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Insecticide resistance surveillance systems for vector-borne diseases are crucial for early detection of resistance and the implementation of evidence-based resistance management strategies. While insecticide susceptibility bioassays are typically conducted under controlled laboratory conditions, mosquitoes in the field experience varying environmental conditions, with temperature being a key determinant. Understanding the relationship between temperature and insecticide toxicity is essential for interpreting and extrapolating assay results across different climate zones or more locally across days with different weather conditions. In this study, we examined Aedes aegypti mosquitoes with different genetic backgrounds of insecticide resistance. Mosquitoes were homozygous for the knockdown resistance (kdr) F1534C mutation, plus either (1) homozygous for the kdr 1016V wildtype allele, (2) homozygous for the kdr V1016I mutant allele, or (3) heterozygous genetic crosses. These three genotypes were exposed to deltamethrin using WHO tube tests at three temperatures (22 °C, 27 °C, and 32 °C) and varying dosages. LC50 values were determined for each genotype and temperature combination. A negative temperature coefficient was observed exclusively in female mosquitoes homozygous for the 1016V wildtype allele, indicating reduced pyrethroid toxicity at higher temperatures. No temperature–toxicity relationship was found in males of this genotype or in other genotypes of either sex. These findings suggest that temperature may interact with kdr mutations and possibly even sex, highlighting the complex interactions between genetic mutations and environmental factors, such as temperature, in determining the insecticide resistance phenotype. Given the wide distribution of Ae. aegypti, understanding how local climate conditions influence insecticide performance will help improve control strategies and slow resistance evolution, protecting public health efforts against mosquito-borne diseases 

West Nile virus (WNV) is the leading mosquito-borne disease causing-pathogen in the United States. Concerningly, there are no prophylactics or drug treatments for WNV and public health programs rely heavily on vector control efforts to lessen disease incidence. Insecticides can be effective in reducing vector numbers if implemented strategically, but can diminish in efficacy and promote insecticide resistance otherwise. Vector control programs which employ mass-fogging applications of insecticides, often conduct these methods during the late-night hours, when diel temperatures are coldest, and without a-priori knowledge on daily mosquito activity patterns. This study’s aims were to 1) quantify the effect of temperature on the toxicity of two conventional insecticides used in fogging applications (malathion and deltamethrin) toCulex tarsalis, an important WNV vector, and 2) quantify the time of host-seeking ofCx.tarsalisand other local mosquito species in Maricopa County, Arizona. The temperature-toxicity relationship of insecticides was assessed using the WHO tube bioassay, and adultCx.tarsalis, collected as larvae, were exposed to three different insecticide doses at three temperature regimes (15, 25, and 35°C; 80% RH). Time of host-seeking was assessed using collection bottle rotators with encephalitis vector survey traps baited with dry ice, first at 3h intervals during a full day, followed by 1h intervals during the night-time. Malathion became less toxic at cooler temperatures at all doses, while deltamethrin was less toxic at cooler temperatures at the low dose. Regarding time of host-seeking,Cx.tarsalis,Aedes vexans, andCulex quinquefasciatuswere the most abundant vectors captured. During the 3-hour interval surveillance over a full day,Cx.tarsaliswere most-active during post-midnight biting (00:00–6:00), accounting for 69.0% of allCx.tarsalis, while pre-midnight biting (18:00–24:00) accounted for 30.0% ofCx.tarsalis. During the 1-hour interval surveillance overnight,Cx.tarsaliswere most-active during pre-midnight hours (18:00–24:00), accounting for 50.2% ofCx.tarsaliscaptures, while post-midnight biting (00:00–6:00) accounted for 49.8% ofCx.tarsalis. Our results suggest that programs employing large-scale applications of insecticidal fogging should consider temperature-toxicity relationships coupled with time of host-seeking data to maximize the efficacy of vector control interventions in reducing mosquito-borne disease burden.