Direct proof of ingested food regurgitation by Spodoptera littoralis caterpillars during feeding on Arabidopsis.

Oral secretions of herbivorous lepidopteran larvae contain a mixture of saliva and regurgitant from the insect gut. Different compounds from the oral secretions can be recognized by the host plants and, thus, represent elicitors that induce plant defenses against feeding herbivores. Exogenously applied oral secretions can initiate the biosynthesis of jasmonates, phytohormones involved in the regulation of plant defense. However, it is not known (a) whether or not non-manipulated insects indeed release oral secretions including gut-derived compounds into a leaf wound during the natural feeding process, or (b) whether they adjust the release of gut components to the state of plant defense. We addressed these questions by using Arabidopsis thaliana as host plant and larvae of the generalist herbivorous insect Spodoptera littoralis. We investigated the conversion of the plant-derived jasmonate precursor, cis-12-oxophytodienoic acid (cis-OPDA), to iso-OPDA by the larvae. This enzymatic reaction is mediated by a specific glutathione-S-transferase in the insect gut, but not in the plant. Any presence of iso-OPDA in plant tissue, thus, indicated that gut content had been regurgitated into the plant wound. Our study demonstrates that the plant is the only source for the substrate cis-OPDA by using aos (allene oxide synthase) mutants that are unable to synthesize OPDA. The fact that iso-OPDA accumulated over time on feeding-damaged leaves shows that the feeding larvae are constantly regurgitating on leaves. Although the larvae provided the signaling compounds that were recognized by the plant and elicited defense reactions, the larval regurgitation behavior did not depend on whether they fed on a defensive wild type plant or on a non defensive coi1-16 plant. This suggests that S. littoralis larvae do not adjust regurgitation to the state of plant defense.