A simple PCR-based method for detecting Anagyrus lopezi (Hymenoptera: Encyrtidae) and Prochiloneurus pulchellus (Hymenoptera: Encyrtidae), primary and hyper parasitoids of the cassava mealybug Phenacoccus manihoti (Hemiptera: Pseudococcidae)

Estimating parasitism rates in the field is essential for developing and evaluating biocontrol strategies using parasitoids. In this study, we developed a simple polymerase chain reaction (PCR)-based method for detecting parasitism of the cassava mealybug Phenacoccus manihoti Matile-Ferrero (Hemiptera: Pseudococcidae) by the primary parasitoid Anagyrus lopezi De Santis (Hymenoptera: Encyrtidae) and its hyperparasitoid Prochiloneurus pulchellus Silvestri (Hymenoptera: Encyrtidae). Primers were designed to amplify partial cytochrome c oxidase subunit I genes of each species, and their sensitivity was evaluated with mealybugs that had been parasitized by A. lopezi 0, 3, and 6 days earlier, and mummified mealybugs containing A. lopezi pupae that had been parasitized by P. pulchellus 0, 3, 6, 9, and 12 days earlier. The detection rate of parasitism by A. lopezi was 100% for all ages of A. lopezi. The detection rate of parasitism by P. pulchellus ranged from 94.1% to 100%, depending on its developmental stage. For P. pulchellus, template DNA was diluted 10 times before PCR because PCR with the original concentration showed low detection rates, presumably due to the presence of PCR inhibitors. Overall, our primers can be considered sufficiently sensitive to be used for detecting each species.