Physiological Mechanisms Of Crustacean Hyperglycemic Hormone In White Shrimp Litopenaeus Vannamei

Crustacean hyperglycemic hormone (CHH) is an important neuroendocrinehormone which synthesized in and secreted from the X-organ/sinus gland (XO/SG)complex in the eyestalk, it plays crucial roles in many biological processes. In thisstudy, we isolated the full-length cDNA of CHH gene from the eyestalk andsuccessfully expressed in E.coli. In order to study the effect of CHH in prawnphysiological metabolism regulation, We inject it into Litopenaeus vannamei bydifferent strengths, the mainly contents including:(1) Molecular cloning,characterization and recombinant expression of crustacean hyperglycemic hormone inwhite shrimp Litopenaeus vannamei;⑵The effects on shrimp gill filament iontransport, blood cell defense, carbohydrate metabolism of recombinant CHHinjections.1. Molecular cloning and expression analysis of crustacean hyperglycemichormone in white shrimp Litopenaeus vannameiCrustacean hyperglycemic hormone (CHH) plays plays an important role incrustacean. In the present study, a full-length cDNA of CHH was cloned from theeyestalk of Litopenaeus vannamei by RACE (approach) for the first time. Thefull-length cDNA of LvCHH was846bp, containing a5’ untranslated region (UTR)of65bp, a3’ UTR of436bp with a canonical polyadenylation signal-sequenceAATAA and a poly (A) tail, and an open reading frame (ORF) of345bp. The ORFencoded a polypeptide of114amino acids including a24amino acid signal peptides.The calculated molecular mass of the mature protein (74amino acids) was8.76kDawith an estimated pI of6.78. The sequence of LvCHH was submitted in NCBIGenBank under the accession number HM748790.2. Phylogenetic analysis revealedthat LvCHH was clustered with CHH of other crustaceans. Specific primerscontaining Xho â… a nd Ndeâ…  restriction sites respectively, were designed based onthe obtained ORF sequence of LvCHH gene and the cloning sites of expression vector pET-32a (+), the recombinant plasmid LvCHH-pET32a, were used to transformE.coli BL21(DE3). LvCHH was successfully expressed by means of SDS-PAGE andwestern blot analyze. We detected gill Na+/K+-ATPase activity after rLvCHH proteininjection and found that All the experimental group Na+/K+-ATPase activity presentedpeak change among0-6h, and the peaks of all treated groups occurred in1h.20and30μg/shrimp-1groups showed significant increase (P<0.05) in1h post-injection.L.vannamei were exposed for96h to hypo-and hyper-salinity challenge. All theseresults indicate that LvCHH was an important hormone involved in the osmosisresponses of swimming shrimps, and can provide further information of crustaceasosmoregulation physiological mechanism.2. Injection of recombinant CHH modulates gill filament signaling pathwaysand ion transport of Litopenaeus vannameiInjection of recombinant CHH modulates Gill filaments signaling pathways andion transport of Litopenaeus vannamei were studied in this part. The experiment wascomposed of three groups (control,10μg recombinant CHH/shrimp-1,20μgrecombinant CHH/shrimp-1), every group has three reduplications, and8samplingtimes (0h,0.5h,1h,3h,6h,12h,24h and48h). The sesults revealed that afterinjection of recombinant CHH, contents of biogenic amine (DA,5-HT, NE) in everytreatment group have significant change, and NE was the the most obvious, thecontent of DA was significantly higher than the control group only at6h, and thecontent of5-HT was significantly higher than the control group only at3h and6h.The results show that NE is the mainly biogenic amine response to the CHH signal,furthermore, biogenic amine content between different treatment group did notchange significantly; cAMP content of each treatment group is significantly higherthan the control group only at1h, and cGMP content is significantly higher than thecontrol group between1h and48h, the results show that the cGMP plays the leadingrole, and And there is a certain dose effect relationship; While PKA is the mainlyprotein kinase response to cGMP; Among three kinds of ion transport enzymes,Na+/K+-ATPase and HCO—3ATPase are the first two response to protein kinase, andthe enzyme activity of V-ATPase was significantly higher than the control group after 12h among the sample time. And haemolymph osmotic pressure also began a significant rise in after12h. Through the above results can be obtained that after injection of CHH, the signal transduction path is NEâ†'cGMPâ†'PKA,PKCâ†'three kinds of ion transport enzymesâ†'haemolymph osmotic pressure.3. Injection of recombinant CHH modulates carbohydrate metabolism of Litopenaeus vannameiInjection of recombinant CHH modulates carbohydrate metabolism of Litopenaeus vannamei were studied in this part. The experimental design was same as above. The sesults revealed that after injection of recombinant CHH, contents of cAMP and cGMP in hepatopancreas have the same trends, they are all significantly higher than the control group between3h and48h, the results show that cAMP and cGMP in hepatopancreas are all on the same level in response to CHH; the activity of GS in muscle has little change among the sample time, and the activity of GP in muscle is significantly higher than the control group between3h and48h, while the activity of GS and GP in hepatopancreas have significant change among the sample time; The activity of FBPase and PEPCK in hepatopancreas and muscle are all significantly higher than the control group among the sample time. Through the above results can be obtained that muscle is the mainly organization in respone to CHH, on the premise of GS activity remains the same, the activity of GP increase, through this way, glucose generated will be greater than the speed of the rate of glucose consumption, it can result in the increase of glucose content in the haemolymph. The gluconeogenesis has a role in the two organizations, but mainly in hepatopancreas. Through the common role of glycogen decomposition and gluconeogenesis, eventually lead to the rise of blood sugar levels.4. Injection of recombinant CHH modulates immune defense indicators of Litopenaeus vannameiInjection of recombinant CHH modulates immune defense indicators of Litopenaeus vannamei were studied in this part. The experimental design was same as above. The sesults revealed that after injection of recombinant CHH, the content of DA in haemolymph is significantly higher than the control group only at6h, and the content of5-HT in haemolymph is significantly higher than the control group between0.5h and12h, and the content of NE in haemolymph is significantly higher than thecontrol group between3h and12h, the results show that5-HT and NEare mainlybiogenic amine in response to CHH. The changes of biogenic amines lead to thechanges of number of blood cells, the number of blood cells is significantly higherthan the control group between0.5h and12h, and this is the same trend as5-HT. Theactivity of prophenoloxidase is significantly higher than the control group between3hand48h, and the activity of phenoloxidase is significantly higher than the controlgroup between1h and6h, through the above results can be obtained that the changesof number of blood cells lead to the change of the activity of prophenoloxidase, butthe change of the activity of prophenoloxidase lag behind of the activity change ofphenoloxidase, this may because signal stimulate phenoloxidase from the condition oflow activity into a highly active state in the haemolymph, along with the increase ofthe activity of phenoloxidase, it stimulate the activity of prophenoloxidase forproviding more phenoloxidase. The antibacterial activities of Litopenaeus vannamei issignificantly higher than the control group between0.5h and6h, but bacteriolyticactivities have little change among the sample time, this show that the role of CHH inimmune defense remains only partial.