الفهرس 
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Abstract
Mosquito vectors transmit wide variety of pathogens that cause severe human illness. The currently applied vector control programs are mostly depending on using pyrethroid insecticides. The intensive exposure of mosquito vector to pyrethroid insecticides has led to arising of pyrethroid resistant population. Resistance in Cx. pipiens mosquitoes was documented as a global problem. Monitoring and measuring the development of resistance in Cx. pipiens populations to pyrethroid is of paramount for vector control management. Hence the present study shed light on understanding insecticide resistance mechanisms.
In the present study, Culex pipiens mosquito larvae were collected from breeding sites in Mansourya canal, Giza governorate, Egypt. Laboratory susceptible Cx. pipiens population was used as a control in the toxicology, biochemical and molecular genomics assays.
Field-collected Cx. pipiens mosquitoes that exhibit pyrethroid resistance were used as a model to investigate the role of miRNAs in regulating pyrethroid resistance through P450s target genes. MicroRNAs expression profiles were determined and linked to its putative P450s targets transcript expression levels, to give a better understanding of their function in the development of insecticide resistance. This may provide an innovative approach with regard to development of more targeted control technologies.
Susceptibility bioassay, kdr mutation detection, and biochemical assay were used to determine the pyrethroid resistant status among Cx. pipiens field population. Molecular genomics assays were conducted to detect P450 genes through cloning followed by sequencing. RT-qPCR was used to detect the expression profile of P450 genes associated with insecticide resistance, while colorimetric microarray and stem-loop RT-qPCR were used to detect the expression profile of corresponding miRNAs.
In pyrethroid susceptibility bioassay, 100 female Cx. pipiens each from field population and laboratory susceptible population (25 in four replicates) were exposed to 6 different dose concentrations of deltamethrin insecticide for a fixed period of time to determine the median lethal concentration (LC50). For the following concentrations (0.001%, 0.025%, 0.075%, 0.1%, and 0.25%) mortality rates of 10%, 28%, 68%, 92%, and 100% respectively, were recorded. The results revealed significant LC50 variations between laboratory and field populations.
For screening the presence of kdr mutation, a total of 50 unfed adult females field population Cx. pipiens mosquitoes, 2 days post adult emergence were tested using PCR followed by sequencing. The kdr allele was individually diagnosed in 50 adult females, sequencing results showed the presence of homozygous kdr resistant alleles TTT (L1014F) (RR) in 32 samples (64 %), heterozygous kdr susceptible/resistant alleles TTA/TTT (L1014/L1014F) (RS) in 14 samples (28 %) and homozygous kdr susceptible alleles TTA )L1014( (SS) in 4 samples (8 %).
In the biochemical assay, 75 adult field population Cx. pipiens mosquitoes (25 in three replicates) and 75 laboratory population (25 in three replicates) were used to determine the cytochrome P450 activity level using monooxygenase assay. Significant differences in the levels of monooxygenase (P450) activity between field and laboratory populations were observed of Cx. pipiens mosquito (t = 10.87, df = 148, P< 0.001).
Cytochrome P450 family gene was amplified using degenerate primers from field-resistant populations of Cx. pipiens mosquito. A DNA fragment of size 490 bp was excised from the gel, purified, cloned and sequenced. The generated sequence was blasted and compared to the P450 sequence database retrieved from the GenBank. Phylogenetic analysis revealed that the obtained sequence was closely related to CYP4J19 gene. The sequence was a variant from CYP4J19 gene and it was given a name of CYP4J19v by Cytochrome P450 Nomenclature Committee.
The RT-qPCR was used to determine the expression level of seven P450 genes associated with insecticide resistance in the field resistant-population compared with the laboratory population of Cx. pipiens mosquito. ِPrimers for CYP4J19v were designed for expression level detection. Results showed significant up-regulation of the seven P450 genes in the field-resistant population. CYP6N23, CYP6AG11, CYP325BG3, CYP9J35, CYP4J19v, CYP6Cp1 and CYP4J19 genes were shown 20, 27.9-, 12.8, 47.6, 31.5, 27.2, and 15.4-fold, respectively higher in field-resistant population compared to the laboratory population.
The expression levels of targeted miRNAs were analyzed with colorimetric microarray based on gold–silver enhancement method. A biotin-labeled miRNA hybridized with microarray designed probes were bounded to gold nanoparticle (gold-streptavidin) with silver enhancement. Stem-loop RT-qPCR was also used for confirmation, where miRNA-specific reverse transcription stem-loop primer and specific forward primer were designed for each miRNA. Results revealed significant down-regulation of miR-2, miR-71, miR-258 and miR-278 and significant up-regulation of miR-309, miR-13 and miR-2951-5P including the positive controls miR-9, miR-7, and miR-92 in field-resistant population compared to laboratory population.
The expression results of miR-2, miR-71, miR-258 and miR-278 displayed a significant negative correlation relationship with the expression of their corresponding target P450s, while miR-13, miR-2951 and miR-309 showed a positive correlation relationship with the expression of their corresponding target P450s.
The present study elucidated the pyrethroid resistance development and its relation to the metabolic and target site modification mechanisms with a first report of L1014F-kdr mutation detection in Cx. pipiens population, which may assist in decision-making processes taken by the government concerning the selection of alternative vector control methods. Monitoring resistance in field mosquitoes should help in shaping national vector control strategies and provide an alarming alert to eliminate the insecticides that show partial resistance in mosquito population before resistance becomes fixed, a step that may result in recovery of the susceptible phenotype.
To our knowledge, the expression profile of miRNAs and its corresponding P450s targets have not been previously reported in field population of Cx. pipiens mosquito in Egypt. The results of this research highlight the need to direct more research efforts towards identifying miRNAs and P450 genes associated with pyrethroid resistance in field Culex mosquito population targeting different geographical area in Egypt, in order to understand their roles in the development of resistance and participate in global vector control future research studies, that aiming to use small RNA and P450 inhibitor approach as an alternative control method.