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# Goal/Experiment:
To isolate individual ticks and control their environment in order to examine how different humidity levels affect the survival and host-seeking behavior of three medically important tick species.

# A Novel Laboratory Method to Simulate Climatic Stress with Successful Application to Experiments with Medically Relevant Ticks V.2

**Authors:**  
Sang Hyo Kim¹, Caleb Nielebeck¹, Lauren Dedmon¹, Mark Pangilinan¹, Jahred Quan¹, William Ota¹, Javier D. Monzón¹  
¹Pepperdine University

**DOI:**  
[dx.doi.org/10.17504/protocols.io.rm7vzyo8rlx1/v2](dx.doi.org/10.17504/protocols.io.rm7vzyo8rlx1/v2)

**Citation:**
Sang Hyo Kim, Caleb Nielebeck, Lauren Dedmon, Mark Pangilinan, Jahred Quan, William Ota, Javier D. Monzón. 2022. A Novel Laboratory Method to Simulate Climatic Stress with Successful Application to Experiments with Medically Relevant Ticks. *protocols.io*. DOI:10.17504/protocols.io.rm7vzyo8rlx1/v2

## Introduction

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. 

## Materials

### Required Ticks
- 72 adult female *Amblyomma americanum*
- 72 adult female *Dermacentor variabilis*
- 72 adult female *Ixodes scapularis*

### Equipment
- 1 Climate Chamber (e.g. Percival I-41VL)
- 216 - 20 cm x 2.5 cm Clear PETG plastic tubes
- 216 - 20 cm Wooden skewers
- 36 - 2 L Airtight containers
- 12 - 32% RH Boveda Two-Way Humidity Control Packs
- 12 - 58% RH Boveda Two-Way Humidity Control Packs
- 12 - 84% RH Boveda Two-Way Humidity Control Packs
- 1 Temperature/Relative Humidity Data Logger (e.g. ONSET UX100-003)
- 70% Ethanol
- Colored dot stickers
- Sharpie

### Other Tools
- Entomology forceps
- 30 cm ruler
- White surface (e.g. lab bench diaper)

> **Note:** Always handle ticks with blunt entomology forceps, as regular forceps can injure them. Always handle ticks over a white surface so that they can easily be spotted in case they are dropped.

### Alternative Methods
For hard-to-find supplies such as specific humidity control packs, these can be made using saturated salt solutions to maintain different humidity levels:
- 32% RH: Saturated solution of magnesium chloride
- 58% RH: Saturated solution of sodium bromide
- 84% RH: Saturated solution of potassium chloride

## Experimental Setup

### Preparation
1. **Incubator Setup:**
   - Program the climate chamber to cycle between 20°C and 30°C with specific temperature increments.
   - Ensure a 12:12 light:dark photoperiod.

2. **Setup Time: 2h**
   - Place a single tick with one wooden skewer in each tube and seal with a cap, labeling each tube with an individual identifier.
   - Place six tubes in each airtight container along with a humidity pack, labeling each container.
   - Confirm the humidity in one container of each RH level with the data logger.
   - Program the climate chamber.

### Program the Climate Chamber
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

### Data Collection: 5w 5d
3. Place all the airtight containers, filled with ticks and humidity packs, into the climate chamber and start the program.

4. Each day thereafter, during the 9:00 to 12:00 or 30°C increment, assess each tick for survivorship and questing behavior. Only take one container out of the chamber at a time. Collect a binary outcome for survivorship and questing, and measure the tick's height (to the nearest 0.5 cm) in the tube if it is found questing.

5. Periodically move the data logger to a new bin to confirm that no unexpected changes to the climate inside the containers have occurred.

6. Repeat steps 4 and 5 for 30 days or until all ticks have died.

### Questing Qualifications
- The individual is not walking but is still with its front legs extended.

![](https://example.com/amblyomma_questing.jpg)
*Example of an **Amblyomma** tick questing.*

![](https://example.com/dermacentor_questing.jpg)
*Example of a **Dermacentor** tick questing.*

### 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.

## Results
This method allows for in-depth analysis of tick behavior under controlled humidity conditions, providing valuable insights into their survivorship and questing behavior.

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