Building an Enhanced Flight Mill for the Study of Tethered Insect Flight

This protocol can be used to build an affordable and enhanced flight mill using makerspace technology, such as laser cutters and 3D printers. Laser cutters and 3D printers can handle intricate designs so you can easily customize and reproduce your flight mill to fit your experimental requirements. To construct the acrylic supports in a makerspace, open and appropriate vector graphics editor, and create file lines in RGB mode with line stroke of 0.0001 point at which the RGB red cuts lines and the RGB blue edges lines.

As a precaution design the curve key as illustrated. Follow the makerspace guidelines on powering up, using and maintaining the laser cutter. In the laser software, select plastic for the material and acrylic for the material type.

For extra precision use the caliper to measure the material thickness and enter its thickness into the material thickness field. Place the material in the printer cavity and cut the curve key. Use the curve key to determine the curve width, then account for curve for all the slit and hole measurements in the acrylic support design as needed before laser cutting the acrylic supports.

For 3D printing of the plastic supports, click 3D Designs and Create to create a new design. To replicate this studies exact 3D printed designs, download the archive, 3D_Prints. zip, and move the folder onto the desktop.

Open the folder. In the online 3D modeling program, work plain webpage, click Import and select all of the stl files from the folder. To self create or make adjustments to the designs, follow the website's tutorials.

Make edits and export the new designs as stl files. To obtain the mirror of a design, click on the object, click M and select the arrow corresponding to the objects width. For 3D printing, double click on the 3D printing, slicing software icon and select the object file to be printed.

Select Print and Machine Type to select the 3D printer and double click the move icon to adjust the object position. Click on platform to ensure that the model is on the platform. And click move and center to place the object at the center of the build area.

When the object is ready, click Print to save the print as a gx file. Next calibrate the extruder of the 3D printer according to standard protocols and confirm that there is enough filament for printing. When all of the objects have been modified transfer the gx files to the 3D printer and print all of the supports and enhancements.

For each printing check that the filament is sticking properly to the plate. In total eight 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 lineage guide rail supports should be 3D printed for this design. After assembling the acrylic walls, insert a 30 millimeter long plastic tube into the top tube support and a 15 millimeter long plastic tube into the bottom tube support of each cell.

Insert a 14 millimeter long plastic tube into the top tube and a 20 millimeter long plastic tube into the bottom tube, making sure that there is strong enough friction between the tubes to hold the tubes in place without allowing the inner tube to slide up and down if pulled. If any tubes are warped, submerge the warped tube segments in boiling water for one minute and straighten the tubes on a towel, allowing the materials to reach room temperature before inserting the straightened segments into the tubes. Placed two low friction neodymium magnets and an inner tube into each magnet support.

Lodge the inner tube firmly into each magnet support such that the gravity acting on the magnets and the magnets support is not strong enough to dislodge the materials from the inner tube. Check whether each pair of magnets repels each other. With the linear guide rail blocks both facing upwards, slide the blocks into the linear guide rail and lodge the linear guide rails and blocks upright into the windows on the outer vertical walls.

Use two short linear guide rails supports, two long linear guide rail supports, four 10 millimeter long iron screws, two 20 millimeter long iron screws, and two hex nuts to secure one linear guide rail in place. To construct the pivoting arm, glue 19 gauge non-magnetic hypodermic steel tubing to the pipette tip axle and the two low friction neodymium magnets to the bent end of the pivot arm to tether the metal painted insect for flight. Wrap a piece of aluminum foil on the unbent end of the pivot arm to create a flag counterweight and to break the infrared beam sent from the infrared sensor transmitter to the receiver.

To set up the infrared sensor and data logger, place the infrared sensor transmitter inside the top linear guide rail block with the emitter of the beam facing downward and place the infrared sensor receiver inside the bottom block facing up. To magnetically tether insects to the flight mill arm for a flight trial, apply magnetic paint to the pronotum of the insect and let the paint dry for at least 10 minutes. Once dry, attach the insect to the flight mill arm magnets.

After attaching up to eight insects, click File and Record in the Flight Analysis software, select the location of the recording file in the first pop-up window, making sure the file name includes the recording set number and the channel letter and click OK.In the next pop-up window, enter the anticipated length of the flight recording. When the insects are in position click OK to begin recording. At the end of the recording, press Control S to finalize the file.

To make an event marker comment, click on the channel number and click Edit and Insert Commented Mark, define the comment with the identification number of the new insect entering the chamber, then click OK, and load the insect into the chamber. After converting the WDH recording files to text format and splitting the text files by event marker comments, open the trough_diagnostic. png file generated in the Flight_scripts folder and check all of the records are robust to changes in the minimum and maximum voltage value of the mean standardization interval.

If the records are okay, specify all of the user settings and Save and Run the flight_analysis. py script. If the script run is successful, the corresponding ID number, chamber and calculated flight statistics of the insect will be printed in the Python Shell.

A comprised flight_stats_summary. csv file of the information will also be printed in the Python Shell in the flight_scripts directory data folder. These representative flight data were obtained experimentally during the winter of 2020 using field collected J Hemmat Aloma from Florida as the model.

In this set of trials, the flight data was successfully recorded for all of the channels without noise or disruption. In this analysis however, the recorded signal was lost in channel three, which dropped the voltage immediately to zero volts, possibly due to the crossing over of open wires or the loosening of wires. As observed for this trial, the trough diagnostic data generated by each revolution of the flight mill arm were robust, indicating that they largely deviated from the files mean voltage.

In this analysis as the standardization interval around the mean increased, the number of troughs identified exhibited little change, suggesting minimal voltage noise and an accurate standardization. In contrast for this flight, the troughs were either too sensitive or had extremist 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.

Individual flight behaviors can be further characterized into four flight categories, bursts, bursts to continuous, continuous to bursts and continuous. Thus the user can use this graphic output to assess an identify a general flight behavior patterns despite unique variations in individual tracks. The more insects that can be tested, the more ways the field can understand how insects move.

These methods also encourage ecologists to use emerging technologies so that they can build their own tools.