| As a kind of effector molecule in crustacean immune response,antimicrobial peptides(AMPs)play important roles in innate immunity.Anti-lipopolysaccharide factor(ALF)as one member of AMPs,has received much more attention for its broad-spectrum activities against bacteria and virus.The prawn Exopalaemon carinicauda is deemed to be a model animal in innate immune research of crustacean,for its short breeding cycle,vulnerable to bacterial and viral infection,easy to operate in experiment.In this study,we used Exopalaemon carinicauda as a model animal,isolated and identified different transcripts of ALFs in E.carinicauda.We analyzed their tissue expression profiles,and investigated their functions.The present study laid a foundation for Exopalaemon carinicauda to be a model animal in drug discovery research.The main progresses are as follows:1.Different isoforms of EcALF showed different expression profiles.EcALF1,EcALF3,EcALF5 were expressed in all the tissues detected in present study,and showed the highest expression level in hemocytes,high expression was also detected in gill and epidermis.EcALF2 showed the highest expression in stomach,high expression was also detected in epidermis.EcALF4 showed the highest expression in epidermis,high expression was also detected in stomach,gill,and hemocyte.EcALF6 showed the highest expression in gill,high expression was also detected in epidermis and stomach.In summary,EcALFs are mainly expressed in hemocytes,stomach,gill,and epidermis.Hemocytes are essential for crustacean immune system and play important roles in recognizing the pathogens and in phagocytosis.Gill is the organ which directly contacts with environmental pathogens.Stomach is a target tissue for WSSV infection.Epidermis is one of the physical barrier in shrimp.The high expression level of EcALFs in these tissues indicated their important role in immunity.2.The peptide,Ec LBDs,synthesized based on the sequence of the LBD domain of EcALFs,showed antibacterial activity against some Gram-negative and Gram-positive bacteria.EcALF1,EcALF2,and EcALF3 showed broad-spectrum activity.The MIC ranges of EcLBD1 for Gram-negative bacteria V.alginolyticus and V.harveyi were 2-4?M and 16-32?M,respectively,and it was 4-8?M for Gram-positive bacteria M.luteus;.The MIC ranges of Ec LBD2 for Gram-positive bacteria S.epidermidis and M.luteus were 16-32?M and 8-16?M,and it was 4-8?M for negative bacteria V.alginolyticus.The MIC ranges of Ec LBD3 for Gram-negative bacteria V.harveyi and P.damselae were 8-16?M and 32-64?M,respectively,and it was 32-64?M for Gram-positive bacteria M.luteus.Different isoforms of Ec ALF with different antibacterial activity indicated that EcALFs might act to defense pathogens synergistically.3.The antiviral antivities of different EcALFs were detected through injecting WSSV pre-incubated with the peptide of Ec LBDs into the prwan and the WSSV copy numbers were recorded.After pre-incubating with Ec LBD1,Ec LBD2,Ec LBD3,Ec LBD4,Ec LBD5,the replication of WSSV in vivo was reduced significantly,which indicated the strong antiviral activity of Ec ALF,while no variation of WSSV copy number in the prawn was detected for Ec LBD6 incubation group.4.In order to study the function of EcALFs in E.carinicauda,ds RNA mediated RNA interference was performed on living prawn.The result showed that after different Ec ALF was silenced,the prawn became unhealthy.Comparing with the EGFP ds RNA injected prawn and untreated prawn,the amount of bacteria in hepatopancreas of Ec ALF-silenced prawn was increased in different level.The amount of bacteria in Ec ALF1,Ec ALF3,Ec ALF4,Ec ALF5,Ec ALF6-silenced prawn was 38-fold,66-fold,78.6-fold,75-fold,and 42.7-fold higher than that in the prawn injected with EGFP ds RNA.After one kind of Ec ALF was silenced,the expression level of other EcALFs changed remarkably,and most of EcALFs expression level were down-regulated.The increase of bacteria number in prawn were probably caused by the decreased expression level EcALFs. |
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