RNA was prepared from bees’ midgut tissue as previously described (21 (link)). Midgut tissue was manually crushed with a disposable pestle in Trizol Reagent (Invitrogen, San Diego, CA, USA), and RNA was then extracted as per the manufacturer’s instructions. RNA was then DNase treated using RQ1 RNase-Free DNase I (Promega, Madison, WI, USA) and cDNA was synthesized using approximately 1 µg of RNA with the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). For qPCR reactions to determine the expression levels of genes of interest, 1 µL of cDNA was used as a template in conjunction with PowerUP SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and appropriate primers in a 20 µL reaction. Reactions were run in a LightCycler 480 thermal cycler (Basel, Switzerland) or Bio-Rad CFX Opus (Bio-Rad, Hercules, CA, USA). Primer sequences targeting transcripts of genes of interest are from references (21 (link), 22 (link)). The difference between the threshold cycle number for β-actin and that of the gene of interest was used to calculate the level of that gene relative to β-actin using the typical 2(-ΔCT) method (17 (link)). All qPCR data represent expression values from individual bees (sample sizes found in figure legends) and is displayed as mean ± SEM.
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