Published: March 9th, 2021
Here, a low-cost, accessible protocol is described to evaluate cold shock recovery of butterflies under ambient environmental conditions.
Ecological physiology, particularly of ectotherms, is increasingly important in this changing world as it uses measures of species and environmental traits to explore the interactions between organisms and their surroundings to better understand their survival and fitness. Traditional thermal assays are costly in terms of time, money, and equipment and are therefore often limited to small sample sizes and few species. Presented here is a novel protocol that generates detailed data on individual behavior and physiology of large, volant, terrestrial insects, using the example of butterflies. This paper describes the methods of a cold shock recovery assay that can be performed in the field under ambient environmental conditions and does not require costly laboratory equipment. This method has been used to understand the response and recovery strategy to cold shock of tropical butterflies, generating individual level data across entire butterfly communities. These methods can be employed in both remote field settings and classrooms and can be used to generate ecologically relevant physiological data and as a teaching tool.
The integration of thermal physiology and ecology in the late 1970s and early 1980s1,2 launched the field of ecological physiology. Extensive thermal studies conducted on ectotherms highlight ecological-physiological synergies across diverse eco-evolutionary contexts3,4,5. Research on thermal physiology of ectothermic organisms has regained attention recently in the face of climate change and altered thermal landscapes across the world6,7. In addition to informing studies in the academic field of ecological physiology, thermal physiology assays can be broadly accessible to researchers and can serve as a hands-on teaching approach for all levels. Components of thermal performance, including thermal limits and effects of temperature shocks, are fundamental to the ecology, behavior, and life history of animals8,9.
Specifically, ecototherms are used to address questions of physiology, as endothermy dictates an inextricable link between ambient and organismal temperature. The temperature range that organisms can withstand (their critical thermal minimum to maximum-thermal range) and the temperatures at which their behaviors and fitness are maximized (thermal optima) are often rooted in ecological and evolutionary processes. These physiological traits are of increasing importance as temperatures, both means and extremes, are increasing10. For example, the abiotic changes, including temperature increases, that accompany habitat destruction and fragmentation has affected communities of ectotherms, including anurans, limiting physiologically fragile species (with narrow thermal tolerance) to small remnant habitat patches11,12.
Assessing key components of thermal performance can be expensive both in terms of time and resources and traditionally requires laboratory equipment and standardized conditions. Moreover, conventional assays often do not reflect the breadth of ambient conditions experienced in nature by a given animal13 as temperature in similar physiology experiments is carefully controlled and often unrelated to ambient conditions experienced by an animal. This temperature control can diminish the understanding of variation in individual responses2,14. Physiologists have relied on laboratory-based heating and cooling experiments, using programmable water baths to steadily heat or cool an animal's environment to inform thermal performance curves15.
Typically, animals are placed in vials with a thermocouple, and their ambient temperature is changed steadily by controlling the temperature of the surrounding water bath. Researchers measure the time it takes to achieve an altered physiological state (e.g., chill coma, knockdown) and the temperature at which the status change occurred16,17. Starting at a minimum of USD $500, these tools are large, heavy, and require additional technical equipment (e.g., computer, thermocouples). Consequently, the basic tools to carry out classic methods of assessing thermal performance are 1) not economically accessible to all, 2) not suitable for assaying animals too large to be contained in customary vials used for small dipterans, and 3) not portable for use in remote field settings. Adherence to common practice has resulted in limited representation across taxonomy and experimental conditions18,19,20.
While complete thermal performance curves can inform species distribution, life history traits, and behavior, among other traits, the quantification of fewer and simpler thermal metrics can be more efficient and still extremely informative. Physiological assays, measuring chill coma onset and subsequent cold shock recovery, cold-hardening, and righting behavior, are effective and executable proxies for the critical thermal minimum of an organism8. Described here is a cold shock assay useful for eliciting physiological data from large terrestrial ectothermic insects. The assay is affordable, accessible, and easy to execute under field conditions or in the classroom. Data on cold shock recovery generated by this protocol can be coupled with species or individual-level trait data to pursue questions regarding ecological physiology and/or used to teach students about physiological principles.
1. Identification of species of interest
2. Conducting a pretrial
3. Collection of insects
4. Set up the cold shock experiment
5. Start cold shock experiment
6. Data processing
The data collected in this protocol allow for examination and partitioning of variables important to organismal physiology. For example, both temperature and light conditions contribute to the recovery of butterflies from cold shock (Figure 1). The plot is intended to explore the interaction between ambient conditions and cold shock recover. Using wild-caught butterflies from both traps and netting, 181 species of butterflies demonstrated distinct recovery from chill coma induced by cold shock (Figure 2). Data presented in Figure 2 were collected by three observers over approximately five months (January, February, May-July 2020) in the Colombian Andes. Experiments were always conducted on the morning after butterfly collection. At maximum efficiency, it was possible for two observers to simultaneously observe four butterflies each, repeated seven times (minimum 7.37 hours), resulting in the testing of 56 individuals on a single morning. This allowed for a great deal of data collection across entire butterfly communities while including and considering data on individual variation. As assays can occur under ambient environmental conditions, recovery conditions are representative of their habitats and reflect the natural variation experienced by organisms in nature. Figure 3 illustrates the overlap between temperature and light conditions of the cold shock recovery experiment and conditions in a pasture from which some tested butterflies were collected.
Figure 1: Scatterplots of recovery time (in seconds) of butterflies after cold shock. (A) Mean temperature and (B) mean LUX (light intensity) during their recovery. Species are organized and colored by Family. Overall, as light and temperature increase, cold shock recovery time decreases, showing variability across taxa. Please click here to view a larger version of this figure.
Figure 2: Example of results from the cold shock recovery assay on 181 species of butterfly from the Colombian Andes. The data represent the number of seconds that elapsed from removing the butterfly from cold and when it was able to fly. Species are organized and colored by Family. This figure demonstrates the taxonomic breadth across which this experiment can be successfully applied, and the variety of cold shock recovery responses across species. Please click here to view a larger version of this figure.
Figure 3: Ambient temperature and LUX during cold shock recovery trials. Plot of ambient temperature (blue) and LUX (light intensity, red) as recorded by data loggers placed in the pastures where butterfly collection took place (light colors, conditions span entire day) and conditions during cold shock recovery trials (dark colors, only morning hours). The ambient field conditions and experimental conditions plotted show the range of and average conditions experienced by butterflies over one week of field sampling and experimentation. Experiments were only conducted in early hours (07:00-13:00 h), while the dataloggers were deployed in the field for one week (daylight hours, 06:00-18:00 h shown). Shown here is the overlap between experimental conditions and ambient conditions experienced by butterflies, demonstrating the ecological relevancy of conducting physiology assays under ambient conditions. Please click here to view a larger version of this figure.
Supplementary Figure 1: Procedure for collecting focal insects-in this case, butterflies-using baited Van-Someren traps and active netting. Traps were baited with both rotting fish and rotting fruit baits. Trap (without bait) in background, in the foreground is a specimen in its unique envelope against a blue plastic collection box. Please click here to download this File.
Supplementary Figure 2: Bags with up to four individual butterflies submerged in ice water in a cooler. Plastic bags were marked with the time they were placed in the ice water, so that cold shock experiments could be staggered through the morning. Plastic bags should be sealed to prevent specimens from getting wet; however, flooding of the bags and envelope in this case had no measurable effect on the recovery of the butterflies. Please click here to download this File.
Supplementary Figure 3: Two observers collect data in the field. Each mesh cage contains four unique butterflies recovering from cold shock. The polyvinyl chloride T-joint in the cage houses the data logger to prevent direct sun or rain exposure. Each observer has a stopwatch that was started immediately upon butterfly release into the cage. The cages are elevated by benches, permitting observers to agitate the base of the cage to ensure that the butterflies responded behaviorally as quickly as physiologically possible. Please click here to download this File.
Supplementary Table 1: Example data sheet. The sheet shows each butterfly's unique ID as assigned in the field and distinguishing characters (species name, key colors) in notes. Also recorded is the dominant position of the butterfly (i.e., which side of the wing was exposed to the sun) during the recovery period, noted as D (dorsal) or V (ventral). Please click here to download this Table.
Supplemental Video 1: Tapping of the cage for cold shock recovery. As butterflies recover, the observer taps the base of the cage gently to induce behaviors as soon as the butterflies are capable. Please click here to download this Video.
The study of thermal physiology incorporates measures of species and environmental traits to better understand the interactions between organisms and their surroundings that are key to survival and fitness. While always integral to understanding the natural history and ecology of plants and animals, thermal traits are of increasing importance in the face of landscape and climate change11,21. Several groups of ectothermic terrestrial insects, in particular, lepidoptera and odonatan, are relatively large and abundant, exhibit distinct behaviors, and are amenable to manipulation. Outlined here is an efficient and low-cost assay to effectively measure physiological responses of such insects. This protocol requires a source of healthy organisms to assay, whose handling time prior to the experiment is limited. While flexible in the number of organisms assayed at one time, the number of focal individuals per experiment will vary based on the purpose of data collection and/or number of observers.
For example, this protocol was developed to collect detailed individual data on butterflies across entire communities. As such, the representative results illustrate an effort to maximize the data collection for individuals of as many species as possible and under a variety of conditions relevant to the local environment. Regardless of the number of focal species, it is crucial for the observer to be able to identify each individual in the cage experiencing the recovery. If the goal is to collect data from only one species, then only one or two individuals (if identifiable based on different wing wear or if individually marked) should be assayed at once. The study subjects must be chosen in accordance with a specific research question or plan of study. Based on the question posed and the purpose of data collection (research or classroom, for example), sample size and collection of other traits will differ.
To illustrate the fundamental components of physiology elucidated by this protocol (induction of chill coma, steps of recovery, role of ambient conditions), a classroom instructor may choose two distinct species or morphs of a single species. If the focal individuals differ only in one key trait (e.g., color), a smaller sample size will be necessary, and students can closely study the relationship of that trait and organismal physiology. Researchers interested in ecological physiology may use their experimental data to explore complex ecological and evolutionary questions. Researchers must be sure to carefully choose focal insects that directly address their questions (e.g., based on life stage, age, sex, location), and, based on the number of variables involved, determine the appropriate sample size. Sample sizes for complex models will be larger than those described above.
While collecting behavioral recovery data, it is key that the cage rest above the ground because the observer must be able to tap the bottom of the cage to elicit recovery behaviors. This ensures that the organism responds (stands, flies) as soon as it is physiologically capable of doing so, and the terminal recovery behavior (flight) is documented. Recording ambient conditions during the cold shock recovery is integral to the study of thermal physiology, as this protocol is designed to study and disentangle the role of environment in organismal physiology. Data loggers (see the Table of Materials) are useful to record standardized measures of relevant conditions (e.g., temperature, light, and even humidity). However, if these tools are unavailable, relevant conditions can be measured in other ways like with a digital thermometer or by simplifying the variable of environmental conditions and using distinct environments such as shade and sun. This protocol gives the researcher options to measure the conditions during cold shock recovery based on the purpose and scope of the study.
Although this method can be modified to better suit specific taxonomic groups, it is recommended that large, volant insects be used. Flying insects that regain their ability to fly independently may be considered to have accomplished a full recovery. The method, as described, was successfully used on butterflies in the tropics and subtropics. Based on the thermal trends of a given area (i.e., the range of temperatures experienced at a site that will vary, thus influencing expectations based on elevation, latitude, canopy cover), an organism may require more or less than one hour in an ice water bath to enter a chill coma. The size of the organism may also affect the time necessary to enter a chill coma. It is key to find the time of cold exposure necessary to induce a chill coma (not moving), but not kill focal species. The time required to induce a chill coma will depend on the size, location, and natural history/behavior of the individuals. Based on results from the cold shock experiment described herein and using knowledge of the ecology of the focal insects, choose a time at which to conclude the trial if a given individual does not make a full recovery.
Based on the specific questions of the researcher, this method can be employed either in the field or the laboratory to allow for both natural environmental variation and control for important variables, respectively. This assay is simple and inexpensive and helps to fill existing gaps in the field of thermal physiology. The ease of this protocol makes it accessible to employ for a diverse array of taxa, opening the field to more than lab-friendly organisms. The novelty of performing a standardized yet ambient thermal assay fills the gap between laboratory and field results22. Leveraging ambient conditions for organism recovery will help researchers partition the role of environmental and species factors in physiology14,22. Finally, because of its low cost and lack of required materials, this protocol can be used in remote locations in the field with little equipment-ideal for many field biologists-as well as in classrooms to allow young students a hands-on learning experience.
The author has no competing financial interests or other conflicts of interests.
Thanks to Jaret Daniels, Isabella Plummer, Brett Scheffers, and Dan Hahn for input on the protocol as it was first developed. Additional gratitude to Jaime Haggard, Sebastián Durán, and Indiana Cristóbal Róis-Málaver for implementing several iterations of this protocol and for input on key components. Thanks also to an anonymous reviewer for feedback on the manuscript as a whole. Support was provided by the McGuire Center for Lepidoptera and Biodiversity's publication fund, the College of Agricultural and Life Sciences, School of Natural Resources and Environment, and Wildlife Ecology and Conservation department at UF.
|24 x 24 x 36" Popup Rearing & Observation Cage
|Ensure that the cage is slightly elevated from the ground to be able to tap the floor of the cage during experiments.
|HOBO Pendant Temperature/Light 8K Data Logger
|If a datalogger is not accessible, researchers may choose to use a digital thermometer to record ambient temperatures at regular intervals. See protocol step 4.5 for additional information.
|HOBO Optic USB Base Station
|Insects (focal taxa)
|Collect sufficient samples to test, ensuring replication of experimental groups (e.g. species, sampling location)
|Sealable plastic bag
|Large coins or small rocks to weigh down the plastic bags will ensure that specimens are submerged in ice water. A standardized weight is ideal.
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