Experiment 1: pure glyphosate and glyphosate-based herbicides
The purpose of this experiment was to test the impact of pure glyphosate and glyphosate-based herbicides (formulation), on mosquito life history traits and susceptibility to malaria parasite infection. For this purpose, one day old mosquito larvae were randomly assigned to five experimental groups (120 larvae/ per group). Two groups corresponded to 2 different concentrations of pure glyphosate, two groups corresponded to the same 2 concentrations of glyphosate but in formulation. The remaining group was reared without glyphosate (control). Larvae were fed daily with 0.5 mg of food, which is consistent with our standard colony maintenance diet (1:1 Fish food and rabbit pellets). For pure glyphosate solution, rearing solutions were prepared from a stock solution of 2 mg/l produced with solid glyphosate (96% purity; provided by Sigma-Aldrich, Switzerland) dissolved in mineral water. From the stock solution, 2 derived solutions were prepared, with the following concentrations: 0.05mg/L and 0.1mg/L. For the glyphosate-based herbicides, a formulated glyphosate solution (glyphosate concentration: 360g/L; Sintagro®) was diluted ten times with mineral water to obtain a stock solution (glyphosate concentration: 3.60 g/L). From this stock solution, 2 derived solutions were prepared to obtain two glyphosate concentrations: 0.05mg/L and 0.1mg/L. The concentrations of glyphosate used in our study (0.05 and 0.1mg/L) were chosen based on concentrations observed in aquatic systems (Giesy et al., 2000) as well as in previous studies on the impact of sub-lethal doses of pesticides on aquatic invertebrates species (Baglan et al., 2018; Kibuthu et al., 2016; Riaz et al., 2009).
Stock solutions were renewed every 6 days, to avoid any degradation effect (Baglan et al., 2018). Rearing solution were replaced every 3 days to ensure that the larvae were exposed to the same concentration of glyphosate during all their development.