The Functional Study Of The Ions Transport Related Molecules In Scvlla Paramamosain During The Molt Cycle

1.Classification of molt stages of Scylla paramamosainWith the aim of investigating selected molt-linked biochemical and inorganic factors in these animals,two rapid molt-staging techniques were developed for the mud crab(Scylla paramamosain)by observing the third maxilliped and the end of dactyl segment in the swimming leg under microscope.Seven molt stages were characterized and clear images were presented.The molt cycle of the crab was divided of four distinct molting periods(postmolt,intermolt,premolt and ecdysis)and the further classification of the premolt period into three substages(D0,D1 and D2).These findings are consistent with the classical scheme elaborated by Drach and Tchernigovtzeff(Drach and Tchernigovtzeff,1967).The present study reveals that the third maxilliped of S.paramamosain was far more visuable than the end of dactyl segment in the swimming leg.Consequently,the methodology of determination of molt stage based on maxilliped is practically and easily performed.It is a useful technical tool for S.paramamosain aquacultural research.2.A structure-function analysis of ion transport in S.paramamosain gillsOsmotic and ionic regulation in the Crustacea is mostly accomplished by the multifunctional gills.However,the morphology of gill structures is not well described in S.paramamosain.This study focuses on structure-function relationships in crustacean gills by using HE,TEM and SEM examination from the organ level of organization.In each gill lamella of S.paramamosain,two symmetrical single-layered epithelia facing each other across the hemolymph space occasionally occupied by a central intralamellar septum and mechanically sustained by the pillar cells.Each lamella is covered by thin cuticle.The posterior gills have a number of distinct ultrastructural features that are characteristic of ion transporting epithelia.These include abundant mitochondria in the sub-apical cytoplasm,a greatly elaborated membrane surface(particularly in the basolateral region facing hemolymph space),and extensive foldings in the apical surface of the leaflets.In contrast,anterior gills that are believed to play roles in both respiration and acid-base regulation are equipped with relatively larger hemolymph space,much thinner epithelial cells with limited membrane elaboration and a few mitochondria.These findings were in line with the previous descriptions of the currently accepted phyllobranchiate gill model,where ion transport mechanisms seem to be located in functionally different gills.3.Exoskeletal cuticle differentiation during molt cycle of S.paramamosainExoskeletal crustacean cuticle is a calcified apical extracellular matrix of epidermal cells,illustrating the chitin-based organic scaffold for biomineralization.Studies of cuticle formation during molting reveal significant dynamics and complexity of the assembly processes,while cuticle formation during the molt cycle is poorly investigated.This study reveals in the S.paramamosain,the ultrastructural organization of the differentiating exoskeletal cuticles during the molt cycle.The results show that the cuticle of the intermolt crab is composed of four sub-layers from exterior to interior including an epicuticle,an exocuticle,an endocuticle and a membranous layer.The differences of cuticular depositions reflect the different functions of each cuticular sub-layer.During the premolt period,after exuviations(post-ecdysis),the syntheses of the endocuticle and membranous layer of the new cuticle progressed chronologically.When the synthesis of the membranous layer is complete,the crabs then enter the intermolt stage.We also investigated the the composition of the cuticles throughout the molting cycle.SEM-EDX analysis showed that the most abundant mineral in cuticle is carbon(40.91%),oxygen(36.59%)and calcium(12.58%),on which crystals of CaCO3 are deposited.At the post-molt stage,calcium content was lowest with a level of 0.42%.At stage C,the cuticle was highly calcified(13.94%),and calcium was increased there after from the D2(21.35%)to stage E(29.75%,discarded cuticle).This result indicates that cuticle composition and structure is influenced by molt cycle.4.Calreticulin regulates Ca2+ storage of S.paramamosain in moultingCalreticulin(CRT),a well conserved endoplasmic reticulum-resident protein for Ca2+-binding,is widely expressed in multicellular eukaryotes.CRT plays a key role in many cellular processes,including Ca2+ homeostasis.To address the role of CRT underlying the Ga2+ homeostasis alternation during the mineralization cycle of the mud crab(Scylla paramamosain),we cloned the full-length cDNA of CRT gene(SpCRT)from S.paramamosain,and its protein contains all signature domains of CRT.Tissue distribution showed the SpCRT transcript was far more abundant in hepatopancreas than in others.During the molt cycle,the total calcium levels in the hepatopancreas were determined using ICP-AES.It decreased significantly at stage A and B(P<0.05),and was relatively steady between intermolt C and early premolt Do(P>0.05).Then it was gradually increased thereafter by nearly 274.2%up until stage E(compared with stage C)(P<0.05).Meanwhile,Ca2+ distribution in hepatopancreas of S.paramamosain was increased from postmolt to premolt,which was determined by potassium pyroantimonate precipitation and electromicroscopy.Additionally,the expression of SpCRT in the hepatopancreas was the lowest in the molt stage E and postmolt stage A,and then it maintained at a high level in other stages.Taken together,the relative retard timing of calcium incrument corfesponding to the higher expression of SpCRT may suggest CRT plays a critical role in Ca2+storage in hepatopancreas during the molt cycle of crustaceans.5.Na+/K+/2C1-(NKCC)regulates ion uptake of mud crab in postmoltAlthough iono-regulatory processes are critical for survival of crustaceans during the molt cycle,the mechanisms involved are not known in detail.The Na+/K+/2Cl-(NKCC),a SLC12A family protein that transports Na+,K+and 2Cl-into cells,is essential for cell ionic and osmotic regulation.To better understand the role of NKCC in the molt osmoregulation,we cloned and functionally characterized a NKCC gene from the mud crab,Scylla paramamosain.Predicted NKCC protein is well conserved,and phylogenetic analysis revealed this protein was clustered with crustaceans NKCC.Tissue distribution showed the NKCC transcript to be highly expressed in the posterior gills.During the molting cycle,hemolymph osmolality and ion concentrations(Na+ and Cl')levels significantly increased over the postmolt period,remained stable in intermolt and premolt stages and then decreased at ecdysis.In parallel,expression of SpNKCC mRNA was significantly elevated(29 to 291-fold)at ion reestabilishing stages(postmolt)as compared with baseline molt levels,a pattern consistent with the coordinated regulation of Na+/K+-ATPase a-subunit(NKA a),carbonic anhydrase cytoplasmic(CAc)isoform and Na+/H+ exchanger(NHE)genes in the posterior gills.These data suggest that SpNKCC may be important in mediating branchial ion uptake during the molt cycle,especially at the postmolt stages.