Bioassays

The F₁ adults were exposed to the insecticides using a modified CDC bottle bioassay (Centers for Disease Control) [16]. Sigma brand technical grade insecticides were used to determine the lethal concentrations that killed 50% (LC₅₀) at each site. The pyrethroids permethrin (Type I) and deltamethrin (Type II), the organophosphates malathion and chlorpyrifos, and the carbamate bendiocarb were used to represent the toxicological groups used by vector control programs in Mexico.

To determine the LC₅₀, we tested five to six insecticide concentrations, which caused 10 to 90% mortality, in four replicates. Each insecticide LC₅₀ required approximately 500 mosquitoes. Table 2 shows the insecticide concentrations (μg/bottle) used to coat 250 ml Wheaton bottles using acetone as the solvent. During the bioassay, 15 to 20 (2–3 day old) females were gently aspirated into each bottle. The knockdown effect was recorded every 10 minutes for 1 hour. After 1 hour of exposure, the mosquitoes were transferred to plastic containers and maintained in the insectary to observe the mortality at 24 hours. The LC₅₀ of each insecticide was also determined for the susceptible New Orleans reference strain (NO) using a different set of insecticide concentrations (Table 2). Each insecticide LC₅₀ was replicated at least five times during a 7-month period. As control, a bottle impregnated only with acetone was used each time a test with field or susceptible mosquitoes was run.

PYRs = pyrethroids, OPs = organophosphates, CARBs = carbamates.

The LC₅₀, 95% confidence intervals, slope, intercept, and p values were determined using the binary logistic regression model with QCal software [17]. The null hypothesis (Ho) assumes the observed mortality curve adjusts to a binary logistic regression model. Thus, we expected p values higher than 0.05 to accept the Ho. When the Ho was rejected, the bioassay was repeated.

To estimate the level of resistance among sites, a resistance ratio (RR) was calculated by dividing the LC₅₀ of the field sites by the LC₅₀ of the NO strain. The RR criterion according to Mazzarri and Georghiou [18] classifies high resistance as RR values greater than 10, moderate resistance as RR values between 5 and 10, and low resistance as RR values less than 5.