After 24 h, the larvae mortality was determined by counting the total number of dead and alive individuals. Larvae that were paralysed or moving only their appendices without the capability to walk were considered dead. Thirty-two and three tests were performed in triplicate with the strains Mozo and ZOR, respectively. The software Intercooled Stata 10 (Stata Corp., 2007) was used to analyse the data obtained from the standardisation of bioassays with larvae and adults of the Mozo strain.

For AIT, analysis was conducted as proposed by Castro-Janer et al. (2009). The following variables were studied: (1) mortality (engorged females that produced eggs were considered alive, and females that did not

produce any eggs were considered dead); (2) egg mass weight (EW), 7 and 14 days after treatment; (3) index of fertility (IFER), 7 and 14 KU-55933 in vivo days after treatment, calculated as egg mass weight (g)/weight of females (g); and (4) index of fecundity (IFEC), 14 days after treatment, calculated as IFER × percentage of larval hatching. For the larvae tests, a probit analysis was run on the mortality results using the software Polo-Plus (LeOra Software, 2003). For each test, the following parameters were determined: lethal concentrations for 50% and 90% (LC50 and LC90) with confidence intervals of 95% (CI 95%), and the slope of the regression line. The resistance ratios (RR50 and RR90) and their CI 95% were generated with the software Polo-Plus using the formula described by Robertson et al. (2007). AZD0530 ic50 The significance of each comparison was determined when the calculated confidence intervals (CI 95%) did not overlap. For the diagnosis of resistance, the three categories established by Castro-Janer et al. (2011) were used: (1) susceptible, when the LC50 (CI 95%) of the field population is not statistically different from the reference strain; (2) incipient resistance, when the LC50 (CI 95%) of the field population is statistically different from the reference strain with medroxyprogesterone RR50 < 2; and (3) resistant, when the LC50

(CI 95%) of the field population is statistically different from the reference strain with RR50 ≥ 2. The AIT results at different immersion times, obtained with the Mozo strain, are presented in Table 1. In all of the tests, the mortality, EW and IFER parameters presented a small CI 95% amplitude. Higher coefficients of regression were obtained for the variables EW and IFER, and the LC50 of these two variables were not significantly different. The calculated LC50 for mortality and IFEC were significantly different from those determined for EW and IFER. IFEC exhibited higher variation independently of the immersion time, with a high CI 95%. Higher mortality of engorged females was observed as the time of immersion increased.

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