Name: quantification of drosophila grooming behavior
Uploaded: 2017-07-19T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Drosophila Grooming Behavior at JoVE.com

We wish to thank Brian Shepherd, Tat Udomritthiruj, Aaron Willey, Ruby Froom, Elise Pitmon, and Rose Hedreen for early work in testing and establishing this methodology and chamber designs. We thank Kelly Tellez and Graham Buchan for reading and editing the manuscript. We thank Andrew Seeds and Julie Simpson for their pioneering work and their advice and support in suggesting the use of Brilliant Yellow Dye (Sigma).&#160;This work is supported in part by the Mary E. Groff Surgical and Medical Research and Education Charitable Trust, the Bronfman Science Center, and the Hellman Fellows Program.

1. Preparation
<ol>
	<li>Prepare an aspirator for moving live Drosophila from a culture vial to the grooming chamber. Aspirators allow transfer of conscious animals to the behavioral chambers to ensure that anesthesia does not affect subsequent behavioral observation.
	<ol>
		<li>Using scissors cut 1.5 feet of tygon tubing ID &#8539;&#34;, OD &#188;&#34;.Cut at least 1 inch off of the tip of a 1 mL disposable micropipette tip. Hold a 1 cm square piece of mesh (opening 0.196 inch) over the cut ...

<ol>
	<li>Szebenyi, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+Behaviour+in+Drosophila-Melanogaster.">Cleaning Behaviour in Drosophila-Melanogaster.</a> Animal. Behaviour. 17, (1969).</li><li>Corfas, G., Dudai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+and+dishabituation+of+a+cleaning+reflex+in+normal+and+mutant+Drosophila.">Habituation and dishabituation of a cleaning reflex in normal and mutant Drosophila.</a> J Neurosci. 9 (1), 56-62 (1989).</li><li>Yanagawa, A., Guigue, A. M. A., Marion-Poll, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hygienic+grooming+is+induced+by+contact+chemicals+in+Drosophila+melanogaster.">Hygienic grooming is induced by contact chemicals in Drosophila melanogaster.</a> Frontiers in Behavioral Neuroscience. 8, (2014).</li><li>Dawkins, R., Dawkins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+Organization+and+Postural+Facilitation+-+Rules+for+Grooming+in+Flies.">Hierarchical Organization and Postural Facilitation - Rules for Grooming in Flies.</a> Animal Behaviour. 24 (Nov), 739-755 (1976).</li><li>Seeds, A. M., 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+suppression+hierarchy+among+competing+motor+programs+drives+sequential+grooming+in+Drosophila.">A suppression hierarchy among competing motor programs drives sequential grooming in Drosophila.</a> Elife. 3, e02951 (2014).</li><li>King, L. B., 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=Neurofibromin+Loss+of+Function+Drives+Excessive+Grooming+in+Drosophila.">Neurofibromin Loss of Function Drives Excessive Grooming in Drosophila.</a> G3-Genes Genomes Genetics. 6 (4), 1083-1093 (2016).</li><li>Tauber, J. M., Vanlandingham, P. A., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+Levels+of+the+Vesicular+Monoamine+Transporter+and+a+Novel+Repetitive+Behavior+in+the+Drosophila+Model+of+Fragile+X+Syndrome.">Elevated Levels of the Vesicular Monoamine Transporter and a Novel Repetitive Behavior in the Drosophila Model of Fragile X Syndrome.</a> Plos One. 6 (11), e27100 (2011).</li><li>Kaur, K., Simon, A. F., Chauhan, V., Chauhan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+bisphenol+A+on+Drosophila+melanogaster+behavior--a+new+model+for+the+studies+on+neurodevelopmental+disorders.">Effect of bisphenol A on Drosophila melanogaster behavior--a new model for the studies on neurodevelopmental disorders.</a> Behav Brain Res. 284, 77-84 (2015).</li><li>Chang, H. 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=Overexpression+of+the+Drosophila+vesicular+monoamine+transporter+increases+motor+activity+and+courtship+but+decreases+the+behavioral+response+to+cocaine.">Overexpression of the Drosophila vesicular monoamine transporter increases motor activity and courtship but decreases the behavioral response to cocaine.</a> Molecular Psychiatry. 11 (1), 99-113 (2006).</li><li>Yellman, C., Tao, H., He, B., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+sexually+dimorphic+behavioral+responses+to+biogenic+amines+in+decapitated+Drosophila.">Conserved and sexually dimorphic behavioral responses to biogenic amines in decapitated Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (8), 4131-4136 (1997).</li><li>Feng, G. P., 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=Cloning+and+functional+characterization+of+a+novel+dopamine+receptor+from+Drosophila+melanogaster.">Cloning and functional characterization of a novel dopamine receptor from Drosophila melanogaster.</a> Journal of Neuroscience. 16 (12), 3925-3933 (1996).</li><li>Gotzes, F., Balfanz, S., Baumann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Structure+and+Functional-Characterization+of+a+Drosophila+Dopamine-Receptor+with+High+Homology+to+Human+D(1/5).">Primary Structure and Functional-Characterization of a Drosophila Dopamine-Receptor with High Homology to Human D(1/5).</a> Receptors. Receptors &amp; Channels. 2 (2), 131-141 (1994).</li><li>Han, K. A., Millar, N. S., Grotewiel, M. S., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAMB,+a+novel+dopamine+receptor+expressed+specifically+in+Drosophila+mushroom+bodies.">DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies.</a> Neuron. 16 (6), 1127-1135 (1996).</li><li>Sugamori, K. S., Demchyshyn, L. L., Mcconkey, F., Forte, M. A., Niznik, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Primordial+Dopamine+D1-Like+Adenylyl+Cyclase-Linked+Receptor+from+Drosophila-Melanogaster+Displaying+Poor+Affinity+for+Benzazepines.">A Primordial Dopamine D1-Like Adenylyl Cyclase-Linked Receptor from Drosophila-Melanogaster Displaying Poor Affinity for Benzazepines.</a> Febs Letters. 362 (2), 131-138 (1995).</li><li>Pitmon, E., 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=The+D1+family+dopamine+receptor,+DopR,+potentiates+hind+leg+grooming+behavior+in+Drosophila.">The D1 family dopamine receptor, DopR, potentiates hind leg grooming behavior in Drosophila.</a> Genes Brain and Behavior. 15 (3), 327-334 (2016).</li><li>Phillis, R. 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=Isolation+of+mutations+affecting+neural+circuitry+required+for+grooming+behavior+in+Drosophila+melanogaster.">Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster.</a> Genetics. 133 (3), 581-592 (1993).</li><li>Hampel, S., Franconville, R., Simpson, J. H., Seeds, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neural+command+circuit+for+grooming+movement+control.">A neural command circuit for grooming movement control.</a> Elife. 4, e08758 (2015).</li><li>Kays, I., Cvetkovska, V., Chen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+analysis+of+single+neurons+to+correlate+synaptic+connectivity+with+grooming+behavior.">Structural and functional analysis of single neurons to correlate synaptic connectivity with grooming behavior.</a> Nature Protocols. 9 (1), 1-10 (2014).</li></ol>

The grooming assay is relatively straightforward, but we would caution experimenters to pay special attention to the following issues. Maintaining a tight seal by tightening the screws on the top and bottom plates after introducing flies and dye is essential for reproducible results. The Brilliant Yellow dye is very fine and loose joints will allow losses of dye from the edges of the chamber. The irregularity in dye content for each well could easily throw off grooming quantification as non-uniform dusting will increase ...

Grooming in Drosophila melanogaster&#160;(D. melanogaster&#160;)&#160;is a robust innate behavior that involves the coordination of multiple independent motor programs1. Fruit flies clean their bodies of dust, microbes, and other pathogens which could inhibit normal physiological function such as vision and flight, or lead to significant immune challenges. In sensing and responding to both mechanical2 and immune activation3, flies repetitively rub their legs together or on a targeted body region until it is sufficiently clean and grooming progresses to another part of the body. Flies perform grooming movements in distinct bouts that largely occur in stereotyped patterns1,4. A behavioral hierarchy becomes apparent as grooming signals are prioritized. Circuits and patterns of activity have been identified in support of a model that grooming programs at the top of the hierarchy occur first and suppress parallel signals from areas of the body that are groomed subsequently5. Highest priority is given to the head, then the abdomen, wings, and finally the thorax5.
The grooming program in D. melanogaster is an ideal system for studying neural circuits, modulatory molecular signals, and neurotransmitters. For instance, compromise of neurofibromin function6, loss of Drosophila fragile X mental retardation protein (dfmr1)7, and exposure to bisphenol A (BPA)8 all cause excessive grooming and other behaviors that are analogous to discrete human symptoms of neurofibromatosis, fragile X syndrome, and aspects of autism spectrum disorders and Attention-deficit Hyperactivity Disorder (ADHD), respectively. Grooming behavior can also be habituated differentially across mutant strains2, lending this motor program to studies of behavioral plasticity. The breadth of neurological phenomena that can be modeled by Drosophila demands a novel comparative approach to measure the ability of flies to groom themselves.
The combined action of vesicular monoamine transporters and the relative abundance of dopamine and other biogenic amines in the body have been shown to mediate fruit fly grooming behavior 9,10. Octopamine and dopamine stimulate comparable hindleg grooming activity in decapitated flies, while tyramine, the precursor of octopamine, also triggers grooming to a lesser extent7. Four dopamine receptors have been identified in D. melanogaster11,12,13,14. By using the grooming assay method described in this protocol, we determined a role for the Type I family Dopamine Receptor DopR (DopR, dDA1, dumb) in hindleg grooming behavior15.
Grooming can be indirectly quantified by looking at the extent of cleanliness by which an animal can fully groom after dusting the entire body with a marker dye or fluorescent dust5,16. The remainder of dust left on the body can be used as a relative marker for the overall behavior. Dusty flies after being given sufficient time to groom may be manifesting a specific deficit in grooming behavior. As grooming investigations have become more extensive, protocols have incorporated such practices as decapitation to add pharmacological treatments onto the neck connective nerves10, tactile stimulation of bristles to elicit the grooming response2 , and video recording of behavior15. Direct observation of grooming can be easily studied by using visual observation and manually recording the frequency and duration of specific grooming events4.
We designed a fifteen-well grooming chamber that can be constructed with a 3D printer or laser cutter, and the blueprint designs are available for reproduction15. The design uses two joined central plates with openings matched and separated by mesh and two additional sliding top and bottom plates, from which flies and/or dye is loaded, respectively. After allowing dusted flies time to groom, we deposit them in ethanol to solubilize the dye and measure the absorbance of this solution at the wavelength of the dye. A plate reader can be used for multiple parallel samples or a single-read spectrophotometer can be used for individual samples. This method minimizes the error induced by handling and allows for grooming assays to be run on a smaller, cost-efficient scale. This method is derived and modified from the methods pioneered by Julie Simpson and Andrew Seeds, who use larger grooming chambers with heating elements for temperature sensitive circuit manipulations5. The following protocol showcases the quantification of grooming of the whole body as well as showing alternate methods for quantitation of dye accumulation on individual body parts. We also present sample comparison data between WT and DopR mutants, as well as methods for calculating a simple performance index for grooming behavior.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>High-Flex Tygon PVC Clear Tubing</td>
			<td>McMaster-Carr</td>
			<td>5229K54</td>
			<td>ID 1/8&#34;, OD 1/4&#34;, used with micropipettor tips and mesh to construct mouth aspirators</td>
		</tr>
		<tr>
			<td>Micropipette tips (1ml and 200ul)</td>
			<td>Genesee Scientific</td>
			<td>24-165, 24-150R</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Screen, 2&#34; x 2.6&#34;</td>
			<td>McMaster-Carr</td>
			<td>9318T44</td>
			<td>Used to construct grooming chamber and mouth aspirators</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Yellow Dye</td>
			<td>Sigma-Aldrich</td>
			<td>201375-25G</td>
			<td>we recommend use of nitrile gloves while handling this product</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Fisher Scientific</td>
			<td>12-812</td>
			<td>set to &#34;touch&#34;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carolina Biological Supply</td>
			<td>86-1282</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>VWR International</td>
			<td>10025-726</td>
			<td></td>
		</tr>
		<tr>
			<td>0.65 ml microcentrifuge tubes</td>
			<td>VWR International</td>
			<td>20170-293</td>
			<td>tubes can be reused with successive assays</td>
		</tr>
		<tr>
			<td>UV 96 well plate</td>
			<td>Corning</td>
			<td>26014017</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy HTX Platereader</td>
			<td>BioTek</td>
			<td>need to download catalog to access product number</td>
			<td>http://www.biotek.com/products/microplate_detection/synergy_htx_multimode_microplate_reader.html?tab=overview</td>
		</tr>
		<tr>
			<td>Gen5 Microplate Reader and Imager Software</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>https://www.microsoftstore.com/store/msusa/en_US/pdp/Excel-2016/productID.323021400?tduid=(65d098c0e83b86c952bdff5b0719c83f)(256380)(2459594)(SRi0yYDlqd0-LI..ql4M2LoZBEhcBljvIA)()</td>
		</tr>
		<tr>
			<td>Drosophila Incubator</td>
			<td>Tritech</td>
			<td>DT2-CIRC-TK</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34; acrylic plastic</td>
			<td>McMaster-Carr</td>
			<td>8473K341</td>
			<td></td>
		</tr>
		<tr>
			<td>8-32 nuts</td>
			<td>McMaster-Carr</td>
			<td>90257A009</td>
			<td></td>
		</tr>
		<tr>
			<td>8-32 x 1&#34; hex cap screws</td>
			<td>McMaster-Carr</td>
			<td>92185A199</td>
			<td>the bottom plate needs to be tapped for this size screw</td>
		</tr>
		<tr>
			<td>8-32 x 1/2&#34; hex cap screws</td>
			<td>McMaster-Carr</td>
			<td>92185A194</td>
			<td>the second plate from the top needs to be tapped</td>
		</tr>
		<tr>
			<td>2-56 3/8&#34; flat head phillips machine screws</td>
			<td>McMaster-Carr</td>
			<td>91500A088</td>
			<td>these hold the two middle plates together</td>
		</tr>
		<tr>
			<td>0.175&#34; ID, 1/4&#34; OD, 0.34&#34; aluminum pipe</td>
			<td>McMaster-Carr</td>
			<td>92510A044</td>
			<td>Manufactured in-house; product listed is approximately the same dimensions and should work for size 8 screws.&#160; These act as sheaths for the 1&#34; screws and set the hex cap up slightly from the surface of the top plate</td>
		</tr>
	</tbody>
</table>

quantification of drosophila grooming behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

The grooming assay yields quantitative data to assess behavioral performance based on the relative remainder of accumulated dye left on the bodies of flies after a set time of measurement for grooming (30 min). Sample images of the sliding grooming chamber design and major steps of the assay are highlighted in Figure 1. Flies aggregate a significant amount of dye from immediate dusting by vortexing in the presence of dye (Figure 2d, 2e). Dusted flies can ...

Watch this Scientific Journal Video about Quantification of Drosophila Grooming Behavior at JoVE.com

Quantification of Drosophila Grooming Behavior

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

The overall goal of this method is to measure the grooming behavior of Drosophila by quantifying and comparing the remainder of colored brilliant yellow dye on the body of the animals in groomed and ungroomed conditions. This method can help answer key questions in the field of neurobiology and behavioral genetics such as the coordination of fine locomotive skills and the associated molecular regulators and circuits. The main advantage is as of this technique, are that it's rapid, robust and easy to perform by many experimentalists and students at a variety of skill levels.Demonstrating this procedure will be Frankie Barradale, a student in my laboratory. To begin the experiment, prepare an aspirator to move live Drosophila from a culture vial to the grooming chamber. Using scissors, cut 1.5 feet of Tygon tubing.Cut at least one inch of the tip of a one milliliter disposable micro-pipette tip and hold the one centimeter square piece of mesh over the 196 inch opening of the cut tip. Snugly place a fresh, one milliliter micro-pipette tip over the mesh and cut tip to create a holding chamber for flies. Cut the extreme tip of the new micro-pipette tip to widen the opening enough to allow the passage of a single fly.The hole will roughly match the opening of the grooming chamber. Snugly fit the nested tips onto the end of the Tygon tubing. Push the tips into the tube to ensure a tight fit to allow for vacuum pressure.On the other end of the tubing, cut the tip of a 200 milliliter micro-pipette tip and fit the narrow cut end into the tubing to form the mouth side of the aspirator. To prepare dust aliquot tubes, weigh five milligrams of brilliant yellow dye on weighing paper. Pour the dust into a 8 milliliter micro-centrifuge tube and tightly close the cap.Next, to prepare ethanol tubes, pipette one milliliter of 100%ethanol into 15 milliliter micro-centrifuge tubes. Label the tubes, cap and save the ethanol tubes for completion of the grooming assay. Also include at least one blank sample with only one milliliter of ethanol without a fly for negative controls.Assemble each grooming chamber by screwing the sliding top plate with the smaller holes, but leaving the bottom plate with the larger holes removed for the addition of the dust. Place each necessary grooming chamber flat on a table with the top face-down. Load five milligrams of brilliant yellow dye aliquot into each chamber, by tapping the tube against the surface of the chamber above the desired well.Tap the chamber against the table to ensure that all the dust falls through the mesh to the top of the plate. Then screw the bottom plate onto each chamber, so the wider ends of the conical openings face outward and the holes do not rest over the wells. Secure the bottom plate tightly, so that it does not slide and release dye.Flip the chamber over and knock it flat against the table a few times so that the dust falls through the mesh to rest on the bottom plate. Slide the top plate of the chamber into the open positions that the small holes align with the 15 wells, allowing the introduction of flies into the individual wells. Load one fly into the mouth aspirator by gently sucking through one end of the aspirator as if using a straw.Deposit the flies by lightly blowing and directing the opening toward the target. After loading a well, slide the top plate so that the fly is trapped in the chamber and place tape over the opening of the well during the loading of the hole chamber, to prevent the fly's escape while loading the other wells. If multiple flies are aspirated into a single well, leave that opening uncovered until only one fly remains.After all of the necessary wells are filled with flies, flip the chamber so that the bottom plate is upward and the dust has the potential to coat the flies by falling through the mesh. Tightly secure the top and bottom plates by tightening the screws and vortex the chamber to dust the flies for four seconds. Knock the chamber, top plate face-down against the table twice, so that the dye falls through the other side.Then, flip the chamber over, top plate face-up and knock the chamber twice, to ensure dye settles on the floor of the bottom chamber, away from the fly, held in the top chamber. For control flies, which are not allowed to groom, immediately proceed to sample preparation. Rest the chamber flat on the table or countertop, with the top plate upward for 30 minutes to allow the flies to groom.Place the chamber in a noise vibration free space, to keep your environment constant for all grooming experiments. Keeping the chamber level with the top plate upward, gently unscrew the bottom plate and remove it from the chamber, taking care not to lose any dye. Place the chamber on a CO2 pad with low air flow until the flies are anesthetized.Unscrew the top plate from the chamber, carefully use forceps to grab a leg and remove one dusted fly from each chamber and deposit it in a 1.5 milliliter micro-centrifuge tube containing ethanol. Once each micro-centrifuge tube contains one fly, close the cap and invert the tubes three times to agitate and mix the solution of dye and ethanol. Incubate the tubes containing ethanol and flies for five hours at room temperature to ensure full removal of the dye from the fly into solution.After five hours, briefly vortex each tube for two seconds to ensure clearance of the dyes from the flies. Aliquot 50 microliters of the experimental 1.5 milliliter tube into a well of a 96 well plate, taking note of the location of each sample on the plate. Add 200 microliters of ethanol to dilute the sample five times to avoid sealing effects of heavily dusted flies or large grooming defects.Use a plate reader to analyze each sample at 397 nanometers. Record the sample through the plate reader and save the spreadsheet with the recording samples on the plate. We used this method to study DopR by comparing the grooming performance of a strong hypomorph and to null DopR mutants to a wild type strain.The DopR mutants retained significantly more dye than their wild type of rescued counterparts, indicating that the DopR mutants were less efficient at grooming behavior. In a parallel experiment of Rutabaga flies, mutants display the higher dye accumulation levels than DopR mutants, suggesting a significant role of DopR in grooming behavior. After watching this video, you should have a good understanding of how to handle flies for behavioral assays and also measure the grooming behavior.

Research

JoVE Journal

Neuroscience

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Methods to Assay Drosophila Behavior

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 ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

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 ...

A Single-fly Assay for Foraging Behavior in Drosophila

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 ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...