Study Of Immune Responses In Aedes Aegypti And Culex Quinquefasciatus Against Romanomermis Wuchangensis

Insect immunity is an important defense mechanism against invasion by pathogens.Insect innate immunity is mainly composed of humoral immunity and cellular immunity,and the Toll-Spatzle and Imd(immune deficiency)pathways are the key components of humoral immunity to regulate expression of many effector genes,including antimicrobial peptide(AMP)genes.Insects can recognize different pathogens to activate the Toll and Imd pathways,such "Non-self" recognition is accomplished by pattern recognition receptors(PRRs)that can recognize pathogen-associated molecular patterns(PAMPs).The Toll and Imd signaling pathways as well as PRRs have been well investigated in the fruit fly Drosophila melanogaster,with a few studies in mosquitoes.Mosquitoes are major vectors for several deadly diseases in humans,and the nematode Romanomermis wuchangensis can infect mosquito larvae and may serve as a biological control agent for mosquito control.However,little is known about whether mosquitoes can recognize R.wuchangensis,which mosquito PRRs are involved in the recognition process,and whether R.wuchangensis infection can activate immune responses in mosquitoes.In this study,we used the mosquitoes Aedes aegypti and Culex quinquefasciatus with R.wuchangensis to investigate mosquito immune responses activated by R.wuchangensis,mosquito C-type lectins(CTLs)as PRRs for recognition of R.wuchangensis,and CTLs as co-receptors to interact with PGRP-LC in the Imd pathway.In this study,samples of A.aegypti and C.quinquefasciatus were collected at 2,4,6 and 8 days with and without infection by R.wuchangensis.qRT-PCR was used to analyze the expression of PRRs,key genes in the Toll and Imd pathways as well as antimicrobial peptide genes after R.wuchangensis infection.Recombinant AaCTLGA5 and CqCTL12 were expressed in bacteria and purified,and purified recombinant CTL proteins were used for binding to R.wuchangensis.Recombinant AaCTLGA5,CqCTL 12,AaPGRP-LC and CqPGRP-LC ectodomains were expressed in Drosophila S2 cells,and co-immuneprecipitation assays were performed to study interactions between AaCTLGA5 and AaPGRP-LC,CqCTL12 and CqPGRP-LC.The following results were obtained:(1)When mosquitoes were infected by R.wuchangensis,the expression of most A.aegypti AMP genes,including AaAttB,AaDefA,AaDefB,AaCecA and AaCecB,was significantly up-regulated compared to the non-infection control groups.Expression of C.quinquefasciatus CqAtt and CqCec was significantly up-regulated compared to the control groups,but expression of CqCecA and CqDef was significantly down-regulated.(2)When mosquitoes were infected by R.wuchangensis,expression of A.aegypti AaCTLGA5 and AaCTLMA15 genes at 2,4 and 6 days post-infection was significantly down-regulated compared to the control groups,but was significantly up-regulated at 8 days post-infection;expression of AaCTL24 and AaGNBP3 genes was significantly down-regulated compared to the control group.Expression of C.quinquefasciatus CqCTL2 and CqCTL24 was significantly up-regulated at 6 and 8 days post-infection,respectively;expression of CqCTL12 was significantly down-regulated,while expression of CqCTLMA15 did not change significantly compared to the control groups except for 8 days post-infection.(3)When mosquitoes were infected by R.wuchangensis,expression of A.aegypti AaPGRP-LC and AaPGRP-S 1 genes at 4 days post-infection was significantly down-regulated,expression of AaPGRP-LE and AaPGRP-SC2 was significantly up-regulated at 4 days post-infection but down-regulated at 6 days post-infection.Expression of C.quinquefasciatus CqPGRP-LC was significantly down-regulated only at 4 days post-infection,and CqPGRP-LE was significantly down-regulated except for 6 days post-infection.Expression of CqPGRP-S1 and CqPGRP-S6 genes were significantly down-regulated at 4 and 6 days post-infection.(4)When A.aegypti larvae were infected with R.wuchangensis,expression of AaToll-1A and AaImd genes was up-regulated to 80-100 folds at 8 and 6 days post-infection,respectively,suggesting that both the Toll and Imd pathways are involved in defense against R.wuchangensis infection.(5)Recombinant AaCTLGA5 and CqCTL12 proteins were expressed in bacteria and purified.In vitro binding assays showed that both purified recombinant AaCTLGA5 and CqCTL12 proteins bound to R.wuchangensis.(6)AaCTLGA5,CqCTL 12,AaPGRP-LC and CqPGRP-LC ectodomains were expressed in Drosophila S2 cells.Co-immunoprecipitation assays showed that AaPGRP-LC can interact with AaCTLGA5,and CqPGRP-LC can interact with CqCTL12.