The Physiological Mechanisms Of Key Factors In IGF/ILP Signaling System In The Mud Crab,Scylla Paramamosain

Insulin-like growth factor(IGF)/insulin-like peptide(ILP)signaling system holds a central position in regulating growth and metabolism in vertebrates.In recent decades,ILPs have been also proved to exist extensively in invertebrates and play critical roles in the regulation of growth,metabolism,reproduction,molt and other behavioral and physiological processes.However,there is little information about the system,biological functions and signal transduction pathway of IGF/ILP in crustaceans.The mud crab Scylla paramamosain is a commercially important portunid species,commonly found in the coastal area of Southeast China.In this paper,we used the S.paramamosain as a model.With the application of molecular biological,cell biological and biochemical technique,a series of key factors involved in the IGF/ILP were cloned out,including the insulin-like androgenic gland factor(IAG),single IGF binding domain protein(SIBD),IGF binding protein-related protein 1(IGFBP-rP1),four neuroparsins(NP1?4),PI3K,Akt,Rheb and TOR.Their nucleotide and deduced amino acid sequences were analyzed and characterized.Further,we examined the spatial and temporal distribution of these genes and investigate their possible physiological mechanisms.The main research findings are as follows:1)The full-length 1269 bp IAG cDNA(termed Sp-IAG)was obtained from the androgenic gland(AG)of S.paramamosain using PCR with degenerate primers and RACE technique.Genomic DNA(gDNA)of Sp-IAG was also cloned.Four exons are separated by three introns.In situ hybridization indicated that the positive signals were localized to the AG cells(both type ? and type ? cells)when using an antisense probe.RT-PCR analysis showed that the Sp-IAG gene was extensively expressed,with its expression being detected in various tissues of male and female crabs,including the ovary.The qPCR was employed to determine the expression levels of ovarian Sp-IAG along the cycle of ovarian maturation.Data showed that Sp-IAG expression levels were maintained at low levels from undeveloped stage to late vitellogenic stage,and then increased significantly at mature stage.Further,the levels of Sp-IAG transcript were also detected at the level of individual crabs during the molting cycle of early juvenile mud crab for the first time.During the molting cycle of the first stage crab,Sp-IAG had a high expression at 0 h(postmolt),its expression then gradually decreased until 96 h and 120 h(premolt),before increasing significantly in the newly molted second stage crab(postmolt).The combined results suggested Sp-IAG might be involved in ovarian maturation and somatic growth in the mud crab.IAG has been considered as the sole ILP identified in crustaceans for a long time,which is produced by the AG in males and controls the sexual differentiation.The results here suggested that functions of IAG differ by species,and functions other than regulating sexual differentiation exist in crustaceans.2)A 143 bp cDNA fragment of SIBD(termed Sp-SIBD)was obtained from the transcriptome database of the brain dissected from female S.paramamosain at the early vitellogenic stage.The 1284 bp full-length Sp-SIBD cDNA was cloned using RACE.Tissue distribution analysis by qPCR revealed that Sp-SIBD was mainly expressed in the hepatopancreas and nervous tissues.In situ hybridization analysis further indicated that the positive signals were mainly localized to the secretory cells when using an antisense probe,which suggested that Sp-SIBD might be an endocrine factor released into the hemolymph.Subsequently,we examined the effects of various stresses,including hyperosmotic stress,hyperthermia,activated stress and fasting,on glucose levels in the hemolymph and Sp-SIBD expressions in the hepatopancreas.Interestingly,we found that Sp-SIBD expression was strongly upregulated in response to these catabolic circumstances.The combined results suggested,by analogy with the IGF binding protein 1(IGFBP1)from vertebrates,for the first time that SIBD might play a key physiological role by sequestering IGF/ILP to inhibit energy-expensive growth until conditions are more favorable.Given the previous findings of insulin-like peptides(ILPs)in invertebrates,we speculate that invertebrate ILPs and SIBDs promise to serve as a pair of counterparts of IGFs and IGFBPs from vertebrate species respectively.3)Based on the transcriptome database of the brain dissected from S.paramamosain females at early vitellogenic stage,we firstly reports the identification of three kinds of invertebrate IGF binding(IB)domain-containing protein genes from the mud crab,including the Sp-SIBD,IGFBP-rP1(termed Sp-IGFBP-rP1)and four NPs(termed Sp-NP1?4).Gene expression analysis showed that transcript distributions of six IB domain-containing genes were relatively concentrated in ovary and hepatopancreas.Also,expressions of Sp-Np1-4,Sp-SIBD and Sp-IGFBP-rPl showed diversity in the ovary and hepatopancreas along with vitellogenesis,suggesting that they might be closely involved in ovarian development,but with separate roles.Subsequently,in vitro explant culture system was used to study the effect of IGF-I and insulin on the gene expressions.Results showed that all these six genes were regulated in response to insulin/IGF-1 administration at certain concentrations.Taking all these findings together,we inferred that IB domain-containing proteins,including NPs,SIBDs and IGFBP-rPs,are likely to serve as endogenous ILP-specific binding proteins in invertebrates.4)The cDNA samples derived from the ovary and hepatopancreas of females at early vitellogenic stage were examined for vitellogenin(vtg)(termed Sp-vtg)expression using a qPCR.The results indicated that the hepatopancreas is the major site of Sp-vtg synthesis in S.paramamosain.The ovary,serving as a minor site,produces Sp-vtg less than 1%of that in hepatopancreas.Given this,the hepatopancreas was further used in in vitro culture system to examine the effect of exogenous insulin on Sp-vtg expression.The levels of Sp-vtg expression were increased with higher dosages of insulin at 200,400 and 800 ng/ml.By searching the transcriptome database of the brain from S.paramamosain,we identified and cloned the partical cDNA sequences of the ORF regions of PI3K(termed Sp-PI3K),Akt(termed Sp-Akt),Rheb(Sp-Rheb)and TOR(termed Sp-TOR).Further experiments revealed that the RNAi-mediated Sp-Akt gene knockdown and the inhibitors of PI3K and TOR could block the stimulation of Sp-vtg expression by insulin.The combined results showed in vitro stimulation of Sp-vtg expression by insulin in the mud crab,S.paramamosain,mediated through PI3K/Akt/TOR pathway,which implicateed the involvement of endogenous ILP and its corresponding signaling in the regulation of Sp-vtg synthesis in S.paramamosain.