The authors thank Mike Crickmore, Dragana Rogulja, and Michelle Frank for comments on the manuscript. Pavel Gorelik provided technical support for manufacturing the behavioral arenas. This work was conducted in Mike Crickmore's lab and is also supported by the Whitehall Foundation (Principal Investigator: Dragana Rogulja). S.X.Z. is a Stuart H.Q. and Victoria Quan Fellow at Harvard Medical School.

NOTE: This protocol describes the preparation (Sections 1 - 3), execution (Section 4), and analysis (Section 4) of 2-D satiety assays. Then, using dopaminergic stimulation as an example, Section 5 shows how to combine thermogenetic stimulation with 2-D satiety assays to induce hypersexuality. Section 6 describes 3 ways to verify the results of 2-D satiety assays. Finally, Section 7 shows how to measure the recovery of mating drive in male flies.
1. Fabricating 8- and 32-chamber Behavioral Arenas
NOTE: Each behavioral arena consists of several layers of laser-cut plastic sheets held together by hex screws and thumb nuts at each of the four corners (Figure 1A).
<ol>
	<li>Fabricate the layers of the arena. Fabricate each layer using a laser cutter to cut sheets of acrylic plastic to the appropriate shape (see Figure 1B, 1C for products). 
	NOTE: Many research institutions have laser cutters in their machine shops or maker spaces. If the institution has access to a laser cutter, continue with the following steps. If not, then follow the instructions in Step 1.2 instead.
	<ol>
		<li>For each layer, open the design pattern in the laser cutter&#39;s software. 
		NOTE: The designs can be found in appropriately named PDF files (e.g., 8-Chamber Arena - Layer 2 - Doors.pdf) in Supplemental Material 1. This file also contains annotations for the type of plastic to be used (e.g., 1/8 inch (i.e.&#160;3.175 mm) clear acrylic). The exact thickness of each layer is not critical. The main requirement is to allow 0.12 inches (3 mm) or more of free vertical space in the chambers.</li>
		<li>Adjust the laser settings (power, focus, etc.) for the type and thickness of plastic to be used. Place plastic in the laser cutter bed and run the laser cutter. 
		NOTE: These settings will vary widely based on the laser cutter model and the strength of its laser. Find appropriate settings based on past experience or by making test cuts on scrap plastic. The deep hexagonal engravings are included in the design to recess the hex screw heads. This feature allows for one-handed tightening of the thumb nuts and allows the arenas to rest flat on the bench (without protruding screw heads). Nevertheless, making deep engravings is often hard to achieve, and time-consuming, using a laser cutter. This step is optional and can be skipped without affecting the behavioral results.</li>
		<li>When cutting the door frame (Layer 2, Figure 1A), also use appropriate laser settings for engraving the chamber numbers included in the design (as in Figure 1B, 1C).</li>
	</ol>
	</li>
	<li>Place an order with an online laser cutting fabricator. Upload all the PDF design files for the 8- and/or 32-chamber arena and specify the material type (e.g., 1/8 inch (i.e.&#160;3.175 mm) clear acrylic) based on the annotations in the files. The results will appear as in Figures 1B (8-chamber arena) and&#160;1C (32-chamber arena).</li>
	<li>Assemble arenas using four hex screws and four thumb nuts to align and hold together the plastic layers (Figure 1A). The arenas will appear as in Figure 1D, 1E (8-chamber) and Figure 1F, 1G (32-chamber). 
	NOTE: The food wall (Layer 4 in Figure 1A) is used for 2-D satiety assays only and is omitted in 32-chamber arenas, which are used in courtship assays.</li>
</ol>
2. Food Preparation for 2-D Satiety Assay
NOTE: Because a 2-D satiety assay spans 4.5 h, fly food is used in the arena to provide nutrition and water. This protocol uses, but is not restricted to, the conventional cornmeal-agar fly food.
<ol>
	<li>Partially assemble the 2-D satiety arena (8-chamber) with Layers 3 - 6 (see Figure 1A for layer assignments) and tighten it with thumb nuts and hex screws.</li>
	<li>Place fresh fly food into a clean, microwave-safe bottle. Add just enough water to cover the bottom surface of the bottle (Figure 2A).</li>
	<li>Microwave food on high for 30 - 45 s. Check progress and stir every 15 s until melted (Figure 2B). CAUTION! Fly food easily boils over.</li>
	<li>Use a blunted 1,000-&#181;L pipette tip (Figure 2C) to slowly transfer ~1 mL melted food into each chamber (Figure 2D).</li>
	<li>Allow food to resolidify at 4 &#176;C for ~10 min (Figure 2E) before completely assembling the arena (Figure 2F).</li>
	<li>Use the completed arena for experiments (see Section 4) or store at 4 &#176;C.</li>
	<li>Store the leftover food at 4 &#176;C and reuse.</li>
</ol>
3. Preparing Virgin Females for Behavioral Assays
NOTE: 2-D satiety assays use large numbers of virgin female flies (~120 - 160 for a full behavioral arena) that are difficult to collect using standard methods. This section describes an alternative approach using the hs-hid transgene on the Y chromosome28, which has been used successfully in courtship assays25,29. The stock used to generate white-eyed virgin females has the genotype w1118(X)/hs-hid(Y);+/+;+/+ (Bloomington stock number 24638).
<ol>
	<li>Flip flies into a new bottle once 20 - 50% of the pupae have eclosed (see Figure 3A for example) and there are enough males (5 - 10) to propagate the stock.</li>
	<li>Heat shock the original bottle in a 37 &#176;C hot water bath for 1 h to sufficiently activate hs-hid to kill males (Figure 3B). After the heat shock, only females will eclose from these bottles and so will remain virgin until the assays. 
	NOTE: Make sure all sides of the bottle are evenly heated (see Figure 3A for water line). Extend this time for up to 2 h if 1 h is not sufficient to euthanize all males.</li>
	<li>Isolate collected females for at least 3 d before experiments to ensure that they are old enough to be sexually receptive25,29. 
	NOTE: Y-chromosome-less males occasionally arise from this stock. These males have white eyes and are unaffected by the heat shock because they do not contain the hs-hid transgene. They cannot produce sperm and therefore cannot decrease receptivity in females30,31.</li>
</ol>
4. Performing and Scoring 2-D Satiety Assays
NOTE: It is important to control the age of the male flies as mating drive changes with age. This protocol uses males that are 3 - 4 d old25.
<ol>
	<li>Set the incubator to the desired temperature (e.g., 23 &#176;C for standard, RT experiments) and humidity (&#62;30%). For incubators without humidity control, leaving a cup of water in the incubator is sufficient.</li>
	<li>Place a 2-D satiety assay arena in the incubator for ~30 min to equilibrate the temperature and to prevent condensation in the chambers during the course of the experiment. The rotating doors should be in the open position.</li>
	<li>Use a fly aspirator32 to aspirate 15 - 20 virgin females into each chamber of the arena (Figure 4). 
	NOTE: It is important that the females and males are not anesthetized on the same day as the assay. In our experience, doing so may impact female receptivity and male mating behavior.</li>
	<li>Aspirate 1 male into each chamber.</li>
	<li>Place the loaded arena in the incubator under a standard consumer camcorder (Figure 5) and film for 4.5 h.</li>
	<li>Score videos by noting when each male fly starts and ends each mating (i.e. copulation). This step may be time-consuming at first. With practice, one can score the videos at 5x speed or higher. 
	NOTE: Since flies mate for ~20 min at ~23 &#176;C (shorter duration at higher temperatures)29, one can skim through the first 15 min of each mating.</li>
	<li>After logging all mating times, sum the number of matings for each fly (see representative Results).</li>
	<li>Use the provided code to generate an ethogram. See Supplemental Material 2 for the code, instructions, and example data.</li>
	<li>Use CO2 or cold temperature to anesthetize the flies before removing them.</li>
</ol>
5. Using Thermogenetic Manipulation to Reverse Satiety in 2-D Satiety Assays
NOTE: This protocol tests whether thermogenetic stimulation of a defined neuronal population can overcome satiety. The experimental flies express the heat-sensitive cation channel TrpA1 in defined populations of neurons (UAS-TrpA1). The steps below apply to the stimulation of dopaminergic neurons (TH-Gal4), which have been shown to promote mating drive25. These steps can be used in conjunction with other neuronal drivers to discover other populations that promote mating drive.
<ol>
	<li>To thermogenetically induce mating behaviors in satiated flies, increase the temperature in the incubator (e.g., from 23 &#176;C to 28.5 &#176;C) after completing a full 4.5 h 2-D satiety experiment.</li>
	<li>After the incubator reaches the desired temperature, continue heat stimulation and score the matings (i.e. copulations) of the males for 1 h (see Representative Results). 
	NOTE: The temperature and length of stimulation may vary depending on the genotype so it is necessary to troubleshoot the optimal conditions that prevent overstimulation of neurons while still producing a robust phenotype.</li>
</ol>
6. Using Courtship and Locomotion to Verify Satiety
NOTE: This section describes 3 optional methods that can be used to verify the results of 2-D satiety assays. They are not required for every assay.
<ol>
	<li>Use courtship assays to verify satiety.
	<ol>
		<li>Aspirate one male and one virgin female into each of the 32 chambers in a courtship arena.</li>
		<li>Film the flies for ~20 min using a standard consumer camcorder.</li>
		<li>Score the courtship index (CI) to quantify courtship25; this is the fraction of time spent performing courtship behavior (singing, following, etc.) and mating (i.e. copulation) over a 5 min window. This window starts from the first instance of courtship behavior. If a male fly does not start courtship in the first 15 min, its CI is 0. Na&#239;ve WT flies should show a CI greater than 0.9, whereas a fully satiated fly should have a CI below 0.2. Other courtship measures may also be considered23,33.</li>
	</ol>
	</li>
	<li>Scoring courtship in 2-D satiety assays
	<ol>
		<li>Score the amount of time that a male fly spends courting, over a time period of a 2-D satiety assay (e.g., the first h). Courtship is defined as the male performing any step of the ritual (singing, following, etc.). Unlike in Section 6.1.3, this step does not include time the male spends mating.</li>
		<li>Divide the courtship time above by the total time the male was not mating (i.e.&#160;not copulating) in the same time period. For example, if a male fly courts for 15 min and mates for 20 min over the first h, the ratio is 15 min / (60 min - 20 min) = 0.375. The ratio is shown as &#34;Fraction of nonmating time in courtship&#34; in Representative Results.</li>
	</ol>
	</li>
	<li>Using locomotion assay to evaluate exhaustion
	<ol>
		<li>Partially assemble the 32-chamber arena with all parts but the contrast sheet.</li>
		<li>Aspirate one male fly (no female flies) into each of the 32 chambers in a courtship arena.</li>
		<li>Film the flies for ~10 min using a standard consumer camcorder.</li>
		<li>Track the flies with the MTrack2 plugin in ImageJ (http://valelab.ucsf.edu/~nstuurman/ijplugins/MTrack2.html).</li>
	</ol>
	</li>
</ol>
7. Recovering Mating Drive after 2-D Satiety Assay
<ol>
	<li>Perform a 2-D satiety assay as described in Section 4, but aspirate the male flies out at the end of the assay.</li>
	<li>Isolate the male flies at 23 &#176;C for the desired number of days.</li>
	<li>Perform a 2-D satiety assay with the recovered males and score their matings (i.e.&#160;copulations) for just 1 h(see Representative Results).</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Manipulation+of+Genes+and+Cells+in+the+Nervous+System+of+the+Fruit.">Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit.</a> Neuron. 72 (2), 202-230 (2011).</li><li>Klapoetke, N. C., Murata, 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nature methods. 11 (3), 338-346 (2014).</li><li>Bellen, H. J., Levis, 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=The+BDGP+gene+disruption+project:+single+transposon+insertions+associated+with+40%+of+Drosophila+genes.">The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.</a> Genetics. 167 (2), 761-781 (2004).</li><li>Spradling, A. C., Stern, D., 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+Berkeley+Drosophila+Genome+Project+gene+disruption+project:+Single+P-element+insertions+mutating+25%+of+vital+Drosophila+genes.">The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes.</a> Genetics. 153 (1), 135-177 (1999).</li><li>Parks, A. L., Cook, K. 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The authors have nothing to disclose.

Motivational states can be satiated, maintained, and recovered34. We present a 2-D satiety assay that quickly and robustly measures all of these aspects of mating drive in the fly. This assay opens up the possibility of using advanced fly genetic manipulations to study the molecular and circuit components of a motivated behavior.
The satiety assay relies on the male&#39;s ability to successfully court and copulate, and to terminate copulations at the appropriate time. T...

Work in Drosophila has provided deep and pioneering insight into many biological phenomena, including the nature of the gene1, principles of embryonic development2, circadian rhythms3, and the development and wiring of the nervous system4,5,6. Motivation remains far less well understood than these phenomena, perhaps because of the limitations on the systems that have been studied thus far. Motivation in the fly is primarily studied in the context of hunger, which presents many challenges due to their vanishingly small food intake per feeding bout and exoskeleton which precludes overt signs of fat deposition. Consequently, there is a need to expand the systems used to study motivation in the fly.
We describe a behavioral framework for the study of mating drive in Drosophila. This system takes advantage of the neurogenetic tools in the fly as well as the accessibility7,8,9,10,11,12 and the putative connectome of its sexually dimorphic circuitry8,13. In addition, much of the innate14,15,16,17,18,19,20,21 and learned22,23,24 sensory-motor circuitry controlling courtship has been worked out in detail, providing a rare opportunity to locate the exact circuit node upon which motivation impinges. We recently reported that, in the fly, as in humans, dopamine levels are central to mating drive25,26,27. We have gained genetic access to the relevant dopamine-producing and receiving neurons in the fly, facilitating detailed molecular- and circuit-level analyses of this conserved phenomenon using the assays we describe here25.
We add to the behavioral assays in Zhang et al.25 a new flat behavioral arena that allows video scoring, which we call a 2-dimensional (2-D) satiety assay, an important improvement over previous methods. Consequently, the new assay is more scalable and quantifiable, and therefore more suitable for genetic screens of genes and neurons involved in motivation. We use this new assay, together with courtship assays and neurogenetic manipulations, to demonstrate how to measure and alter mating drive in the fly.

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measuring and altering mating drive in male drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

To characterize Drosophila mating drive, 3-day-old, WT Canton-S males were tested in a 2-D satiety assay. Over the course of the assay (4.5 h), males mate an average of 4.8 &#177;0.3 (mean &#177;standard error of mean, SEM) times. Matings initiate mostly in the first 2 h (78%) (Figure 6A, 6B) and become less frequent as the assay progresses (Figure 6A, 6B). This decrease is not due to the lack of mating partners (74% females remain unmated throug...

Watch this Scientific Journal Video about Measuring and Altering Mating Drive in Male Drosophila melanogaster at JoVE.com

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

measuring-and-altering-mating-drive-in-male-drosophila-melanogaster

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11.0K Views.  Boston Children’s Hospital. This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Video: Measuring and Altering Mating Drive in Male Drosophila melanogaster

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 in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (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 ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

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 stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction 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

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

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

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

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

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...