Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

Summary

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

Abstract

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Introduction

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis has been under investigation, especially in the past 50 years^{1,2,3,4,5,6,7,8}. In recent years, despite the fact that 1) many neurons mediating larval light avoidance have been identified^{4,5,9,10,11,12} and 2) the complete connectome of larval visual system at the resolution of synapses has been established¹³, the neural mechanisms underlying larval phototaxis remain largely unclear.

A number of behavioral assays have been used in studying larval phototaxis. They can be largely divided into two classes: one involving spatial light gradients and the other involving temporal light gradients. For spatial light gradient assays, the arena is divided into equal number of sections in light and dark. The arena can be divided into light and dark halves^2,4 or light and dark quadrants^14,15, or can even be separated into alternate light and dark squares like on a checkerboard⁷. Usually, agar plates are used for spatial light gradient assay, but tubes that are divided into alternate light and dark sections can also be used^10,14.

In older version of assays, light illumination generally originates from below the larvae. However, illumination in newer versions largely originates from above, since larval eyes (e.g., the Bolwig's organs that are sensitive to low or medium light intensities¹⁶) are contained in the opaque cephalopharyngeal skeleton with openings towards the upper front. This makes larvae more sensitive to light from upper front directions than from below behind directions⁷. For temporal light gradient assays, the light intensity is spatially uniform in the arena, but the intensity changes over time. In addition to temporal square wave light (i.e., flashing on/off or strong/weak light^3,7), temporally varying light that conforms to a linear ramp in intensity is also used⁸ to measure the sensitivity of larvae to a temporally changing light stimulus.

A third type of phototaxis assay is the directional light scape navigation, which involves illumination from above at an angle of 45°⁷. Before the work of Kane et al.⁷, only coarse parameters such as the number of larvae in light and dark regions, frequency of turning, and trail length were calculated in larval phototaxis assays. Since the work of this same group, with the analysis of high temporal resolution video record for larval phototaxis, detailed dynamics of larval movement during phototaxis (i.e., instant speeds of different parts of larval body, heading direction, turning angle and corresponding angular velocity) have been analyzed⁷. Thus, more details of larval phototaxis behavior have been able to be discovered. In these assays, larvae are tested in groups so that group effects are not excluded.

This protocol introduces a light-spot assay for the investigation of larval behavioral responses to individual light stimulation. The main experimental set-up consists of a visual stimulation system and infrared light-based imaging system. In the visual stimulation system, an LED light source generates a round 2 cm-diameter light spot on an agar plate, where the larva is tested. The light intensity can be adjusted using an LED driver. The imaging system includes an infrared camera that captures the behavior of the larva in addition to three 850 nm infrared LEDs that provide illumination for the camera. The lens of the camera is covered by an 850 nm band-pass filter to block light from the visual stimulation system from entering the camera, while the infrared light is allowed to enter the camera. Thus, interference of visual stimulation on imaging is prevented. In this assay, the behavioral details of the fast responses of individual larvae within a period including before, during, and after entering light are recorded and analyzed.

Protocol

1. Preparation of Drosophila larvae

1. Prepare standard medium consisting of boiled corn meal (73 g), agar (5.6 g), soybean meal (10 g), yeast (17.3 g), syrup (76 mL) and water (1000 mL).
2. Raise all flies at 25 °C on standard medium in a room with a 12 h/12 h light/dark cycle.

2. Preparation of agar plates

1. Prepare 1.0% agar solution. Weigh 3 g of agar in a 500 mL beaker with a balance, then add 300 mL of distilled water. Place a foil paper over the breaker to prevent the water from evaporating. Heat the beaker in a microwave to boil.
2. Take out the beaker and stir well with a glass rod, then heat in a microwave oven to boil. Repeat until the liquid is completely transparent and fluid.
   NOTE: The final hot agar solution should be free of air bubbles; otherwise, pouring the agar into the plate will result in uneven and dented surface of the agar plate, which will affect subsequent larval behavior testing. The concentration of the agar solution should not be too high or low. If the concentration is too high, the light diffuse reflection on the agar plate will be strong and smear the light/dark boundary. If the concentration is too low, the larvae will leave traces on the plate. Both of these errors will hinder video processing later in the protcol.
3. Slowly pour the hot agar solution into a Petri dish (diameter 15 cm) until the bottom is evenly covered with a layer of agar (~4 mm in thickness), and cool at room temperature (RT) until the agar solution is solidified.
4. Agar plate should be used when it is freshly prepared. If it is not, pour a layer of water onto the surface and store it in the refrigerator at 4 °C for later use.

3. Set-up of visual stimulation system

1. Select the LED light source: collimated LED blue light at 470 nm or warm white light.
   NOTE: The light source can be replaced with LED light of any other wavelength. In this experiment, blue light LED is used as an example.
2. Roll a thick piece of aluminum foil or black cardboard (ensure opacity) to form an open cylinder of 12 cm in length with a diameter similar to that of the blue-light LED with a diameter of 3 cm. Let the top end of the cylinder cover the front end of the blue-light LED. Cover the bottom end with a black cardboard with a small round hole (0.5 cm diameter) in the center. This constitutes the light source system set-up.
3. Fix the prepared light source onto the iron frame with a clip, making sure the LED light projects down towards the desktop. Tilt the cylinder slightly. The angle between the cylinder plane and vertical plane is about 10° (see Figure 1). Connect the 470 nm blue LED light to the "LED1" plug of the high-power LED driver. Turn on the driver, turn the knob in the upper right corner of the driver to select channel 470 nm, then click LED. Then, when the screen displays "√", a blue light spot will appear on the desktop.
   NOTE: If the cylinder leaks light in addition to the small round hole, it is recommended to use black tape on the leaking parts to make sure that only the hole can pass light through.
4. Click OK and turn the knob to adjust the intensity of the light. Rotate the knob to a higher light intensity of 50 mA. Measure and record the spectrum of the light with a spectrometer.
5. Move the position of the light source up and down to adjust the diameter of the light spot to 2 cm. The desktop should be black for a better contrast effect.
6. Rotate the knob to choose the light intensity according to experimental needs. Use a compact power meter console with a standard photodiode power sensor to measure the maximal and minimal light power in the spot, record it, measure three times, and take the average value.
   NOTE: It is recommended to use a photodiode power sensor to measure the light intensity for light of a specific wavelength and use a thermal power sensor to measure the light intensity for white light.
7. Calculate the light intensity in the light spot by dividing the measured light power by the area of the sensor.
   NOTE: For example, if the measured light power in step 2.6 is 20 pW and area of the sensor is 0.81 mm², the light intensity is 24.69 pW/mm² (dividing 20 pW by 0.81 mm²).

4. Set-up of the imaging system

1. Clamp a high-resolution web camera with an iron clip, at about 10 cm above the light spot on the desktop (Figure 1).
2. Adjust the orientation of the camera lens towards the desktop. Connect the camera to a computer through a USB interface.
3. Place an agar plate on the desktop right beneath the camera.
4. Open the "Amcap9.22" software on the computer with Windows 7, and the light spot will be automatically shown in the window of AMcap. Move the camera slightly left or right to ensure that the light spot is near the center of the window. Ensure that the camera does not block the light path. The light spot should be complete and round.
   NOTE: The software can be found at http://amcap.en.softonic.com/.
5. Fix an 850 nm ± 3 nm band-pass filter with a clip at 5-7 mm right below the camera.
   NOTE: The diameter of the filter is about 2.5 cm, and the camera lens is less than 1 cm in diameter, so the filter can cover the visual field of the camera. With the filter below the camera, the light spot should not be seen in the window of AMcap.
6. Place three infrared-light-generating LEDs (central wavelength = 850 nm) evenly around the agar plate. Each LED should be about 5 cm away from the edge of the agar plate, and the lens face of the LED should be at a 70° downwards angle towards the agar plate. Connect the LEDs to the power through the AC-to-DC converter.
   NOTE: It is better to fix the positions and angles of the infrared light LEDs to ensure consistency of the brightness of the field in various experimental trials and facilitate later video processing.
7. Put a black board between the computer and the device. Set down the brightness of the computer screen to prevent the computer screen light from affecting the experiment.
   NOTE: Keep the environment dark when measuring wavelength or intensity of the light.

5. Setting parameters of imaging

1. On the menu of the AMcap software, choose Options | Video Device | Capture format, and set the pixel size of the captured video to 800 x 600 and frame rate to 60 fps.
2. Remove the filter from beneath the camera, put a ruler under the camera and adjust the camera focus to make the scale line clear and parallel to the width of the video field of view.
3. Click Capture | Set up | Video capture to select the save path, click Start recording, record the actual distance corresponding to 600 pixels, and calculate the ratio of each pixel to the actual distance.

6. Video recording of light avoidance behavior

1. Maintain a temperature of 25.5 °C through all experiments. Control room temperature with an air conditioner if required. Keep the humidity constantly at 60% with a humidifier.
2. Take a short video of the light spot position named "lightarea1". Then, move the 850 nm ± 3 nm filter back to cover the camera lens.
   NOTE: When recording larval behavior, the camera lens is covered by the 850 nm ± 3 nm filter so that the light spot is not shown in the video. The light spot can be reconstructed in videos with larvae later with Matlab. Do not change the position of the camera, and avoid changing the ratio of each pixel to the actual distance measured in step 4.3.
3. Turn on a light (i.e., a room light) far away from the experimental device. Turn down the light as low as possible, as long as the larvae can be clearly seen with the eyes. Take the larvae out of the culture medium with a spoon, gently pick a third-instar larva, and wash it clean with distilled water. Be careful to wash larva one at a time to avoid interference from hunger. A single experiment requires at least 20 larvae.
4. Transfer the larva to the center of the agar plate placed beneath the camera during step 3.3. Gently remove excess water from the larva with a brush or use blotting paper to remove water from the larva to prevent reflection of light under the lens. Turn off the room light and allow the larva to acclimate for 2 min in the dark environment.
5. Turn on the LED light to generate infrared light, and gently brush the larva to the center of the plate. When the larva begins to crawl straight, rotate the plate to make the larva head towards the light spot. Make sure that it crawls straight from the start, or else it may not obtain access to the light spot.
6. Click Capture | Set up | Video capture to select the save path, then click Start recording to record. Allow the larva to crawl towards the light spot, enter the light spot, then leave the light spot until it is nearly out of the field of view. Click Stop recording. If the larva turns away from the light spot before getting close, directly click Stop recording.
7. Move the filter away from the camera. Take a short video of the position of the light spot named "lightarea2" and compare it to "lightarea1" to ensure that the light spot position is not changed. If an obvious position change is observed, discard the data.

7. Data analysis

1. Use SOS¹⁷ to extract animal body contour and movement parameters from videos using image processing methods as previously described¹⁷.
   NOTE: Parameters including headSpeed (velocity of larval head), tailSpeed (velocity of larval tail), midSpeed (velocity of larval midpoint of skeleton line), and cmSpeed (velocity of larval centroid) were used to measure larval movement speed. Parameters including headTheta (the angle between the lines of head-midpoint and midpoint-tail) and headOmega (the changing speed of headTheta) were used to measure larval body bending and the angular speed of bending.

Results

According to the protocol, the light spot assay was used to investigate light avoidance behavior of third instar larva that were raised at 25 °C on standard medium in a room with a 12 h/12 h light/dark cycle. A single w¹¹¹⁸ larva was tested using the light spot assay at 25.5 °C. The average light intensity of the light spot generated by a 460 nm LED was 0.59 µW/cm². The whole process of larval entering and exiting the light spot was recorded and analyzed using SOS software an...

Discussion

This protocol presents the light spot assay to test the ability of Drosophila larvae to escape from light. This assay allows tracking of the behavior of larvae before entering, during encountering, and after leaving a light spot. Details of larval movement can be captured and analyzed. The light spot assay is very simple and possesses strong practicability. The cost of the whole device is not high. In the experiment, LED light is used as the light source. It can be replaced with light sources of different wavele...

Materials

Name	Company	Catalog Number	Comments
850 nm ± 3 nm infrared-light-generating LED	Thorlabs, USA	PM100A	Compatible Sensors: Photodiode and Thermal Optical Power Rangea: 100 pW to 200 W Available Sensor Wavelength Rangea: 185 nm-25 μm Display Refresh Rate: 20 Hz Bandwidtha: DC-100 kHz Photodiode Sensor Rangeb: 50 nA-5 mA Thermopile Sensor Rangeb: 1 mV-1 V
AC to DC converter	Thorlabs, USA	S120VC	Aperture Size: Ø9.5 mm Wavelength Range: 200-1100 nm Power Range: 50 nW-50 mW Detector Type: Si Photodiode (UV Extended) Linearity: ±0.5% Measurement Uncertaintyc: ±3% (440-980 nm), ±5% (280-439 nm), ±7% (200-279 nm, 981-1100 nm)
band-pass filter	Thorlabs, USA	DC2100	LED Current Range: 0-2 A LED Current Resolution: 1 mA LED Current Accuracy: ±20 mA LED Forward Voltage: 24 V Modulation Frequency Range: 0-100 kHz Sine Wave Modulation: Arbitrary
Collimated LED blue light	ELP, China	USBFHD01M	Max. Resolution: 1920X1080 F6.0 mm Sensor: 1/2.7" CMOS OV2710
Compact power meter console	Ocean Optics, USA	USB2000+(RAD)	Dimensions: 89.1 mm x 63.3 mm x 34.4 mm Weight: 190 g Detector: Sony ILX511B (2048-element linear silicon CCD array) Wavelength range: 200-850 nm Integration time: 1 ms – 65 seconds (20 seconds typical) Dynamic range: 8.5 x 10^7 (system); 1300:1 for a single acquisition Signal-to-noise ratio: 250:1 (full signal) Dark noise: 50 RMS counts Grating: 2 (250 – 800 nm) Slit: SLIT-50 Detector collection lens: L2 Order-sorting: OFLV-200-850 Optical resolution: ~2.0 nm FWHM Stray light: <0.05% at 600 nm; <0.10% at 435 nm Fiber optic connector: SMA 905 to 0.22 numerical aperture single-strand fiber
High-Power LED Driver	Minhongshi, China	MHS-48XY	Working voltage: DC12V Central wavelength: 850nm
high-resolution web camera	Thorlabs, USA	MWWHL4	Color: Warm White Correlated Color Temperature: 3000 K Test Current for Typical LED Power: 1000 mA Maximum Current (CW): 1000 mA Bandwidth (FWHM): N/A Electrical Power: 3000 mW Viewing Angle (Full Angle): 120˚ Emitter Size: 1 mm x 1 mm Typical Lifetime: >50 000 h Operating Temperature (Non-Condensing): 0 to 40 °C Storage Temperature: -40 to 70 °C Risk Groupa: RG1 – Low Risk Group
LED Warm White	Mega-9, China	BP850/22K	Ø25.4(+0~-0.1) mm Bandwidth: 22±3nm Peak transmittance:80% Central wavelength: 850nm±3nm
Spectrometer	Noel Danjou	Amcap9.22	AMCap is a still and video capture application with advanced preview and recording features. It is a Desktop application designed for computers running Windows 7 SP1 or later. Most Video-for-Windowsand DirectShow-compatible devices are supported whether they are cheap webcams or advanced video capture cards.
Standard photodiode power sensor	Super Dragon, China	YGY-122000	Input: AC 100-240V~50/60Hz 0.8A Output: DC 12V 2A
Thermal power sensor	Thorlabs, USA	M470L3-C1	Color: Blue Nominal Wavelengtha: 470 nm Bandwidth (FWHM): 25 nm Maximum Current (CW): 1000 mA Forward Voltage: 3.2 V Electrical Power (Max): 3200 mW Emitter Size: 1 mm x 1 mm Typical Lifetime: 100 000 h Operating Temperature (Non-Condensing): 0 to 40 °C Storage Temperature: -40 to 70 °C Risk Groupb: RG2 – Moderate Risk Group
Thermal power sensor	Thorlabs, USA	S401C	Wavelength range: 190 nm-20 μm Optical power range:10 μW-1 W(3 Wb) Input aperture size: Ø10 mm Active detector area: 10 mm x 10 mm Max optical power density: 500 W/cm2 (Avg.) Linearity: ±0.5%