Species-specific real-time PCR assays to identify arthropod predators of <i>Eucalyptus</i> pests, the 2 paropsine leaf beetles <i>Paropsis charybdis</i> and <i>Paropsisterna cloelia</i> (both Coleoptera: Chrysomelidae)

The paropsine leaf beetles Paropsis charybdis (Stål) and Paropsisterna cloelia (Stål) are 2 major defoliators on eucalypts in New Zealand. In Australia, arthropod predators can contribute to suppressing paropsine populations. Identifying key predators of paropsine beetles from the wild and confirming predation events can be challenging but it is an essential step in the selection of biocontrol agents. Molecular gut content analysis using real-time polymerase chain reaction (RT-PCR) offers an efficient tool to study predation. We developed 2 species-specific RT-PCR assays with primers that amplify 180 bp DNA fragments of the cytochrome oxidase subunit I of P. charybdis and Pst. cloelia. Specificity of the assays was validated using 5 closely related, nontarget paropsine species, 13 predator taxa, and 4 species of alternative prey. Both assays have a high specificity and sensitivity, can distinguish DNA of the 2 paropsine species from nontarget species and detect as little as 10 copies of DNA within the bodies of field-collected arthropod predators. Further, we provide a rigorously tested surface decontamination method for predator samples collected with a beating tray to avoid compromising molecular results with the detection of trace prey DNA on the exterior of the predator. These 2 RT-PCR assays represent the first ready-to-use molecular approach that can reveal the relationship between paropsines and arthropod predators in the field. They could be used to evaluate potential biocontrol agents, assess their post-release control effectiveness, and study spatial and seasonal variation of predator-prey dynamics to understand the effectiveness of management strategies.