The Effect Of KK-42 On The Molt Cycle Of Juvenile Macrobrachium Nipponense And Its Mechanisms

The Macrobrachium nipponense,a member of the Palaemonidae family of decapod crustaceans,is naturally distributed throughout almost freshwaters in China.Owing to its excellent adapt ability and high economic value,M.nipponense has becomean important breeding species for freshwater aquaculture in China.Our previous study demonstrated that KK-42,a kind of imidazole derivative,could improve growth of Penaeus schmittiand juvenile M.nipponense significantly.However,the mechanism remains unclear.It was reported that the growth of crustaceans is closely related to the molting cycle.We assume that KK-42 probably improves growth of crustaceans by affecting molting cycle.In this study,bioassay was performed to explore the mechanism of KK-42 on the molting cycle of juvenile M.nipponense.The molting cycle were measured,the expression levels of the three genes related to molting cycle were analyzed and the cuticle ultrastructure of the juvenile M.nipponense were observed after KK-42 treatment.The methods and the results are specified as follows:The two months-old juvenile prawns(1.2-2.0 cm in length)were collected from our aquaculture ground,and acclimated at 26±1 °C,in running-water tanks in the laboratory and fed twice daily for 1 week before the experiments.The prawns were administered with KK-42 at a concentration of 0(control group)or 1.95 × 10-4mol/L(experiment group)for 1 min,respectively.A portion of them were used to measure the growth rate and molt cycle;and the rest,at different stage,were sacrificed to RNA extraction,cDNA clone,analysis of chitinase(Chi)and N-acty1-β-D-glucosaminidase(NAGase)mRNA expression levels,enzyme assay and cuticle ultrastructure observation.The average weight of KK-42 treatment group was significantly higher than that of the control group,and its growth rate increased significantly in the first two weeks after KK-42 treatment.The molting period tended to prolong as the growth of juvenile prawns,and that was shorten by KK-42 treatment at the first two cycles among four consecutively-determined molt cycles,decreased from(8.70 ± 1.07),(9.81 ± 0.43)d/ molt cycle to(6.93 ± 0.97),(8.11 ± 1.20)d/ molt cycle,respectively.Two M.nipponense chitinase genes(MnChi)were cloned from cuticle: MnChi-1 and MnChi-3.The full-length cDNA of Mn Chi-1 and MnChi-3 were 1532 bp and 1413 bp,containing a 1230 bp and 1140 bp open reading frame(ORF),that encode proteins of 410 and 380 amino acids(aa)and with a 66 pb,26 pb 5’-untranslated region(UTR),a 236 bp,247 pb of 3’UTR,respectively;The calculated molecular masses and estimated isoeletric point of translated MnChi-1 and MnChi-3 proteins were 76.65 KDa,31.33 kDa and 5.62,7.12,respectively.They shared almost the same architecture characteristics,a signal peptide,a catalytic domain and a GH18 conserved domains,whereas S/T-rich and chitin-binding domains(CBDs)were absent.MnChi-1was expressed in the cuticle,gut and hepatopancreas,but highestexpression was observed in hepatopancreas.Expression of MnChi-1in the cuticle or hepatopancreas were highest during premolt(D).MnChi-3 was expressed in the cuticle,hepatopancreas,eyestalk and gut,but highest in the gut.MnChi-3 shared almost the same expression pattern with MnChi-1during molting cycle.The expression of MnChi-1 was up-regulated in the cuticle of intermoult(C)and the culticular Mnchi-1 mRNA level as well as the chitinase activity in the stage C increased by more than 10 and 2-fold than that in control group,respectively,at 3,6 and 9 h after KK-42 treatment.KK-42 treatment had relatively weak effects on the stage D,only had a modestrise at 12 h.Mn Chi-1 had almost the same expression pattern in the hepatopancreas.Mn Chi-3 shared almost the same expression pattern with MnChi-1 in the cuticle and hepatopancreas whether on the stage C or stage D.A full-length of M.nipponense N-acty1-β-D-glucosaminidase gene(MnNAGase)in the cuticle was cloned and characterized for the first time.The full-length cDNA contained 2536 bp with a 1854 bp open reading frame encoding 617 amino acids,a 382 pb 5’UTR,a 300 bp 3’UTR.The predicted molecular mass and isoeletric point were 69.72 k Da and 5.43.The gene possessed a signal peptide,a catalytic domain,a GH20 conserved domains and nine catalytic domain conserved sites which shares 90.4 % amino acids sequenceidentity with that of Fenneropenaeus chinensis.Real-time PCR analysis revealed that the MnNAGase expression werehighest during premolt(D).The expression of MnNAGase was up-regulated significantly in the cuticle of intermoult(C),increased rapidly at 3 h,reached peak at 6 h and more than 4-fold than that in control group,after KK-42 treatment.The MnNAGase activity had almost the same pattern in the cuticle.The expression of MnNAGase was up-regulated slightly in the cuticle of premolt(D),only increased at 6 h,the MnNAGase activity increased significantly at 3,6,and 9 h than that in control group after KK-42 treatment.Epithelial cells are the target tissue of ecdysone(MH).In view of the significant regulatory effect of KK-42 on chitinase,NAGase activity and gene expression in the stage C,the ultrastructural changes of epithelial cells in the stage C were observed by transmission electron microscope.The results showed that epithelial cells were single layer,flat and arranged regularly.Epithelial cells began to extend and many organelles such as microtubules,Golgi element,rough endoplasmic reticulum and ribosome had emerged at 9 h after KK-42 treatment.In conclusion,the results of this study demonstrate that KK-42 administration can shorten molt cycle and promote growth of juvenile M.nipponense,up-regulate MnChi-1,MnChi-3 and MnNAGase mRNA expression and enzyme assay in the cuticle or hepatopancreas,activate the epithelium to secrete variety enzyme,accelerate degradation of old cuticle,which is likely one of KK-42 growth-promoting and shortening molt cycle mechanisms on juvenile M.nipponense.