The Function Study Of Ficolin-like, Toll And IMD In The Innate Immunity Of Macrobrachium Nipponense

Macrobrachium nipponense(commonly known as-freshwater prawn or river prawn)is a kind of important freshwater prawn aquaculture,its delicious taste,high nutritional value,can create enormous economic value.However,in recent years,with the frequent outbreaks of disease,mainly invading bacteria and viruses and other pathogens,the prawn culture suffered huge economic losses,so the prevention of prawn disease,study the innate immune defense mechanism urgently.Ficolins have crucial functions in recognizing and eliminating pathogens in innate immunity.Tolls have an essential role in initiating innate immune responses against pathogens.The immune deficiency(IMD)signal-pathway mediates innate immunity against Gram-negative bacteria in crustaceans.In this paper,M.nipponensis used as the research,to study the role of ficolins,Toll and IMD signaling pathway in the immune system.1.The role of two ficolin-like protein in the innate immune defenseIn this study,we identified two ficolin-like genes from the oriental river prawn,Macrobrachium nipponense.These genes were designated as MnFicol and MnFico2.MnFicol cDNA has 1600 bp,whereas MnFico2 has 1486 bp.In addition to a coiled-coil region or a low complexity region,the two ficolins both contained a signal peptide and a fibrinogen-related domain.qRT-PCR results showed that the highest expression level of MnFicol expression was in the gills,whereas that of MnFico2 was in the heart.The transcripts of MnFicol and MnFico2 could both respond to bacteria challenge.The transcription of antilipopolysaccharide facto,rs ALFs(MnALFl and MnALF2)and crustin genes(MnCru4,MnCru5,MnCru6,and MnCru7)was inhibited in the gills of MnFico1 or MnFico2 knockdown prawns at 24 h Vibrio parahaemolyticus challenge.Recombinant proteins of rMnFicol and rMnFico2 could bind toward diverse bacteria and agglutinate Gram-negative and Gram-positive bacteria with the presence of calcium(Ca2+).rMnFicol and rMnFico2 proteins also have lipopolysaccharide and peptidoglycan binding activity.Both recombinant ficolin proteins could help the prawn to facilitate the clearance of V.parahaemolyticus in vivo.MnFico1 and MnFico2 may play an important role as pattern recognition receptors in the innate immune defense of M.nipponense against bacterial invasion.2.Toll receptor regulates the expression of antimicrobial peptide(AMP)in the gillsIn this study,a novel Toll gene,MnToll1,was first identified from the freshwater prawn,M.nipponense identified.The MnToll1 cDNA is 3186 bp long with-a 2867 bp open reading frame that encodes a 955-amino acid protein.MnTolll contains a signal peptide,12 LRR domains,3 LRR_TYP domains,2 LRR_CT domains,a low complexity region,a transmembrane region,and a TIR domain.qRT-PCR analysis revealed that MnToll1 mRNA was detected in all tested tissues,and the expression of MnToll1 in the gills was significantly upregulated after V.parahaemolyticus challenge.Results of RNA interference by dsRNA also showed that MnToll1 regulates the expressions of 2 crustins in the gills of prawns.This study shows that M.nipponense plays an important role in inducing an innate immune response to expression of antimicrobial peptides.3.The role of immune deficiency homolog(IMD)in innate immunityIn the present study,the IMD receptor(MnIMD)from the oriental river prawn,Macrobrachium nipponense was identified.The full-length cDNA of MnIMD was 782bp with an open reading frame of 549 bp that encodes a putative protein of 182 amino acids including a death domain at the C-terminus.Multiple alignment analysis showed that IMDs in prawn M.nipponense and other crustaceans had high similarity.They were clustered together in the phylogenetic tree.The recombinant protein of MnIMD was furtherly expressed and purified.Western blot analysis with the antiserum prepared from the purified mature protein indicated that MnIMD was present in most tissues,but with the highest level in the gills,which was consistent with the qRT-PCR results.After V.parahaemolyticus challenge MnIMD was significantly induced in gills.RNA interference analysis showed that the IMD pathway was involved in regulating the expression of different antimicrobial peptide(AMP)genes,including Cru4 and Cru6.In summary,studies on immune genes such as ficolin-like,Toll and IMD are helpful to deepen the understanding of the innate immune mechanisms of M.nipponense,and also provide theoretical guidance and practical significance for the prevention and control of prawns diseases.