Determining Temperature Preference of Mosquitoes and Other Ectotherms
September 28th, 2022
Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.
This two chamber apparatus setup is used to study the temperature preference of insects and can be modified to study other binary choice preference situations such as light versus dark or high versus low humidity. The main advantages of this technique are the simple setup, the spacious area that allows the insects to exhibit their flying nature when choosing their preferred temperature, and easy scoring without external equipment. The most difficult part of the protocol is removing the acrylic breach after the experiment.
This is because there is a high chance of the insects escaping. Thus, it is crucial for us to use the carbon dioxide to anesthetize the insects. Demonstrating the procedure will be Kinga Stobierska and Lorna Glenn, both master students from my laboratory.
To begin, prepare 2 empty mosquito cages with 12 centimeter sleeve openings and ensure there are no holes or other damages. Then using a mechanical aspirator, transfer 30 insects to a separate cage for easier handling and disposal after the experiment. Set the incubators to the desired temperatures and allow them to heat up and stabilize.
It takes approximately 30 minutes to achieve a temperature in the range of 25 to 30 degrees Celsius. Then place an empty mosquito cage in an incubator and feed the sleeves of the cage through the front hole of the incubator. Confirm the inside air temperature of the incubator with a temperature probe.
Next, place an openable flap prepared with duct tape over the hole in the acrylic tube. Roll up the sleeve protruding through the hole in front of the first incubator, and secure it over one end of the acrylic tube with an elastic band. Then place the incubators facing each other and insert one end of the acrylic tube into the sleeve of second incubator hole.
Make sure that the diameter of the acrylic tube is larger than the hole in the front of the incubators so that it completely covers the hole. Ensure the acrylic tube is not loose and dangling between the incubators. If it is, pull the cage sleeves to remove excess material between the cage and rubber band.
To put the mosquitoes inside, open the duct tape flap. Place a funnel into the hole of the acrylic tube and then empty the insects into the tube through the funnel. After emptying the insects, remove the funnel and cover the tube hole with a duct tape flap.
Leave for 30 minutes for the insects to select the preferred chamber. At the end of the settling time, visually observe and write down the number of insects seen in the acrylic tube bridge. Then tap or blow all the insects in the bridge to either side of the incubator or by releasing carbon dioxide into the bridge.
Next, pinch and close the sleeves from the acrylic tube on both sides by quickly fastening them with a tight knot. Then remove the cages from both incubators and visually count the insects in each cage. Make sure the numbers from both incubators and the bridge add up to 30.
In the present study the temperature preference of the mosquitoes and fruit flies was determined using the two-chamber model. The results showed no significant difference when both the cages were maintained at 27 degrees Celsius, indicating the mosquitoes ability to freely choose between cages exhibiting their natural behaviors. Conversely, when the two chambers were set at different temperatures the mosquitoes consistently selected the optimal temperature of 27 degrees Celsius over 30 degrees Celsius.
Similarly, when the setup was tested with Drosophila Melanogaster, another ectoderm model the fruit flies avoided the warmer chamber and preferred their optimal temperature of 25 degrees Celsius. Ensure that the setup is completely sealed with no hiding spaces and no mesh disrupting the flying area so that the insects can freely choose between the 2 chambers.
Overview
This study presents a simple and cost-effective method for assessing temperature preferences in insects using a two-chamber apparatus. The method allows insects to engage in natural behaviors, revealing their preferred environmental temperature.
Key Study Components
Research Area
- Insect behavior
- Temperature preference
- Habitat choice
Background
- Insects thrive within specific temperature ranges.
- Various factors influence their temperature preferences.
- Understanding temperature choices impacts ecological and evolutionary studies.
Methods Used
- Two-chamber apparatus for temperature choice
- Mosquitoes and Drosophila melanogaster as model organisms
- Visual scoring and carbon dioxide anesthesia for insect handling
Main Results
- Mosquitoes preferred 27°C over 30°C under varying conditions.
- Drosophila melanogaster also favored 25°C over warmer temperatures.
- Methodology showcased the insects' ability to exhibit natural behaviors in temperature selection.
Conclusions
- This study successfully demonstrates an effective way to study temperature preference in insects.
- The findings have implications for understanding insect behavior in changing environments.
Frequently Asked Questions
Executive Industry Relevance
Quantitative assessment of temperature preference in mosquitoes and other ectotherms enables robust hypothesis testing for environmental adaptation and vector biology. This two-chamber apparatus supports predictive confidence in behavioral phenotyping by allowing natural behaviors, such as flight, critical for accurate target validation in early discovery. The method's flexibility and scalability position it as a reusable platform for comparative studies across diverse ectothermic models in biopharma R&D.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables rigorous interrogation of thermosensory pathways and behavioral phenotypes.
- Supports functional target validation by isolating temperature as an independent variable.
- Facilitates mechanistic de-risking for vector-borne disease research.
- Provides a platform for evaluating genetic or environmental manipulations affecting temperature preference.
Screening & Assay Development
- Prepares validated behavioral assays for downstream screening of modulators or interventions.
- Ensures reproducibility and standardization through controlled environmental conditions.
- Delivers quantitative outputs for robust comparison of experimental groups.
- Enables scalable, high-throughput behavioral screening in small ectotherms.
Translational & Preclinical Research
- Aligns behavioral phenotyping with disease-relevant environmental exposures.
- Supports translational continuity from discovery to preclinical vector competence studies.
- Enables risk-adjusted advancement of candidate interventions targeting vector behavior.
- Provides predictive de-risking for environmental and genetic manipulation strategies.
Pipeline & Workflow Integration
This apparatus integrates into the discovery-to-preclinical continuum by enabling hypothesis-driven behavioral assays, supporting both target validation and screening workflows.
- Discovery Biology: Facilitates null hypothesis testing and pathway clarification for thermosensory mechanisms.
- Screening: Provides standardized, reproducible behavioral assays with quantitative readouts.
- Analytics: Enables statistical comparison of group preferences and behavioral responses.
- Translational Research: Bridges laboratory findings to field-relevant environmental exposures in vector biology.
- Enterprise Reuse: Adaptable for diverse ectothermic species and environmental manipulations, supporting broad R&D needs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in behavioral phenotyping.
- Operational Value: Promotes assay standardization, reproducibility, and scalability across research teams.
- Strategic Value: Informs go/no-go decisions for vector-targeted interventions and environmental adaptation studies.
- Portfolio Impact: Supports risk-adjusted prioritization of behavioral and environmental intervention strategies.
Implementation Considerations
- Requires expertise in behavioral assay design and ectotherm handling.
- Needs temperature-controlled incubators and basic analytical infrastructure for quantitative scoring.
- Demands cross-team standardization of assay setup and scoring protocols.
- Adaptable to various small ectotherms and environmental manipulations with minimal modification.
- Careful containment and anesthetization procedures are critical to prevent escape and ensure safety.
