The Expression Characteristics Of Seven New Toll-like Receptors From Mud Crab Scylla Paramamosain And The Comparative Study On Their Response To Bacterial Infection

The mud crab,Scylla paramamosain,is an important economic crab for marine aquaculture in China.It lacks adaptive immunity,and mainly relies on pattern recognition receptors(PRRs)to recognize pathogen-associated molecular patterns(PAMPs)to activate innate immune responses and resist infection by external pathogens.Toll-like receptors,as an important PRR,can widely recognize pathogenic microorganisms.However,the mechanism of how crab Toll-like receptors recognize pathogens and activate immune pathways has not been well studied.In this study,the sequences of seven new Toll-like receptors from S.paramamosain,named SpToll3-9 were obtained based on the previous transcriptome database of our laboratory.In view of the existence of so many Toll-like receptors,whether their expression characteristics in vivo and their response to pathogen are consistent or have their own characteristics deserve further exploration.This study explained the SpToll3-9 mRNA expression profiles in normal tissues and expression characteristics at different developmental stages of S.paramamosain using real-time quantitative PCR.The expression profiles of SpToll3-9,the immune-related genes in Toll signaling pathway and AMPs in S.paramamosain after challenge with Gram-negative bacteria Vibrio alginolyticus and Gram-positive bacteria Staphylococcus aureus were also analyzed by qPCR.Primary culture of hemocytes in vitro were used to investigate the effects of PAMPs on the transcriptional level of SpTol13-9.At the same time,the expression characteristics of SpToll1 and SpToll2 were also analyzed.This study revealed the respective immune characteristics of Toll-like receptors in crabs and the immune signaling pathways that may be involved,which lays the foundation for the subsequent research on the immune regulation mechanism of Toll-like receptors involved in pathogenic microorganism infection.The main results are as follows:1.cDNA cloning and bioinformatics analysis.According to the the transcriptome database established in our laboratory,partial cDNA sequences were obtained and seven new SpTolls genes from S.paramamosain were cloned by PCR and RACE-PCR.These SpTolls genes were designated as SpToll3,SpToll4,SpToll5,SpToll6,SpToll7,SpToll8,SpToll9 based on high homology(30%-80%)with the reported SpToll1-2 and Toll genes of other shrimps and crabs.The full-length cDNAs of SpToll3-9 are 5033 bp,4825 bp,6504 bp,4932 bp,4427 bp,5245 bp,and 4421 bp,encoding 1229,1371,1555,1370,980,1196,and 976 amino acids,respectively.SMART analysis showed that SpTolls contains a typical Toll structure,a Toll/interleukin-1 receptor,transmembrane region and leucine-rich repeat,all of which carry signal peptides except SpToll5.Phylogenetic tree showed that SpToll3-9 clustered with Toll genes found in other shrimps and crabs.2.The SpToll3-9 mRNA transcripts at different developmental stages of S.paramamosain and the tissues distribution in female and male S.paramamosain.As determined by qPCR,we found that SpToll3-9,including the SpTolll and SpToll2,were all expressed in the early developmental stages of S.paramamosain.The expression of SpToll4 and SpToll5 were lower than other SpTolls.SpToll1-9 transcripts were significantly reduced from zoea V to megalopa(p<0.01),suggesting that the transcriptional expression of these 9 SpTolls during the early development stage in S.paramamosain has its regularity.The results of tissue distribution show that the transcript level of SpToll3-9 had no gender differences other than gonads.SpToll3、SpToll4、SpToll7、SpToll8 were highly expressed in gills while SpToll5、SpToll6、SpToll9 were highly expressed in hepatopancreas.Among gonad related organs,SpToll5,SpToll1,SpToll8 were highly enriched in ovaries.SpToll3-9 were constitutively expressed in all male gonadal organs,especially in testis higher than most other tissues.3.Expression profiles of SpTolls in primary cultured crab hemocytes.The primary culture of mud crab hemocytes was exposed to LPS,LTA,Poly(I:C),PGN and Glucan.The results of qPCR analysis showed that the transcript levels of SpToll2,SpToll3,SpToll6 and SpToll9 in hemocytes were significantly induced by LPS and LTA.Poly(I:C)and Glucan challenge also resulted in a significant up-regulation of SpToll2 expression.The significant up-regulation of SpToll9 expression in response to LTA and LPS stimulation in vitro was consistent with the results induced by V.alginolyticus and S.aureus challenge in vivo.By the way,the SpToll2 could be significantly induced by LPS,LTA,Poly(I:C)and Glucan.4.Expression profiles of SpToll3-9,the immune-related genes in Toll signaling pathway and antimicrobial peptides in S.paramamosain megalopa post LPS and V.alginolyticus challenge.The results showed that the transcript level of SpToll4 was significantly down-regulated only after V.alginolyticus infection,while SpToll3,SpToll6,SpToll8 and SpToll9 were all down-regulated after LPS and V.alginolyticus challenge.Myd88 was significantly up-regulated at 12 h after V.alginolyticus challenge.In addition,the expression levels of Crustin2 and Lysozyme were significantly decreased,while Arasin was significantly increased after LPS stimulation.The expression patterns of Crustinl,ALF2,and Hyastatin were similar,all of which were significantly up-regulated at the early stage post infection(3 h,6 h),and then significantly inhibited(18 h,24 h).V.alginolyticus infection could significantly increase the expression of Crustinl,Crustin2 and ALF2,but significantly inhibit the transcript levels of Hyastatin,Lysozyme and Arasin.All these results indicated that the regulatory mechanisms of different AMPs may be through but not limited to the signaling pathways involved in Toll,and the bacterial components and live bacteria will induce different immune responses in mud crabs.5.Expression profiles of SpToll3-9,the immune-related genes in Toll signaling pathway and AMPs in male S.paramamosain upon V.alginolyticus challenge.qPCR analysis revealed that the expression pattern of SpToll3-9 in different tissues after infection with V.alginolyticus was different.The expression of SpToll6,SpToll8,SpToll9 in gills were significantly up-regulated with V.alginolyticus challenges.The expression of SpToll6 and SpToll9 can be significantly induced in hepatopancreas.In the case of the hemocytes,the expression level of SpToll9 was significantly elevated,while SpToll6 was significantly down-regulated.No significant changes was observed in SpTolls expression in testis.In addition,SpToll1 were significantly up-regulated in gills and hepatopancreas,and down-regulated in hemocytes.The expression of SpToll2 in gills and hemocytes was up-regulated.As a key factor in the Toll signaling pathway,the expression of Dorsal was significantly up-regulated in hemocytes.Myd88 expression was significantly enhanced in hepatopancreas,but down-regulated in hemocytes.At the same time,the results showed that the relative expression levels of Crustin,Lysozyme,ALF were significantly up-regulated and Hyastatin was down-regulated upon V alginolyticus challenges.All these results suggested that after being infected with gram-negative bacteria V alginolyticus,S.paramamosain might activate the expression of AMPs such as Crustin,Lysozyme,ALF through the Toll signaling pathway mediated by SpToll1,SpToll2,SpToll6,SpToll8 and SpToll9.6.Expression profiles of SpToll3-9,the immune-related genes in Toll signaling pathway and AMPs in male S.paramamosain upon S.aureus challenge.For S.aureus challenge,SpToll7 and SpToll9 expression in gills were significantly up-regulated.In hemocytes,the expression of SpToll4 and SpToll9 were significantly elevated compared with the controls.However,there was no significant changes in the transcript level of SpToll3-9 in hepatopancreas.Compared with the results of expression profiles upon V.alginolyticus challenges,we found that both bacteria could significantly induce SpToll9 expression in hemocytes and gills,and SpToll1 expression was significantly down-regulated in hemocytes;while SpToll6 expression was only stimulated after challenge with Gram-negative bacteria V.alginolyticus.SpToll7 only responded to the Gram-positive bacteria S.aureus.SpToll3 and SpToll5 showed no significant changes in both bacteria infected crabs,that is,they did not response to infection of these two bacteria.In addition,both bacteria could inhibit the expression of SpToll1 in hemocytes.After S.aureus infection,there was no significant changes in the transcript level of Dorsal and Myd88,which are considered to be the key factors in Toll signaling pathway.But the expression of AMPs such as ALF and Crustin were significantly changed.The above results indicated that the regulatory pathways of the immune response of AMPs against pathogen infection is not limited to the signaling pathway involved by SpTolls,and there may also have other regulatory mechanisms.