Aphids (Hemiptera: Aphididae) are pests of a vast array of crops globally, contributing to significant yield loss annually. Biological control, utilizing natural enemies to suppress focal pest species, has often been integrated into aphid management practices. To ensure that arthropod predators are consuming the target aphid, and ultimately have an impact on pest population dynamics, it is important to gather information on consumption patterns in space and time. Molecular gut content analysis can be used to assess the strength of trophic linkages between predators and prey by detecting the presence of species-specific DNA (including aphid DNA) in their gut. However, many generalist predators readily consume aphid honeydew as an additional nutritive resource beyond direct consumption of prey. This raises the important question of whether aphid honeydew is responsible for false-positive predation events being recorded using DNA-based gut content analysis, should honeydew contain detectable quantities of aphid DNA. To evaluate this potential source of error, we examined honeydew of three species of aphid, Acyrthosiphon pisum, Sitobion avenae, and Rhopalosiphum padi, for aphid DNA using standard gut content analysis methods. After extensive testing, we revealed that aphid honeydew did not contain any detectable aphid DNA for any of the species, nor did honeydew inhibit the amplification of aphid DNA in positive control experiments. These data indicate that aphid predation rates previously reported are not influenced by direct consumption of honeydew. This study, therefore, shows that using DNA-based gut content analysis is a reliable method to measure aphid predation in the field, even when predators are exposed to and consume, honeydew.
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