An integrative approach combining molecular analyses and experiments to investigate predation of insect eggs by a mite

Uncovering the predation habits of small arthropods such as mites can be a major challenge, as direct observations are not always feasible in natura. Therefore, PCR‐based analyses of gut content, or body content when gut dissection is not applicable, are being increasingly used to detect remains of prey items. Here, we present an integrative approach to investigate predation of eggs of the viburnum leaf beetle Pyrrhalta viburni, an invasive pest in North America, by the oribatid mite species Trichoribates trimaculatus, to assess the potential of this mite as a biological control agent. We combined (1) manipulative laboratory experiments exposing beetle eggs to mites under controlled conditions and (2) body content molecular analyses using quantitative PCR (qPCR) to detect traces of consumed P. viburni egg DNA in mites. To account for risks of false‐negative and false‐positive results, mainly due to detectability problems, body surface contamination, and non‐specific DNA amplification, we developed a stepwise procedure to analyze qPCR results. Egg integrity and survivorship were unaffected by mites in all experiments. However, traces of P. viburni DNA were detected in the body of mites exposed to P. viburni eggs, suggesting that they consumed either fragments of the chorion or fragments of the egg cap secreted by P. viburni females to protect the eggs, which was shown to contain P. viburni DNA. In conclusion, T. trimaculatus does not directly impact P. viburni eggs and should therefore not be considered as a biocontrol agent. The fact that mites did not directly predate eggs but contained traces of P. viburni DNA shows that detection of an organism's DNA within a presumed predator does not necessarily equal predation and that results of molecular analyses should be interpreted with caution in studies attempting to reconstruct trophic interactions.