Characterization And Function Analysis Of The CHH-? Genes In Tiger Shrimp (Penaeus Monodon)

Penaeus monodon,is an important aquaculture animal belonging to Penaeus,Penaeidae,Decapoda,Crustacea,Arthropoda.It is also the subject in our laboratory.Based on a thorough understanding of the achievements and progress in the field for some time in the past,we used three genes selected from the transcriptomics bank of P.monodon.A series of research methods including bioinformatics analysis,real-time fluorescence quantification,RNA interference,fluorescence in situ hybridization were used in this study for the identification of CHH-? type and their function analysis."Multifunction" and tissue-specific reported recently were focused on,and the relationship between CHH-? gene sequence and their function such as blood glucose regulation,gonadal regulation,and metamorphosis development was also our target.It was showed that PmHHLPP plays an important role in the regulation of blood glucose and the development of metamorphosis,probably involved in energy regulation.Its injection of dsRNA significantly increases blood glucose levels in shrimp.PmCHH had been proven its functions in ovarian development and larval metamorphosis,but no blood glucose regulation activity appeared.A significant tissue-specificity was observed in PmHHLP,and it was found in the spermatophore sacs and vas deferens of P.monodon,playing an important role in the formation of spermatophore sacs wall.The functions of the three CHH-? genes covered the functions of CHH-?I gene like gonad regulation,larval development,showing a clear multifunctional tendency.Details are shown as follow:PmHHLPP had not only the basic characteristics of the CHH-? gene,but also showed similarities to other CHH genes in its genome structure and amino acid sequence structure,including several amino acid residue sites and conservation that might be related to blood glucose regulatory activity.PmHHLPP was expressed in all tissues and had relatively stable expression levels in the nervous system,intestine,stomach and other tissues,considering a certain fluctuation observed in different developmental stages.PmHHLPP was also believed participating in the process of larval metamorphosis,since its expression level significantly increased during the important stage of metamorphosis of larval molluscs,such as the stages III and IV of nauplius,the transitional stages between nauplius and zoea,and the stages of mysis,which were relative with energy metabolism.Further glycemic control studies had shown that loss of PmHHLPP resulting in an increase in blood glucose levels in shrimp,which was more than 30%.When dopamine injections were added after 24 hours,blood glucose levels would remain a high level for 12 hours.In conclusion,PmHHLPP was presumed a CHH-? gene involved in the regulation of blood glucose.Although PmCHH exhibited similar features to multiple CHH genes in genome structure and amino acid sequence,the gene is also present in lots of sites,such as W⁷⁰,N⁷⁴,N⁷⁵,Q⁸¹,I⁸³,I⁹⁰and K¹¹²,together with another two tissue-specific CHH-?I genes from Rimicaris exoculate and L.vannamei,showed different sequence characteristics from other CHH genes,and these three genes were clustered independently.In terms of expression levels,there was no significant difference in the expression of PmCHH in other tissues except for gill and gut,including the eyestalk ganglions,hepatopancreas,epidermis,and muscle.The expression levels of PmCHH in the eyestalk ganglions,muscles,and epidermis of hepatopancreas were significantly decreased after the unilateral ocular stalk cutting off.During the larval metamorphosis,PmCHH showed a distinctly different expression trend from that of PmHHLPP.Besides,the expression level of PmCHH increased significantly in the phase III of the ovarian development,and maintained a high level,indicating its role in the development of oocytes.Studies of blood glucose regulation reflect the weak correlation between PmCHH and blood glucose regulatory activity:gene silencing did not result in significant changes in blood glucose concentrations in P.monodon.It demonstrated that PmCHH acts as a CHH-? gene in the process of multifunctional potential.PmHHLP has the basic structural features of the CHH-? gene,including the "signal peptide-CPRP-mature peptide" structural model and six highly conserved cysteine residues in the mature peptide.However,the similarity of the two tissue-specific HHLP genes in Fenneropenaeus chinensis was much higher,reaching 67%and 63%,and their clustering results were independent of one another.Subsequent functional expression also confirmed this similarity.PmHHLP showed obvious tissue-specificity:it did not expressed in tissues like eyestalk ganglions,hepatopancreas,and muscles,but only specifically expressed in spermatophore sacs and vas deferens,and only a small amount existed in the testis and ovary.The FISH results showed its specific expression sites:the epithelium of spermatophore sacs and vas deferens,and PmHHLP was detected in the inner columnar epithelial cells in the early stage of formation.The regeneration experiments also showed an important relationship with spermatophore sacs:as the expression level of spermatophore sacs grows significantly,there was also a phenomenon of late expression in the vas deferens formed in the spermatophore sacs;it is weakly related to the increase in the number of sperms.In the gene silencing experiment of PmHHLP,it was confirmed that in the case of maintaining the silencing efficiency of more than 80%of the gene,the weight gain of the spermatophore sacs' wall,but the number of sperm was only significantly different on the 15 th day,which indicated that the PmHHLP played an important role of the formation of the spermatophore sacs,and that Pm HHLP and the two other specific genes in F.chinensis,is part of a new branch of CHH-?.In conclusion,the three CHH-? genes exhibit difference in function,including their original function of blood glucose-regulating activities,but some various processes such as regulation of spermatophore sacs in P.monodon,indicating the phenomenon of“multifunction”of crustacean hyperglycemic hormones in P.monodon.It is also an important manifestation of gene structure and function diversification,which further enriches our understanding and improvement of the CHH family.The research on the regulation network of peptide hormones provides a basis for further understanding of the formation and regulation of the male reproductive system of P.monodon.