Study On The Immune Function Of Lectin, Dorsal And Ras In Main Economic Shrimps

Shrimp aquaculture,throughout the world,gets heightened attention because of the great economic value it brings.With the increasingly stressful environment the development of intensive culture,the gradual imbalance of nutrition supply,and the invasion of various pathogens including bacteria,viruses,fungi,and parasites,result in frequently occurred diseases which cause the huge economic losses in the whole aquaculture industry.In view of this,it is of profound significance to study the immune response mechanism of shrimp against pathogen invasion.Lectins play important roles in the process of pathogen recognition and clearance,but the current studies on the function of C-type lectin and L-type lectin in innate immune defense are still limited.Toll signaling pathway plays an important role in humoral immunity in response to Gram-positive bacterial and fungal infections through regulating the expression of antimicrobial peptides.Dorsal,as a key transcription factor in the Toll signaling pathway,is involved in regulating the expression of antimicrobial peptides.Small GTPases participate in a variety of cellular life activities,and previous studies showed that small G proteins regulate the phagocytosis of haemocytes in shrimp during virus infection.To clarify the specific functions and mechanisms of the above immune-related genes in the innate immune system of crustacean,we used Macrobrachium nipponense,Macrobrachium rosenbergii,and Marsupenaeus japonicus as experimental animals to study the roles of C-type lectin contain Clip domain(Mn-clip-Lec),L-type lectin(MrVIP36),the key transcription factor Dorsal(MrDorsal)in the Toll signaling pathway and three members of Ras small G proteins family(MjRap,MjRas and MjRal)in host innate immune defenses including antibacterial and(or)antiviral responses.The results of this study are described as follows:1.Functional study of C-type lectin with Clip domain from M.nipponense in bacterial infectionWe discovered a novel and unreported C-type lectin contains a Clip domain from the oriental river prawn,M nipponense,designated as Mn-clip-Lec.It consists of 1315 bp clone with an open reading frame of 1098 bp,encoding a polypeptide of 365 amino acids.The Mn-clip-Lec sequence possesses a Clip domain at the N-terminal and two carbohydrate recognition domains at the C-terminal.Mn-clip-Lec was higher expressed in hemocytes than other tissues.The expression levels of Mn-clip-Lec,proPO-activating system members(MnPPAF,MnPPAE and MnPo),and antimicrobial peptides(MnALF and MnCRU)were significantly up-regulated after Staphylococcus aureus infection.RNA interference(RNAi)-mediated suppression of the Mn-clip-Lec gene in S.aureus-challenged prawns significantly reduced the transcript levels of proPO-activating system members and antimicrobial peptides.Furthermore,Mn-clip-Lec gene silencing resulted in a significant decrease in the total hemolymph PO activity infected with S.aureus.The purified recombinant Mn-clip-Lec protein(rMn-clip-Lec)could bind five tested bacteria with different activities.rMn-clip-Lec could agglutinate S.aureus in the presence of calcium.rMn-clip-Lec could bind to glycoconjugates on the bacteria surface,such as lipopolysaccharide(LPS)and peptidoglycan(PGN).rMn-clip-Lec was also able to accelerate the clearance of S.aureus in vivo.These findings suggest that Mn-clip-Lec,a C-type lectin contain Clip domain,participate in innate immune responses including recognizing pathogen,activating the proPO system,and regulating the expression of antimicrobial peptides.Based on the above study,proteins from cDNA Library of M nipponense that interacted with Mn-clip-Lec were obtained by yeast two-hybrid screening,which need to be further validated.These results from screening may serve as a basis for further study on the mechanism of body's innate immune response against pathogen invasion.2.Study on the immune responses of L-type lectin to bacterial and viral infections in M rosenbergiiWe cloned a novel L-type lectin from freshwater prawn M rosenbergii,designated as MrVIP36.The full-length cDNA of MrVIP36 is 1687 bp with a 972 bp open reading frame encoding a putative protein of 323 deduced amino acids.The deduced MrVIP36 protein contains an LTL-like domain(LTLD)and a transmembrane domain.Phylogenetic tree analysis indicated that MrVIP36 has a closer evolutionary distance with L-type lectins from invertebrates than from vertebrates.MrVIP36 was expressed widely in different tissues in healthy M rosenbergii,especially in the hepatopancreas and intestine.The transcription of MrVIP36 was significantly up-regulated in hemocytes of prawns after S.aureus,Vibrio parahaemolyticus,and White spot syndrome virus(WSSV)infections.The recombinant protein MrLTLD(rMrLTLD)could bind all tested bacteria.It could agglutinate S.aureus and V.parahaemolyticus in the presence of calcium.rMrLTLD could also bind to LPS and PGN.Moreover,rMrLTLD could inhibit the growth activities of microorganisms in vitro and accelerate the bacterial clearance in vivo.Meanwhile,survival rate analysis showed that rMrLTLD can reduce the death of M.rosenbergii caused by WSSV infection.Our results indicate that MrVIP36 may functioned as a pattern recognition receptor playing crucial roles in the antibacterial and antiviral immune responses of M.rosenbergii.3.Dorsal transcription factor is involved in regulating expression of crustin genes during WSSV infection in M.rosenbergiiWe identified a dorsal homolog(named MrDorsal)from freshwater prawn M.rosenbergii.The full-length cDNA of MrDorsal comprises 2533 bp with an open reading frame of 1986 bp,which encodes apeptide of 661 amino acid residues.Amino acid sequence analysis showed that MrDorsal contains a Rel homolog domain(RHD)and an IPT/TIG(i.e.,Ig-like,plexin,and transcription factors)domain.The signature sequence of dorsal protein FRYMCEG existed in the deduced amino acid sequence.MrDorsal was expressed higher in the hemocytes and gills than that of other tissues.The expressions of MrDorsal and crustin genes(MrCrustin2 and MrCrustin4)were significantly upregulated after challenged with WSSV.Knockdown of MrDorsal could suppress the transcription of above-mentioned crustin genes in gills of prawns after WSSV challenge.Results indicated that MrDorsal was involved to regulate the expression of crustin genes and it might play potential important roles during WSSV infection in M rosenbergii.4.Identification of three members of small GTPases superfamily from M japonicas challenged with WSSVThree Ras small G proteins(named MjRap,MjRas,and MjRal)were cloned and identified from kuruma shrimp M japonicus.The full-length cDNA of MjRap,MjRas,and MjRal are 1236,1330,and 2074 bp with 570,609,and 597 bp open reading frame encoding the proteins of 189,202,and 198 amino acids respectively.Phylogenetic analysis showed that Rap,Ras,and Ral from different species gather together.The MjRap,MjRas,and MjRal genes were ubiquitously expressed in the hemocytes,hepatopancreas,gills,stomach,and muscle of healthy shrimp.MjRal in the gills was up-regulated 48 and 72 h post-WSSV challenge.No change in the MjRap or MjRas transcript was observed in the gills under the WSSV challenge.Further research indicated that the RNAi of MjRal could obviously increase the copies of WSSV.In addition,after injection of WSSV with recombinant MjRal(rMjRal)protein,the expressions of both VP28 and WSSV copies in gills were tested at 48 h.The results showed that no evident change in the VP28 expression could be detected.However,the WSSV copies were distinctly reduced.So,it could be concluded that only MjRal was involved in shrimp antiviral innate immune response by inhibiting the WSSV replication.