Island Assay: A High-Throughput Method to Evaluate Drosophila Locomotor Behavior

Overview

This video describes the island assay, a behavioral method used to study Drosophila locomotion. The sample protocol features a setup for the assay compatible with semi-automated image analysis.

Protocol

This protocol is an excerpt from Eidhof et al., High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay, J. Vis. Exp. (2017).

1. Construction of the Island Assay Box

1. Prepare a tray made of a robust material, such as poly methyl methacrylate (PMMA), to contain a layer of water (i.e. a bath). Ensure that the material is not white.
   NOTE: Dimensions of 40 x 35 x 2.5 cm³ are recommended (Figure 1A).
2. Prepare a box (42 x 38 x 25 cm³) made of a robust, transparent material, such as PMMA, to be placed around the bath to prevent the flies from escaping into the laboratory space. Place a hole (20 x 30 cm²) on the lateral side that is large enough for the experimenter to easily handle the vials with flies and drop them on the island (Figure 1A).
3. Prepare an elevated platform 10 x 15 x 2.5 cm³ in dimension, made of an impermeable material (PMMA or plastic) that is watertight.
   NOTE: This platform is required to have a uniform white surface to ensure good contrast for image analysis. The size is not necessarily fixed, but it should be large enough to make sure that all flies initially land on the platform and get the chance to walk on it (Figure 1A).
4. Fix the platform by either gluing it in to the bath or placing weights or other heavy objects inside the platform box to prevent positional changes of the platform during the experimental/filming session.

2. Software Requirements and Installation

1. Installation of image-recording software.
   1. Download image-recording software to record the island image series (see the Table of Materials) and install the software on a computer.
      NOTE: The imaging-recording software described in this protocol is only supported by Windows. An alternative for other users has been added to the Table of Materials.

3. Preparation of the Flies to Be Tested in the Island Assay

1. Collect staged flies for each experimental condition under cold or carbon dioxide (CO₂) anesthesia, as previously described (over a period of 1 - 2 days).
   1. Prepare a minimum of 3 sample vials per experimental condition, each containing approximately 15 staged flies. Only use flies with intact wings and that are of similar age and gender.
      NOTE: For the experiments described here, 5 sample vials containing 15 male flies of the genotypes w^-; Actin-Gal4/+ (control) and w^-; Actin-Gal4/GD11950 (tefu RNAi) were collected on the day of eclosion, aged for 4 days, and were used to perform the island assay. RNAi, control strains, and the Actin-gal4 driver were obtained from a commercial source (see the Table of Materials).
2. Let the flies recover to avoid effects from the anesthetics (at least 1 day when using CO₂) before testing the collected flies in the island assay at the age of interest.

4. Experimental Setup

NOTE: See Figure 1B.

1. Add cold water with a small amount of soap to the bath tray and position the platform in the middle.
   NOTE: The soap decreases the surface tension of the water; flies that touch the water will drown. This prevents increasing amounts of flies flying in the box during the progression of the experimental session.
2. Place the transparent box on top of the tray and illuminate the platform from above using a lamp.
   NOTE: Illumination of the platform is mainly required to ensure proper video contrast. An ordinary 12-V LED light is appropriate for this.
3. Position the webcam directly above the platform (outside the box) and connect it to a computer.
4. Create new folders on the computer to store the different experimental data prior to the experiments.
   1. Follow the structure of the example illustrated in Figure 2A to create the folders. For example, if the experimental design requires testing two genotypes with five replicates each, first create a main folder containing the date of the experiment. Inside the main folder, create two subfolders (one per genotype). Inside the genotype folders, create five new subfolders, one per replicate.
      NOTE: For further analysis, it is essential that the image series corresponding to individual experiments are saved in folders with unique names.

5. Video Settings Setup in the "Capture Device" Section of the Interface

1. Open the image-recording software and, under the "Capture" tab, click on "Time-Lapse Images…" and select the appropriate webcam as the "Capture Device".
2. Place a dead fly on the island; adjust the video settings by clicking on the "Video Settings" box. Scroll through the tool bars, adjusting brightness, contrast, and color in a manner such that the dead fly appears black on a white background (Figure 2B). When the adjustments are complete, click "Ok".

6. Recording and Video Saving Settings Setup in the "Time-lapse" Section of the Interface

1. Adjust the settings in the "Time-Lapse Movie Setup" to save the experiment as an .avi file. Click on "Browse…", select the directory where the movie will be stored, define the name of the video, and press "save".
   NOTE: The video file is not used for the quantification of data; however, it might be useful to get an overall idea about the experiment.
2. Adjust the settings in the "Time-Lapse Movie Setup" section.
   1. In the compression box, select "Intel IYUV code" for video compression and choose "Take one frame every 0.1 seconds", with the "Play-back rate (images per second):" at 10.
   2. Save the experiment time series as .bmp frames by clicking on "Advanced…" Select "Create a .bmp image for each captured frame". Click on "Browse". Select the same directory in the "During AVI movie capture" window (as chosen in step 6.1), click "Open", and press "Ok".
      NOTE: Notice that, during the experiment, the frames are stored as .bmp files (required for data analysis). The program names the frames as "A" followed by the number corresponding to the frame capture sequence (e.g., "A_number.bmp") (Figure 2C). Always save the images belonging to different experiments in a new folder to ensure that previously recorded image series will not be overwritten. Be aware that the images are not automatically saved in the same folder as the video file unless this folder is selected under "Browse…" upon clicking the "Advanced" box.

7. Island Assay and Data Collection

1. Press the start button in the time-lapse image interface of the image-recording software to start the recording.
2. Tap the experimental vial containing flies (step 3) 2-3 times to ensure that flies are at the bottom of the vial. Quickly remove the plug of the vial and, with a vigorous movement, tap the vial on the platform so that all flies fall onto the platform at the same time (Figure 1C).
3. After approximately 30 s, press the "Stop" button to stop recording.
   NOTE: If all flies vanish from the platform, the recording can be stopped earlier.
4. Remove the flies that remain for 30 s on the platform by hand after stopping the image recording.
5. Before proceeding to recording the next experiment, change the target directory for the .bmp files and movies (see section 6).

Representative Results

Figure 1: Flowchart outlining the requirements, experimental procedure, and analysis of the island assay. (A) Island assay equipment. (B) Experimental setup for the island assay. (C) Island assay. (D) Processing of island assay data with the "Drosophila Island Assay" macro. The "Drosophila Island Assay" macro is composed of 3 sub-macros: 1) make stack and projection, 2) define platform, and 3) analysis. (E) Processing and statistical evaluation of data using the "Island Assay Analysis" script. Please click here to view a larger version of this figure.

Figure 2: Examples of different adjustments required during the protocol. (A) The required directory structure in which island assay experiments must be stored for data processing and analysis. (B) When adjusting the video settings, flies must appear black on a white background. (C) Image frame output files as saved by the image-recording software described in this manuscript. (D) The yellow outline shows the platform selection. The stored platform selection in the "ROI Manager" is highlighted in blue. (E) Flies are represented as white dots during the adjustment of the "Minimum fly size setting." The results window shows the area of the flies in pixels. (F) Example of a single recorded image frame (on the left) and the corresponding frame in the resulting image stack, obtained with the "Drosophila Island Assay" macro (on the right). Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
25 x 95 mm Drosophila vials	Flystuff	32-116SB	-
Logitech C525 HD Webcam	Logitech	-	Any webcam with USB connection is suitable.
Stand to hold webcam	-	-	-
Lamp	-	-	12 V LED lights are appropriate
Pounding pad	-	-	Any mouse pad works
Island Assay box	-	-	Dimensions 40x35x2.5 cm. Hole 20x30 cm. Transparent.
Island Assay bath	-	-	Dimensions 42x38x25 cm. Non white.
Island/platform	-	-	Dimensions 42x38x25 cm. Uniform white.
Soap	-	-	Standard dishwashing detergent is suitable.
Computer	-	-	Scripts run both on Windows and Mac
Image-recording software: HandiAvi®	AZcendant®	-	HandyAvi is only compatible with Windows and has been described throughout the manuscript. It can be downloaded from: http://www.azcendant.com/DownloadHandyAvi.html (version 5.0)
Image-recording software: WebcamCapture	-	-	Fiji/ImageJ plugin that can be used on Mac alternative to HandyAvi for image-recordings and can be downloaded from: https://imagej.nih.gov/ij/plugins/webcam-capture/ When using this method, the user has to use the same folder setup and image-recording settings indicated in this manuscript, with the exception that for each experimental replicate, the captured image stack should be exported as Stack.tiff to the corresponding experimental replicate folder. Upon running the "Drosophila Island Assay" macro on this data, no text should be present in the "First frame identifier" setting.