الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 154 |  |  |
| | | from | 154 | | |
Abstract
VI. Summary
Culex mosquitoes are the primary vectors of an extensive range of diseases. Culex pipiens L. and Cx. quinquefasciatus Say are closely related species that make up the Cx. pipiens complex. Using chemical insecticides is considered a fundamental way to control mosquito populations. The most frequently used class of insecticides are pyrethroids. Repeated applications of insecticides will lead to the evolution of insecticide resistant populations. characterization of genes involved in the inheritance of insecticides resistance is essential for understanding the development of resistance mechanisms.
There are two major mechanisms of insecticide resistance: target site resistance: in which the insecticide is unable to bind to its target; and metabolic resistance: in which detoxification enzymes are preventing or reducing insecticide action. In general, there are three important families of detoxification genes: 1) Cytochrome P450 (P450) genes; 2) carboxyl/cholinesterases (CCEs), and 3) glutathione S-transferases (GSTs). Glutathione S-transferases have been hypothesized to be involved in the metabolism and detoxification of organophosphates, organochlorines, and pyrethroid insecticides.
105
The GSTs are a large gene family that consists of three classes according to their location within the cell. The majority of GSTs are cytosolic proteins of which the Delta and Epsilon subclasses are insect-specific, and many members of these subclasses are associated with detoxification and resistance to insecticides.
Only few GSTs have been well characterized in Cx. quinquefasciatus. The study of specific GST genes in Cx. quinquefasciatus may facilitate our understanding of insecticide resistance in this important vector species.
Although elevated GST activity and gene expression levels are found in pyrethroid-resistant insects, there is no direct evidence for GSTs to metabolize permethrin. In the present study, two genes (CPIJ002663 and CPIJ002681) were selected for using in a comprehensive analysis of insecticide resistance in field collected Cx. pipiens and Cx. quinquefasciatus collected in Egypt and the United States of America, respectively (5 governorates in Egypt and 3 states in the USA). Two sets of field collected Cx. quinquefasciatus from insecticide treated and untreated areas from New Orleans (Louisiana), Dallas (Texas) and Florence (Mississippi) in the USA, were assayed using the CDC bottle assay for
106
determining insecticide phenotype status. Tested mosquitoes from treated areas displayed variable degrees of mosquito resistance to permethrin, whereas mosquitoes from untreated areas displayed susceptibility to that same insecticide, with the exception of NOU mosquitoes which exhibited a degree of resistance despite no insecticide was applied in that area. All tested mosquito populations showed resistance to malathion whether originating from treated or untreated areas, with the exception of the TXT population which was susceptible to malathion.
A sequence analysis of the two GST genes was conducted using bio-assayed mosquitoes for providing complementary genetic data. Primer pairs were newly designed, according to the published Cx. quinquefasciatus genome, to amplify CpGSTD5, CqGSTD5 and CqGSTD11. Results indicated that the amplified products from mosquitoes collected from both Egypt and the USA were of ≈ 556 bp and ≈ 828 bp long, respectively. The reliable sequences obtained after editing the sequencing files for CpGSTD5 and CqGSTD5 are 491 bp (encoding for a 163 aa polypeptide) and 498 bp (encoding for a 166 aa polypeptide), respectively. The similarity of the two homologues to that of the reference gene was 79.8 and 80.9%, respectively. The CqGSTD11 yielded 694 bp (encoding for a 231 aa polypeptide) with 98% of the
107
reference gene. The sequence identity of the translated amino acid sequences of the two obtained genes were 99% for CpGSTD5 and CqGSTD5, and 96% for CqGSTD11.
Two major domains (N-terminal and C-terminal) were exposed from our sequence analysis, the positions and ranges of each differ according to the studied gene. For CpGSTD5 and CqGSTD5, either domain extended through the amino acids as following: (aa 1-66) and (aa 84-173) in CpGST5 and (aa 1-66) and (aa 84-175) in CqGSTD5. While for CqGSTD11, the domain positions are (aa 1-94) and (aa 108-225).
Alignment of the obtained amino acids sequences in NCBI conserved domains detected polymorphic loci, in CpGSTD5. All five detected polymorphisms were nonsynonymous, but did not occur in functional conserved regions and did not show any heterozygosity. However, in CqGST5, there were nine polymorphic loci, all of which did not occur in the functional conserved residues, five positions were heterozygous. In CqGST11, there were 31 polymorphic loci, of which 15 were nonsynonymous mutations. Two of the latter occurred in functional conserved sites, both of them being heterozygous in few tested individuals “F8L (TTT-CTT/YTT)” and “F52A (TTT-GCG/KYK)”.
108
Overall results revealed that no genetic differences were demonstrated to be associated with any resistance phenotype. The studied genes appeared not to play major roles in permethrin or malathion resistance in examined mosquitoes of the Cx. pipiens complex. Also, no specific molecular marker was detected for differentiation between susceptible and resistance individuals through sequence alignment and phylogenetic analysis. As all individual sequences were laid in a single clade, this suggests that they have the same origin with no significant difference for separation into different clades.
Further studies are needed to designate a specific molecular marker that would differentiate susceptible and resistant individuals regarding tested insecticides