Aggregation-seeking assay

In this assay, fresh fully fed bed bugs were placed in the top jar of the bioassay assembly and challenged to cross the bed net to reach a dark resting site. Newly molted second-instar bed bugs were starved 7–10 days and fed for 1 h in a 5.5 × 4.8-cm jar at 20–40% relative humidity and 25–26 °C without exposure to a bed net. Only fully fed bed bugs were selected and placed in a new jar that contained a fresh file folder paper walkway. This jar was connected as the top jar to the bed net-sandwich assembly. The bottom jar served as the target for aggregation-seeking fed bed bugs. To make this jar attractive to fed bed bugs, we “conditioned” file folder paper by exposing it to a colony of bed bugs, which impregnated it over time with feces and aggregation pheromone. A section of the aggregation-pheromone impregnated paper was placed within the target (bottom) jar, and the jar was wrapped completely in aluminum foil to create an attractive darkened aggregation site [38]. Furthermore, the whole bioassay assembly was placed horizontally in a 25 °C incubator on a photoperiod of 12:12 light:dark h at 35–45% relative humidity. The combination of the attractive paper and darkness served to attract bed bugs toward the aggregation jar, while the incubator lights repelled bed bugs away from the exposed starting jar. We counted and removed bed bugs from the target aggregation jar after 1 h, and then every 24 h, during the photophase (daytime) for 7 days. At each check, we removed the aggregation jar from the assay assembly, counted and removed the bed bugs (including dead bugs) and reconnected the aggregation jar to the bioassay system.