Study On Immune Related Genes Of Crayfish And Crabs

The Chinese mitten crab(Eriocheir sinensis) as one of the commercially important crab, also was known as the fresh water crab、hairy crab and so on, belongs to Athropoda, Malacostraca, Decapoda, Grapsidae, Eriocheir, Eriocheir sinensis. The Chinese mitten crab not only is prestigious food, but also has better nutritive value and raising value because of its rich-nutrition and delicious taste. The Red claw crayfish (Cherax quadricarinatus), that also is known as the Australian freshwater lobster or red claw crayfish, belongs to Crustacea, Decapoda, Parastacidae. Cherax quadricarinatus, that is native to Australia and introduced into China in1991for the first time. It is breeding successfully and widely cultured in the next year because of its fast growth, delicious taste, high yield and adaptability. With the development of the intensive aquaculture and the enlargement of the cultivation scale, the frequent outbreaks of diseases have caused decreased production and catastrophic losses in the past decade. Crustacean can induce rapid and effective immune responses to clear the intruding pathogens relying largely on innate immunity, which attracts more and more attention. Upon this backdrop, studying the structure and transcriptional responses of potential immune-related genes may facilitate a better understanding of the crustacean immune defense and recognition mechanisms and support the sustainable development of better disease management strategies in the crustacean farming industry.The main research contents and results are as follows:1) Two novel Toll genes(EsTolll and EsToll1) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosanTolls/Toll-like receptors (TLRs) play an essential role in initiating innate immune responses against pathogens and are found throughout the insect kingdom but have not yet been reported in the crustacean, Eriocheir sinensis. For this purpose, we cloned two novel Toll genes from E. sinensis, EsTolll and EsToll2. The full-length cDNA of EsTolll was3,963bp with a3,042-bp open reading frame (ORF) encoding a1,013-amino acid protein. The extracellular domain of this protein contains17leucine-rich repeats (LRRs) and a139-residue cytoplasmic Toll/interleukin-1receptor (TIR) domain. The cDNA full-length of EsToll2was4,419bp with a2,667-bp ORF encoding an888-amino acid protein with an extracellular domain containing10LRRs and a139-residue cytoplasmic TIR domain. By phylogenetic analysis, EsTolll and EsTolll clustered into one group together with Tolls from other crustaceans. Quantitative RT-PCR analysis demonstrated that a) both EsTolll and EsToll2were constitutively expressed in all tested crab tissues; b) EsTolll and EsToll2were differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsToll2expression was significantly upregulated at almost all time intervals post-challenge with LPS, PG and GLU. Our study indicated that EsTolll and EsToll2are differentially inducibility in response to various PAMPs, suggesting their involvement in a specific innate immune recognition mechanism in E. sinensis.2) Molecular cloning and expression analysis of Tube from Eriocheir sinensisAs a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but have not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was2,247-bp with a1,539-bp open reading frame (ORF) encoding a512-amino acid protein. The protein contained a116-residue death domain (DD) at its N-terminus and a272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in ovary and testis, and moderately expressed in thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2(November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (β-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis.3) Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensisDorsal as a crucial component of Toll signaling pathway, played important roles in induction and regulation of innate immune responses. In this study, we cloned a NF-κB-like transcription factor Dorsal from Eriocheir sinensis and designated it as EsDorsal. The full-length cDNA of EsDorsal was2,493bp with a2,022-bp open reading frame (ORF) encoding a673-amino acid protein. This protein contained a171-residue conserved Rel homology domain (RHD) and a102-residue Ig-like, plexins and transcription factors domain (IPT). By phylogenetic analysis, EsDorsal was clustered into one group together with other invertebrate Dorsals or NF-κBs, and then clustered with vertebrate NF-κBs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that a) EsDorsal had higher expression level in immune organs; b) EsDorsal differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsDorsal was more responsive to LPS than GLU and PG. Collectively, EsDorsal was differentially inducibility in response to various PAMPs, suggesting its involvement in a specific innate immune regulation in E. sinensis.4) A novel Crustin involves in antibacterial responses in the red claw crayfish, Cherax quadricarinatus Antimicrobial peptides are important immune effectors and play important roles in mediating innate immune responses against intruding pathogens. Here, we successfully isolated and characterized a novel Crustin from Red claw crayfish Cherax quadricarinatus and named it as CqCrustin. The deduced amino acid sequence of CqCrustin exhibited the typical configuration similar to other crustacean Crustin orthologs, including one signal peptide region at N-terminus between1and16and a long whey acidic protein domain (WAP domain) at C-terminus between60and107along with a WAP-type "four-disulfide core" motif. Phylogenetic analysis showed that CqCrustin was clustered with other crustacean Type Ⅰ Crustins firstly, and then with other crustacean Type Ⅱ Crustins, finally with other crustacean Type Ⅲ Crustins. CqCrustin showed higher sequence similarity (69%) with Crustin2from Procambarus clarkii (Pc-Crustin2). Transcription of CqCrustin-Ⅰ were both1) detected in all tissues, especially in hemocytes and gill; b) differentially induced in hemocytes post β-1,3-glucans (GLU), lipopolysaccharides (LPS) and peptidoglycans (PG) injection at selected time points. To understand its biological activity, the recombinant CqCrustin protein was constructed and expressed in Escherichia coli BL21(DE3). Recombinant protein rCqCrustin exhibited distinct bacterial binding activities against different Gram-positive bacteria, Gram-negative bacteria and fungus. Furthermore, bacterial growth inhibition assays demonstrated that rCqCrustin responds positively to the growth inhibition of different Gram-positive bacteria, Gram-negative bacteria and fungus. These findings suggested CqCrustin may involve in a specific innate immune recognition and defense mechanism against bacteria and fungus in Cherax quadricarinatus.5) Identification and characterization of Dscam isoforms isolated from the red claw crayfish Cherax quadricarinatusThe Down syndrome cell adhesion molecule (Dscam) belongs to the immunoglobulin superfamily (IgSF) member and has been identified and isolated from some vertebrates and invertebrates. Recently, many studies have confirmed the important role of Dscam in mediating innate immune response against intruding pathogens and wiring of the nerve system in invertebrates, especially in Arthropod. Here, we successfully isolated and characterized the Dscam from Red claw crayfish Cherax quadricarinatus and named it as CqDscam. The deduced amino acid sequence of CqDscam exhibited the typical configuration similar to other invertebrate Dscam orthologs, including one signal peptide,10immunoglobulin (Ig) domains,6fibronectin type III (FNIII) domains and one transmembrane (TM) domain and cytoplasmic tail domain. Phylogenetic analysis showed that CqDscam was clustered with other invertebrate Dscams firstly, and then with vertebrate Dscams. In the extracellular region, the variable regions of CqDscam were located in N-terminal half of Ig2and Ig3domains and the complete Ig7domain. The CqDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events and could generate more than37,620different unique isoforms. Transcription of CqDscam were both1) detected in all tissues, especially in immune tissues and nerve tissues; b) differentially induced in hemocytes post glucans (GLU), lipopolysaccharides (LPS) and peptidoglycans (PG) injection at selected time points. Importantly, we had detected membrane-bound and secreted Dscam isoforms in C. quadricarinatus and showed that secreted isoforms were extensively transcribed post different PAMPs challenge, respectively. Results from immuno-localization assay revealed that CqDscam evenly distributed in the cell surface of hemocytes. In addition, the PAMPs specific isoforms of CqDscam were shown to be associated with bacterial clearance and phagocytosis in Red claw crayfish. These findings suggested CqDscam may involve in a specific innate immune recognition and defense mechanism in Cherax quadricarinatus.