## 2 Materials and Methods
### BPA Treatment  
Sterile, purified and pathogen-free BPA (4,4\({}^{\prime}\)-Isopropylidenediphenol) was supplied as dry white pills (Acros Organics, Geel, The Netherlands) with purity > 96%, according to the manufacturer's information. BPA was first dissolved in a small volume of DMSO, and then H\({}_{2}\)O was added to prepare the stock solution (DMSO was carefully kept at 0.1% or lower throughout the experiments). For each treatment, this solution was diluted and mixed with \"Carolina Drosophila Medium Formula 4-24\({}^{\oplus}\)\" (Carolina Biological Supply Company 2016, Burlington, NC, Canada). For the longevity, prolificacy and development time analysis, 5 concentrations of BPA were tested (0.5, 1, 2, 5 and 10 mM). These concentrations were chosen based on two previous studies where it was assumed that 1 mM of BPA corresponds, in flies, to the approximate human lowest-observed-adverse-effect level (LOAEL) of 50 mg/kg body weight/day recommended by the U.S. Environmental Protection Agency (EPA) [48; 49; 50]. The selected concentration range, although not environmentally relevant at first glance, was designed to examine dose-response patterns and potential thresholds of effect, and to dissect mechanistic aspects of toxicity. High-dose exposure is a standard practice in model organism toxicology studies to simulate chronic or cumulative stress scenarios. All  
Figure 2: Representative images of some of the experimental groups used in this study. assays were performed in duplicate and, for the negative control group, a solution of 0.1% DMSO was added to the instant \"Carolina\" medium. After the treatment, all flies were kept in a non-treated standard medium. To avoid possible contamination of BPA from plastic material, only glass material was used in this study. Two types of treatment were used: acute, where we evaluated the effects of BPA exposure over a short period of time (72 h); and chronic, where the individuals were exposed for a longer period (in this case, throughout larval development). Exposure times were kept consistent in all experiments. For the acute treatment, 10 couples (2-5 days old) were mated in vials containing either non-treated (control group) or treated instant \"Carolina\" medium, and were allowed to feed and lay eggs. After 72 h, the flies were transferred to new vials containing a non-treated standard medium, where they continued to lay eggs. F\({}_{1}\) subjects born in the non-treated (F\({}_{1}\)-N) medium were then used to evaluate the effects of parental BPA acute exposure in the offspring. The F\({}_{1}\) progeny born in the treated (F\({}_{1}\)-T) medium was exposed to BPA throughout the larval development, and was used to evaluate the effects of chronic treatment after parental acute treatment. After hatching, 10 couples of the F\({}_{1}\)-T group were transferred into new vials containing BPA-treated medium, and the treatment process was repeated to obtain the F\({}_{2}\) progeny. This transgenerational study allowed us to evaluate, in the offspring, the effects of acute and/or chronic treatment of the parental generation (F\({}_{1}\)-N and F\({}_{2}\)-TN respectively), the effects of acute treatment (F\({}_{0}\)), the effects of chronic treatment throughout larval development (F\({}_{1}\)-T) and the effects of consecutive chronic treatments (F\({}_{2}\)-TT). A scheme of this procedure is depicted in Figure 3A. For the behavioural assay, only the F\({}_{1}\)-T generation was used (Figure 3B). As for the genotoxicity assays (SMART and Comet), heterozygous individuals were treated with 1, 10, 20 and 50 mM of BPA. Higher concentrations were used in these assays  
Figure 3: (**A**) BPA treatments used to evaluate longevity, prolificacy and development time. Ten couples of flies (n = 20; 2–5 days old) were crossed in a BPA-treated medium and were allowed to feed and lay eggs, first in that medium, and then in a non-treated standard medium.