Secondary predation by omnivores: Cereal aphid consumption bears no risk of misinterpretation in DNA ‐based diet analysis

Many omnivorous arthropods act as important pest control agents in agroecosystems. Assessing their prey choices helps to better understand their biocontrol potential in different environmental settings. To this end, multi-target approaches of molecular gut content analysis allow studying their prey choices. Yet, DNA-based analysis of trophic interactions is challenged by the question of whether the detected food DNA was ingested primarily as direct prey, or secondarily via the gut content of a consumed animal. Experimental data are needed to understand the potential bias derived through secondary predation. Here we address this issue for a phloem-feeding aphid consumed by omnivorous carabids. In feeding experiments, Pseudoophonus rufipes beetles were offered grain aphids Sitobion avenae as primary prey, which had fed on barley seedlings. Gut content samples of carabids collected after 0, 3, 9, 12, 24, 32 or 64 h of digestion time and whole-body aphids were screened for the presence of aphid and plant DNA. Samples positive for plant DNA were subsequently tested with Poaceae-specific primers. None of the 7.8% gut content samples that tested positive for general plant DNA amplified with the Poaceae primers. Moreover, none of the whole-body extracted aphids tested positive for plant DNA, neither with general nor with family-specific primers. We, therefore, conclude that the plant DNA detections in our experiment can be ascribed to remnants of plant material in the guts of the field-collected carabids. Our findings demonstrate that these phloem-feeding aphids are an unlikely cause of false trophic link assignment because no secondarily consumed plant DNA, derived either from aphid gut content or host-plant environmental DNA on aphid bodies, could be detected in carabid gut content samples.