Expression And Functional Analysis Of Immune Genes In Culex Quinquefasciatus Infected By Romanomermis Wuchangensis

Mosquitoes, the vector of many pathogens and parasites, can cause serious infectious diseases including malaria, dengue fever and lymphatic filariasis, and is an important public health pest. Romanomermis wuchangensis can parasitise and kill more than ten kinds of Culex and Aedes larvae, and is considered as a promising biological pesticide. R. wuchangensis infections will cause a series of immune responses of its host which are involved in many kinds of molecules such as pattern recognition receptors (PRRs). Insect through PRRs recognize and bind pathogen associated molecular patterns of pathogens (PAMPs) to activate innate immune responses. C type lectins (CTLs), as one of the important PRRs, have a variety of functions and play an important role in the identification and removal of pathogens. In this research, survival rate of Culex quinquefasciatus infected with R. wuchangensis was analyzed, and using qRT-PCR analyzed relative mRNA expression levels of 19 immune genes in different stage larvae of C. quinquefasciatus. These 19 genes include PRRs genes—CTL12 (C-type lectin 12), PGRPLC (Peptidoglycan recognition protein LC), PGRPLE(Peptidoglycan recognition protein LE), PGRPS1(Peptidoglycan recognition protein S1) and PGRPS6 (Peptidoglycan recognition protein S6); genes in Toll pathway—TOLL9A and CACT (Cactus); genes in IMD (Immune deficiency) pathway—IMD (Immune deficiency) and REL1A (Relish 1A); genes in JAK/STAT (Janus kinase/signal transducer and activator transcription) pathway—DOME (Domeless), HOP (Hopscotch) and STAT1A (Signal transducer and activator of transcription 1A);genes in coagulationm and melanization pathway—SRPN6 (Serine proteinase inhibitor 6) and CLIPC2 (Clip-domain serine protease C2); immune effector molecule genes—PPO2 (Prophenoloxidase 2), PPO5 (Prophenoloxidase 5) and CECA2 (Cecropin 2); and genes in autophagy pathway—ATG5 (Autophagy-related protein 5) and ATG8 (Autophagy-related protein 8). We screened out the immune gene CTL12 whose relative mRNA expression level was significantly down regulated and obtained its recombinant protein by prokaryotic expression. The functional experiment of recombinant protein rCqCTL12 in vitro were carried out, which preliminarily studied the function and role of CqCTL12 gene in immune response of C. quinquefasciatus against bacteria and R. wuchangensis. The results are as follows:1. Survival rate analysis of C. quinquefasciatus infected with R. wuchangensis showed that survival rate of treatment group larvae was significantly lower than that in the control group after 6 days (p<0.001), indicating that R. wuchangensis has a high lethal rate to C. quinquefasciatus.2. Expression analysis of immune genes in different stages of C. quinquefasciatus infected with R. wuchangensis showed that CTL12 and PGRPLC were significantly down regulated expression (p<0.05) in initial stages (2 days,4 days) of nematode parasitism, suggesting that they may play an important role in immune response of C. quinquefasciatus against R. wuchangensis. While ATG5 and ATG8 were significantly up regulated expression (p<0.05) in late stages (6 days) of nematode parasitism, inferring that at this time a large number of autophagy phenomenon was occuring in vivo of C. quinquefasciatus.3. Sequence analysis of CqCTL12 and CqATG8 genes showed that CqCTL12 gene contains 486 nucleotides, encodes 162 amino acids, protein molecular weight is about 16.04kDa and contains a typical CRD domain. The CRD domain contains 4 conserved cysteine and 1 Ca2+ binding site which contains a QLD motif binding to galactose and a WND motif associated with Ca2+dependent activity. The length of CqATG8 gene is 357bp, encodes 118 amino acids and its protein molecular weight is about 14.056kDa.4. Recombinant protein rCqCTL12 and rCqATG8 were obtained by prokaryotic expression and purification. Agglutination assay of rCqCTL12 toward bacteria showed that rCqCTL12 could significantly aggregate Escherichia coli and Staphylococcus aureus and has a typical Ca2+ dependent effect.5. Binding assay of rCqCTL12 toward nematodes showed that rCqCTL12 could bind to both living and formaldehyde-killed nematodes and this combination has nothing to do with the existence of Ca2+ or not.The above results suggest that many immune genes of C. quinquefasciatus such as CqCTL12 play an important role in the process of resisting the invasion of nematodes and bacteria. The research results provide a basis for the subsequent application of R. wuchangensis in biological control of sanitary mosquito pests.