This protocol is an extremely low cost method of collecting data on organismal physiology, in an accessible and standardized way. This tool can be used to explore physiology at several levels. It can be employed by intrepid field biologists, as well as young school children.

The main advantage of this technique is that it relies on readily available materials and is very low cost. It can be performed nearly anywhere and by researchers with a range of expertise and goals. This method can provide insight into physiological, behavioral and ecological processes.

As the key metrics for eliciting a physiological response are standardized, the method can increase our ability to understand and compare key traits across groups of individuals, species, and ecological communities. Handling insects without damaging them takes practice, but practice makes perfect. Defining the parameters of your experiment, Like how long to keep insects in ice water, can take time and it requires experimentation and understanding of your focal taxa.

Demonstrating the procedure with me will be Heather Rohrer and Matt Standridge, two technicians from the Daniel's lab. Begin with identifying the species of interest, keeping in mind that each group will differ in the time it takes to induce a chill coma. Based on the organism and use of data, choose different cutoff points for the experiment if the focal individual does not fly.

Conduct a pretrial on a small sample to determine the key parameters, like time required on ice, to induce a chill coma. Choose a cutoff time based on the ecology of the species. Keeping in mind that after many minutes of being incapable of flight, some insects are taken by predators.

Collect insects using appropriate methods, such as baited traps and entomological nets. Place each individual in a separate glassine envelope with a unique ID and store the animals in a shaded cool place protected from direct sun, wind and predators. Always expose the animal to the experimental treatment within 24 hours of being captured to standardize this time as much as possible across trials.

Fill a cooler with water and sufficient ice to maintain the environment in the water at zero degrees Celsius. Choose between one and four focal individuals for a round of experimentation, making sure that each individual is identifiable. When using multiple species, use only one of each to avoid confusion.

If the experiment is not related with wing coloration, mark the wings with unique IDs with a fine felt tipped marker to distinguish individuals. If the experiments meet neither of the above criteria, conduct the experiment on one individual at a time. Populate the rows of data sheet with the information about each insect assayed, including their unique ID and a useful identifier, in the notes.

Place all focal individuals in a sealed plastic bag with a weight and place the bag in ice water for 60 minutes to induce chill coma. Use a simple thermometer to record ambient temperature at short intervals by hand. If available, you can record ambient conditions using a data logger.

First, connect the data logger, go to device, launch and select the attached device by clicking Okay. Enter the trial name, select Temperature and Light Intensity and enter the units. Set the logging interval to 10 seconds.

Enter date and time for the experiment and select Delayed Start to start the data logger at the selected time. Ensure that the data logger information, including time of day, is synchronized with experiments so that data on ambient conditions can later be matched with each individual focal insect. Decide on the experimental parameters, like whether recovery will take place in shade or sun, and note these treatments on the data sheet.

Place a mesh cage for the insects in an appropriate location so that the temperature and light environments are homogenous within the cage. Keep the base of the cage elevated so that it can be tapped by the observer and place the data logger just outside or inside the cage where it can be kept undisturbed. Remove the animals from the ice water bath after 60 minutes and immediately remove the insects from the plastic bag, then remove each individual from its envelope quickly with minimal handling and start the stopwatch as soon as the animals are in the mesh cage.

Tap the base of the cage with a pencil frequently and strongly enough just to agitate the recovering insects without causing a response. Mark the trial as complete once an individual has flown. End the trial and consider the insect to have not recovered if it has not moved after 30 minutes.

Remove the insects from the mesh cage and place the individuals back into their labeled glassine envelopes. Liberate the animals or keep them for further data collection and process the data as described in the text manuscript. To access the recorded environmental data, plug the HOBO data logger back into the computer.

Go to device readout, select Stop Logging and save the files in the desired folder as HOBO files. After a pop-up is displayed, confirm the units of the parameters and select Plot to obtain a graph for experimental conditions. To export the data table, select File Export Table Data and export it as a CSV file to save in the appropriate folder.

In this protocol, the interaction between ambient conditions important to organismal physiology and cold shock recovery was explored for variable species of butterflies. It was observed that as average temperature of trial increased, cold shock recovery time decreased showing variability across taxa. Similarly, the inverse relation between the mean light intensity of the trial and cold shock recovery time was observed, indicating that both temperature and light conditions contribute to the recovery of the butterflies.

181 wild species of butterflies collected by three observers over five months showed distinct recovery from chill coma induced by cold shock, highlighting the taxonomic breadth across which this experiment can be successfully applied. The ambient field conditions and experimental conditions were plotted, demonstrating the ecological relevancy of conducting physiology assays under ambient conditions. When performing this protocol, be sure to pay close attention to all individuals in the mesh cage as changes in behavior can be subtle and happen quickly.

The data generated by this protocol shed light on the eco physiological traits of organisms tested. Many other traits contribute to organismal physiology, like size, color, and phylogeny. Data on additional traits will further explain and contextualize measures of cold shock recovery, adding to the ecological and life history context of this information.

I've used this method to characterize the eco physiology of entire communities of butterflies in the Colombian Andes. Because the protocol is accessible, cheap and simple enough to implement in rural settings, data was collected on hundreds of individuals in a short period of time.

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