Sample type drives dietary reconstruction in the endangered alpine bush-cricket Anonconotus italoaustriacus

Understanding the trophic ecology of threatened insects is critical for developing effective conservation strategies, as diet directly affects survival, reproduction, and population stability. In this case study, we investigated the food plants exploited by Anonconotus italoaustriacus (Orthoptera: Tettigoniidae) by reconstructing its diet and comparing midgut- and feces-derived dietary data. This flightless alpine bush-cricket is endemic to the Eastern Alps, where it survives in four distinct populations, and is currently classified as Endangered by the IUCN. To evaluate the effect of sample type on the accuracy of dietary reconstruction, total DNA was extracted from intestinal and fecal samples and sequenced using the Illumina MiSeq platform, targeting the ITS2 region. Intestinal samples showed higher sequencing success and yielded more ASVs (32±5) than fecal samples (14±4). Beta diversity analysis revealed that samples clustered by matrix rather than by sampling site, suggesting that differences in taxonomic composition are primarily driven by sample type rather than environmental factors. Intestinal contents reflected a broader dietary spectrum integrated over digestion, whereas fecal samples showed a narrower dietary signal. Despite these differences, the two matrices provided partially complementary information: 45% of the detected taxa were shared, 40% were exclusive to intestinal samples, and 15% to feces. Across intestinal samples, Rhododendron spp. was consistently detected in all populations, suggesting either widespread availability or preferential exploitation. In contrast, fecal samples were dominated by a single plant genus, whose identity varied among sampling site, highlighting matrix-specific biases linked to physiological processes. Thus, using various sample types provides a more complete picture of the diet. These findings underscore the importance of selecting and, when feasible, combining different sample types to better capture dietary diversity. However, feces seem to represent a valuable, non-lethal source of dietary information, offering a practical approach for ecological studies on endangered species, where minimizing harm is essential.