DOI: 10.5061/dryad.7sqv9s4xz
title: Snow flies self-amputate freezing limbs to sustain behavior at sub-zero temperatures

description: All living things are profoundly affected by temperature. In spite of the
 thermodynamic constraints on biology, some animals have evolved to live
 and move in extremely cold environments. Here, we investigate behavioral
 mechanisms of cold tolerance in the snow fly (Chionea spp.), a flightless
 crane fly that is active throughout the winter in boreal and alpine
 environments of the northern hemisphere. Using thermal imaging, we show
 that adult snow flies maintain the ability to walk down to an average body
 temperature of -7 °C. At this supercooling limit, ice crystallization
 occurs within the snow fly’s hemolymph and rapidly spreads throughout the
 body, resulting in death. However, we discovered that snow flies
 frequently survive freezing by rapidly amputating legs before ice
 crystallization can spread to their vital organs. Self-amputation of
 freezing limbs is a last-ditch tactic to prolong survival in frigid
 conditions that few animals can endure. Understanding the extreme
 physiology and behavior of snow insects is important at this moment when
 the alpine ecosystems they inhabit are rapidly changing due to
 anthropogenic climate change.
description: This dataset was collected using the FLIR T860 thermal camera and
 the FLIR ResearchIR program to record body temperatures and behavior of
 snow flies and other crane flies during experimental trials.
description: Microsoft Excel is required to open data files. 
description: # Data for: Snow flies self-amputate freezing limbs to sustain motor
 behavior at sub-zero temperatures Authors - Dominic Golding, Katie L.
 Rupp, Anne Sustar, Brandon Pratt, John C. Tuthill Institution - Department
 of Physiology and Biophysics, University of Washington, Seattle, WA 98195,
 USA Correspondence - John C. Tuthill (tuthill@uw.edu) Date of data
 collection - 2020 to 2023 Publication DOI -
 [https://doi.org/10.1016/j.cub.2023.09.002](https://doi.org/10.1016/j.cub.2023.09.002) Dryad dataset DOI - https://doi.org/10.5061/dryad.7sqv9s4xz *** ## Description of the Data and file structure General description - Microsoft Excel spreadsheets of freezing point temperatures and behavioral data from snow fly and other crane fly cold tolerance experiments. General structure - Each file is a Microsoft Excel spreadsheet containing raw data and/or excel-based "table" sheet used for analysis. Raw data sheets were used for figure generation using Python, while Excel table were used for statistical analyses. Excel-based table sheets should be only be used in Excel or a comparable spreadsheet program. The equations used for each cell can be found by clicking the numerical value of interest in Excel. Cells with "null" values in data sheets indicate when data was not recorded. Cells with "null" values in tables indicate cells that were not used for analysis. File list: 1. 2020\_21\_22\_cold\_tolerance\_autotomy\_analysis\_reformatted\_v2.xlsx - Excel sheet used to record snow fly collection information, cold tolerance data, autotomy data, and subsequent analysis. 1. Number of sheets: 7 2. Name of sheet and description of sheet variables: 1. Snow fly Summary - 2021 adjusted - data sheet used for snow fly collection information and cold tolerance experiments. Can be downloaded as a .csv file for further analysis using Python. 1. column A (date) - date of trial 2. column B (video) - video file name of trial 3. column C (image\_mode) - image mode on FLIR T860 used for each trial. Macro mode expands field of view. 4. column D (fly\_id) - snow fly ID 5. Column E (sex) - sex of snow fly 6. Column F (size) - relative size of fly. Actual size of each fly was not quantified. 7. Column G (species (morph ID)) - morphological identification of species for selected flies based upon criterion listed in Byers, 1984 (reference in paper) 8. Column H (species (DNA ID)) - DNA identification of species for each fly as described in Method details 9. Column I (trial) - trial number 10. Column J (cold\_room) - whether trial took place in cold room. All trials for this paper took place in a cold room as described in Method details 11. Column K (number of legs) - number of legs of fly when trial took place. Note that for trial 1, this refers to the number of legs that fly had when collected from their environment 12. Column L (death) - whether snow fly died during trial 13. Column M (origin\_of\_freezing) - bodily location on snow fly when bodily freezing was first observed 14. Column N (fp\_ambient\_temp) - temperature (°C) of the cold plate when bodily freezing was first observed 15. Column O (fp\_fly\_temp\_pre) - temperature (°C) of the snow fly (minimum body temperature) when bodily freezing was first observed 16. Column P (fp\_fly\_temp\_post) - temperature (°C) of the snow fly (minimum body temperature) within 5 seconds of freezing observed in the body 17. Column Q (fp\_frame) - frame number when bodily freezing was first observed 18. Column R (collector) - Individual who collected snow fly 19. Column S (collection\_date) - date of collection for snow fly 20. Column T (location) - name of collection location 21. Column U (latitude) - snow fly collection latitude 22. Column V (longitude) - snow fly collection longitude 23. Column W (elevation) - snow fly collection elevation (ft) 24. Column X (notes) - notes taken prior to, during, and after each snow fly trial. Unique behaviors and notable occurrences during cold tolerance experiments listed here. 25. Column Y (Froze to plate) - whether the legs of each snow fly froze to the cold plate during cold tolerance trials 26. Column Z (Frame movement stopped) - frame when all snow fly movement ceased after freezing within body was observed. 27. Column AA (number of lost legs during trial) - number of legs that were lost during the trial 28. Column AB (number of legs that visibly froze & detach) - number of legs that were observed to freeze and detach for each fly during a cold tolerance trial 29. Column AC (time of trial fly supercooled (s)) - time when fly froze during a cold tolerance trial (in seconds) 30. Column AD (Post detachment survival frame) - number of frames that fly survived after detachment of first limb during a cold tolerance trial 2. SF\_tempdata\_parameters - temperature data from sheet 1 reformatted by parameters listed in Row 1. The purpose of this sheet is to organize temperature data by looking at specific variables of interest i.e. column A lists all cold plate temperature values at the freezing point for male flies, column B lists all cold plate temperature values at the freezing point for male flies etc. 3. SF\_tempdata\_parameters\_analysis - table used to quantify the minimum, 1st quartile, median, 3rd quartile, maximum, average and standard deviation of each column of data from sheet 2. Column I calculates significant differences using t-tests between parameters (indicated in Column J notes section). 4. SF\_cold\_tolerance\_analysis - table used to calculate average cold plate temperatures , freezing point temperatures, post-freezing temperatures, frame when freezing occurred, and elevation of collection for snow flies using data from sheet 1. 5. SF\_origin\_freeze - table used to count the total number of freezing events within the leg, abdomen and thorax. 6. SF\_autotomy\_additional\_analysis - Sheet used for additional analyses. Each column in row 1 indicates the subsequent calculation in row 2. Each column is independent from one another i.e. A1 & B1 ("number of flies in total" & "77") is independent from A2 & B2 ("number of female flies" & "39"). Using Excel is necessary to see equations used for these calculations. 3. Missing data: none 4. Abbreviations used: fp = freezing point 2. 2021\_SF\_additional\_observations\_leg\_reformatted\_v2.xlsx - Excel sheet used to record and analyze freezing events within each snow fly leg throughout cold tolerance trials. The key (column AH) describes each type of freezing event recorded in the data sheet. 1. Number of sheets: 2 2. Name of sheet and description of sheet variables: 1. 2021\_SF\_additional\_obs\_legs - sheet used for data collection. Can be downloaded as a .csv file for further analysis using Python. 1. Column A (date) - date of trial 2. Column B (video) - video file name of trial 3. Column C (fly\_id) - snow fly ID 4. Column D (L1) - type of event (described by key in Column AH) that occurred within left front leg 5. Column E (L1\_frame) - frame when event occurred 6. Column F (L1\_leg\_temp) - temperature (°C) when event occurred 7. Columns G-U (L2 - R3\_leg\_temp) - Same as Columns D, E & F for legs L2, L3, R1, R2, & R3 8. Column V (notes) - any additional notable events observed during trial 9. Column W (Leg detached) - whether any legs detached during trial 10. Column X (KEY) - key for events noted for each leg 2. 2021\_SF\_condition\_count - table used to count the total number of events across all trials for L1, L2, L3, R1, R2, & R3. Counts used to quantify the percentage of legs that detached after freezing was observed within the leg (Column M2). 3. Missing data: none 4. Abbreviations used: L1 = Left front leg, L2 = left middle leg, L3 = left hind leg, R1 = right front leg, R2 = right middle leg, R3 = right hind leg 3. Snow\_fly\_crane\_fly\_temp\_analysis\_reformatted.xlsx - Excel sheet used for statistical analysis of SCP temperature differences between snow flies and other crane flies using raw data from 2020\_21\_22\_cold\_tolerance\_autotomy\_analysis\_reformatted.xlsx and Crane\_flies\_2022\_small\_medium\_reformatted.xlsx 1. Number of sheets: 4 2. Name of sheet and description of sheet variables: 1. SF\_CF\_data - Cold plate, freezing point, and post-freezing point temperature data for crane flies and snow flies from cold tolerance trials. All data and variables are from raw data sheets previously indicated. 2. SF\_CF\_avg\_stdev - table with calculations for average, stdev using data from sheet 1. 3. SF\_CF\_avgtempdiff - sheet used to calculate the total temperature increase (°C) of crane flies and snow flies after bodily freezing had occurred using data from sheet 1. 4. SF\_CF\_T\_test - sheet used to test for significant differences between cold plate, freezing point, and post-freezing point temperatures (°C) of snow flies and crane flies using data from sheet 1. 3. Missing data: none 4. Abbreviations used: fp = freezing point 4. Leg\_freeze\_time\_reformatted.xlsx - Excel sheet used to record time of freezing from tarsus to tibia in FLIR videos. 1. Number of sheets: 2 2. Name of sheet and description of sheet variables: 1. Leg\_freeze\_time\_data - sheet used for data collection. Can be downloaded as a .csv file for further analysis using Python. 1. Column A (date) - date of trial 2. Column B (video) - video file name of trial 3. Column C (fly\_id) - snow fly ID 4. Column D (fly\_freeze\_initialization) - frame when freezing first observed 5. Column E (fly\_leg detachment) - frame when leg detached 6. Column F (time until detachment) - time lapsed from freeze initialization to detachment in seconds ((fly\_leg detachment - fly\_freeze\_initialization)/30) 7. Column G (time to reach tibia) - frame when freezing reached tibia 8. Column H (tarsus to tibia freeze time) - time (ms) taken for freezing to reach tibia from tarsus ((G-D)/30\*1000) 9. Column I (time to reach femur) - frame when freezing reached femur 10. Column J (tarsus to femur freeze time) - time (ms) taken for freezing to reach femur from tarsus ((I-D)/30\*1000) 11. Column K (move in response to freezing) - visual movement of fly in response to freezing detected in tarsus 2. Freeze\_time\_analysis - table with calculations for average, stdev, median, and n values using data from sheet 1. 3. Missing data: none 4. Abbreviations used: none 5. Crane\_flies\_2022\_small\_medium\_reformatted.xlsx - Excel sheet used to record crane fly collection information and data from cold tolerance FLIR experiments. Can be downloaded as a .csv file for further analysis using Python. 1. Number of sheets: 1 2. Name of sheet and description of sheet variables: 1. Crane\_flies\_2022\_small\_medium 1. All variables are the same described in "2020\_21\_22\_cold\_tolerance\_autotomy\_analysis\_reformatted.xlsx" sheet 1. 3. Missing data: none 4. Abbreviations used: See "2020\_21\_22\_cold\_tolerance\_autotomy\_analysis\_reformatted.xlsx" 6. Snow\_fly\_crane\_flies\_activity\_timepoints\_reformatted.xlsx - Excel sheet used to record snow fly and crane fly activity on the cold plate at different time points. 1. Number of sheets: 2 2. Name of sheet and description of sheet variables: 1. Snow fly crane flies activity - sheet used for data collection. Can be downloaded as a .csv file for further analysis using Python. 1. Column A (CF\_ID) - crane fly ID 2. Column B-G (CF\_activity\_3c to CF\_activity\_-17c) - scored activity value at each temperature indicated by column header (0, 1, or 2) 3. Column H (SF\_ID) - snow fly ID 4. Column I-N (SF\_activity\_3c to SF\_activity\_-17c) - scored activity value at each temperature indicated by column header (0, 1, or 2) 5. Column O - description of the analysis along with key for scoring criteria 2. SF\_CF\_activity\_count - table with calculations to count total number of flies within scored criteria. Equations for calculations can be found by clicking each numerical value in Excel. 1. Columns B-G and I-N correspond to column headers in "Snow fly crane flies activity" sheet 1 2. Row 2 indicates total number of flies inactive at each temperature 3. Row 3 indicates total number of flies conscious at each temperature 4. Row 4 indicates total number of flies active at each temperature 3. Missing data: none 4. Abbreviations used: CF = crane fly, SF = snow fly 7. Ambient\_temp\_data\_visual\_crossing.xlsx - Excel sheet of average daily temperature data obtained from the Visual Crossing Weather Query Builder (https://www.visualcrossing.com/weather/weather-data-services) for collected snow flies. Can be downloaded as a .csv file for further analysis using Python. 1. Number of sheets: 1 2. Name of sheet and description of sheet variables: 1. Ambient\_temp\_data\_visual\_crossing 1. Column A (Latitude) - snow fly collection latitude 2. Column B (Longitude) - snow fly collection longitude 3. Column C (Species\_ID) - snow fly ID 4. Column D (Species) - snow fly species identification (through morphological and DNA identification techniques) 5. Column E (Temperature) - average daily temperature (°C) when snow fly was captured as recorded in the Visual Crossing Weather Query Builder 3. Missing data: none 4. Abbreviations used: none ## Sharing/access Information Python code used to determine snow fly temperature, body velocity, and trajectory, as well as, for generating plots is located at https://github.com/tuthill-lab/Golding\_2023