The Role And Expression Regulatory Mechanism Of Ecdysis Triggering Hormone Signal Pathway In Molt Of Mud Crab Scylla Paramamosain

Currently,studies in the regulation of crustacean molting were mostly focused onupstream neuropeptides such as MIH and the molecular mechanisms involved in 20E synthesis.However,the molecular mechanism of 20E signal-regulating a series of downstream physiological processes such as cortical dissolution,new epidermal formation,appendage shaking,and cuticle coloration through its receptor Ec R is still unclear.As an important economic species,it is of great significance in economic and ecological value to study the regulation mechanism of ecdysis downstream of 20E in the crab Scylla paramamosain.In this study,several key molecules involved in the signal transduction of"20E-Ec R～ETH～molting"of mud crab were investigated,and the key role of ETH in the regulation of molting in mud crab was investigated.Ec R-2and ATF-4 were identified as ETH transcriptional activators,and ETH regulated the molting of mud crab through its receptor ETHR.These results provide a new approach for the prevention and treatment of ecdysplasia in crustaceans such as mud crab and the development of specific insecticides.The main research contents of this paper are as follows:1.ETH and its related neuropeptides co-regulate the molting of Scylla paramamosainRNA interference,q PCR,immunohistochemistry,and other techniques were used for the research of ETH,EH,and CCAP.The results showed that ETH,EH,and CCAP were highly expressed in the thoracic ganglia,and the expression levels were interrupted with periodic ecdysis.RNAi showed that knockdown of these three genes could inhibit ecdysis significantly,and the ecdysis failures can be rescued in neuropeptide compensation experiments.q PCR and immunohistochemistry results showed that ETH and EH had different regulatory relationships in early and late premolt D stages.The results showed that ETH was an important neuropeptide,and it also work closely with EH and CCAP in the normal ecdysis of mud crab.The result has laid a foundation for further development of the relationship between 20E signal and ETH.2.Coactivators ATF-4 and Ec R-2 activate ETH transcription and regulate molting of mud crabThrough molecular cloning and bioinformatics analysis,the results showed that different regions of the three Ec R isomers in mud crab were in the D-hinge region and the E ligand-binding domain.ATF-4 had a typical leucine zipper structure,and 31potential transcription factor binding sites were predicted in the ETH promoter region.The potential ATF-4 binding sites were located upstream of ATG（-470～-480bp）,and the transcription levels of the above genes were distributed periodically.In vivo experiment results showed that the expressions of ETH,EH,Ec R-2,Ec R-3,and ATF-4in the thoracic ganglia were significantly regulated by 20E.Ec R-1,Ec R-2,and ATF-4had different phenotypic characteristics on ecdysis inhibition,and Ec R-2 and ATF-4had significant regulatory effects on ETH transcription and expression level.The interaction between Ec R-2 and ATF-4 and between Ec R-2 and ATF-4 and between ATF-4 and pro ETH was obvious.These results indicate that ATF-4 and Ec R-2 are the key factors in the regulation of the molting of mud crab.As important transcription factors,ATF-4 and Ec R-2 activate the transcription of ETH through 20E titer regulation and then regulate the occurrence of molting of mud crab3.The ETH receptor was identified as ETHR,which plays an important role in the molting of mud crabThrough molecular cloning and bioinformatics analysis,the results showed that ETHR obtained by transcriptome screening had a typical seven-fold transmembrane structure of GPCR.Rose TTAFold modeling,molecular blind docking prediction and molecular dynamics simulation predicted that the ETHR binding with ETH mature peptide had good stability,and ligand binding sites would have a good stability.The strong interaction between the two proteins was further confirmed by yeast two hybridization.The expression distribution characteristics showed that ETHR was highly expressed in thoracic ganglia in the early premolt stage D,showing significant periodic expression characteristics.In vivo experiment results showed that the expression of ETHR and downstream gene CCAP were significantly regulated by 20E.ETH,ETHR,and CCAP were also significantly regulated by each other,and ETH and ETHR were very important to regulating normal molting and hemolymph apoptosis of mud crab.These results indicate that ETH and its receptor ETHR were important in molt regulation and participate in hemolymph apoptosis and transcriptional regulation of downstream gene CCAP.4.Transcriptomic analysis of ETH gene by RNA interference in D₀ and D₂ was performedComparative transcriptome analysis result showed that the transcription levels of RNA polymerase complex and other biological macromolecules in D₀ were significantly higher than those in D₂.In D₀,ds ETH resulted in significant expression differences in genes related to carbohydrate metabolism,folic acid synthesis,and chitin binding pathway.The candidate genes include riosephosphate isomerase gene,methylenetetrahydrofolate reductase gene,formimidoyltransferase-cyclodeaminase gene,peritrophin gene,and etc.At D₂,ds ETH resulted in significant expression differences in the genes related to calcium channel inhibitor activity,fat digestion,and absorption,and myocardial contraction pathway.Candidate genes included fibrocystic genes,secretory phospholipase A2 genes,tropomyosin 1 genes,and etc.These results indicate that ETH has different physiological regulation pathways in D₀ and D₂.