Thermo-tolerance comparisons among bed bug strains

The procedures used for step-function thermo-tolerance comparison experiments were similar to those used for determining LT₇₅ estimates for the Harlan nymphs. For each population, ten mixed sex adult insects (1:1 ratio) were placed into a 15-mL glass test tube with a strip of filter paper as harborage. Test tubes were sealed with Parafilm, placed in a 12x6 plastic holding rack and then transferred to a water bath heated to 45°C. Insects were exposed at 45°C for 10, 12, 13, 14, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 mins to generate exposure time-mortality data. Three to four replicates (10 adults per replicate) were performed for each time point. Additional time points that provided 75–100% mortality were included in step-function heat exposure experiments to increase the precision of LT₉₉ estimations [34]. Test tubes were removed from the water bath after the exposure period had elapsed and bed bugs were placed in a 35x10mm Petri dish with a Whatman No. 1 filter paper disc. Petri dishes were held in an environmental chamber with temperature, humidity, and light conditions identical to those used for rearing. Mortality was scored 24 h after exposure using the parameters described under determination of lethal time estimates for late instar nymphs. Control insects were held in test tubes at room temperature and then transferred to Petri dishes.

Procedures for the ramp-function heat exposure bioassay that utilizes a gradual or incremental increase in temperature were somewhat similar to those used for the step-function bioassays explained above. Briefly, 15-mL glass test tubes with 10 mixed sex adult bed bugs (1:1 ratio) per tube were placed in a 12x6 plastic holding rack that was transferred to a water bath at room temperature. The water bath was then turned on and the bed bugs were exposed to gradually rising temperatures at the rate of 0.57 °C/min until the water temperature reached 45°C. Once the water bath reached 45°C (~37-min heating time), insects were held in the water bath for a time that corresponded with the LT₉₉ time for the Harlan strain. After the ramp-up heat exposure period was completed, insects were placed into Petri dishes with filter paper and mortality was scored 24 hours later using previously described criterion under determination of the lethal time estimates for late instar nymphs’ section. Three replicates of ten mixed sex bed bugs per replicate were performed for each population, including the Harlan strain, which was used as a positive control for all bioassay tests. Test tubes containing control insects were held at room temperature during the ramp-up heat exposure experiments.