| The coelomocytes are the main effectors of immune response and play key roles in resisting foreign body invasion in Apostichopus japonicus.It has a special defense mechanism of evisceration and regeneration in A.japonicus.Although the evisceration does not directly cause the death,it will affect the growth rate of the sea cucumber.At the same time,as a result of the discharge of a large amount of coelomic fluid and coelomocytes,the sea cucumber temporarily loses the main immune defense barrier and is more susceptible to infection by pathogens.At present,more attentions have been paid to the regeneration process and mechanism of evisceration such as digestive tract,while less attention has been paid to the regeneration of coelomocytes,especially the mechanism of immune response and the reconstruction of immune defense barrier after evisceration in sea cucumber.In this paper,the position of "hematopoietic tissue" and the source of coelomocytes of A.japonicus has been characterized by using histological observation,monoclonal antibody tracing,hematopoietic transcription factor detection and EdU proliferation assay.The resumption regulations and immune response of coelomocytes after evisceration were also researched by using cell count,immune enzyme detection and fluorescence quantitative PCR.These results were helpful to analyze the mechanism of coelomocytes and the evolution of the immune system of the organism,and laid a foundation for further research on the reconstitution mechanism of the immune defense barrier after evisceration.Localization and characterization of hematopoietic tissues(HPTs)in sea cucumber A.japonicus.In the present study,histological features of the rete mirabile,the respiratory tree,the polian vesicle and the coelomic epithelium were characterized by using tissue section techniques.Two homologs of GATA1 were detected with molecular weight of 43 kDa and 90 kDa in coelomocytes,rete mirabile,respiratory tree and polian vesicle by using GATA1 antibody of human hematopoietic transcription factor through western-blot.The large GATA1-positive were detected in coelomocytes,whereas no signals were detected in the coelomic epithelium by using immunofluorescence technique.Immunohistochemistry analysis by mAb 3F6 against A.japonicus coelomocytes showed that positive signals were observed in the tube wall of the rete mirabile,respiratory tree,cyst wall of the polian vesicle and in the coelomocyte antrum of coelomic epithelium.The numerous EdU-positive positive signals were observed in the tube wall of the rete mirabile,respiratory tree,cyst wall of the polian vesicle using EdU detection technique.These results suggest that the rete mirabile,respiratory tree and polian vesicle were the HPTs of A.japonicus.Resumption regulations of coelomocytes after evisceration in the sea cucumber A.japonicus.The coelomocytes and coelomic fluid were nearly exhausted after evisceration.With the regeneration of internal organs,the coelomocytes and coelomic fluid would gradually recover.In this experiment,the test results showed that coelomic fluid volume was restored to the pre-evisceration level at 2 hours post-evisceration(2 hpe).Total coelomocyte count reached a peak at 6 hpe,followed decreased and then increased with a slight fluctuation,restored to the pre-evisceration level at 35 days post-evisceration(35 dpe).The change in different subtypes of coelomocytes was consistent with that of total coelomocytes.However,there were some variations in the regeneration of coelomocyte subtypes.At the end of the study,only the counts of amoebocytes and morula cells recovered to the pre-evisceration level.DNA replication assay showed EdU-positive cells accounted for 9.5% of the total coelomocytes counts at pre-evisceration and accounted for 4.7% at 6 hpe.However,the percentage of EdU-positive cells significantly increased,reaching 18.6% at 3 dpe,then declined.Therefore,we analyzed the observed increase in coelomocytes at 6 hpe,which may be due to coelomocytes migration from the water-vascular system into the coelom.Immune responses post-evisceration in the sea cucumber A.japonicus.Previous studies in our laboratory have suggested that although the cell types were same,the total cell number and percentages of each type were different.Furthermore,the polian vesicle was reserved when the sea cucumber was eviscerated.In this study,the differences of immune function were first compared between the coelomic fluid and coelomocytes in coelom and polian vesicle from the normal sea cucumber,the results suggested that there were significant differences on immune enzymes and the expression levels of immune genes.Therefore,this study focuses on the coelom and polian vesicle to further study the early immune response of the sea cucumber after evisceration.The results showed that the immunoenzymes activities of ACP,AKP,CAT and SOD were decreased first and then recovered gradually in the coelomic fluid supernatants both from the coelom and polian vesicle within 7 days post evisceration.The activities of ACP and AKP in coelomic fluid supernatant from coelom was restored to the pre-evisceration level within 7 dpe.However,the activities of CAT and SOD didn't recover to the pre-evisceration levels at 7 dpe.The ACP and AKP activities in coelomic fluid supernatant from polian vesicle showed a trend of decreasing first,then rising and then falling,except a transitory peak at 36 hpe.However,the activities of the two enzymes didn't recover to the pre-evisceration level at 7 dpe.The activities of CAT and SOD rose first,then decreased,and finally recovered to the pre-evisceration level at 7 dpe.After evisceration,the expression of the six genes in the coelomocytes from coelom were maintained at low levels.The genes expression reached a peak at 3 dpe except the Aj-iLys.Especially,the expression levels of AjToll and AjTLR3 at 3 dpe up-regulated to 1.8-fold and 14.0-fold compared to the pre-evisceration.In the polian vesicle,the expression levels of AjAP,AjMKK3/6,AjToll and AjTLR3 decreased first,then rose,and reached the peak at 7 dpe.However,no significant difference in AjC3-2 and Aj-iLys was observed within 5 days post evisceration.But the expression levels reached a peak at 7 dpe,with 15.0-fold and 4.45-fold compared to the pre-evisceration level,respectively.These results suggested that: the rete mirabile,respiratory tree and polian vesicle were the HPTs of A.japonicus.Total coelomocyte count increased sharply at 6 hpe,and then increased with a slight fluctuation,restored to the pre-evisceration level at 35 dpe.There existed difference of non-specific immune factors in coelomic fluid from the polian vesicle and coelom of healthy A.japonicus,and the immune response to the evisceration were different,except a transitory peak in the polian vesicle.Besides,the expression peak of immune-related genes in the coelomocytes from the coelom occurred earlier than that from the polian vesicle. |
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