Biological Analysis Of Clip Domain Serine Proteases Gene Of Aedes Aegypti And Their Function In Innate Immunity

Clip domain serine proteases(CLIPs),a very diverse group of proteolytic enzymes,play a crucial role in insect innate immunity.The innate immune response is the first barrier for mosquitoes to fight against the invasion of pathogenic microorganisms,in which Toll pathway,immunodeficiency(IMD)pathway and the melanin-forming process are the main means of innate immunity of Aedes aegypti.In Ae.aegypti,CLIPs are classified into five subfamilies(CLIPA,CLIPB,CLIPC,CLIPD,and CLIPE)based on their sequence specificity and phylogenetic relationship.In this study,the similarity of CLIPs was analyzed by Clustal Omega,an online software.Using SOPMA to predict their secondary structures.Using MEGA 7 software to construct a phylogenetic tree.By q RT-PCR,the expression levels of CLIPs in different tissues(Malpighian tubule,midgut,ovary,fat body and thoax)of adult female mosquitoes and the various stages(larva,female,male)was studied,and the induction kinetics of CLIPs to different bacterial infections was studied by infecting female adult mosquitoes.To determine whether CLIPB15 and CLIPB22 respond to microbial challenge and tissue injury,RNA interference(RNAi)was performed to explore their functions in defense against bacterial and fungal infections of Ae.aegypti.In addition,the expression levels of NF-κB transcription factors REL1 and REL2 in Toll pathway and IMD pathway It were investigated after bacterial infection.Finally,changes of phenoloxidase(PO)activities were investigated in the CLIPB15 and CLIPB22 knockdown adults.Eight CLIPs genes of Ae.aegypti were obtained from Gen Bank database of national center for biotechnology information.Through bioinformatics analysis,it was found that the amino acid sequences of the eight CLIPs had high similarity in the carboxyl terminal protease domain,and random curl accounted for a high proportion in the secondary structure of these proteins.The phylogenetic analysis showed that there were three different branches of the eight CLIPs genes,one branch had high homology with PPAF1 in Anopheles gambiae,one had high homology with two CLIPs in An.gambiae,and the other had high homology with the SP5 of Drosophila melanogaster.The temporal and spatial expression profiles showed that the expression levels of the six CLIPs genes in Ae.aegypti were abundant in the4 th instar larvae and adults,and low in pupae.The expression levels were similar in different tissues of female adult mosquitoes,with high expression levels in thorax and fat body,but low in Malpighian tubule,midgut and ovary.In bacterial infection experiments,the six CLIPs genes had different induction kinetics,suggesting that CLIPs in Ae.aegypti has a specific response to microbial infection.The m RNA levels for CLIPB15 and CLIPB22 were also up-regulated by the fungus,Beauveria bassiana infections as well as in the tissue injury experiments.RNAi-mediated knockdown of CLIPB15 showed a significant decrease of PO activity in the hemolymph of Ae.aegypti,while RNAi experiments revealed that both genes were involved in immune defense against bacterial and fungal infections.At the same time,the m RNA expression of NF-κB transcription factors REL1 and REL2 in Toll pathway and IMD pathway was different between knocked down and wild-type mosquitoes after infected with bacteria.In conclusion,the functions of eight CLIPs genes of Ae.aegypti are mainly related to the innate immunity.CLIPB15 and CLIPB22 affect the ability of adults to fight against microbial infection,and the Toll and melanization pathways to a certain extent.