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
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