| As a marine arthropod,molting is an important growth and development process for Tachypleus tridentatus.If the molting fails,the mortality rate will increase considerably.So far,however,studies on physiology,biochemistry and gene expression related to molting of juvenile horseshoe crabs are not sufficient.Moreover,the impacts of future marine environmental changes on molting and physiology are lacking,which need to be clarified for better protecting horseshoe crabs.In this paper,the molting mechanism of juvenile horseshoe crab and physiological response to ocean acidification stress were studied by using ELISA,transcriptome,and quantitative real-time PCR（qRT-PCR）technologies.The main results are as follows:1.To explore the physiological changes during the molt cycle of juvenile horseshoe crabs,chitinase andβ-NAGase activity were measured through the absorption spectrophotometry,and ecdysone content and Ec R gene expression changes were detected by ELISA and qRT-PCR technologies,respectively.The results showed that from the intermolt to the postmolt,chitinase,β-NAGase activity and ecdysone content increased firstly and then decreased,and reached the maximum value in the premolt,which were 7.26 U/g,502 U/gprog,77.9 ug/m L.The highest value of Ec R gene expression level was observed in the intermolt,and no significant difference was found among premolt,ecdysis and postmolt.Through correlation analysis,it was found that chitinase andβ-NAGase were positively correlated with ecdysone,whileβ-NAGase was negatively correlated with Ec R gene expression.These results suggested that the molting of juvenile horseshoe crabs was directly regulated by ecdysone,and chitinase andβ-NAGase are the key enzymes during the molt cycle,which play a synergistic role with ecdysone.However,the specific regulation mechanism of Ec R gene during the molt cycle needs further study.2.Based on the Illumina HiSeqTM 2500 sequencing platform,transcriptome sequencing was performed at different molting stages of juvenile horseshoe crabs for the first time.A total of 6482 differentially expressed genes（DEGs）were found at four different molting stages.There are 5763 DEGs（3588 up,2175 down）in the Pr-vs-In stage,344 DEGs（153 up,191 down）in the Ec-vs-Pr stage,and 375 DEGs（100 up,275down）in the Po-vs-Ec stage.In addition,59 shared DEGs were identified in four consecutive molting stages,among which the highly expressed DEGs were mainly cuticle proteins,crustacean hyperglycemic hormone and glycine-rich cell wall structural protein,which involve in chitin binding,structural constituent of cuticle and neuropeptide signaling pathway GO terms.KEGG pathways showed that in the Pr-vs-In stage,the signal transduction and endocrine system were significantly enriched,such as insect hormone biosynthesis,protein digestion and absorption,and PPAR signaling pathways.In the Ec-vs-Pr stage,ether lipid metabolism,peroxisome,and apoptosis were significantly enriched.In the Po-vs-Ec stage,the DEGs involved in transport and catabolism were the most,and the phagosome,lysosome,and platelet activation pathways were significantly enriched.By comparing the molting-related genes of juvenile horseshoe crabs at different molting stages,it was found that the cytochrome P450 family genes,Ec R,RXR,E74,CHH and chitinase genes are important for the molting activities.These results suggest that the key biological processes and signaling pathways involved in hormone regulation,cuticle structural proteins,catabolism,apoptosis,and innate immune response are important regulation mechanisms in the molting process of horseshoe crabs.3.In this study,the 1stinstar horseshoe crabs were exposed to acidified seawater（pH 7.3,pH 8.1 as control）for 28 days to determine the effects of ocean acidification（OA）on their growth,molting,oxidative stress and gut microbiota.Results showed that there were no significant differences in growth index and molting rate between OA group and control group,but the chitinase activity,β-NAGase activity,and ecdysone content in OA group were significantly lower than those of the control group at the 7thday.Compared to the control group,ROS and MDA contents in OA group were significantly increased at the end of the experiment.SOD,CAT,and AKP activities increased first and then decreased,GPX decreased first and then increased,and GST activity changed little during the experiment.According to the result of 16S r RNA sequencing of gut microbiota,microbial-mediated functions predicted by PICRUSt showed that“Hematopoietic cell lineage”,“Endocytosis”,“Staphylococcus aureus infection”,and“Shigellosis”pathways significantly increased in OA group.The above results indicate that OA had no significant effect on growth index and molting rate,but interfered with the activity of chitinolytic enzymes and ecdysone expression of juvenile horseshoe crabs,and caused oxidative stress.In addition,OA had adverse effects on the immune defense function and intestinal health. |
