|
```markdown |
|
# 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. |
|
|
|
--- |
|
endofoutput |
|
``` |