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 ove...

<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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>850 nm &#177; 3 nm infrared-light-generating LED</td>
			<td>Thorlabs, USA</td>
			<td>PM100A</td>
			<td>Compatible Sensors: Photodiode and Thermal 
			Optical Power Rangea: 100 pW to 200 W 
			Available Sensor Wavelength Rangea: 185 nm-25 &#956;m Display Refresh Rate: 20 Hz 
			Bandwidtha: DC-100 kHz 
			Photodiode Sensor Rangeb: 50 nA-5 mA 
			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: &#216;9.5 mm 
			Wavelength Range: 200-1100 nm 
			Power Range: 50 nW-50 mW 
			Detector Type: Si Photodiode (UV Extended) 
			Linearity: &#177;0.5% 
			Measurement Uncertaintyc: &#177;3% (440-980 nm), &#177;5% (280-439 nm), &#177;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 
			LED Current Resolution: 1 mA 
			LED Current Accuracy: &#177;20 mA 
			LED Forward Voltage: 24 V 
			Modulation Frequency Range: 0-100 kHz Sine Wave 
			Modulation: Arbitrary</td>
		</tr>
		<tr>
			<td>Collimated LED blue light&#160;</td>
			<td>ELP, China</td>
			<td>USBFHD01M</td>
			<td>Max. Resolution: 1920X1080 
			F6.0 mm 
			Sensor: 1/2.7&#34; CMOS OV2710</td>
		</tr>
		<tr>
			<td>Compact power meter console&#160;</td>
			<td>Ocean Optics, USA</td>
			<td>USB2000+(RAD)</td>
			<td>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 &#8211; 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 &#8211; 800 nm) 
			Slit: SLIT-50 
			Detector collection lens: L2 
			Order-sorting: OFLV-200-850 
			Optical resolution: ~2.0 nm FWHM 
			Stray light: &#60;0.05% at 600 nm; &#60;0.10% at 435 nm 
			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 
			Central wavelength: 850nm</td>
		</tr>
		<tr>
			<td>high-resolution web camera</td>
			<td>Thorlabs, USA</td>
			<td>MWWHL4</td>
			<td>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&#730; 
			Emitter Size: 1 mm x 1 mm 
			Typical Lifetime: &#62;50 000 h 
			Operating Temperature (Non-Condensing): 0 to 40 &#176;C 
			Storage Temperature: -40 to 70 &#176;C 
			Risk Groupa: RG1 &#8211; Low Risk Group</td>
		</tr>
		<tr>
			<td>LED Warm White</td>
			<td>Mega-9, China</td>
			<td>BP850/22K</td>
			<td>&#216;25.4(+0~-0.1) mm 
			Bandwidth: 22&#177;3nm 
			Peak transmittance:80% 
			Central wavelength: 850nm&#177;3nm&#160;</td>
		</tr>
		<tr>
			<td>Spectrometer&#160;</td>
			<td>Noel Danjou</td>
			<td>Amcap9.22</td>
			<td>AMCap&#160;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&#160;Video-for-Windowsand&#160;DirectShow-compatible devices are supported whether they are cheap webcams or advanced video capture cards.</td>
		</tr>
		<tr>
			<td>Standard photodiode power sensor&#160;</td>
			<td>Super Dragon, China</td>
			<td>YGY-122000</td>
			<td>Input: AC 100-240V~50/60Hz 0.8A 
			Output: DC 12V 2A</td>
		</tr>
		<tr>
			<td>Thermal power sensor&#160;</td>
			<td>Thorlabs, USA</td>
			<td>M470L3-C1</td>
			<td>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 &#176;C 
			Storage Temperature: -40 to 70 &#176;C 
			Risk Groupb: RG2 &#8211; Moderate Risk Group</td>
		</tr>
		<tr>
			<td>Thermal power sensor&#160;</td>
			<td>Thorlabs, USA</td>
			<td>S401C</td>
			<td>Wavelength range: 190 nm-20 &#956;m 
			Optical power range:10 &#956;W-1 W(3 Wb) 
			Input aperture size: &#216;10 mm 
			Active detector area: 10 mm x 10 mm 
			Max optical power density: 500 W/cm2 (Avg.) 
			Linearity: &#177;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 ...

Using the light spot assay, the behavior for larvae can be tracked before, during, and after entering the light area allowing the detail of the larval movement to be analyzed. This assay is simple and easy to perform. The light response is tested only one time, so that any possible effect of light adaptation can be excluded.To prepare the agar plates, slowly and evenly cover the bottom of a 15 centimeter Petri dish with an approximately four millimeter thick layer of hot 1%agar. Allow the plate to cool at room temperature until the agar has solidified. To set up the visual stimulation system, clip a prepared LED light source onto an iron frame, taking care that the light projects down toward the desktop.Tilt the cylinder slightly until the angle between the cylinder plane and the vertical plane is about 10 degrees. Connect the 470 nanometer blue LED light to the LED one plug of the high power LED driver and turn on the driver. Turn the knob in the upper right corner of the driver to select channel 470 nanometers and click LED.When the screen displays a check, a blue light spot will appear on the desktop. Click OK and turn the knob to adjust the intensity of the light. Move the position of the light source up and down to adjust the diameter of the light spot to two centimeters.Rotate the knob to select the light intensity according to the experimental requirements. Use a compact power meter console with a standard photo diode power sensor to measure the maximal and minimal light power in the spot. The light power needs to measured in the dark in the actual experiment.Then record the light power, measure it three times and use the average value. To set up the imagining system, clamp a high resolution web camera with an iron clip about 10 centimeters above the light spot on the desktop and adjust the orientation of the camera lens toward the desktop. Connect the camera to a computer running Windows 7 through a USB interface and place the agar plate on the desktop right below the camera.Open the AMCap 9.22 software, the light spot will automatically be shown in the AMCap window. Move the camera slightly left or right to ensure that the light spot is near the center of the window. Use a clip to fix an 850 plus or minus three nanometer bandpass filter at five to seven millimeters immediately below the camera.Place three infrared light generating LEDs with a central wavelength of 850 nanometers evenly around the agar plate about five centimeters away from the edge of the plate and with LED lenses at a 70 degree angle facing toward the plate. Then connect the LEDs to a power source through their AC to DC converters. In the menu of the AMCap software, select options, video device, and capture format and set the pixel size of the captured video to 800 by 600 and the frame rate to 60 frames per second.Remove the filter and place a ruler under the camera. Adjust the camera focus until the scale line is clear and parallel to the width of the video field of view and click capture, setup, and video capture to select the save path. Then click start recording to record the actual distance corresponding to 600 pixels and calculate the ratio of each pixel to the actual distance.Before introducing the larva to the imaging setup, acquire a short video of the light position and name the unfiltered control background video light area one. Place the filter back on the camera lens and turn on a light far from the experimental device at as low a setting as possible while still allowing the larvae to be clearly observed. Use a spoon to remove some larvae from the culture medium and gently select a third instar larva.Wash the larva in distilled water and place the larva in the center of the agar plate. Gently remove any excess water from the larva and turn off the room light. Allow the larva to acclimate for two minutes in the dark environment before turning on the infrared LED light.The following light avoidance behavior experiment needs to be carried out in the dark. For the convenience of demonstration we filmed in the bright light. 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. Click capture, setup, and video capture to select the save path and click start recording to record. Allow the larva crawl toward, enter, and leave the light spot until it is nearly out of the field of view, and click stop recording.Move the filter away from the camera and acquire a short video of the position of the light spot. Then name the post assay unfiltered video light area two and compare this video with the light area one video to ensure that the light spot position is not changed. Here, time curves of the tail speed, body bending angle and angular speed of body bending of a representative larva can be observed.The size of the larval head cast and, therefore, maximal body bending angle is significantly reduced in third instar larvae with octopaminergic neurons inhibited by tetanus toxic expression compared to parental controls indicating that Tdc2-Gal4 neurons are necessary for a normal larval light response. For successful image processing, make sure that the camera doesn't block the light and that the light spot is round and near the center of the window. Following this procedure, optogenetics can be performed to observe how the larvae react when specific neurons are activated.Using this assay, the details of larval movement can be analyzed allowing the far impact of environmental and of internal factors on larval light avoidance behavior to be studied.

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6.1K visualizações.  Zhejiang University. Usando o ensaio de ponto de luz, o comportamento das larvas pode ser rastreado antes, durante e depois de entrar na área de luz permitindo que os detalhes do movimento larval sejam analisados. Este ensaio é simples e fácil de executar. A resposta à luz é testada apenas uma vez, de modo que qualquer possível efeito da adaptação da luz pode ser excluído.Para preparar as placas de ágar, cubra lentamente e uniformemente o fundo de uma placa de Petri de 15 centímetros com uma cam...