Name: Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae (Video) | JoVE Journal
Uploaded: 2013-04-20T14:00:00.000+00:00
Duration: 7 min 14 s
Description: Learn a light-dark preference assay to study innate and circadian photobehavior in Drosophila larvae. This method enables researchers to investigate sensory processing and circadian regulation.

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5 minutes

We thank our colleagues at the Department of Biology, University of Fribourg for fruitful discussions. We thank the Bloomington Stock Center for providing fly stocks. This work was financially supported by the Swiss National Science Foundation (PP00P3_123339) and the Velux Foundation to S.G.S.

1. Larval Rearing
<ol>
 <li>Keep fly strains or genetic crosses in mass culture at 25 &deg;C on corn meal medium under a 12-hr light-12-hr dark cycle in a fly incubator equipped with light and timer.</li>
 <li>Dilute backer yeast in water to form a fluid paste (10 g of backer yeast diluted with 3-4 ml distilled H2O). Add a small drop to the corn meal food and cover the vials. Let dry for at least one hour to avoid adult flies sticking to the yeast paste. Putting a small drop of yeast diluted in water on the surface of the food can enhance oviposition. Alternatively baker yeast paste, 20% acetic acid (AcOH) (Sigma Aldrich, Switzerland A6283-100ML) can be used. For this, dip the tip of a Q-tip on the solution and spread on the corn meal food surface. </li>
 <li>Put adult flies (at least four days old, but not older than ten days) in corn meal food vials. Put enough adults into each vial in order to obtain sufficient larvae to repeat the preference test several times and allow statistical comparison. For genetic crosses, we typically use a minimum of 20 females and 5-10 males per vial, but some lines require more females to obtain a sufficient larval offspring.</li>
 <li>Allow adults to lay eggs for 12 hr and transfer them to new vials. Adults can be transferred in the morning and the evening. We usually transfer adults for seven to ten days and if required take new adults. </li>
 <li>Keep the egg collections taken every 12 hr in the incubator and let grow the larvae at 25 &deg;C, 12-hr light-12-hr dark cycle and 60% humidity. To test for circadian components of visual behavior move larvae to constant darkness (DD) 48 hr after egg collection (corresponding to two-light-dark cycles). For this, use a separate incubator without light but keep all other conditions such as temperature and humidity identical. Be sure to transfer the vials to the DD incubator just before the switch to the dark phase. Before moving the vials to another incubator put vials in a cardboard box or wrap the vials with aluminum foil to prevent light exposure during the transfer (packing in cardboard box or wrapping has to be done during the "lights-on phase" prior to the shift between incubators). Prevent the animals from any light exposure after this point and until experiments start.</li>
 <li>After 4 days (84 to 108 hr from egg laying) collect early L3 (feeding larvae) at the time points to be tested (see 4. Light preference test, point. 4.4.). </li>
</ol>
2. Test Setup
<ol>
 <li>Perform experiments in a dark room with constant temperature and humidity conditions. </li>
 <li>To keep two quadrants of the Petri dish in darkness, cover the lid with black tape and aluminum foil. In order to do so, mark the center of the lid circumference. Also mark four points in the border of the lid with 90 &deg; of separation among them. To make it easier, draw a 20 cm square divided in four quadrants of 10 cm length on a sheet of paper or with help of a computer. Cut 10 cm squares of aluminum foil and black tape (Figure 1A). Cover two opposite quadrants by gluing a square of aluminum foil, as first layer and black tape covering it, be careful to also cover the border of the lid. 
 In the past two slightly distinct forms of this assays were used. We here use the "quarter-plate" assay, in which the Petri dish is divided in four equal quarters10. In the alternative "half-plate" assay the Petri dish is divided half (half exposed to light, half in darkness)1,7,8. Until today no significant differences between the two assays has been published.</li>
 <li>Glue a quadrant sheet such as the one used for marking the lids right under the light source on the table. Mark the circumference of a Petri dish centered on the quadrants intersection. Use the marks as reference to place the test plate always in the same position under the light source. </li>
 <li>Install a lamp, either a simple white light bulb (Phillips, Softtone 5W) or a LED lamp (LED lamp, 80012 White, Osram) above the Petri dish with the help of an iron support stand (Fisher Scientific, S47808). Adjust the height in a way that the whole test plate is homogenously illuminated. We strongly recommend the use of LED lamps since they emit more specific wavelengths than conventional light bulbs. Moreover LEDs emit less heat. </li>
 <li>Adjust light intensity with the help of a photometer (Environment Meter PCE EM882). To achieve the required light intensity of 350-760 lux, move the lamp up or down as required. Plugging the lamp to an adjustable power supply gives more flexibility to change intensities without moving the lamp (Figure 1B). </li>
</ol>
3. Plates Preparation 
<ol>
 <li>Make 200 ml of 2.5 % agar (Sigma-Aldrich, Switzerland A5093-500G) with double distilled water (Millipore). </li>
 <li>Place Petri dishes on a table (90-mm diameter; Greiner Bio-One GmbH, 4550 Kremsmeinster, Austria) in rows to allow pouring hot agarose. </li>
 <li>Boil the agarose in a microwave until the solution is completely transparent and fluid. Make sure that liquid solution does not contain bubbles. CAUTION: Transport carefully to the table since the solution can be very hot! </li>
 <li>Pour hot agarose into the Petri dishes until the whole surface is homogenously covered with a thin layer of the solution, about two or three millimeters are enough. Be quick to prevent agarose solidifying before coating all plates. Let the plates cool down. Store plates at room temperature and use them only on the same day of preparation. </li>
</ol>
4. Light Preference Test 
<ol>
 <li>Work under red light conditions in the experiment room to prevent light influence before the test since Drosophila is not able to sense red wavelengths of light. Use a red light bulb (Phillips, PFE712E*8) mounted in a lamp to illuminate the place to work.</li>
 <li>Maintain temperature through all experiments at 25 &deg;C. Control room temperature by means of a heater or cooling device if required.</li>
 <li>Take some food from the vials reared for two-day cycles under constant darkness (from section 1). Since larvae are usually digging on the surface of the food, take the uppermost layer (about 5 mm deep) with help of a spatula (Fisher Scientific, 14-373-25A). Spread the food on the outer side of a Petri dish lid, add some water and mix gently with the spatula. </li>
 <li>Collect feeding L3 larvae from the food. As light preference will be tested, choosing only early/feeding L3 larvae is crucial since the negative phototaxis is assured. Late/wandering L3 larvae or big larva crawling on the food presumably already switched their photobehavior. Feeding L3 larvae (negative phototactic) can be recognized because their anterior spiracles are open and protruded to the outside in a finger-like form. The posterior spiracles have three openings each, and four groups of large branched hairs. The salivary glands extend to the second abdominal segment11. Staging larva under a microscope is convenient whereas no white light should be used. A red light lamp is necessary to illuminate the larvae if staging under stereoscopic microscope before experiments is required.</li>
 <li>Wash larvae briefly in tap water and collect early-feeding third-instar larvae (still in red light). Before transferring the larvae to the test plate, take the larvae with a wet paintbrush and carefully absorb excess water with a paper towel or filter paper. Do not dry larvae too excessively since it could harm the animal and influence its behavior.</li>
 <li>Using the wet paintbrush carefully transfer the larvae to plates. Place a group of approximately 30 larvae in the center of the plate. Cover the plate with the lid already prepared with the quadrants and set the plate under the light source ready for experiment (see section 2). </li>
 <li>Turn on the white light lamp and start the timer. Let the larva move freely on the plate for 5 min, then quickly remove the lid and count the number of larvae in the dark and in the light quadrants. Marking the position of each larva with a marker can ease the counting. Alternatively take a picture of the plate and count a posteriori. Larvae showing unclear light preference, like larvae crawling on the walls or burrowing into agar should be considered as neutral preference and just be included in the total number of larvae when the light preference index is calculated. </li>
 <li>After counting, discard the plate with larvae and replace with a new test plate for the next experiment. Collect used plates in an autoclave plastic bag for later handling and disposal.</li>
 <li>Once you have performed a sufficient number of experiments a new genotype can be tested. 10-15 trials per genotype are sufficient for analysis. </li>
</ol>
5. Data Analysis 
<ol>
 <li>For convenience transfer the data to a datasheet as Excel (Microsoft) or Origin (Origin Lab) on a computer for further statistical analysis. Arrange in one column the number of animals in darkness, in a second column the number of animals in light quadrants and in the third the total of animals in the plate (including "neutral" larvae).</li>
 <li>Calculate the preference index (PREF) for the darkness for each experiment using the following formula: 
 PREF(darkness) = (number of larvae in dark - number of larvae in light)/total number of larvae </li>
 <li>Compare statistically a set of data with an appropriate analysis for groups. Here, we use a Wilcoxon test to compare statistically two groups. An ANOVA test with a Tukey's multiple-comparison post hoc test can be done, if normal distribution assumption is fulfilled in a manifold group comparison. </li>
 <li>Make graphs that show clearly comparisons among lines and time points along the day cycle. We use Origin Software (Origin Lab) to test statistical significance and generate appropriate graphs, but any other statistical software can be helpful.</li>
</ol>

<ol><li>Sawin-McCormack, E. P., Sokolowski, M. B., Campos, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+and+genetic+analysis+of+Drosophila+melanogaster+photobehavior+during+larval+development%5BTitle%5D)+AND+%22J.+Neurogenet%22%5BJournal%5D)">Characterization and genetic analysis of Drosophila melanogaster photobehavior during larval development.</a> J. Neurogenet. 10, 119-135 (1995).</li>
<li>Sprecher, S. G., Pichaud, F., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+and+larval+photoreceptors+use+different+mechanisms+to+specify+the+same+Rhodopsin+fates%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Adult and larval photoreceptors use different mechanisms to specify the same Rhodopsin fates.</a> Genes Dev. 21, 2182-2195 (2007).</li>
<li>Sprecher, S. G., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Switch+of+rhodopsin+expression+in+terminally+differentiated+Drosophila+sensory+neurons%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons.</a> Nature. 454, 533-537 (2008).</li>
<li>Xiang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light-avoidance-mediating+photoreceptors+tile+the+Drosophila+larval+body+wall%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall.</a> Nature. 468, 921-926 (2010).</li>
<li>Diaz, N. N., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photoreceptors%3A+unconventional+ways+of+seeing%5BTitle%5D)+AND+%22Curr.+Biol%22%5BJournal%5D)">Photoreceptors: unconventional ways of seeing.</a> Curr. Biol. 21, R25-R27 (2011).</li>
<li>Emery, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+CRY+is+a+deep+brain+circadian+photoreceptor%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Drosophila CRY is a deep brain circadian photoreceptor.</a> Neuron. 26, 493-504 (2000).</li>
<li>Mazzoni, E. O., Desplan, C., Blau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+pacemaker+neurons+transmit+and+modulate+visual+information+to+control+a+rapid+behavioral+response%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Circadian pacemaker neurons transmit and modulate visual information to control a rapid behavioral response.</a> Neuron. 45, 293-300 (2005).</li>
<li>Keene, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+visual+pathways+mediate+Drosophila+larval+light+avoidance+and+circadian+clock+entrainment%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Distinct visual pathways mediate Drosophila larval light avoidance and circadian clock entrainment.</a> J. Neurosci. 31, 6527-6534 (2011).</li>
<li>Gong, Z. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+Pairs+of+Neurons+in+the+Central+Brain+Control+Drosophila+Innate+Light+Preference%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Two Pairs of Neurons in the Central Brain Control Drosophila Innate Light Preference.</a> Science. 330, 499-502 (2010).</li>
<li>Lilly, M., Carlson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((smellblind%3A+a+gene+required+for+Drosophila+olfaction%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">smellblind: a gene required for Drosophila olfaction.</a> Genetics. 124, 293-302 (1990).</li>
<li>Bodenstein, D. The postembryonic development of Drosophila. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBodenstein%2C+D+%22Biology+of+Drosophila%22">Biology of Drosophila</a>. Demerec, M. , John Wiley & Sons. 275-367 (1950).</li>
<li>Pittendrigh, C. S. Circadian systems: Entrainment. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPittendrigh%2C+CS+%22Biological+Rhythms%22">Biological Rhythms</a>. 4 Handbook of Behavioral Neurobiology, Plenum. 95-124 (1981).</li>
<li>Collins, B., Kane, E. A., Reeves, D. C., Akabas, M. H., Blau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Balance+of+Activity+between+LN%28v%29s+and+Glutamatergic+Dorsal+Clock+Neurons+Promotes+Robust+Circadian+Rhythms+in+Drosophila%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Balance of Activity between LN(v)s and Glutamatergic Dorsal Clock Neurons Promotes Robust Circadian Rhythms in Drosophila.</a> Neuron. 74, 706-718 (2012).</li>
<li>Keene, A. C., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Seeing+the+light%3A+photobehavior+in+fruit+fly+larvae%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Seeing the light: photobehavior in fruit fly larvae.</a> Trends Neurosci. 35, 104-110 (2012).</li>
<li>von Essen, A. M., Pauls, D., Thum, A. S., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capacity+of+visual+classical+conditioning+in+Drosophila+larvae%5BTitle%5D)+AND+%22Behav.+Neurosci%22%5BJournal%5D)">Capacity of visual classical conditioning in Drosophila larvae.</a> Behav. Neurosci. 125, 921-929 (2011).</li>
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The authors have nothing to disclose.

The light preference test described takes advantage of the larval innate photobehavior. The assay is easy to establish, allows many repetitions at low cost and delivers valuable information about light sensing and processing. The experimental paradigm allows relatively quick quantification of how many individuals prefer light or dark. Such preference can be displayed as crude percentages or alternatively as Preference Index (PREF). The PREF is expressed as the difference of animals that preferred light and animals that p...

Here we describe a behavioral assay based on the larval preference for dark (or light). Larvae react with a strong and stereotypic photonegative response during foraging stages (L1 to early L3)1. The assay is aimed to assess the photophobic behavior of the larva and compares the light or dark preference of a group of larvae moving freely in a Petri dish coated with agar. This behavioral assay not only provides information about the sensitivity, integration and temporal plasticity of the visual system, it further provides hints on how light sensitivity and its process is controlled by the circadian system.
The Drosophila larval eye (also termed Bowlig Organ; BO), is the main organ for light perception. Each eye is composed of 12 photoreceptors (PR), eight PRs express the green-sensitive rhodopsin6 (rh6) and four PRs express the blue-sensitive rhodopsin5 (rh5)2,3. In addition to PRs, also class IV multidendritic neurons, which cover the larval body wall, have been identified to respond to noxious light intensities4,5. It is also known that pacemaker neurons situated in the central larval brain express the light sensitive protein Cryptochrome (Cry) that acts as clock intrinsic blue light sensor within the brain6,7. Intriguingly photophobicity of wild type animals shows a circadian component at different time points during the course of day and night when testing with this assay. Responses to light of foraging L3 larva showed stronger photophobicity at dawn and lower photophobicity at dusk when tested for light-dark preference7. Interestingly only Rh5-PRs are required for light avoidance, while Rh6-PRs are dispensable. Both, Rh5-PRs and Rh6-PRs are involved in resetting the molecular clock by light8. The Cry pathway must be coordinated with the other light-sensing pathways to orchestrate an appropriate behavioral output in the course of the day. Acetylcholine in PRs plays an essential role in light avoidance behavior as well as entrainment of the molecular clock. Blocking acetylcholine neurotransmission from PRs to circadian pacemaker neurons reduces the photophobic response in the light-darkness preference assay8. Employing the same assay, two symmetrical pairs of neurons have been recently identified to switch the light preference of the third larval instar of Drosophila9. These two pairs of neurons may be functioning during late larval stages, when animals leave the food to presumably find an appropriate pupariation site. However, the question of how the visual pathways interact and control larval visual behavior in a circadian manner remains largely unanswered. The light preference assay allows comparisons among circadian time points, fly lines and circadian state under different light qualities. The assay is easily prepared and inexpensive and has been useful previously in several labs to describe and study light derived behavior in the larva.

<table><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>A5093-500G</td><td>2.5%; Sigma-Aldrich, 9471 Buchs, Switzerland </td></tr><tr><td>Petri dishes</td><td>Greiner Bio-One GmbH</td><td>633180</td><td>90-mm diameter; Greiner Bio-One GmbH, 4550 Kremsmeinster, Austria</td></tr><tr><td>LEDs Lamp</td><td>OSARAM</td><td>80012 White</td><td>LED lamp, 80012 White</td></tr><tr><td>Environment Meter</td><td>PCE</td><td>PCE EM882</td><td>Lux, Temp, RH%</td></tr><tr><td>Thermostatic cabinet</td><td>Aqua Lytic (Liebherr)</td><td>ET636-6</td><td></td></tr><tr><td>Light timer</td><td>Timer T</td><td>6185.104</td><td>230V/50HZ (check specifications for your country)</td></tr><tr><td>Universal thermostat</td><td>Conrad</td><td>UT200</td><td></td></tr><tr><td>Humidifier</td><td>Boneco</td><td></td><td></td></tr><tr><td>Balck tape</td><td>Tesa</td><td></td><td>5 cm </td></tr><tr><td>Glue</td><td>Uhu</td><td></td><td></td></tr><tr><td>lncubator lamp</td><td>Phillips</td><td>Softtone</td><td>5W</td></tr><tr><td>Timer clock</td><td>Ziliss</td><td></td><td>Ziliss, Switzerland</td></tr><tr><td>Excel Software</td><td>Microsoft</td><td></td><td>Excel</td></tr><tr><td>Origin Software 8.5</td><td>OriginLab</td><td></td><td></td></tr><tr><td>Backer Yeast</td><td>Migros Switzerland</td><td></td><td></td></tr><tr><td>Iron support stand 17X28CM</td><td>Fisher Scientific</td><td>S47808</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma Aldrich</td><td>A6283-100ML</td><td>20% acetic acid dilluted in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Red light lamp</td><td>Phillips</td><td>PFE712E*8C</td><td></td></tr><tr><td>Spatula </td><td>Fisher Scientific</td><td>14-373-25A</td><td></td></tr><tr><td>Power supply</td><td>EA</td><td>EA PS 2042-06B</td><td>Optional</td></tr><tr><td>Aluminium foil</td><td>Prix Coop</td><td></td><td></td></tr><tr><td>Heater</td><td>GOON</td><td>NSB200C</td><td></td></tr><tr><td>Microwave Oven</td><td>Intertronic</td><td></td><td></td></tr><tr><td>Standard corn meal fly food</td><td></td><td></td><td></td></tr><tr><td>Destilled water </td><td></td><td></td><td></td></tr></tbody></table>

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

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.

Following the protocol described above, we tested light-dark preference in early third larval stadium of wild type Canton-S flies at two different circadian times CT0 and CT12. Adults were reared 12-hr light-12-hr dark and left to lay eggs for 12 hr. Larvae grow the first two days under the same light-dark regime. Since we wanted to test circadian modulation under constant conditions (free running of the circadian clock), larvae were then transferred to constant darkness for the next three days until test was performed ...

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 ...

The overall aim of the following experiment is to test the innate and circadian photo behavior of drosophila lava by means of a light dark preference test. This is achieved by first rearing the experimental larvae under defined light dark conditions to establish an initial circadian clock in the larvae. Next, the larvae is switched to constant darkness, which allows the larval circadian clock to free run.Once at the L three feeding stage, the lava is sorted into groups to test their light preference at different circadian time points. Results are obtained that show innate and circadian light preference based on calculating a preference index and comparing between time points and experimental groups. Though this method can provide insight into the innate and circadian regulation of light sensing and the processing of photo behavior, it can also be applied to other systems such as olfaction, cassation, chemo, thermo taxis.Likewise, it can be modified for learning and memory tests. Demonstrating the procedure will be Alina and BMA To graduate students from my laboratory To collect eggs, allow mating adult flies to lay for 12 hours on fresh files and transfer them to new vials. Keep transferring the adults every 12 hours for a 12 hour light, 12 hour dark cycle.Keep the 12 hour egg collections in the incubator. For the experiment, raise the animals the early L three larva stage. These are feeding lae available 84 to 108 hours from egg.Laying Correct staging of LAE is critical for the success of the experiment. Since late L three, LAE changed the photo behavior To test for circadian components of visual behavior. 48 hours before testing and just before the larvae subjective, dawn, put the collected larvae into a cardboard box.Then transfer the box to a constant darkness incubator for 48 hours until the experiment, these larva must remain undisturbed in their dedicated space. Prepare the lid of a nine centimeter Petri dish using a 20 centimeter square divided in four quadrants. As a guide.Glue 10 centimeter squares of aluminum foil on opposite quadrants and cover them in black tape. Be sure to cover the border of the lid. Next glue, A 20 centimeter square divided in four quadrants under the light source on the table.Mark the circumference of a test dish on the sheet to act as a guide for consistent displacement. Install an LED lamp above the Petri dish using an iron support stand. Adjust the height in a way that the whole test plate is homogenously illuminated.Adjust light intensity at a test site to between 350 and 760 luxe by either further adjusting the lamp height or by using an adjustable power supply. The experiment room needs red light illumination and must have the same temperature as where the animals are reared from. The vials reared two days under constant darkness.Collect the lava in their food with a spatula from the top five millimeters of the food. Spread the food on the outer side of a Petri dish lid. Add some water to the food and gently mix it with the spatula.Add a drop of tap water to a new dish using a paintbrush. Transfer the larvae to the drop of water to wash them. Then use a wet paintbrush to collect the larvae using the wet paintbrush.Select only the early feeding L three larvae and avoid late wandering L three larvae. Transfer the selected larvae to the center of a test plate. Feeding L three larvae have anterior sphericals that are open and protruded to the outside.In a finger-like form. The posterior sphericals have three openings each and four groups of large branched hairs. The saliva glands extend to the second abdominal segment.Once 30 larvae have been placed at the center of the test plate, cover it with the prepared lid. Turn on the LED lamp. Start a timer and let the larvae move freely on the plate for five minutes.Then quickly remove the lid and count the number of larvae in the dark and in the light quadrants. Marking the position of each lava can ease the counting. A quick photograph can also help larvae showing unclear light preference like larvae crawling on the walls or borrowing into agar should be counted as neutral preference and added to the total number of larvae when the light preference index is calculated.After counting the larvae, discard the plate and use a new test plate for the next experiment. After performing 10 to 15 trials per genotype, proceed with analysis following the protocol. The light dark preference of Canton ES lava was compared at two different circadian times.CT zero and CT 12, subjectively, dawn and dusk larvae or groan for 48 hours under standard conditions and then switch to constant darkness for three days. Light dark testing under 350 luxe revealed that light photosensitivity was higher in larvae tested at CT zero compared to larvae tested at CT 12. Using this formula, the darkness preference of CT zero lava was 0.52 compared to 0.36.The CT 12 larvae comparing the indexes using a wilcoxon test produces significant P value of 0.023. After watching this video, you should have a good understanding of how to test the light preference of troph larva at different circuit and time points to this goal. You should be able to entrain on stage F three oph larva, test their photophobic behavior and calculate the light preference index for statistical comparison.

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Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology.

Circadian Entrainment of Drosophila Melanogaster

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

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.

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Learn a light-dark preference assay to study innate and circadian photobehavior in Drosophila larvae. This method enables researchers to investigate sensory processing and circadian regulation.

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

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