This work is supported by natural science foundation of China (31671074) and Fundamental Research Funds for the Zhejiang Provincial Universities (2019XZZX003-12).

1. Preparation of Drosophila larvae
<ol>
	<li>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).</li>
	<li>Raise all flies at 25 &#176;C on standard medium in a room with a 12 h/12 h light/dark cycle.</li>
</ol>
2. Preparation of agar plates
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C for later use.</li>
</ol>
3. Set-up of visual stimulation system
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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&#176; (see Figure 1). Connect the 470 nm blue LED light to the &#34;LED1&#34; 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 &#34;&#8730;&#34;, 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
4. Set-up of the imaging system
<ol>
	<li>Clamp a high-resolution web camera with an iron clip, at about 10 cm above the light spot on the desktop (Figure 1).</li>
	<li>Adjust the orientation of the camera lens towards the desktop. Connect the camera to a computer through a USB interface.</li>
	<li>Place an agar plate on the desktop right beneath the camera.</li>
	<li>Open the &#34;Amcap9.22&#34; 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/.</li>
	<li>Fix an 850 nm &#177; 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.</li>
	<li>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&#176; 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.</li>
	<li>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.</li>
</ol>
5. Setting parameters of imaging
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Video recording of light avoidance behavior
<ol>
	<li>Maintain a temperature of 25.5 &#176;C through all experiments. Control room temperature with an air conditioner if required. Keep the humidity constantly at 60% with a humidifier.</li>
	<li>Take a short video of the light spot position named &#34;lightarea1&#34;. Then, move the 850 nm &#177; 3 nm filter back to cover the camera lens. 
	NOTE: When recording larval behavior, the camera lens is covered by the 850 nm &#177; 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Move the filter away from the camera. Take a short video of the position of the light spot named &#34;lightarea2&#34; and compare it to &#34;lightarea1&#34; to ensure that the light spot position is not changed. If an obvious position change is observed, discard the data.</li>
</ol>
7. Data analysis
<ol>
	<li>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.</li>
</ol>

<ol>
	<li>Grossfield, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+distribution+and+light-dependent+behavior+in+Drosophila.">Geographic distribution and light-dependent behavior in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 68, 2669 (1971).</li><li>Godoy-Herrera, R. C. L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behaviour+of+Drosophila+melanogaster+larvae+during+pupation.">The behaviour of Drosophila melanogaster larvae during pupation.</a> Animal Behaviour. 37, (1989).</li><li>Busto, M., Iyengar, B., Campos, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior:+modulation+of+locomotion+by+light+in+the+Drosophila+melanogaster+larva+requires+genetically+distinct+visual+system+functions.">Genetic dissection of behavior: modulation of locomotion by light in the Drosophila melanogaster larva requires genetically distinct visual system functions.</a> Journal of Neuroscience. 19, 3337 (1999).</li><li>Mazzoni, E. O., Desplan, C., Blau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+pacemaker+neurons+transmit+and+modulate+visual+information+to+control+a+rapid+behavioral+response.">Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response.</a> Neuron. 45, 293 (2005).</li><li>Keene, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+visual+pathways+mediate+Drosophila+larval+light+avoidance+and+circadian+clock+entrainment.">Distinct visual pathways mediate Drosophila larval light avoidance and circadian clock entrainment.</a> Journal of Neuroscience. 31, 6527 (2011).</li><li>Keene, A. C., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+light:+photobehavior+in+fruit+fly+larvae.">Seeing the light: photobehavior in fruit fly larvae.</a> Trends in Neurosciences. 35, (2012).</li><li>Kane, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+structure+of+Drosophila+larva+phototaxis.">Sensorimotor structure of Drosophila larva phototaxis.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, E3868 (2013).</li><li>Humberg, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedicated+photoreceptor+pathways+in+Drosophila+larvae+mediate+navigation+by+processing+either+spatial+or+temporal+cues.">Dedicated photoreceptor pathways in Drosophila larvae mediate navigation by processing either spatial or temporal cues.</a> Nature Communications. 9. 1260, (2018).</li><li>Gong, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+pairs+of+neurons+in+the+central+brain+control+Drosophila+innate+light+preference.">Two pairs of neurons in the central brain control Drosophila innate light preference.</a> Science. 330, (2010).</li><li>Yamanaka, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroendocrine+Control+of+Drosophila+Larval+Light+Preference.">Neuroendocrine Control of Drosophila Larval Light Preference.</a> Science. 341, 1113 (2013).</li><li>Zhao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+disinhibitory+mechanism+biases+Drosophila+innate+light+preference.">A disinhibitory mechanism biases Drosophila innate light preference.</a> Nature Communications. 10, (2019).</li><li>Gong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Neuronal+Pathway+that+Commands+Deceleration+in+Drosophila+Larval+Light-Avoidance.">A Neuronal Pathway that Commands Deceleration in Drosophila Larval Light-Avoidance.</a> Neuroscience Bulletin. Feb. 27, (2019).</li><li>Larderet, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+Drosophila+larval+visual+circuit.">Organization of the Drosophila larval visual circuit.</a> eLife. 6, (2017).</li><li>Sawin-McCormack, E. P., Sokolowski, M. B., Campos, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+genetic+analysis+of+Drosophila+melanogaster+photobehavior+during+larval+development.">Characterization and genetic analysis of Drosophila melanogaster photobehavior during larval development.</a> Journal of Neurogenetics. 10, (1995).</li><li>Farca, L. A., von Essen, A. M., Widmer, Y. F., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+preference+assay+to+study+innate+and+circadian+regulated+photobehavior+in+Drosophila+larvae.">Light preference assay to study innate and circadian regulated photobehavior in Drosophila larvae.</a> Journal of Visualized Experiments. 74 (74), e50237 (2013).</li><li>Xiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-avoidance-mediating+photoreceptors+tile+the+Drosophila+larval+body+wall.">Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall.</a> Nature. 468, 921 (2010).</li><li>Gomez-Marin, A., Partoune, N., Stephens, G. J., Louis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+tracking+of+animal+posture+and+movement+during+exploration+and+sensory+orientation+behaviors.">Automated tracking of animal posture and movement during exploration and sensory orientation behaviors.</a> PLoS ONE. 7, e41642 (2012).</li></ol>

The authors have nothing to disclose.

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...

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 years1,2,3,4,5,6,7,8. In recent years, despite the fact that 1) many neurons mediating larval light avoidance have been identified4,5,9,10,11,12 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 halves2,4 or light and dark quadrants14,15, 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 used10,14.
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&#39;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 light3,7), 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&#176;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.

<table><tbody><tr><td>850 nm &amp;plusmn; 3 nm infrared-light-generating LED</td><td>Thorlabs, USA</td><td>PM100A</td><td>Compatible Sensors: Photodiode and Thermal&lt;br/&gt; Optical Power Rangea: 100 pW to 200 W&lt;br/&gt; Available Sensor Wavelength Rangea: 185 nm-25 &amp;mu;m Display Refresh Rate: 20 Hz&lt;br/&gt; Bandwidtha: DC-100 kHz&lt;br/&gt; Photodiode Sensor Rangeb: 50 nA-5 mA&lt;br/&gt; Thermopile Sensor Rangeb: 1 mV-1 V</td></tr><tr><td>AC to DC converter</td><td>Thorlabs, USA</td><td>S120VC</td><td>Aperture Size: &amp;Oslash;9.5 mm&lt;br/&gt; Wavelength Range: 200-1100 nm&lt;br/&gt; Power Range: 50 nW-50 mW&lt;br/&gt; Detector Type: Si Photodiode (UV Extended)&lt;br/&gt; Linearity: &amp;plusmn;0.5%&lt;br/&gt; Measurement Uncertaintyc: &amp;plusmn;3% (440-980 nm), &amp;plusmn;5% (280-439 nm), &amp;plusmn;7% (200-279 nm, 981-1100 nm)</td></tr><tr><td>band-pass filter</td><td>Thorlabs, USA</td><td>DC2100</td><td>LED Current Range: 0-2 A&lt;br/&gt; LED Current Resolution: 1 mA&lt;br/&gt; LED Current Accuracy: &amp;plusmn;20 mA&lt;br/&gt; LED Forward Voltage: 24 V&lt;br/&gt; Modulation Frequency Range: 0-100 kHz Sine Wave&lt;br/&gt; Modulation: Arbitrary</td></tr><tr><td>Collimated LED blue light&amp;nbsp;</td><td>ELP, China</td><td>USBFHD01M</td><td>Max. Resolution: 1920X1080&lt;br/&gt; F6.0 mm&lt;br/&gt; Sensor: 1/2.7&quot; CMOS OV2710</td></tr><tr><td>Compact power meter console&amp;nbsp;</td><td>Ocean Optics, USA</td><td>USB2000+(RAD)</td><td>Dimensions: 89.1 mm x 63.3 mm x 34.4 mm&lt;br/&gt; Weight: 190 g&lt;br/&gt; Detector: Sony ILX511B (2048-element linear silicon CCD array)&lt;br/&gt; Wavelength range: 200-850 nm&lt;br/&gt; Integration time: 1 ms &amp;ndash; 65 seconds (20 seconds typical)&lt;br/&gt; Dynamic range: 8.5 x 10^7 (system); 1300:1 for a single acquisition&lt;br/&gt; Signal-to-noise ratio: 250:1 (full signal)&lt;br/&gt; Dark noise: 50 RMS counts&lt;br/&gt; Grating: 2 (250 &amp;ndash; 800 nm)&lt;br/&gt; Slit: SLIT-50&lt;br/&gt; Detector collection lens: L2&lt;br/&gt; Order-sorting: OFLV-200-850&lt;br/&gt; Optical resolution: ~2.0 nm FWHM&lt;br/&gt; Stray light: &amp;lt;0.05% at 600 nm; &amp;lt;0.10% at 435 nm&lt;br/&gt; Fiber optic connector: SMA 905 to 0.22 numerical aperture single-strand fiber</td></tr><tr><td>High-Power LED Driver</td><td>Minhongshi, China</td><td>MHS-48XY</td><td>Working voltage: DC12V&lt;br/&gt; Central wavelength: 850nm</td></tr><tr><td>high-resolution web camera</td><td>Thorlabs, USA</td><td>MWWHL4</td><td>Color: Warm White&lt;br/&gt; Correlated Color Temperature: 3000 K&lt;br/&gt; Test Current for Typical LED Power: 1000 mA&lt;br/&gt; Maximum Current (CW): 1000 mA&lt;br/&gt; Bandwidth (FWHM): N/A&lt;br/&gt; Electrical Power: 3000 mW&lt;br/&gt; Viewing Angle (Full Angle): 120?&lt;br/&gt; Emitter Size: 1 mm x 1 mm&lt;br/&gt; Typical Lifetime: &gt;50 000 h&lt;br/&gt; Operating Temperature (Non-Condensing): 0 to 40 &amp;deg;C&lt;br/&gt; Storage Temperature: -40 to 70 &amp;deg;C&lt;br/&gt; Risk Groupa: RG1 &amp;ndash; Low Risk Group</td></tr><tr><td>LED Warm White</td><td>Mega-9, China</td><td>BP850/22K</td><td>&amp;Oslash;25.4(+0~-0.1) mm&lt;br/&gt; Bandwidth: 22&amp;plusmn;3nm&lt;br/&gt; Peak transmittance:80%&lt;br/&gt; Central wavelength: 850nm&amp;plusmn;3nm&amp;nbsp;</td></tr><tr><td>Spectrometer&amp;nbsp;</td><td>Noel Danjou</td><td>Amcap9.22</td><td>AMCap&amp;nbsp;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&amp;nbsp;Video-for-Windowsand&amp;nbsp;DirectShow-compatible devices are supported whether they are cheap webcams or advanced video capture cards.</td></tr><tr><td>Standard photodiode power sensor&amp;nbsp;</td><td>Super Dragon, China</td><td>YGY-122000</td><td>Input: AC 100-240V~50/60Hz 0.8A&lt;br/&gt; Output: DC 12V 2A</td></tr><tr><td>Thermal power sensor&amp;nbsp;</td><td>Thorlabs, USA</td><td>M470L3-C1</td><td>Color: Blue&lt;br/&gt; Nominal Wavelengtha: 470 nm&lt;br/&gt; Bandwidth (FWHM): 25 nm&lt;br/&gt; Maximum Current (CW): 1000 mA&lt;br/&gt; Forward Voltage: 3.2 V&lt;br/&gt; Electrical Power (Max): 3200 mW&lt;br/&gt; Emitter Size: 1 mm x 1 mm&lt;br/&gt; Typical Lifetime: 100 000 h&lt;br/&gt; Operating Temperature (Non-Condensing): 0 to 40 &amp;deg;C&lt;br/&gt; Storage Temperature: -40 to 70 &amp;deg;C&lt;br/&gt; Risk Groupb: RG2 &amp;ndash; Moderate Risk Group</td></tr><tr><td>Thermal power sensor&amp;nbsp;</td><td>Thorlabs, USA</td><td>S401C</td><td>Wavelength range: 190 nm-20 &amp;mu;m&lt;br/&gt; Optical power range:10 &amp;mu;W-1 W(3 Wb)&lt;br/&gt; Input aperture size: &amp;Oslash;10 mm&lt;br/&gt; Active detector area: 10 mm x 10 mm&lt;br/&gt; Max optical power density: 500 W/cm&lt;sup&gt;2&lt;/sup&gt; (Avg.)&lt;br/&gt; Linearity: &amp;plusmn;0.5%</td></tr></tbody></table>

light spot-based assay for analysis of drosophila larval phototaxis

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.

According to the protocol, the light spot assay was used to investigate light avoidance behavior of third instar larva that were raised at 25 &#176;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 &#176;C. The average light intensity of the light spot generated by a 460 nm LED was 0.59 &#181;W/cm2. The whole process of larval entering and exiting the light spot was recorded and analyzed using SOS software an...

Watch this Scientific Journal Video about Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis at JoVE.com

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

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.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

light-spot-based-assay-for-analysis-of-drosophila-larval-phototaxis

Research

JoVE Journal

Biology

6.2K Views.  Zhejiang University. Met behulp van de lichte spottest kan het gedrag voor larven worden gevolgd vóór, tijdens en na het betreden van het lichtgebied, waardoor de details van de larvebeweging kunnen worden geanalyseerd. Deze test is eenvoudig en eenvoudig uit te voeren. De lichtrespons wordt slechts één keer getest, zodat elk mogelijk effect van lichtaanpassing kan worden uitgesloten.Om de agarplaten te bereiden, bedek je langzaam en gelijkmatig de bodem van een petrischaal van 15 centimeter met een on...

Video: Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Neuroscience

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Behavior

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

Light Spot Assay: A Method to Investigate Drosophila Phototactic Behavior

Source: Sun, Y., et al. <a href="https://www.jove.com/video/60235/" target="_blank">Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis</a>. J. Vis. Exp. (2019).
Drosophila larvae are light-sensitive and respond to changes in surrounding light. This video describes an assay that tests their ability to avoid light, called the light spot assay. The featured protocol demonstrates how to set up the assay with a blue light LED and shows how to record the animal&#39;s behavior in response to the light encounter.

Source: Sun, Y., et al. Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis. J. Vis. Exp. (2019).
Drosophila larvae are light-sensitive ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

Samenvatting

Meer video's verkennen