Study On The Immune Function Of Six Crustacean Peptides From Macrobrachium Japonicus

Macrobrachium nipponense is an important freshwater-cultured prawn.It is rich in nutrition and has a big market in China.In recent years,due to environmental pollution and the pursuit of high-yield intensive aquaculture,many diseases infected by microorganisms have appeared in the farming of M.nipponense.A number of diseases reported in prawns are caused by pathogens,including viruses and bacteria.Therefore,research on the prawn innate immune mechanism against pathogen has received significant attention.Crustin is an antibacterial peptide in crustaceans,which regulates its transcription level through transcription factors in various signaling pathways in the innate immune system to achieve the role of defense against foreign pathogens.This study investigated the role of three Crustin immune genes in the innate immune defense mechanism of M.nipponense.The results of the study are as follows:1.Crustin,as antimicrobial peptides,are important to crustacean innate immunity.In this study,two kinds of Gly-rich Crustin genes were cloned from M.nipponense and were referred to as Mn-Gly-Cru1 and Mn-Gly-Cru2.These Crustins belong to type Ⅱ Crustin with typical type Ⅱ Crustin structures.The full-length cDNA of Mn-Gly-Cru1 is 677 bp and contains a 576 bp open reading frame(ORF)encoding191 amino acids.The full-length cDNA of Mn-Gly-Cru2 is 727 bp,with 573 bp ORF encoding 190 amino acids.The constructed phylogenetic tree indicated that Mn-Gly-Cru1 and Mn-Gly-Cru2 belong to the type Ⅱa subfamily.RT-PCR analysis showed that Mn-Gly-Cru1 and Mn-Gly-Cru2 are widely distributed in various tissues.qRT-PCR results indicated that Mn-Gly-Cru1 is mainly expressed in the gills,whereas Mn-Gly-Cru2 is expressed at the highest level in hemocytes.The transcripts of Mn-Gly-Cru1 and Mn-Gly-Cru2 respond to bacterial or white spot syndrome virus(WSSV)stimuli.48 hours after the injection of ds MnRelish,MnRelish was knocked down,and then the Crustin was detected,and it was found that the expression of Mn-Gly-Cru1 and Mn-Gly-Cru2 was subsequently inhibited.After white spot syndrome virus,Vibrio parahaemolyticus,or Staphylococcus aureus challenge,MnRelish,Mn-Gly-Cru1,and Mn-Gly-Cru2 were all upregulated.However,the expression levels of MnRelish,Mn-Gly-Cru1,and Mn-Gly-Cru2 at 6 h bacteria or 36 h WSSV challenge were downregulated in Relish-silenced prawns when compared with the control(bacteria or WSSV challenge only,bacteria or WSSV challenge plus ds GFP injection).Results suggest that Mn-Gly-Cru1 and Mn-Gly-Cru2 play essential roles in M.nipponense innate immunity against bacteria or WSSV,and the expression levels of both genes are regulated by Relish transcriptional factor.2.Carcinin,a member of the Crustin family,plays important roles in crustacean innate immunity.In this study,we identified two Carcinin isoforms(MnCarc1 and MnCarc2)produced by alternative splicing from M.nipponense.The full length of MnCarc1 and MnCarc2 cDNA are 1554 and 1495 bp with 687 and 609 bp ORF-encoding proteins that contain 228 and 202 amino acids,respectively.The genome of Carcinin has nine exons and eight introns.MnCarc1 transcript contains all nine exons,whereas MnCarc2 only contains eight exons and lacks exon 4.MnCarc1 and MnCarc2 proteins contain a signal peptide,cysteine-rich regions,and a whey acidic protein domain.The phylogenetic tree shows that MnCarc1 and MnCarc2 are not grouped with other Crustins and Carcinins.MnCarc1 and MnCarc2 form a subgroup.MnCarc1 and MnCarc2 are widely distributed in various tissues.The expression of MnCarc1 and MnCarc2 were evidently upregulated at multiple time points in hemocytes and the intestine of M.nipponense after WSSV,V.parahaemolyticus,and S.aureus challenges.Further studies showed that knockdown of MnDorsal or MnStat transcription factor could remarkably inhibit the upregulated expression of MnCarc1 and MnCarc2 caused by viral or bacterial challenges.In addition,recombinant MnCarc1 and MnCarc2 proteins could bind to various bacteria and polysaccharides and inhibit the growth of S.aureus and V.parahaemolyticus in vitro.This study indicated that Carcinins from M.nipponense were involved in prawns innate immunity.3.Two Crustins genes were cloned in M.nipponense,named MnCru174 and MnCru191.Their full lengths are 740 and 781 bp,with ORF of 525 and 576 bp,encoding 174 and 191 amino acids,respectively.Both MnCru174 and MnCru191 genome sequences contain two exons and one intron,and their proteins all contain signal peptides,cysteine-rich regions,glycine-rich regions and whey acidic protein domains.The phylogenetic tree shows that MnCru174,MnCru191 and MnCru3 aggregate into one branch,and the evolutionary distance is very close.MnCru174 and MnCru191 are widely distributed in various immune tissues.In hemocytes,gills and stomach tissues of M.nipponense,the transcription levels of MnCru174 and MnCru191 increased significantly at multiple time points after stimulation by WSSV,S.aureus and V.parahaemolyticus.Using RNAi experiments to silence MnCru174 and MnCru191 can significantly increase the copy number of WSSV and the number of S.aureus and V.parahaemolyticus colonies.In addition,recombinant MnCru174 and MnCru191 proteins can be combined with various bacteria and polysaccharides.This study shows that MnCru174 and MnCru191 play an important immune role in the defense of M.nipponense against bacterial and viral invasion.This article takes M.nipponense as the research object and makes an in-depth study on the functions of its six Crustins.There are two aspects of significance: one is to enrich the theory of innate immunity of crustaceans by studying the Crustin genes(Mn-Gly-Cru1,Mn-Gly-Cru2,MnCarc1,MnCarc2,MnCru174,MnCru191)in M.nipponense;the second is to lay a foundation for the interpretation of the molecular regulation mechanism of the innate immune signal pathway of M.nipponense through this research,and also provide theoretical support for the prevention of M.nipponense disease.