Oct 16, 2023
Version 5
A novel laboratory method to simulate climatic stress with successful application to experiments with medically relevant ticks V.5
Peer-reviewed method
- Sang Hyo Kim1,
- Caleb Nielebeck1,
- Lauren Dedmon1,
- Mark Pangilinan1,
- Jahred Quan1,
- William Ota1,
- Javier D. Monzón1
- 1Pepperdine University
External link: https://doi.org/10.1371/journal.pone.0275314
Protocol Citation: Sang Hyo Kim, Caleb Nielebeck, Lauren Dedmon, Mark Pangilinan, Jahred Quan, William Ota, Javier D. Monzón 2023. A novel laboratory method to simulate climatic stress with successful application to experiments with medically relevant ticks. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzyo8rlx1/v5Version created by Caleb Nielebeck
Manuscript citation:
Nielebeck C, Kim SH, Dedmon L, Pangilinan M, Quan J, et al. (2022)A novel laboratory method to simulate climatic stress with successful application to experiments with medically relevant ticks. PLOS ONE 17(9): e0275314.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 13, 2023
Last Modified: October 16, 2023
Protocol Integer ID: 89273
Keywords: Tick questing, tick behavior experiment, Amblyomma, Dermacentor, Ixodes
Abstract
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.
Guidelines
Questing qualifications:
The individual is not walking but it is still with its front legs extended
Example of an Amblyomma tick questing
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
Materials
Ticks:
- 72 adult female Amblyomma americanum
- 72 adult female Dermacentor variabilis
- 72 adult female Ixodes scapularis
Experimental set up:
- 1 - Climate chamber (e.g. Percival I-41VL)
Equipment
Incubator
NAME
Climate Chamber
TYPE
Percival
BRAND
I-41VL
SKU
LINK
- 216 - 20 cm x 2.5 cm Clear PETG plastic tubes
- 216 - 20 cm Wooden skewers
- 36 - 2 L Airtight containers
- 12 - 32 % Boveda Two-Way Humidity Control Packs
- 12 - 58 % Boveda Two-Way Humidity Control Packs
- 12 - 84 % Boveda Two-Way Humidity Control Packs
- 1 - Temperature/relative humidity data logger (e.g. ONSET UX100-003)
Equipment
HOBO Temperature/Relative Humidity 3.5% Data Logger
NAME
Data logger
TYPE
ONSET
BRAND
UX100-003
SKU
LINK
- 70% Ethanol
- Colored dot stickers
- Sharpie
Other tools:
- Entomology forceps
- 30 cm ruler
- White surface (e.g. lab bench diaper)
Safety warnings
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.
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.
Set up
Set up
2h
Place a single tick with one wooden skewer in each tube and seal with a cap, labelling each tube with an individual identifier
PETG plastic tubes with wooden skewers prepared for ticks. Notice that we drilled holes in the caps to allow airflow between the tubes and the containers.
Place six tubes in each airtight container along with a humidity pack, labelling each container
Airtight container with six tubes, humidity pack, and data logger. Example container of Dermacentor variabilis (purple) at 58% RH; every container was replicated four times for a sample size of n=24 per RH group.
Confirm the humidity in one container of each RH level with the data logger
Program the climate chamber
Note
Reference your chamber's user's manual
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
To create a 12:12 light:dark photoperiod, lighting increments should be as follows:
- 9:00 - Lights on
- 21:00 - Lights off
Data collection
Data collection
5w 5d
Place all of the airtight containers, filled with ticks and humidity packs, into the climate chamber and start the program
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)
Note
Only take one container out of the chamber at a time
Note
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
Periodically move the data logger to a new bin to confirm that no unexpected changes to the climate inside the containers has occurred
Repeat steps 4 and 5 for 30 days or until all ticks have died
Spotlight video
Spotlight video