Magnalyser version 1

For each mosquito, protein extraction was performed using head and thorax according to the protocol previously published^{28 (link)}. Abdomen was excluded from analysis in order to increase the reproducibility and quality of spectra and reduce the bias caused by the midgut microbiota^{33 (link)}. Briefly, these were put into individual microtubes and rinsed with 1 mL of 70% ethanol for 60 s, followed by 1 mL of distilled water for 60 s. The remaining water was then eliminated using a micropipette and left to evaporate. Three metal beads, 15 µL of acetonitrile (50%) and 15 µL of formic acid (70%) were subsequently added to the microtube. Each sample was subsequently homogenized (automated method) using a MagnaLyser, version 1.1 (Roche, Mannheim, Germany) with 3 cycles of 30 s at a frequency of 3000 rpm. After homogenization, 1 µL of sample was deposited directly on a steel MALDI plate (Bruker Daltonics, Wissembourg, France) and allowed to dry before adding another 1 µL of the same sample over the first spot^{28 (link)}. To create the reference main spectrum pattern (MSP), a total of 8 spots were spotted of the same sample^{28 (link)}. Conversely, each sample intended to be queried against these reference spectra were deposited in duplicate, as suggested by previous work^{28 (link)}. Matrix solution was also loaded in duplicate onto each MALDI-TOF plate in order to control matrix quality.

Collected mosquitoes were pooled per species, season, and collection location (indoor/outdoor) to a total of 10 mosquitoes maximum per minipool, and homogenized with 500 µL of PBS using a MagnaLyser version 1.1 (Roche, Mannheim, Germany) at 6000 rpm for 1 min. Crushing material was centrifuged for 2 min at 12,000× g and 4 °C, then 167 µL of supernatant was transferred individually to 835 µL of RNA later solution (Invitrogen). The mixture was incubated overnight at 4 °C and stored at −80 °C until shipment to Institut Pasteur in Paris. According to the mosquito species season and collection location (indoor/outdoor), minipools were combined to form large pools that contained a maximum of 100 mosquitoes per pool (Table S1). A total of 6646 mosquitoes were selected and subsequently distributed across 103 large pools. Overall, total RNA was extracted from the 103 large pools of mosquitoes in a Biosafety Level 3 (BSL-3) laboratory using the Maxwell RSC simply RNA tissue kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. RNA extracts were quantified with the Qubit RNA High sensitivity assay (Invitrogen, Waltham, MA, USA) and analyzed using an Agilent BioAnalyzer RNA pico chip (Agilent, Waldbronn, Germany).