*2.4. Organisms Exposures for RNA Extraction*

Two hundred nauplii of *A. franciscana* were exposed to NAP, PHE, FLT, and BkF at 0.26 mg/L, 1.15 mg/L, 0.81 mg/L, and 84.6 mg/L, respectively, whereas 10 *A. franciscana* adults were exposed to NAP, PHE, FLT, and BkF at 1.45 mg/L, 1.15 mg/L, 0.81 mg/L, and 84.6 mg/L, respectively. These concentrations were chosen because they did not cause mortality in acute tests. All the experiments were performed in triplicates.

Samples were collected after 48 h of exposure by centrifugation at 4000× *g* for 15 min in a swing-out rotor at 4 ◦C in a 2 mL tube, kept on ice, and were further homogenized in TRIzol (Invitrogen, Paisley, UK) using a TissueLyser II (Qiagen, Valencia, CA, USA) and steal beads of 7 mm diameter (Qiagen, Valencia, CA, USA). Total RNA was extracted and purified using Direct-zolTM RNA Miniprep Plus Kit (ZYMO RESEARCH). The amount of total RNA extracted was estimated by the absorbance at 260 nm and the purity by 260/280 and 260/230 nm ratios, using a NanoDrop spectrophotometer 2000 (Thermo Scientific Inc., Waltham, MA, USA), to exclude the presence of proteins, phenol, and other contaminants [48].
