High-Throughput Assays of Critical Thermal Limits in Insects
Summary
Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.
Abstract
Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.
Introduction
Thermal limits of insects
Variation in environmental conditions, including temperature, is a major factor influencing the performance, fitness, survival, and geographic distribution of organisms1,2. Upper and lower thermal limits determine the theoretical range of environments an organism can tolerate, and, therefore, these limits are important predictors of plant and animal distributions, especially in the face of climate change3,4. Thus, protocols to accurately measure thermal limits are important tools for ecologists, physiologists, evolutionary biologists, and conservation biologists, among others.
As the most abundant and diverse terrestrial animals, insects are frequently used for measurements of thermal limits. Critical thermal maxima (CTmax) and critical thermal minima (CTmin) are commonly used to assess intra- and interspecific variation in thermal tolerance5,6,7. While CTmax and CTmin can be measured for multiple phenotypes, including growth, reproductive output, and behavior, they are most commonly applied to locomotor function5,6,7. Thus, CTmax (also called heat knockdown temperature) and CTmin are often defined as the high and low temperatures at which insects lose motor function and are unable to remain upright5,6,7,8,9,10,11. CTmin coincides with the onset of chill coma, a reversible paralysis brought on by cold temperatures6. While paralysis at the thermal limits is often reversible, continued exposure to these temperatures leads to ecological death5.
Common methods for measuring thermal limits
A variety of apparatuses have been used to measure thermal limits (summarized in Sinclair et al.)6. Briefly, insects are heated or cooled in incubators12,13, containers submerged in fluid baths11,14,15,16, aluminum blocks10,17, or jacketed containers18, and monitored until locomotion ceases. To monitor insects during the assay, the most common method is direct observation, in which individuals are continuously monitored in real time or retrospectively with recorded video6,9,10,11,15,17. While direct observation methods have minimal equipment requirements, they are labor-intensive and limit throughput. Alternatively, insects can be observed indirectly by collecting individuals at discrete times as they fall from perches6,19,20,21 or using activity monitors13.
Indirect methods for measuring thermal limits are generally higher-throughput and potentially less error prone than direct observation methods. The most common method for indirect monitoring uses a jacketed temperature-controlled column6,8,19,20,21. Insects are placed inside a column with perches, and the temperature of the inner chamber is controlled by pumping fluid from a temperature-controlled fluid bath through the jacketed lining of the column. Individuals that reach their thermal limit fall from their perch and are collected at discrete temperatures or time intervals. While this method works well for CTmin, it has been found unsuitable for CTmax, because flies voluntarily walk out of the bottom of the column when the temperature increases. The new method described here circumvents this issue by individually containing flies during automated measurements.
In addition to the method of observation, two types of temperature regimes are commonly used to assess upper thermal limits. Dynamic assays consist of gradually increasing temperature until motor function is lost; that temperature is the dynamic CTmax7,8,9,13. In contrast, static assays consist of a constant stressful temperature until motor function is lost; that time point is the heat knockdown time (heat KDT), also called the static CTmax (sCTmax) in a recent paper by Jørgensen et al.7,8,9,16,22. Although CTmax and heat knockdown assays (heat KD assays) produce metrics with different units, mathematical modeling of the two traits indicates they give comparable information on heat tolerance and are both ecologically relevant8,9. Dynamic assays yield a temperature that can be compared to environmental conditions, and they are preferable when there are large differences in heat tolerance, such as comparisons between species with widely different thermal niches. However, due to the high Q10 for heat injury accumulation, a static assay may be preferable for detecting small effect sizes, such as intraspecific variation in heat tolerance9. Also, practically speaking, a static assay requires less sophisticated equipment than a dynamic assay.
Objective
The objective of this paper is to formalize methods for CTmin and heat KD assays that can be used in future research to assess the thermal limits of motile insects. The protocols are adapted from previously established methodologies and are designed to be high-throughput, automated, and cost-effective. Both assays can be completed in a short amount of time (~2 h), which means that multiple experiments can be conducted in a single day, producing large amounts of data without sacrificing repeatability or accuracy. With this setup, the heat tolerance of 96 flies can be measured simultaneously, while the column for CTmin can hold more than 100 flies, provided there is adequate surface area for perching.
The high-throughput method for observing CTmin modifies the common jacketed column methodology with the addition of an infrared sensor to automatically count flies. The use of an infrared sensor for counting was first proposed by Shuman et al. in 199623 but has not been widely adopted. The addition of the infrared sensor allows for the generation of continuous data rather than collecting data at discrete intervals. This protocol also minimizes experimenter error by eliminating manual data entry and the need to manually switch collection tubes below the jacked column at discrete time points.
The high-throughput method for recording heat KDT is modified from two previous studies of heat tolerance in insects10,12. Individual flies are stored in a 96 well plate in a temperature-controlled incubator and video is recorded. This protocol minimizes experimenter bias in determining heat KDT because experiments can be reviewed and verified by playing back the recording. This protocol also provides a set of custom Python scripts that can be used to speed up video analysis. The use of individual wells eliminates interference that can occur when other individuals move around or fall over, which can be a problem when groups of individuals are observed in the same arena10,17. Furthermore, the temperature-controlled incubator provides a stable temperature across all 96 wells, unlike the temperature gradient sometimes observed across a temperature-controlled aluminum block10. Also note that the 96 well recording method can be adapted to measure dynamic CTmax and potentially CTmin (see Discussion).
To demonstrate each protocol, the thermal limits of adult Drosophila melanogaster females from select lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) were compared24. These lines were selected because preliminary experiments indicated significant differences in thermal tolerance. These assays proved to be robust methods for discriminating differences in thermal tolerance. The following two protocols, high-throughput CTmin assay (section 1) and high-throughput heat KD assay (section 2), describe the necessary actions to produce CTmin and heat KDT data for any motile insect life stage capable of fitting in the apparatuses, such as adult Drosophila. For CTmin it is also essential that the insect be able to perch. Here, each assay is demonstrated in adult Drosophila melanogaster. However, modifications may be required for other taxa or life stages6. Minor changes might include using perching material with larger openings to accommodate larger specimens in the CTmin assay or using a higher quality camera to discern the subtle KDT of a slow moving insect or life stage in the heat KD assay. This protocol does not describe methods for preparing flies, but it is important to standardize rearing protocols to ensure repeatability25 (see Garcia and Teets26 and Teets and Hahn27). The protocols provided include information on how to build and set up the apparatuses, how to record measurements, and a brief description of data analysis.
Protocol
Representative Results
Discussion
The two methods detailed above generate high-throughput data of ecologically relevant metrics for upper and lower thermal limits. These protocols build upon previously established methodologies common to research on insect thermal limits (summarized in Sinclair et al.)6. Both protocols can be completed in a short amount of time (~2 h each), produce data sets with large sample sizes, do not sacrifice repeatability or accuracy, and minimize experimenter error by eliminating manual data record…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Ellie McCabe for assistance with fly rearing. This work is supported by United States Department of Agriculture National Institute of Food and Agriculture Hatch Project grant 1010996 and National Science Foundation grant OIA-1826689 to N.M.T.
Materials
| ARCTIC A40 Refrigerated fluid circulator (Programable teperature ramps) | Thermo Scientific; Waltham, MA | 153-5401 | |
| C922 Pro Stream Webcam | Logitech; Newark, CA | 960-001087 | |
| Circular adjustable steel clamp – 5.08 cm to 7.62 cm | Any | Any | |
| Clear acrylic tubing – 5.7 cm x 5.1 cm x 0.3 cm | United States Plastic Corp., OH | 44036 | |
| Clear acrylic tubing – 6.35 cm x 5.7 cm x 0.3 cm | United States Plastic Corp., OH | 440515 | |
| Clear acrylic tubing – 7 cm x 6.35 cm x 0.3 cm | United States Plastic Corp., OH | 44041 | |
| Clear silicone sealant | Any | Any | |
| Collection tube (15 ml) | Any | Any | |
| Cordless Drill | Any | Any | |
| Drosophila Funnel Monitor (DFM) | TriKinetics; Waltham, MA | DFM | Used to count the number of flies that fall through the funnel at a given time point |
| DAM data collection software | TriKinetics; Waltham, MA | Records data input from the DFM | |
| Fly Storage Lid | FlySorter; Seatle, WA | FS-96LID-5PK | Used to load flies into the storage plate for the sCTmax assay |
| Fly Storage Plate | FlySorter; Seatle, WA | FS-96PLATE-5PK | Used to hold flies during in the sCTmax assay |
| Fly Food Tray | FlySorter; Seatle, WA | FS-TRAY-5PK | Used to keep flies on food after loading into the 96-well plate until the sCTmax assay |
| Glass funnel | Kimax | 28950-75 | 75mm |
| Gutter guard | Any | Any | ~0.5 cm diameter openings |
| Hacksaw | Any | Any | |
| Heratherm Thermo Scientific incubator | Thermo Scientific; Waltham, MA | OMS100 | |
| Hose nylon adapters (2) – ¼ MNPT x 3/8 | United States Plastic Corp., OH | 61135 | |
| Hot glue gun and glue | Any | Any | |
| Light Source | Any | Any | |
| Magnets | Any | Any | |
| OMEGA TC-08 Recorder and TC-08 Player Software | OMEGA; Norwalk, CT | ||
| OMEGA thermocouple (Type T) | OMEGA; Norwalk, CT | 5LRTC-TT-K-20-36 | |
| Plastic funnel | Any | Any | 2" diameter |
| Plastic tubing – 0.6 cm diameter | United States Plastic Corp., OH | 62852 | |
| Retort ring | Any | Any | 2" diameter |
| Retort stand | Any | Any | |
| Retort three-prong clamp | Any | Any | |
| Rstudio | |||
| Serial port connector (PSIU9) | TriKinetics; Waltham, MA | PSIU9 | Intermediate connection between the DFM and computer, allows for multiple DFM connections |
| Styrofoam (2" thick) | Any | Any | |
| Tape | Any | Any | |
| Uninterrupted Power Supply (PS9-1) | TriKinetics; Waltham, MA | PS9-1 | Power supply for the DFM and PSIU9 |
| Weld-on #4 Acrylic Cement | United States Plastic Corp., OH | 45737 |
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