Investigation Of Embryonic Immunity And Key Molecules Of Lectin Pathway In Portunus Trituberculatus

At present,most research focuses on defenses of adult crabs.However,few studies were conducted to elucidate how the developing embryos protect themselves from pathogen attacks before the immune system is fully mature.This study reported the immune function of the early embryos of Portunus trituberculatus,an important aquaculture crab in China,and explored its immune mechanism.The results showed that the extracts of fertilized egg from P.trituberculatus had significant inhibitory effects on the growth of Gram-negative bacteria Vibrio alginolyticus,Gram-positive bacteria Micrococcus luteus,Staphylococcus aureus and Pichia pastoris,but not on Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli.The protein extracts from fertilized eggs of P.trituberculatus had obvious agglutinating and damaging effect on V.alginolyticus,E.coli and P.pastoris,but had no agglutinating and damaging effects on P.aeruginosa,M.luteus and S.aureus.The activity of lysozyme and Cu/Zn-SOD increased with the development of embryo.ELISA results showed that the C3 and C4,key molecules of complement system,existed in fertilized eggs.Different from the above enzymatic activity experiments,C3 and C4 decreased first and then increased with the development of embryos,and the content of fertilized eggs and cleavage stage was significantly higher than that of blastocyst stage.It is speculated that C3 and C4 may be important maternal immune molecules.The activation of complement agglutinin pathway requires the participation of Ca²⁺.Metal ion chelating experiments show that the antimicrobial activity of protein extracts of crab decreased significantly after adding Ca²⁺chelating agent,while the antimicrobial activity of protein extracts was restored after supplementing sufficient Ca²⁺.On the contrary,the antimicrobial activity of crab fertilized oocyte protein extracts could not be restored by supplementing Mg²⁺by alternative inhibition pathway.The results showed that the protein extracts of P.trituberculatus had inhibiting microbial growth,agglutination and bacterial damage activities.Lysozyme,Cu/Zn-SOD and complement lectin pathways played an important role in the immune protection of early embryos.The key molecules of the complement lectin pathway,mannose-binding lectin?MBL?and ficolin,are key model recognition receptors?PRRs?that play an important role in the innate immune response.In this study,a new mannose-binding lectin?designated as PtMBL?and a new ficolin?designated as Ptficolin?were cloned from P.trituberculatus.The complete cDNA of PtMBL gene was 1,208 bp in length with an open reading frame?ORF?of 732 bp that encoded 244 amino acid proteins.PtMBL shared lower amino acid similarity with other MBLs,yet it contained the conserved carbohydrate-recognition domain?CRD?domain with QPD motif and was clearly member of the collectin family.PtMBL transcripts were mainly detected in eyestalk and gills.The temporal expression of PtMBL in hemocytes showed different activation times after challenged with Vibrio alginolyticus,Micrococcus luteus and Pichia pastoris.The recombinant PtMBL protein revealed antimicrobial activity against the tested Gram-negative and Gram-positive bacteria.It could also bind and agglutinate(Ca²⁺-dependent)both bacteria and yeast.Furthermore,the agglutinating activity could be inhibited by both D-galactose and D-mannose,suggesting the broader pathogen-associated molecular patterns?PAMPs?recognition spectrum of PtMBL.The complete cDNA of Ptficolin gene was 2,183 bp in length with an open reading frame?ORF?of 963 bp that encoded 321 amino acid proteins.Ptficolin shared lower amino acid similarity with other invertebrates ficolins,yet it contained the conserved FBG domain.Ptficolin transcripts were mainly detected in hepatopancreas,eyestalk and thoracic ganglia.The temporal expression of Ptficolin in hemocytes showed rapid activation after challenged with V.alginolyticus,M.luteus and P.pastoris.These results together indicate that PtMBL and Ptficolin could serve as not only a PRR in immune recognition but also a potential antbacterial protein in the innate immune response of crab.