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Background  Feeding habits are central to animal ecology, but it is often difficult to characterize the diet of organisms that are arboreal, nocturnal, rare, or highly mobile. Genetic analysis of gut contents is a promising approach for expanding our understanding of animal feeding habits. Here, we adapt a laboratory protocol for extracting and sequencing plant material from gut contents and apply it to Neotropical forest katydids (Orthoptera: Tettigoniidae) on Barro Colorado Island (BCI) in Panama.    Methods  Our approach uses three chloroplast primer sets that were previously developed to identify vegetation on BCI. We describe the utility and success rate of each primer set. We then test whether there is a significant difference in the amplification and sequencing success of gut contents based on the size or sex of the katydid, the time of day that it was caught, and the color of the extracted gut contents.    Results  We find that there is a significant difference in sequencing success as a function of gut color. When extracts were yellow, green, or colorless the likelihood of successfully amplifying DNA ranged from ~30–60%. When gut extracts were red, orange, or brown, amplification success was exceptionally low (0–8%). Amplification success was also higher for smaller katydids and tended to be more successful in katydids that were captured earlier in the night. Strength of the amplified product was indicative of the likelihood of sequencing success, with strong bands having a high likelihood of success. By anticipating which samples are most likely to succeed, we provide information useful for estimating the number of katydids that need to be collected and minimizing the costs of purifying, amplifying, and sequencing samples that are unlikely to succeed. This approach makes it possible to understand the herbivory patterns of these trophically important katydids and can be applied more broadly to understand the diet of other tropical herbivores.

Applying and refining DNA analysis to determine the identity of plant material extracted from the digestive tracts of katydids