Feeding assays

We conducted insect herbivore feeding assays as previously described^{36, 37} to determine the influence of exposure to the individual E. solidaginis compounds on S. altissima anti-herbivore defenses. As discussed in our previous work, the galling habit of E. solidaginis larvae makes them impractical for conducting such assays; therefore, we conducted these experiments using another goldenrod specialist herbivore, the goldenrod leaf beetle (T. virgata) as a proxy for the fly larvae. We have previously demonstrated that exposure of S. altissima plants to the E. solidaginis emission primes anti-herbivore defenses effective against a suite of herbivores, including T. virgata ³⁶.

After exposing plants to the various treatments for 24 h, we allowed T. virgata larvae to feed on the plants for an additional 24 h. Larvae were starved for 4 h at room temperature before the experiment. After larvae fed on the plants for 24 h, we collected the plants and scanned the leaves to measure the total leaf area consumed. The total area removed was quantified using Adobe Photoshop software.