| Scylla paramamosain is the dominant species distributed and cultured in the southeastern coastal areas of China.At present,the mud crab aquaculture industry is facing the risk of multiple pathogen infections,which pose a serious threat to the sustainable and healthy development of the entire industry.The recurring diseases that often occurred during crab culturing,and the diseases caused by different bacteria are the main threat to crustacean aquaculture.However,due to the lack of a comprehensive understanding of mud crab immunity,It is difficult to develop effective measures to prevent and cure diseases based on immunological knowledge.Therefore,it is extraordinarily necessary to decode the genome information of S.paramamosain and uncover the innate immune characteristics of S.paramamosain based on genomics.Furthermore,sexual dimorphism in immunity has been reported in both males and females,due to the lack of genome information of non-model animals,most studies have used model animals to investigate the sexual dimorphism.To our knowledge,sexual dimorphism of the immune system has not been reported in crustaceans.In this study,high quality whole genome information of S.paramamosain was obtained through De novo sequencing,and the innate immune characteristics of S.paramamosain were uncovered based on genomics.The expression of genes in the 11 main organs and tissues of male and female in the natural state were compared and analyzed by using transcriptome technique,and at the same time,after the male and female crabs were challenged with V.alginolyticus and WSSV,receptively,and transcriptomics was used to analyze the expression of sex-biased in innate immunity of S.paramamosain under pathogen infection,thus revealing the dimorphism of the innate immunity in S.paramamosain.The main research results are as follows.1.The complete genome sequence of S.paramamosain was uncovered:Muscles from healthy adult female crabs were collected in 2016,the S.paramamosain genome was sequenced and assembled using the third-generation sequencing technology,and the second-generation sequencing data was used to correct the obtained genome assembly results.In addition,the assembled contigs/scaffolds sequence was mounted to the chromosome level using the H-iC technology to obtain the pseudo A genomewide map at the chromosome level of the mud crab.The genome size of S.paramamosain wass 1.2 Gbp,the total length of contig was 1,076,451,913 bp,the length of contig N50 was 3,558,762 bp;the total length of scaffold was 1,076,539,814 bp,the length of scaffold N50 was 21,897,065 bp,and the genome mount rate was 91.19%.The CEGMA evaluation results showed that the core eukaryotic gene(Core Eukaryotic Genes)assembly ratio of the S.paramamosain genome reached 94.76%;the BUSCO evaluation results showed that 93.9%of the complete copy genes were assembled by the orthologous single-copy gene assembly of the S.paramamosain genome.Both evaluation results showed that the consistency,completeness and accuracy of the S.paramamosain genome are high-quality.By genome annotation,the analysis results showed that the S.paramamosain genome contains 61.46%of repeat sequences,total of 30,350 genes were predicted,and 96.9%of them were predicted that they have biological function.In addition,phylogentic analysis showed that S.paremamosain and Litopenaeus vannamei are classified into the same phylogenetic branch,and the most recent common ancestor(MRCA)of the two species appeared approximately 416.7 million years ago(297.8-487.9).Ma).Further analysis revealed that S.paramamosain,L.vannamei,Daphnia pulex and 5 insects have a closer evolutionary relationship.These results were consistent with the conclusion that previous studies had shown that crustaceans and hexapods constituted a pancrustacea clade(Pancrustacea or Tetraconata),rather than the previously reported hexapod and polypod had a closer relationship,and they were all Atelocerata.2.Based on comparative genomic analysis,the expanded gene families in the genome of S.paramamosain was uncovered,which were closely related to the reproductive and development processes and environmental adaptation:Through comparative analysis with other 24 species including L.vannamei,D.pulex and D.melanogaster,etc.It was found that 19 gene families in the S.paramamosain genome had expanded,and these gene families were closely related to the physiological processes such as the growth and development,environmental adaptation and its reproduction.A total of seven gene families related to the growth and development,they are 36 BR-C genes,60 serine/threonine-protein kinase genes,4 G-protein coupled receptor Mth2-like genes,43 ankyrin repeat genes 18 peptidyl-prolyl cis-trans isomerase genes,4 male specific gene family testis determining factor genes and 13 putative JHE-like carboxylesterases genes.A total of seven gene families related to environmental adaptation,they are 25 neural-cadherin(N-cadherin)genes,5 neurotrimin-like genes,23 glutamate-gated chloride channel(glycine receptors)genes,106 ionotropic glutamate receptors(olfactory ionotropic receptor)genes,5 metabotropic glutamate receptors genes,24 actin genes and 19 CYP2 genes.A total of five gene families involved in reproductive development,they are 12 SP-10-like genes,5 Scygonadin genes,22 dual specificity testis-specific protein kinase 1 genes,4 vitellogenin genes and 6 ovary development-related protein genes.3.Based on the genome,the characteristics of innate immunity in S.paramamosain were uncovered:By analyzing all annotated genes in the S.paramamosain genome for the first time,692 immunity-related genes with annotable function were predicted.The corresponding genes were categorized into 40 different functional groups,and the number of each group ranged from 1 to 121 genes.Those genes include ten gene families related to pattern recognition receptors(198 genes in total),two gene families related to signal modulation(18 genes in total),gene families related to three signal transduction pathways(51 genes in total),17 gene families related to effector molecules(334 genes in total),three gene families related to cytokines related genes(23 genes in total)and five gene families related to the antioxidant system(68 genes in total).In the genome of the S.paramamosain,through comparison and analysis with the known gene sequences,prediction and analysis of conserved domains,comparison and annotation of PFAM,NR or Uniprot database,phylogenetic analysis and other methods,the immunity-related gene families in the four main immune response processes were analyzed and identified.we conformed 8 gene families of PRRs,they are 121CTLs genes,37 FREPs genes,17 SRs genes,4 DSCAM genes,1 GNBPs gene,8 TEPs genes and 2 Galectins genes,and at the same time,no PGRPs gene was identified;two gene families involved in signal modulation,they are 10 CLIP domain contained serine protease gene families and 8 serpins genes;a total of 51 genes were identified from gene families related to three signaling pathways,they are the 34 genes of the Toll signaling pathway,9 genes of the IMD signaling pathway and 8 genes of JAK/STAT pathway.Furthermore,in the S.paramamosain genome,a total of 5 gene families related to immune effectors were identified,they are 5 Scygonadin genes;a total number of 12 proPO genes,7 PPAE genes,3 proteinase inhibitor genes and 2 LGBP genes of the proPO system;the related genes of the complement system include 5 C1q genes,8 ficolin genes,3 Mannose-binding lectin(MBL)genes and 3 complement component genes of MCRs(mammalian c3-like molecules)related to macroglobulin complementation were identified.In contrast,the pattern recognition molecular genes MASP,protease factor B,and complement component genes C4-C9 were all lost;for the HSPs gene families,12 HSP70 genes,4 HSP90 genes,15 HSP60 genes,39 HSP40 genes and 14 HSP20 genes were identified.Compared S.paramamosain with A.gambiae,D.melanogaster D.pulex and L.vannamei,parts of immune-related gene families expanded in the genome of S.paramamosain and the other 2 crustaceans,including CTL,Sp?tzle and caspase genes.A total of 75 CTL genes,20 Sp?tzle genes and 17 caspase genes were identified in the S.paramamosain genome.In terms of immunity-related genes,in addition to crustaceans,S.paramamosain and A.gambiae also had some things in common.The A.gambiae genome encodes 57 FREP and 20 proPO genes,and the S.paramamosain genome encodes 43 FREP and 12 proPO genes.Compared with the other three pancrustaceans,the FREP and proPO gene families have expanded in the S.paramamosain and A.gambiae genome.In the S.paramamosain genome,gene contraction of PRRs were also found.There was only one gene copy of the Gramnegative binding protein(GNBP)in the genome.Analyses of the S.paramamosain genome and transcriptome results showed that there is no PGRP gene in the genome,and the PGRP gene had also been lost in the genome of D.pulex and L.vannamei.4.Based on genomics and histological transcriptome analysis,sexual dimorphism in innate immunity was found in both male and female crabs:The transcriptome analysis of the main tissues and organs of female and male mud crabs found that there were 23,372 differentially expressed genes in the gonads(T vs Ov)of male and female crabs.Compared with female crabs,there were 23,372 differentially expressed genes in male crabs.17,064 genes were down-regulated and 6,308 genes were down-regulated.The number of differentially expressed genes between male and female crabs in the other 10 tissues or organs was only 11-355.In female ovaries,97 immune-related genes were up-regulated compared with male testes,while in male testes,225 immune-related genes were up-regulated compared with female ovaries.On the contrary,only 2-22 immune-related DEGs were identified in other 10 tissues.Correlation analysis revealed the sex-biased expression characteristics of immunity-related genes in both male and female crabs,and the results showed that more than 90%of the differentially expressed genes were in the gonads.During mating,61 immunity-related genes were differentially expressed between mature and unmated ovaries,while only 9 immunity-related genes were differentially expressed between mated and unmated ovaries.The results of the distribution of immunity-related genes with sex-biased expression on chromosomes showed that 31 male-biased expression genes were mainly distributed on 10 chromosomes,and 14 female-biased expression genes were mainly distributed on 7 chromosomes5.Transcriptomics analysis of the sex-biased expression of differential genes in the gonads of female and male crabs artificially infected with V alginolyticus,the results further confirmed the sexual dimorphism in innate immunity of S.paramamosain:Transcriptomics analyses found that 24 immunity-related genes were significantly differentially expressed in female gland,of which 19 genes were upregulated and 5 genes were down-regulated.While in male gonad,31 immunity-related genes were significantly differentially expressed,including 24 up-regulated genes and 7 down-regulated genes.Compared with healthy individuals,when S.paramamosain were challenged with V.alginolyticus,12 immune effectors changed their expression levels within 3,6 and 12 hours after infection in the female glands,for example,the cactus gene and 2 genes belonging the proPO sytem changed their expression levels after 3 and 6h,one FREP and serpins genes were significantly differential expression after 6h.In contrast,in male gonads,28 immunity-related genes were not significantly induced until 24 hours after infection.Only 5 genes were significantly differentially expressed both in male and female gonads.When the ovary-matured female and male adult S.paramamosain were challenged with V.alginolyticus,the survival rate of female crabs was significantly higher than that of male crabs(P-value<0.05).When S.paramamosain was challenged with white spot syndrome virus(WSSV),it was found that 64 immunity-related genes were induced to be significantly differentially expressed in female gonad,including 47 significantly up-regulated genes and 17 significantly down-regulated genes.While when the male crabs were challenged with WSSV,only 22 genes were induced to be significantly differentially expressed,and none of these genes were immunity-related genes.The results further support the existence of sexual dimorphism in the immune response to V.alginolyticus infection in female and male mud crabs. |
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