Time-course analysis system for leaf feeding marks revealed effect of Arabidopsis thaliana trichomes on herbivore insect feeding behavior.

Bioassay with insect herbivore is a common approach to studying plant defense levels. While measuring insect growth rate as a negative indicator of plant defense levels is simple and straightforward, analyzing more detailed feeding behavior parameters of insects, such as feeding rates, leaf area consumed per feeding event, intervals between feeding events, and spatiotemporal patterns of feeding sites on leaves, is more informative. However, such observations are generally time consuming and labor-intensive. Here, we provide a semi-automated system for quantifying feeding behavior parameters of insects feeding on plant leaves. Automated photo scanners record time-course development of feeding marks on leaves. An image analysis pipeline processes the scanned images and extracts leaf area. By analyzing changes in leaf area over time, it detects insect feeding events and calculates the leaf area consumed during each feeding event, providing quantitative parameters of insects feeding behavior. In addition, it visualizes spatio-temporal changes in feeding sites, providing a measure of the complex behavior of insects on leaves. Using this analysis pipeline, we demonstrate that Arabidopsis thaliana trichomes reduce insect feeding rate, but not feeding duration or intervals between feeding events. Our image acquisition system requires only photo a scanner and a laptop computer and does not require any specialized equipment. The analysis software pipeline is provided as an ImageJ macro and R package and is available at no cost. Taken together, our work provides a scalable method for quantitative assessment of insect feeding behavior on leaves, facilitating understanding of plant defense mechanisms.