Protease extraction and preparation

The midguts of actively feeding fourth-instar H. zea larvae were dissected to extract soluble gut proteases. The food bolus from 10 guts (representing one biological replicate) were pooled together in a microcentrifuge tube, flash frozen in liquid nitrogen, and stored at −80°C until used. Soluble gut proteases were extracted by thawing the gut contents on ice before adding 500 µL of milli-Q water, followed by vortexing for 2 minutes. The tubes were then centrifuged at 27,000 × g for 20 minutes at 4°C, and the supernatants filtered with 0.22-µm filters to obtain the soluble protease sample. Protein concentrations in soluble midgut protease extracts were estimated using a Qubit Protein Assay Kit and adjusted to 1 mg/mL before storing at −80°C.