic traits were screened by comparing the differences in immune-related indicators between four resistant and four susceptible families;then the resistant and control populations were used to test the validity of the screened resistance indicators.Vibrio load,physiological metabolism,hemocytes function,physiological and biochemical indexes and gene expression data were measured by different resistant phenotypes of clams under Vibrio infection.The results of independent-sample t-test analysis showed that 31 physiological and biochemical indices,34 genes in the hepatopancreas and 33 genes in the gill were screened for differences among different resistant phenotypes of clams(P<0.05);then the total number of hemocytes,GPx activity,TG content,Glu content in the hepatopancreas and MT,RIG-I,TLR6,DRD3 in the hepatopancreas were verified by the resistant and control populations,a total of eight indicators,can be used as resistance candidates for V.harveyi infection in clams.The results of the study provide a reference for indirect selection and breeding for high resistance of V.harveyi in T.granosa. |
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