2.2.1. RNA Extraction

A primary homogenate was prepared by grinding individuals pooled by morphotype (Supplementary Materials Table S1) into 500 to 800 µL phenol/guanidine-based QIAzol Lysis Reagent (Qiagen, Courtaboeuf, France) by homogeneization with one 8 mm diam. bead in 2 mL tubes, with tissue lyzer (Qiagen) during 30 s at 30 Hz. This was repeated three more times, after a wait of 30 s. Tubes were then centrifugated at 4 °C for 2 min at 12,000× g, and the supernatant was collected in a new tube to which chloroform was added (100 µL chloroform for 500 µL phenol/guanidine-based QIAzol Lysis Reagent (Qiagen), vortexed for 15 s and incubated at room temperature for 3 min. After 15 min centrifugation at 12,000× g at 4 °C, the aqueous phase was transferred for RNA purification on columns and treated according to the manufacturer’s protocol (RNAeasy minikit, Qiagen). The final suspension volume was 100 µL, and RNA was quantified on Nanodrop.

For A. mellifera samples, 40-individuals were pooled and RNA extracts were processed using standard methods, as described by Dalmon et al. [38]. Large specimens of Xylocopa and Bombus individual wild bees were ground in 5 mL PBS 1× using plastic bags containing filter. Five hundred microliters of this primary homogenate (filtered extract) was added to 900 µL of Quiazol (Qiagen, Courtaboeuf, France). After 5 min at room temperature, 180 µL of chloroform was added and samples were processed, as described above.