Studies On The Attachment Relationship Between Embryo And Mother And Embryo Immunity Of Procambarus Clarkii

The crayfish Procambarus clarkii,belonging to Crustacea,Cabaridae,is commonly known as freshwater lobster and crayfish.It is the most widely distributed and most commonly cultivated freshwater crayfish in the world.It is delicious and nutritious,and is well received by domestic and foreign markets.In recent years,the wild resources of P.clarkii are far from being able to meet the market demand.The rapid increase in people’s consumption demand for P.clarkii has greatly promoted the development of its aquaculture industry.Artificial breeding and seedlings of P.clarkii is gradually being valued by people.Due to the biological characteristics of P.clarkii with few eggs,low hatching rate and unsynchronized hatching time,it limits the factory production of shrimp larvae and restricts the development of large-scale breeding of P.clarkii.Therefore,it is extremely important to grasp the embryonic development process of P.clarkii,understand the conditions required for its embryonic development,analyze how to improve the hatching rate and survival rate,and conduct targeted research on the immune ability and attachment relationship during the embryonic development of P.clarkii.In this study,the healthy,non-injured and basically the same size P.clarkii with egg and the female shrimp without egg were selected from the lobster breeding base of the Lobster Industry Group in Xuyi County,Jiangsu Province as experimental animals.The mother shrimp that had not laid eggs were observed continuously,and the embryos were collected every other day from the time when the mother began to lay eggs to the time when the seedlings were separated from the mother.The embryos were collected for the study of the immune ability of P.clarkii during the embryonic development and the samples were taken by scanning electron microscopy.The hatching fluid secreted during egg laying was collected and analyzed by mass spectrometry.To investigate the effects of different temperatures on the activities of antioxidant enzymes during in vitro incubation of embryos of P.clarkii.In addition,the effects of high p H and salinity stress on the surface morphology of embryos were also observed.The results of this study are as follows:1.Observation on the surface morphology of egg membrane of P.clarkiiThe mature eggs,naturally fertilized eggs attached to the ventral limbs and fertilized eggs under environmental stress of P.clarkii were observed by optical microscopy and scanning electron microscopy.The mature egg only has the primary egg membrane,which is covered with micropores and particles,which is fragile and easily damaged.The fertilized egg is smooth and not easily damaged,and the interior is dense and elastic.The surface morphology of the secondary egg membrane of the fertilized eggs under high p H and high salinity stress changed.The secondary egg membrane plays an important role in embryo development.2.SDS-PAGE gel electrophoresis and mass spectrometry analysis of the hatching solution of P.clarkiiThe hatching fluid secreted by the mucous glands of P.clarkii plays an important role in the process of embryo development.In the early stage of embryo development,the hatching fluid plays an important role in the successful attachment of the embryo,the formation of the secondary egg membrane and the isolation from the damage of the external environment.In this study,the enzymatic hydrolysates of proteins in the hatching solution of P.clarkii were identified by SDS-PAGE and mass spectrometry analysis.Combined with database search,a total of 30 proteins were identified.The subcellular localization and COG functional annotation of the identified proteins were summarized by protein retrieval.3.Changes in the activities of antioxidant enzymes and Na~+K~+-ATPase at different developmental stages of P.clarkii embryos and the effects of temperature on themIn order to investigate the non-specific immune mechanism and osmotic pressure regulation ability of P.clarkii from embryo fertilized egg to seedling stage,the levels of superoxide dismutase(SOD),catalase(CAT),malondialdehyde(MDA)and Na~+K~+-ATPase(NKA)in P.clarkii from embryo fertilized egg to seedling stage were measured by biochemical methods.The results showed that SOD always played an antioxidant role during the embryonic development of P.clarkii,and CAT only played an antioxidant role before and after membrane rupture of P.clarkii embryos.The change of MDA content during the embryonic development of P.clarkii was similar to the change of SOD activity,which continued to increase with the development of embryos.During the whole process from embryo fertilization to membrane breaking,the activity of NKA enzyme was very low and had no significant change.However,after embryo membrane breaking,from the seedling stageⅠto the seedling stage,P.clarkii was in direct contact with the external environment.In order to adjust the osmotic pressure balance in vivo to adapt to the external environment and resist stress,the activity of NKA enzyme increased significantly.In the study of the effect of temperature on the activity of antioxidant enzymes during in vitro incubation of P.clarkii embryos,it was found that temperature had different effects on embryo development in different stages of P.clarkii embryo development.During in vitro incubation of P.clarkii embryos,the temperature set in this study only had a significant effect on CAT activity of P.clarkii embryos atⅠand sub-seedling stages,but had little effect on MDA content.In conclusion,the hatching fluid of P.clarkii plays an important role in the attachment relationship of embryos.The secondary egg membrane is in direct contact with the external environment,and a large number of bacteria will be attached to the surface.At the same time,more damage will occur in the later stage,so that the embryo can break the membrane and emerge.In the embryonic development process of P.clarkii,the early stage mainly relies on the maternal SOD to play a role,and the immune ability is weak.In the later stage,the immunorelated genes of the embryo begin to express,and the immune ability is significantly improved.Among them,the progut stage and the seedlingⅠstage are the key period for the embryo hatching of P.clarkii.In this period,we must strengthen management to avoid the interference of various factors on the embryo.At the same time,we must maintain appropriate water quality conditions to maintain the normal development process of the embryo,and the environmental conditions should not mutate.Temperature had little effect on the activity of antioxidant enzymes during in vitro incubation of P.clarkii embryos.This study provides basic information for further revealing the embryo incubation mechanism of P.clarkii,and also has important reference value for further research on artificial insemination and artificial incubation of this animal in vitro.