The Study On The Immunomodulatory Role Of Glucose Metabolic Hormone In Crustacean

A robust immune response against invading pathogens is crucial for host to survive,which depends greatly on the well balance of metabolism.Increasing evidence has indicated that some glucose metabolic hormones,such as insulin,could modulate immune responses directly.In the present study,the dynamic changes of crustacean hyperglycemic hormone(CHH)in Litopenaeus vannamei and insulin-like peptide(ILP)in Eriocheir sinensis were inestigateed after immune stimulation,the distribution and metabolic role were also identified,and the immunomodulatory role of CHH was further explored in shrimp L.vannamei for better understanding of the immunomodulation of glucose metabolic hormone in invertebrate.CHH family is a group of ecdysozoans-specific peptide hormone involved in glucose metabolism and other biological events.In the present study,two members of CHH family(designated as LvCHH I and LvCHH II)in shrimp Litopenaeus vannamei with one and two crustacean neurohormone domains respectively were chosen to investigate their putative modulatory roles in both glucose metabolism and immune response.LvCHH I and LvCHH II were both expressed in the sinus gland and lamina ganglionalis of eyestalks and were significantly induced after white spot syndrome virus(WSSV)infection.Meanwhile,significant increases of hemolymph glucose levels were observed in shrimp at 12 and 24 h after WSSV infection while the glucose inside the hemocytes decreased at 6 h and then increased at 12 h.Gain-of-function of rLvCHHs was subsequently conducted in vivo by injecting the recombinant proteins(rLvCHH I and rLvCHH II).The hemolymph glucose increased significantly from 0.5 h to 3 h after the shrimps received an injection of rLvCHH I,while it decreased at 0.5 h and increased afterward at 3 h post r LvCHH II injection.At the meantime,significant decreases of reactive oxygen species level in hemocytes were observed at 3 h and 6 h post rLvCHH I injection,while it remained unchanged in rLvCHH II injection group.rLvCHH I and r LvCHH II could bind to the cytomembrane of primary shrimp hemocytes in vitro,and the expressions of superoxide dismutase and LvRelish increased when the hemocytes were incubated with rLvCHH I for 3 hours.Meanwhile,the expression of antimicrobial peptides,crustin and penaeidin-4,were also induced by r LvCHH I and rLvCHH II.These results demonstrated that host immune response,in addition to glucose metabolism,could be directly modulated by LvCHH family.The full-length cDNA of an ILP(designated Es ILP)was identified by the conserved insulin-like growth factor domain(IIGF)from the genome information of Eriocheir sinensis.The 216 bp open reading frame encoded a putative protein of 71 amino acids.Multiple sequence alignment presented four conserved cysteine residues which were critical for the fundamental structure and function of IIGF domain.The EsILP transcripts could be detected in the hepatopancreas,eyestalk,muscle,gill,hemocyte,hemopoietic tissue,stomach,while the Es ILP protein was detected in hepatopancreas and thoracic ganglion mass.Gain-of-function of Es ILP was subsequently conducted in vivo by injecting the recombinant proteins.The hemolymph glucose decreased significantly from 6 h to 12 h,indicating its functional similarity with insulin.Besides,the temporal expression of Es ILP mRNA in different tissue after Aeromonas hydrophila challenge was measured by real-time RT-PCR.It was shown that the EsILP mRNA expression decreased significantly in hepatopancreas while highly increased in eyestalk and hemocytes.In conclusion,the above results demonstrated the direct immunomodulation role of glucose-regulating hormone in crustaceans and would shed new light into understanding the interaction between glucose-regulating hormone and immune response in crustaceans.