TITLE: A novel laboratory method to simulate climatic stress with successful application to experiments with medically relevant ticks
AUTHORS: Sang Hyo Kim, Caleb Nielebeck, Lauren Dedmon, Mark Pangilinan, Jahred Quan, William Ota, Javier D. Monzón

[DESCRIPTION]
This protocol details a novel method to isolate individual ticks and manipulate their environment. We successfully used this method to investigate how humidity affects survival and host-seeking (questing) behavior of three species of ticks: the lone star tick (Amblyomma americanum), American dog tick (Dermacentor variabilis), and black-legged tick (Ixodes scapularis). We placed 72 adult females of each species into individual plastic tubes and separated them into three experimental relative humidity (RH) treatments representing distinct climates: 32% RH, 58% RH, and 84% RH. For 30 days we assessed the survival and questing behavior of each tick.

The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.

[BEFORE_START]
We acquired adult female ticks from the Oklahoma State University Tick Rearing Facility and experimented with one temperature range and three relative humidities. This protocol can be modified for different species, life stages, temperatures, humidities, and other small organisms. 

[GUIDELINES]
Questing qualifications: 
The individual is not walking but it is still with its front legs extended       

Death qualifications: 
If any tick appears dead, lightly blow on it since ticks respond to carbon dioxide exhaled by potential hosts
If the tick does not move at all in 2 minutes, it should be counted as dead and placed in 70% ethanol

[STEPS]
SECTION: Set up
1. Place a single tick with one wooden skewer in each tube and seal with a cap, labelling each tube with an individual identifier  
  
SECTION: Set up
2. Place six tubes in each airtight container along with a humidity pack, labelling each container
   

SECTION: Set up
2.1. Confirm the humidity in one container of each RH level with the data logger 

SECTION: Set up
3. Program the climate chamber
 

SECTION: Set up
3.1. To cycle between 20 °C to 30 °C, the temperature increments should be as follows: 
3:00  - 25 °C 
6:00  - 27.5 °C 
9:00  - 30 °C 
12:00  - 27.5 °C 
15:00  - 25 °C 
18:00  - 22.5 °C 
21:00  - 20 °C 
24:00  - 22.5 °C 


SECTION: Set up
3.2. To create a 12:12 light:dark photoperiod, lighting increments should be as follows:
9:00 - Lights on
21:00 - Lights off
SECTION: Data collection
4. Place all of the airtight containers, filled with ticks and humidity packs, into the climate chamber and start the program
SECTION: Data collection
5. Each day thereafter, during the 9:00 to 12:00 or 30 °C increment, assess each tick for survivorship and questing behavior (see guidelines for qualifications of survivorship and questing)
 

 


SECTION: Data collection
5.1. Periodically move the data logger to a new bin to confirm that no unexpected changes to the climate inside the containers has occurred
SECTION: Data collection
6. Repeat steps 4 and 5 for 30 days or until all ticks have died
SECTION: Spotlight video
7.