Experimental setup

We used 216 adult female ticks from three different species: 72 Amblyomma americanum, 72 Dermacentor variabilis, and 72 Ixodes scapularis, and placed them into individual plastic tubes (20 cm height x 2.5 cm diameter). In each tube, we placed a 20-cm wooden skewer to provide a perching surface for the ticks in addition to the tube’s plastic surface. We secured the opening of the tubes with either a mesh screen fastened with a rubber band or the tube’s cap with drilled holes to permit free circulation of air. We divided the 216 tubes into 36 2-L airtight containers– 12 containers for each species and six tubes per container. For each species, we separated the 12 containers into three groups of four and placed a two-way humidity control pack into each airtight container to create three climate groups: 32%, 58%, and 84% RH. Thus, each container with six ticks was replicated four times for a total of 24 ticks per group and nine total groups (Fig 1). The humidity control packs use a combination of salts and water to set and maintain a predetermined RH inside the containers. We positioned the 12 containers at random into their own designated plastic tray, which we then placed onto one of the three racks of an incubator at random. We programmed the incubator to cycle between 20°C and 30°C in a 24-hour period every day, changing by 2.5° every three hours. We also programmed the incubator with a 12:12 photoperiod to turn the lights on from 9 AM to 9 PM and off from 9 PM to 9 AM. We used a HOBO data logger to independently record temperature and RH inside the containers in hourly intervals.

Each cylinder represents a plastic tube containing one tick, and each rectangle represents an airtight container with a constant RH maintained by a humidity control pack. There were nine distinct groups based on species and RH. Each container with six ticks was replicated four times for a total sample of 24 ticks per group.