A Comparative Proteomic Study On Insecticide Resistance In The Mosquito Of Culex Pipiens Pallens

Mosquito-borne infectious diseases, such as malaria, dengue fever and yellow fever, have caused a serious threat to human health. Mosquito control is considered as an important element in the current global strategies for the control of major mosquito-borne diseases. The control of mosquito populations is often based on chemical insecticides because of its killing speed, long-lasting and convenient features. Unfortunately, long-term intensive and widespread use of insecticides has led to the development of insecticide resistance in important vector mosquito species, which has been the biggest single obstacle in the struggle against vector-borne diseases. It is a serious problem to be solved for researchers how to take a scientific and rational management measures for reducing or avoiding the appearance and development of insecticide resistance. It is very important to understand deeply the mechanism of insecticide resistance for providing a scientific theory of insecticide resistance management, and it is a prerequisite for insecticide resistance management to detect early and real-time monitor the insecticide resistance level in different field mosquito populations. With the development of modern biotechnology, a series of new technologies and methods especially in the field of proteomics research have emerged and been mature, and it is possible to screen out resistance-related proteins by proteomics methods, which is conducive to explain the mechanism of insecticide resistance, and to find some proteins used as markers of insecticide resistance for detecting the resistance level of field mosquito populations.Two lab strains of deltamethrin-sensitive (Lab-DS) and deltamethrin-resistant (Lab-DR) of Culex pipiens pallens were used in our study. We compared protein abundances in the life stage of 4th-instar larvae, pupae, male and female between the Lab-DS strain and Lab-DR strain by isobaric tags for relative and absolute quantitation (iTRAQ) coupled liquid chromatography/tandem mass spectrometric (LC-MS/MS) analysis. Two biological replicates were performed in present study. A total of 30 differentially expressed proteins were identified in the Lab-DR strain compared with the Lab-DS strain. Among them,15 proteins were up-regulated and 15 proteins were down-regulated in the Lab-DR strain. By GO and pathway analysis, it was showed that the differentially expressed proteins were involved in some processes, such as metabolism (including carbohydrate metabolism), oxidative phosphorlation and cuticle formation. Based on the biological function of the differentially expressed proteins, we compared the mRNA expression levels of CYP6AA9, ATP synthase B chain, prag01, CPIJ009715 and anamorsin in female adults between the Lab-DS strain and Lab-DR strain using quantitative real-time PCR method. The results showed that:(1) CYP6AA9, ATP synthase B chain and pragOl were highly expressed in the Lab-DR strain (P<0.05); (2) anamorsin was lowly expressed in the Lab-DR strain, but not statistically significant (P>0.05); (3) CPIJ009715 was highly expressed in the Lab-DR strain (P<0.001).Based on the results mentioned above, three candidate proteins of CYP6AA9, pragOl and CPIJ00971 were validated further by Western blot. Firstly, we separately designed and synthesised two peptides for CYP6AA9 and pragOl. Meanwhile, we purified recombinant proteins of CPIJ009715 expressed in bacteria. Then, the purified peptides and recombinant proteins were used as antigens to separately prepare the polyclonal antibodies of CYP6AA9, prag01 and CPIJ009715 successfully. We compared protein abundances of CYP6AA9, prag01 and CPIJ009715 in four different life stages between the Lab-DS strain and Lab-DR strain by Western blot. The results showed that:(1) CYP6AA9 was highly expressed in four different life stages of the Lab-DR strain compared with in the Lab-DS strain, especially in male and female adults; (2) CPIJ009715 was highly expressed in male adults of the Lab-DR strain compared with in the Lab-DS strain, no difference expression in 4th-instar larvae between the Lab-DS strain and Lab-DR strain, and not detected in pupae and female adults of both strains; (3)the expression of Prag01 was not detected in four different life stages of both strains.Based to the results mentioned above, it was showed that CYP6AA9 was closely associated with pyrethroid resistance and may be used as a marker for insecticide resistance detecting in field mosquito populations. Then, three field mosquito populations of Cx. pipiens pallens were collected from Bengbu City (Anhui Province), Jining City (Shandong Province) and Nanjing City (Jiangsu Province). The insecticide susceptibility test of three field mosquito populations was carried out by the WHO tube bioassay. When exposure with 0.05% deltamethrin for 1 h, the mosquitoes which were knocked down were considered as deltamethrin-sensitive strains, otherwise as deltamethrin-resistant strains. We compared protein abundances of CYP6AA9 between the deltamethrin-sensitive strain and deltamethrin-resistant strain of each field population by Western blot. The results showed that CYP6AA9 was highly expressed in the deltamethrin-resistant strain compared with in the deltamethrin-sensitive strain of each field population, consistent with the results from the laboratory populations mentioned above. CYP6AA9 was confirmed further to be closely associated with pyrethroid resistance.Enzyme-linked immunosorbent assay (ELISA) is used widely to measure all kinds of antigens and antibodies because of its simplicity, higher sensitivity and application for quantitative analysis. Protein abundances of CYP6AA9 in the laboratory populations and field populations were further analysised by ELISA. The results showed that:(1) protein abundances of CYP6AA9 were positive correlation with the insecticide resistance level; (2) the difference of CYP6AA9 expression was not obvious among the deltamethrin-sensitive strains of laboratory populations, the strains exhibited lower resistance level of laboratory populations and the deltamethrin-resistant strains of field populations, which meant that it was difficult to differentiate CYP6AA9 expression between the sensitive strains and the lower resistance strains; (3) CYP6AA9 was highly expressed in the higher resistance strains of laboratory populations compared with in the sensitive strains of laboratory populations, the lower resistance strains of laboratory populations and the resistant strains of field populations. Meanwhile, CYP6AA9 was also shown to be expressed at slightly higher level in the deltamethrin-resistant strain of Jining population than in the deltamethrin-resistant strain of Bengbu population. Taken together, these results indicated that CYP6AA9 may be considered as a marker for detecting the level of pyrethroid resistance and used to compare the resistance level of different field population using ELISA, especially well applicable for the populations exhibited a relatively high level of resistance.The present study is the first report of a proteomic research on insecticide resistance in the mosquito of Cx. pipiens pallens. A total of 30 proteins associated with insecticide resistance were identified by iTRAQ-coupled LC-MS/MS. Some candidate proteins were verified further in the laboratory population and field population of the Culex mosquito by quantitive real-time PCR and Western blot. The results showed that CYP6AA9 was closely associated with pyrethroid resistance. CYP6AA9 may be used as a marker to compare the resistance level of different field mosquito population with ELISA.