Molecular Basis Of Immunity In Embryonic And Larval Stages Of Scylla Paramamosain And The Immune Response Of Larvae To Pathogen Infection

The economical species Scylla paramamosain has been cultured in mainland and Taiwan of China for many years.For the mud crab aquaculture,one of the most critical puzzles hindering the healthy development of crab breeding at early development stages is of the high mortality during larva development and this serious problem has been suspending without any effective solution.The main cause of mass death of crabs during larva development is thought to be relative to the infection caused by pathogenic microorganisms such as bacterium,fungi or virus.Thus,it is urgent to clarify the immunologic features and mechanism of crabs at early development stages and the relationship between the development of immunologic mechanism and the high mortality,by which the bottleneck for crab breeding will be hopefully solved.In the study,the transcriptome and proteome techniques were used to reveal the bio-information of immune-associated genes or proteins and involved signaling pathways present in the normal early developmental stages of mud crab,including several stages of embryo,several stages of zoea,megalopa and juvenile crab.In addition,the transcriptome analyses were also applied for screening and identification of the up-or down-regulated genes at larval stages of zoea I stage,megalopa and the first stage of juvenile crab followed by the challenges with Vibrio alginolyticus or LPS.Totally,we constructed one transcriptome database of embryogenesis,one transcriptome database of larval developmental stages and three bacterial infection databases.The main results are as follows:1.This is the first project covered all early developmental stages of S.paramamosain.In this project,we separated samples into five embryo stages including embryol stage,embryo2 stage,embryo3 stage,embryo4 stage.Zoea stages(Z1-Z5),megalopa and the first stage of juvenile crab were also collected.The zoeaI stage larva,megalopa and the first stage of juvenile crab were under the challenge of LPS or V.alginolyticus2.To obtain a general view of the mud crab early developmental stages,we de novo assembled a cDNA library with an equal mixture RNA isolated from each embryo development stages.Assembly of the transcriptome resulted in 77,643 unigenes of which 23,103 were annotated in the embryo cDNA library with a N50 of 1,465 bp.Then we assembled ovary,each embryo stage,each zoea stage,megalopa and the first stage of crab,mapped them back to the reference databases as mentioned above for expression annotation in different transcripts.Totally,we got 9.42 G data,which obtained 116,370 transcripts and 77,643 unigenes.There were 16,170 unigenes annotated in the NR database,18,556 unigenes in PFAM database and 19,164 unigenes in GO database.3.We performed a proteome analysis on different embryo developmental stages and zoea I stage.Using the iTRAQ technique,we totally obtained 33 8,059 spectra.Through Mascot analysis,16362 spectra were matched,including 6593 unique spectra and 642 identified proteins.In addition,there are 1.14%of genes associated with immune under GO classification annotation.4.We de novo assembled a cDNA library with an equal mixture RNA isolated from each larva stage as reference databases.Assembly of the transcriptome resulted in 418,297 unigenes in larva cDNA library with a N50 of 1,310 bp.Totally,we got 142.94 G data,which obtained 39,293,0129 transcripts and357,535,604 unigenes.There were 85,908unigenes annotated in the NR database,96,993 unigenes in PFAM database and 98,091 unigenes in GO database.5.Transcriptome analysis implicated that there’re four important immune related system existing in early developmental stages of mud crab.Totally,122 transcripts of 44 immune associated genes and 267 transcripts of 56 immune associated genes were manually identified according to annotated sequences against embryo developmental stages database and larval developmental stages database,respectively.It is indicated that different immune associated genes such as AMPs,pathogen recognition receptors and genes involved in proPO cascade,Toll signaling pathway,IMD pathway,complement system and clotting system,play a significant role in early developmental stages of S.paramamosain.6.Revealing the expressions patterns of antimicrobial peptides in embryonic and larval stages.In embryonic stages,there are crustin,ALF,arasin,carcinin,hyastatin,scygonadin and lysozyme.In larval stages,there are crustin,ALF,arasin,carcinin,hyastatin and lysozyme.It is found that scygonadin has the highest expression level in the early embryonic stage,while crustin,ALF and hyastatin highly expressed in later developmental stages.It is indicated that different AMPs would express in different developmental stages to combat the pathogen challenge.Meanwhile,as mud crab developed the quantity of AMPs transcripts increased.Even though the kind of AMPs decrease,the expression level of transcripts increase.It is indicated that since the short of physical protection of egg envelope,mud crab might need a great amount of AMPs to defend itself from the outside attack.7.Revealing the existence of Toll signaling pathway and IMD pathway in early developmental stages of mud crab.In embryonic stages there are Spatzle,Toll,Pelle,TRAF6,Cactus and Dorsal involving in the Toll signaling pathway,while in larval stages there are Spatzle,Toll,MyD88,Pelle,Tube,TRAF6,Cactus,Dorsal and Deaf.It is found that IAP,IKKE,IKKβ and Relish involving in the IMD pathway in the embryonic stages.In larval stages there are IMD,IAP,IKKe,IBKKβ,Dredd and Relish.It is indicated that these two pathways might involve in the regulation of AMPs and other immune factors.In embryo stages,Toll/IMD and Jak/Stat signaling pathway could be detected with a few key components missing,whereas,in larvae stages,most of homologs to the components in these pathways of Drosophila were identified.8.Revealing the existence of proPO cascade in early developmental stages of mud crab,including ppaf,SPH,LGBP,ppae,pacifastin,serpin,serine proteinase,a2m,peroxinectin and QM.LGBP,pacifastin and ppae were firstly identified in mud crab.The components in proPO cascade of embryo stages exhibit high similarity to the components in larvae stages.Besides,these factors show a high expression level in the early embryonic stages,which indicate that proPO cascade play a more important role in the early embryonic stage.Interestingly,these key factors might be maternal transfer.9.108 DGE libraries of zoea I,megalopa and juvenile crab after V.alginolyticus challenge were established.Totally,we got 654.33 G clean reads data.After the challenge,there are 8;356,4,665 and 12,195 differentially expressed genes in zoea I,megalopa and juvenile crab,respectively.10.108 DGE libraries of zoea I,megalopa and juvenile crab after LPS challenge were established.Totally,we got 644.86 G clean reads data.After the LPS challenge,there are 2,117,4,725 and 9,937 differentially expressed genes in zoea I,megalopa and juvenile crab,respectively.11.Comparing the transcriptome data under LPS or V.alginolyticus challenge,there were huge amount of genes differentially expressed between these two test groups.Under the LPS challenge,there were fewer immune related pathways and factors specially expressed in zoea I,megalopa and juvenile crab,including proPO in zoea I stage,TAK1 and galectin in megalopa,pellino and ppae in juvenile crab.Under the V.alginolyticus challenge,there are more immune related genes and factors specially expressed in zoea I,megalopa and juvenile crab,including ppaf,peroxinectin,MyD88,TRAF6,Dorsal and C-type lectin in zoea I stage,MyD88 and Dorsal in megalopa stage,arasin,Spatzle,MyD88,IRAK4,TAK1,a2m,peroxinectin and Techylectin in juvenile crab stage.There were immune related genes and factors under both LPS and V.alginolyticus challenge,including ALF,crustin,Dscam,SCR,TLR,pacifastin,a2m and serpin in zoea I stage;ALF,arasin,crustin,TLR,Spatzle,Relish,pacifastin,serpin,ppaf,ppae,a2m,Dscam,SCR and C-type lectin in megalopa stage;ALF;crustin,TLR,Pelle,IRAK1,Dorsal,Reilsh,serpin,pacifastin,SCR,C-type lectin,Dscam and mannose-binding protein in juvenile crab.It is indicated that mud crab posseses a solid immune system in early developmental stages to combat different challenge.12.Determination of the genomic sequence of SpLGBP.The full-length 1092 bp transcript of SpLGBP with a 364-amino acid open reading frame(ORF).It is predicted that the molecular weight of SpLGBP is 41.41 kDa and the isoelectric point is 4.5.SpLGBP contains two integrin domain RGD(Arg-Gly-Asp),two potential glycosylation sites NRS and NDS and a kinase C phosphorylation sites of SAR.In addition,there’s a 16 glycoside hydrolase family domain(Glyco hydro 16)structure from 79 to 262 amino acids.The recombinant SpLGBP was capable of binding different PAMPs such as LPS(G1)and LTA(G+).Through the microscopy,we noticed that the recombinant SpLGBP could agglutinate bacteria and fungus.