Assessment in Mosquito Populations- Standardized Protocols for Testing Insecticide Susceptibility and Biochemical Resistance, Verified with Culex Quinquefasciatus

Objective: This study aimed to develop and validate a harmonized protocol for the integrated assessment of insecticide resistance, combining phenotypic bioassays and biochemical analyses in field populations of Culex quinquefasciatus. Materials and Methods: A standardized workflow was established, encompassing larval sampling from diverse habitats, insectary rearing under controlled conditions, morphological identification, and susceptibility testing via WHO tube and CDC bottle bioassays using permethrin, deltamethrin, bendiocarb, and malathion. Concurrently, biochemical assays were performed to quantify glutathione-S-transferase (GST) activity and total protein concentration in mosquito homogenates. The protocol was validated using field-collected Culex populations from West Africa. Results: Phenotypic assays confirmed strong pyrethroid resistance, with mortality rates of 32–49% for permethrin and 38–57% for deltamethrin, alongside significantly prolonged knockdown times. CDC bottle bioassays yielded consistent results. Biochemically, resistant populations exhibited markedly elevated GST activity (0.72–1.15 μmol/min/mg protein) and higher total protein levels (2.8–4.1 mg/mL) compared to more susceptible groups. A strong inverse correlation was observed between high GST activity and low mortality in bioassays. Conclusion: The integrated methodological framework proved to be reliable, reproducible, and suitable for field-laboratory application. It effectively detects both the presence and a likely metabolic mechanism of insecticide resistance, providing a critical tool for surveillance programs and evidence-based vector control management.