TITLE: Benefits of Makerspaces: Building An Enhanced Flight Mill for the Study of Tethered Insect Flight
AUTHORS: Anastasia Bernat

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Makerspaces have a high potential of enabling researchers to develop new techniques and work with novel species in ecological research. This protocol demonstrates how to take advantage of the technology increasingly found in makerspaces in order to build a more versatile flight mill for a relatively low cost. Since this flight mill and trial design initially extracts its prototype from flight mills built in the last decade, this protocol instead focuses more on outlining divergences made from the simple, modern flight mill. As already shown, previous studies have made known how advantageous flight mills are to measuring flight parameters such as speed, distance, or periodicity and to allowing researchers to associate these parameters with morphological, physiological, or genetic factors. In addition to these advantages, I discuss the benefits of using the technology in makerspaces like 3D printers and laser cutters in order to build a more flexible, sturdy, and collapsible flight mill design. Most notably, the 3D printed components of this design allow the user to adjust the mill arm and IR sensor heights to test insects of various sizes and enable the user to easily disassemble the machine for quick storage or transportation to the field. Moreover, I emphasize making greater use of magnets and magnetic paint to attach insects with minimal stress during flight trials. Lastly, this protocol details a versatile analysis of flight data by programming how to efficiently take and process continuous but differentiable flight trials. Although more labor-intensive, communally-shared makerspaces and free, online 3D modeling programs can help researchers avoid costly, pre-made products with narrowly adjustable dimensions. By taking advantage of the flexibility and reproducibility of technology in makerspaces, this protocol promotes creative flight mill design and inspires open science.</div></div>

[STEPS]
?. Build the Flight Mill in a Makerspace
?. [Laser cut and assemble the acrylic plastic support structure.]
Use eight 304.8 mm by 609.6 mm, 3.175 mm thick transparent acrylic sheets to construct the acrylic plastic support structure. Ensure that the material is not polycarbonate, which looks similar to acrylic but will melt instead of cut under the laser.
?. 
?. [Laser cut and assemble the acrylic plastic support structure.]
Locate the laser cutter in the makerspace. This protocol assumes the makerspace has a Universal Laser Systems (ULS) Platform Series (PLS) 6.75 Laser Cutter. For other laser cutters, read the laser cutter settings to determine what line color or thickness is needed to set the file lines to be laser cut or engraved (not to be rastered).
?. [Laser cut and assemble the acrylic plastic support structure.]
Open Adobe Illustrator, Inkscape (free), or another vector graphics editor. Prepare a file that reads the acrylic support design in a vector format with the aforementioned lines shown in Figure 1. File lines can be created in Adobe Illustrator in RGB mode with a line stroke of .0001 pt where RGB Red (255, 0, 0) cuts lines and RGB Blue (0, 0, 255) etches lines.
?. [Laser cut and assemble the acrylic plastic support structure.]
As a precaution, test and account for kerf for all slit and hole measurements, which can vary based on the laser cutter’s beam width, the width of the material, and the material type used. This can be done by designing and testing a kerf key (Supplemental Files).
?. [Laser cut and assemble the acrylic plastic support structure.]
Save the acrylic support designs and kerf key as file types that can be read by the laser software such as .AI, .DXF, or .SVG files. To send the job to the laser cutter, the user will need to print the file on the laser cutter’s local machine and then open the laser software. If printed correctly, all the vector cutting lines in the design will appear with the appropriate corresponding colors in the laser software’s control panel.
?. [Laser cut and assemble the acrylic plastic support structure.]
Select the material, in this case Plastic, and then the material type, Acrylic. For extra precision, measure the material thickness with a caliper and enter its thickness in the material thickness field. It is recommended to auto-enable the Z-Axis of the material’s focal point, set the Figure Type to ‘None’, and leave the Intensity at 0%. Changing any advanced metrics on the laser cutter such as the laser % power or % speed will need to be further tested with the kerfkey.Note: The rule of thumb is that the thicker the material, then the more power is required at a lower speed.
?. [Laser cut and assemble the acrylic plastic support structure.]
Before cutting, follow the makerspace’s guidelines on powering up, using, and maintaining the laser cutter. Place materials in the printer cavity and cut the acrylic supports.
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							Note: To prevent possible eye damage, do not look at the laser or leave any acrylic sheet unattended while cutting.
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?. [Laser cut and assemble the acrylic plastic support structure.]
Clean excess material out of the printer cavity and assemble the support structure. Assemble by inserting each horizonal shelf into the open slits of the outside vertical walls and central vertical wall as labeled in Figure 2A.
?. [3D print the plastic supports.]
Open a web browser and create a free account at Tinkercad.com, an online 3D modeling program.
?. [3D print the plastic supports.]
Click on 3D Designs > Create a new design. To replicate this study’s exact 3D printed designs as seen in Figure 3, download the archive 3D_Prints.zip (Supplemental Files) and move the folder onto the desktop. Unzip and open the folder. In Tinkercad’s workplane webpage, click on Import in the top right corner and select the .STL file(s).
?. [3D print the plastic supports.]
To self-create or make adjustments to the designs, follow the website’s tutorials, make edits, and then export the new designs as .STL files. In total, 8 linear guide rails, 16 linear guide rail blocks, 12 to 20 screws, 15 cross brackets, 16 magnet holders, 16 tube supports, 16 short linear guide rail supports, and 16 long linear guide rail supports need to be 3D printed. To obtain the mirrors for all the linear guide rail related designs, click on the object, press M, and select the arrow corresponding to the object’s width.Note: See step 1.24 for more information on the linear guide rail pegs.
?. [3D print the plastic supports.]
Download and install the free 3D printing slicing software, FlashPrint 4.4.0, to convert .STL files to a 3D printer readable .GX file. Other conversion software programs are acceptable, but this protocol assumes the makerspace is using a FlashForge Finder 3D Printer.
?. [3D print the plastic supports.]
Double-click the icon of FlashPrint to start the software. Click Print > Machine Type and select the 3D Printer that is located in the makerspace.
?. [3D print the plastic supports.]
Click the Load icon to load a .STL model file and the object will display on the build area.
?. [3D print the plastic supports.]
Select the object and double-click the Move icon. Click “On Platform” to ensure that the model is on the platform. Click “Center” to place the object at the center of the build area or drag the object with the mouse pointer to position the object on the build area.
?. [3D print the plastic supports.]
Click on the Print icon. Ensure that Material Type is set to PLA, supports and raft are enabled, Resolution is set to Standard, and that the temperature of the extruder matches the temperature suggested by the 3D Printer guide. The temperature can be changed within More Options >> Temperature.
?. [3D print the plastic supports.]
Press OK and save the .GX file in the 3D_Prints folder or on a USB stick if the file cannot be transferred to the 3D Printer through USB cable.
?. [3D print the plastic supports.]
Locate a makerspace’s 3D printing machine and ensure that the extruder is calibrated and there is enough filament for printing. Transfer the .GX file to the 3D Printer and print all types and quantities of plastic supports and enhancements.For each print, check that the filament is properly sticking to the plate.
?. [Assemble 3D prints onto the acrylic support structure.]
To visualize all the supports in place, see Figure 2B.
?. [Assemble 3D prints onto the acrylic support structure.]
Hot glue 3.175 mm thick neoprene sheets onto the interior walls of the cross bracket. When dry, insert the cross brackets at the junctions of the acrylic shelves and the walls at the back of the device. This will help stabilize the flight mill.
?. [Assemble 3D prints onto the acrylic support structure.]
Where possible, use 3D printed screws in order to minimize the magnetic influence of iron screws. Screw in the tube holders onto the bottom and top of each cell.Ensure that the top and bottom tube supports are aligned.
?. [Assemble 3D prints onto the acrylic support structure.]
Insert a 30 mm long plastic tube (inner diameter (ID) 9.525 mm; outer diameter (OD) 12.7 mm) into the top tube support and a 15 mm long plastic tube (ID 9.525 mm; OD 12.7 mm) into the bottom tube support of each cell. Then, insert a 40 mm long plastic tube (ID 6.35 mm; OD 9.525 mm) into the top tube and a 20 mm long plastic tube (ID 6.35 mm; OD 9.525 mm) into the bottom tube. There should be strong enough friction between the tubes to hold the tubes in place, but not too much that the inner tube can still slide up and down if pulled on. If tubes are warped, submerge segments of the tubes for 1 minute in boiling water and insert them after they have been straightened out on a towel and have reached room temperature.
?. [Assemble 3D prints onto the acrylic support structure.]
Place two low-friction N42 neodymium magnets (10 mm diameter; 4 mm length; 2.22 kg holding force) into a magnet support and insert the inner tube into the magnet support. Repeat on the bottom inner tube. Check to see that the magnets are repelling each other. Additionally, check that the inner tube is firmly lodged into each magnet support so that gravity acting on the magnets and magnet support is not strong enough to dislodge them from the inner tube.
?. [Assemble 3D prints onto the acrylic support structure.]
Facing the same direction, slide two linear guide rail blocks into the linear guide rail. Lodge the linear guide rails and blocks upright into the windows on the outer vertical walls. Ensure that the block openings are facing upwards. To secure one linear guide rail in place, use two short linear guide rail supports, two long linear guide rail supports, four 10 mm long iron screws (M5; 0.8 thread pitch; 5 mm diameter), two 20 mm long iron screws (M5; 0.8 thread pitch; 5 mm diameter), and two hex nuts (M5; 0.8 thread pitch; 5 mm diameter). The full assembled linear guide rail is shown in Figure 2C.Note: Open slots in the linear guide rail are intended to be used if and only if the linear guide rail becomes eroded by the repeated sliding of its block. If so, 3D print a small T-shaped peg found in the 3D_Prints folder.
?. [Construct the pivoting arm. Sub-sections 1.25 and 1.26 are equivalent to sub-sections 1.2.2. and 1.2.3. in Attisano et al 2015 methods paper10. Instructions are described here again for ease of assembly.]
Insert an entomological pin into a 20 µL filtered pipette tip to fix the pin in place. Slide the pin down enough such that both ends extend out the pipette tip to form the axis of the flight mill arm.
?. [Construct the pivoting arm. Sub-sections 1.25 and 1.26 are equivalent to sub-sections 1.2.2. and 1.2.3. in Attisano et al 2015 methods paper10. Instructions are described here again for ease of assembly.]
Cut a 24 cm length of 19 gauge non-magnetic hypodermic steel tubing. Hot glue the top of the pipette tip from step 1.25 to the center point of the tubing. Bend one end of the tubing at 2 cm from the end to an angle of 95°, leaving a long arm of 12 cm from the center point and a short arm with a 10 cm radius from the center to the bend.
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							Note: The radius length of the arm can be varied to accommodate different insect sizes. Additionally, the bent ending of the arm can support different angles in order to position the insect in its natural flight orientation.
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?. [Construct the pivoting arm. Sub-sections 1.25 and 1.26 are equivalent to sub-sections 1.2.2. and 1.2.3. in Attisano et al 2015 methods paper10. Instructions are described here again for ease of assembly.]
To test its magnetic suspension, position the arm at the top set of magnets. When properly positioned between the two magnets, the rotating arm should spin freely around the vertically suspended pin.
?. [Construct the pivoting arm. Sub-sections 1.25 and 1.26 are equivalent to sub-sections 1.2.2. and 1.2.3. in Attisano et al 2015 methods paper10. Instructions are described here again for ease of assembly.]
Glue two low-friction N42 neodymium magnets (3.05 mm diameter; 1.58 mm length; 0.23 kg holding force) on the bent end of the pivot arm, so that the metal-painted insect can be tethered for flight. On the unbent end of the pivot arm, wrap a piece of aluminum foil to create a flag. The foil flag will help act as a counterweight, and, due to its highly reflective properties, it will optimally break the IR beam sent from the IR sensor transmitter to the receiver.
?. [Set up the IR sensor and data logger.]
Place the IR sensor transmitter inside the top linear guide rail block with the source of the beam facing downwards. Then, place the IR sensor receiver inside the bottom block facing upwards.
?. [Set up the IR sensor and data logger.]
On a solderless breadboard, connect the IR sensor transmitter and receiver in series with the data logger as shown in the electronic circuit in Figure 4B. Ensure that the IR sensor transmitter (not the receiver) input is connected first following the 180Ωresistor. Place another 2.2kΩresistor before the output of the IR receiver connection. Configure each channel’s electronic circuit in alternate rows along the breadboard to minimize noise in voltage signal from multiple sensors during recording (Figure 4A).
?. Conduct Flight Trials
?. [Magnetically tether insects to the flight mill arm.]
To minimize stress placed on the insect, apply magnetic paint on the insect’s pronotum and let it dry for at least 10 minutes. Once dry, attach the insect to the flight mill arm magnets.
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							Note: For a stronger attraction between the insect and the arm magnets, apply multiple layers of magnetic paint.
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?. [Magnetically tether insects to the flight mill arm.]
Up to 8 insects can be flown at a time in the flight mill. Paint prep at least 16 insects in order to test multiple insects sequentially during a single recording session.
?. [Magnetically tether insects to the flight mill arm.]
To remove the magnetic paint after testing, chip off the paint with fine forceps and dispose of it according to EPA and OSHA regulations.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
Download and install the free WinDAQ Data Recording and Playback Software.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
Download the following Python scripts: split_files.py, standardize_troughs.py, and flight_analysis.py (Supplemental Files). Create a new folder titled Flight_scripts on the desktop and move the scripts into that folder.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
Create 5 new folders with the following exact names inside the Flight_scripts folder: data, files2split, recordings, split_files, and standardized_files. Download the datasheet.xlsx (Supplemental Files) and drag the file into the data folder in the Flight_scripts directory.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
Use the datasheet.xlsx as a manual data-recording template. A minimum of four columns is needed: the identification number of the bug, whether the bug died before being tested, the recording set number, and the chamber comprised of the channel letter and channel number (e.g. “A-1”, “B-4”). One possible chamber configuration is seen in Figure 2A.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
Open the WinDAQ Dashboard, select the data-logger(s) from the checkbox list and press ‘Start Windaq Software’. A new window will open for each data-logger selected and the input signal from each sensor will be shown.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
To start a new recording session, press File > Record. Select the location of the recording file in the first pop-up window. Write the file name carefully. Files have to have at least the following in their names: the recording set number and channel letter. An example of a filename modeled in the Python scripts is the following: “T1_set006-2-24-2020-B.txt". Refer to split_files.py lines 78-87 from the Flight_scripts folder to get further details. Then, press OK.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
In the next pop-up window, enter the anticipated length of the flight recording. Press OK when the insects are in a position to begin flight. Then, press Ctrl-S to finalize the file once the recording time has elapsed. Do not press Ctrl-S unless there is a need to terminate the recording early.Note: If the file terminates too early either by typing Ctrl+S or the aforementioned length of time was too short, append a new recording to an existing file by clicking on File > Record. Select the file to append to and click “Yes” on the following pop-up window.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
When pulling out tested insects during the recording, insert a commented event marker of the incoming insect at its selected chamber. Always manually record the ID, chamber, and recording set of the incoming insect in datasheet.xlsx before swapping insects.
?. [Record multiple insects sequentially without terminating a recording session using WinDAQ’s Event Marker Comment tool.]
To make an event marker comment, select the channel by clicking on the channel number. Then, click Edit > Insert Commented Mark. Define the comment with the identification number of the new insect entering the chamber. Press OK and load the insect into the chamber.
?. [Visualize event marker comments and convert file from WDH to TXT.]
Visualize event marker comments by going to Edit > Compression… and then clicking the “Maximum” button to fully compress the waveform into one window (Figure 5A).
?. [Visualize event marker comments and convert file from WDH to TXT.]
Save the file in a .TXT format by going to File > Save As. Select the location to save the file, select the file type as “Spreadsheet print (CSV)” in the pop-up window, and write the filename with ‘.txt’ at the end. In the following pop-up window, select “Sample Rate”, “Relative Time”, “Date and Time”, and write “1” in between “Channel Number” and “Event Markers”. Deselect all other options and click OK to save the file.
?. Analyze Flight Data
?. [Split files by event marker comments.]
Install the latest version of Python. All scripts in this protocol were developed on Python version 3.8.0.
?. [Split files by event marker comments.]
Ensure that the following libraries are installed and up to date for the installed version of Python: csv, os, sys, re, datetime, time, and matplotlib.pyplot.
?. [Split files by event marker comments.]
Drag all TXT file recordings into the recordings folder of the Flight_scripts directory.
?. [Split files by event marker comments.]
Open the datasheet.xlsx file and save as a CSV by changing the file format to “CSV UTF-8 (Comma delimited)” if on Windows or “Macintosh Comma Separated” if on a Mac.
?. [Split files by event marker comments.]
Open the split_files.py icon with the text editor of choice. If there is no preference, right-click on the script icon and select ‘Open with IDLE’.
?. [Split files by event marker comments.]
Recode lines 133-135 and 232-233 if the user wrote a filename different from the suggested template ("T1_set006-2-24-2020-B.txt"). See lines 116-131 for an example on how to recode the script to accommodate different filenames, using the split( ) function.
?. [Split files by event marker comments.]
In line 266, type the path to the Flight_scripts folder, and run the script. If the script successfully runs, the user will see, within the Flight_scripts directory, intermediate TXT files of mapped insect IDs generated in the files2split folder and TXT files for each insect tested in each recording set in the split_files folder. Additionally, in the Python Shell, users should see print statements of the filename, which insects are being swapped at a numbered event marker, and which files are being split and generated into new files by insect ID.
?. [Standardize and select the troughs in the recorded signal as follows:]
Open the standardize_troughs.py icon with the text editor of choice. If there is no preference, right-click on the script icon and select ‘Open with IDLE’.
?. [Standardize and select the troughs in the recorded signal as follows:]
In line 120, type the path to the Flight_scripts folder, and run the script. If the script successfully runs, the user will see files generated in the standardized_files folder in the Flight_scripts directory. All files should start with the “standardized_” and end with the original filename.
?. [Standardize and select the troughs in the recorded signal as follows:]
Check the quality of the recordings: Open the trough_diagnostic.png generated by the standardize_troughs.py located in the Flight_scripts folder. Ensure that all records are robust to changes in the minimum and maximum voltage value of the mean standardization interval. Recordings may have a lot of noise or have overly sensitive troughs if they exhibit large decreases in the number of troughs identified and counted when the minimum and maximum deviation values are increased.Note: Additional diagnostics for the min-max normalization factor can also be coded, performed, and plotted. An alternative method for checking recording quality is described in steps 2.3.1. and 2.3.2. of the Attisano et al 2015 methods paper10.
?. [Standardize and select the troughs in the recorded signal as follows:]
After diagnostics have been assessed, uncomment out line 159 and specify the minimum and maximum deviation values, which define the minimum and maximum values around the mean voltage used to perform the standardization for all files. The default are deviation values 0.1 V.
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							Note: In line 45, the user can also specify the min-max normalization factor threshold in order to identify a voltage far below the threshold value.
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?. [Standardize and select the troughs in the recorded signal as follows:]
Comment out lines 150 and 168 after inputting the deviation values and then run the script. The script will then run the standardizations efficiently for all files (approximately 40 times faster).
?. [Analyze the flight track using the standardized file.]
Open the flight_analysis.py icon with the text editor of choice. If there is no preference, right-click on the script icon and select ‘Open with IDLE’.
?. [Analyze the flight track using the standardized file.]
In lines 76-78, edit the optional speed correction that suppresses additional rotations of the mill’s arm after an insect stops flying. Determine this threshold value with caution when working with slow flying insects.
?. [Analyze the flight track using the standardized file.]
In line 121, edit the speed thresholds to correct for false speed readings such as extremely fast speeds or negative speeds. In line 130, edit the time gap value to filter out long gaps that occur between two consecutive uninterrupted flying bouts.
?. [Analyze the flight track using the standardized file.]
In line 350, type the path to the folder in which the *.TXT standardized files are saved.
?. [Analyze the flight track using the standardized file.]
In line 353, input the arm radius length used during trials, which defines the circular flight path flown per revolution by the insect.
?. [Analyze the flight track using the standardized file.]
Identify the distance and time SI units as a string in lines 357 and 358.
?. [Analyze the flight track using the standardized file.]
In lines 388-397, use the split( ) function to extract, at minimum, the insect’s identification number and the set number and chamber in which the insect flew from the filename. The script follows the comprehensive filename example of “standardized_T1_set006-2-24-2020-B.txt”. However, this filename can be simplified as suggested in step 2.9 and variables like trial type on lines 392 and 401 can be commented out or deleted if not used.
?. [Analyze the flight track using the standardized file.]
Once all the user settings are specified, save the file and run the script. If the script successfully runs, the insect’s corresponding ID number, chamber, and flight statistics calculated in the script will be printed in the Python Shell. Additionally, a flight_stats_summary.csv file similarly comprised of the information printed in the Python Shell is generated in the data folder of the Flight_scripts directory.
?. Representative ResultsFlight data were obtained experimentally during Winter 2020 using field collected soapberry bugsJadera haematolomafrom Florida as the model insects (Bernat, A. V. and Cenzer, M. L. , 2020, unpublished data). Representative flight trials were conducted in the Department of Ecology and Evolution at the University of Chicago, shown below in Figures 5-8. The flight mill was set up within an incubator set to 28°C/27°C (day/night), 70% relative humidity, and a 14 hour light/10 hour dark cycle (sunrise at 8 AM and sunset at 10 PM). For each trial, the flight track of multiple bugs was recorded every hundredth of a second by the WinDAQ Software for up to 24 hours. Any individuals that did not exhibit continuous flight behavior at least within the last 10 minutes of its 30 minute testing phase were pulled off the flight mill and replaced with a new bug and its accompanying ID in an event marker comment. All bugs that exhibited continuous flight remained on the flight mill beyond 30 minutes until they stopped flying. Bugs were swapped until 4 PM each day. As represented in Figure 8, flight trials of single individuals in one day’s worth of recording varied in length from 30 minutes to 20+ hours and were routinely asynchronous. By inserting event markers at the addition of new individuals, the process of this complex data structure becomes successfully automated in the split_files.py script.The on-screen waveform and graphic diagnostic output also make it possible to identify gaps or resolve inconsistencies in the flight track data.Figure 5A shows the event marker comments made during recording and has successfully collected data for all channels without disruption. Figure 5B shows a moment where the recorded signal was lost in channel 3, dropping the voltage immediately to 0 V. This was possibly due to the crossing over of open wires or the loosening of wires. Figure 6 compares three data files to show how noise or sensitive troughs in the recording data were diagnosed during the standardization process. The first (Figure 6A) is a file whose troughs generated by each revolution of the flight mill arm were robust, meaning they largely deviated from the file’s mean voltage. In turn, as the standardization interval around the mean increased, there was no change in the number of troughs identified. On the other side of the diagnostic spectrum, the third file (Figure 6C) had troughs that were either too sensitive or had extraneous voltage noise that did not deviate largely from the file’s mean voltage. As a result, its number of troughs decreased substantially as the standardization interval around the mean increased. To check this individual’s behavior, the original WDH recording file can be viewed to confirm whether it was truly flying.By plotting the flight speed and duration statistics of the individual, flight behavior can be further characterized into four flight categories: bursts (B), bursts to continuous (BC), continuous to bursts (CB), and continuous (C) as represented in Figure 7. An individual that strictly exhibited continuous flight flew uninterrupted for 10 minutes or more at least by the end of its 30 minute testing phase (Figure 7A). An individual that flew sporadically throughout its 30 minute testing phase exhibited bursting flight (Figure 7B). An individual that initially strongly exhibited continuous flight for more than 10 minutes and then tapered within its 30 minute testing phase into sporadic bursts exhibited bursts to continuous flight (Figure 7C). Finally, an individual that initially demonstrated bursting flight and then transitioned into continuous flight for the remainder of the 30 minute testing phase and beyond exhibited bursting to continuous flight (Figure 7D). Thus, specific to the model insect and experimental framework, the user can use this graphic output to assess and identify general flight behavior patterns despite unique variations in individual tracks.
?. DiscussionThe simple, modern flight mill provides a range of advantages for researchers interested in studying the flight behavior of a model species by delivering a reliable and automated design that tests multiple insects efficiently and cost-effectively10,22. Likewise, there is a strong incentive for researchers to adapt to fast-emerging technologies and techniques from industry and other scientific fields to design and build experimental tools to study ecological systems9,23-24. This protocol takes advantage of two rapidly emerging technologies, the 3D printer and the laser cutter, which are becoming increasingly available in communal makerspaces, in order to enhance the simple, modern flight mill. These enhancements provide a more flexible, adjustable, and collapsible design that accommodates insects of different sizes, minimizes stress placed on the insect, and allows the flight mill to be transported easily to multiple locations or environments. Furthermore, the additional expenses of using the technologies are minimal or even free. However, these technologies can also be a challenge to experiment with if their accessibility or reaching proficiency in using vector graphics editors and 3D image software is not readily available. In turn, the flight mill presented here serves to both encourage researches to incorporate available emerging technologies in their workflow and allow researchers to build a customizable, flexible, and effective flight mill without specialized knowledge of electronics, programming, or computer aided design (CAD) models.The strongest aspects of this protocol are the makerspace’s technologies that expand a user’s flight mill design options, the use of magnetic paint to minimize insect stress, and the automation of flight recordings followed by the processing of multiple insects within a single recording. The laser cutter offers precise and exact cutting capabilities that can handle jobs of almost any complexity. The user can modify the acrylic support structure to mount additional 3D prints or purchased items. The 3D printer allows the user to create customizable flight mill components that can bypass costly, pre-made products with narrowly adjustable dimensions. Finally, the use of automated recording software and Python scripts to differentiate multiple flight trials within a single recording makes it possible to study sporadic bouts of flight to very long bouts of flight. However, given how variable flight activity and duration is across species, it is suggested that the user conducts preliminary trials in order to understand the limits and general patterns of a species’ flight behavior so as to optimize data collection. The user can also assess the integrity of their recordings using the diagnostic graphic output and can account for any necessary speed corrections in the scripts.Researchers should also be aware of the flight mill’s general constraints. Previous studies have made known and have attempted to remediate the limitations of tethered flight including a lack of tarsal contact to allow the insect to rest at will15,22, the absence of energy expended when an insect takes-off25, the additional drag the insect overcomes when pushing the flight mill arm, and the insect needing to compensate for the outward aerodynamic forces experienced due to the centrifugal acceleration of its circular flight track6,26. Additionally, there continues to be inconsistencies on how to categorize or more precisely quantify the short or ‘trivial’ bursts insects display, especially when considering the flight behavior and mechanisms of large migratory insects to those of small insects who exhibit mostly hovering flight21,27-28. Despite these limitations, there has been significant progress in capturing and categorizing flight behavior within insect species and researchers have continued to couple the flight mill with other technologies and methods6-8.Flight mills have played an important role in enabling researchers to understand the dispersal of insects – an ecological phenomenon still essentially intractable in the field. Future advances in the design and application of the flight mill can be achieved as researchers become more proficient in emerging technologies and the software accompanying those technologies. This could include designing flight mill arm bearings that allow vertical lift or gives the insect greater flight orientation flexibility. Additionally, the precision of laser cutters and 3D printers may be necessary for researchers interested in scaling down and calibrating for small insects with mostly hovering capabilities. In turn, the goal of this protocol was to provide an easy entry to these technologies while constructing one of the most common and useful devices in the field of behavioral ecology – the flight mill. If researchers have access to a communal makerspace and are committed to navigating its technologies, the resulting enhancements and improvements of the modern flight mill will lead to creative and collaborative flight mill design and will continue to offer insights into the underlying traits and mechanisms that influence insect species’ variations and patterns in movement.
?. References
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?. Acknowledgements I would like to thank Meredith Cenzer for purchasing all flight mill materials and providing continuous feedback from the construction to the write-up of the project. I would also like to thank Ana Silberg for her contributions to standardize_troughs.py. Finally, I would like to thank the Media Arts, Data, and Design Center (MADD) at the University of Chicago for permission to use its communal makerspace equipment, technology, and supplies free of charge.