Name: measuring and altering mating drive in male drosophila melanogaster
Uploaded: 2017-02-15T14:00:00.000+00:00
Duration: 7 min 2 s
Description: Watch this Scientific Journal Video about Measuring and Altering Mating Drive in Male Drosophila melanogaster at JoVE.com

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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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> Motivational Systems (Problems in the Behavioural Sciences). , (1986).</li><li>Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mating+of+a+fly.">The mating of a fly.</a> Science. 264 (5166), 1702-1714 (1994).</li><li>Simpson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+manipulating+neural+circuits+in+the+fly+brain.">Mapping and manipulating neural circuits in the fly brain.</a> Advances in genetics. 65 (9), 79-143 (2009).</li><li>Venken, K. J. T., Simpson, J. H., Bellen, H. 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. R., 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=Systematic+generation+of+high-resolution+deletion+coverage+of+the+Drosophila+melanogaster+genome.">Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome.</a> Nature genetics. 36 (3), 288-292 (2004).</li><li>Matthews, K. A., Kaufman, T. C., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+resources+for+Drosophila:+the+expanding+universe.">Research resources for Drosophila: the expanding universe.</a> Nature reviews. Genetics. 6 (3), 179-193 (2005).</li><li>Ni, J. Q., Liu, L. 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=A+Drosophila+resource+of+transgenic+RNAi+lines+for+neurogenetics.">A Drosophila resource of transgenic RNAi lines for neurogenetics.</a> Genetics. 182 (4), 1089-1100 (2009).</li><li>Ni, J. Q., Zhou, R., 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+genome-scale+shRNA+resource+for+transgenic+RNAi+in+Drosophila.">A genome-scale shRNA resource for transgenic RNAi in Drosophila.</a> Nature. 8 (5), 405-407 (2011).</li></ol>

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.

<table><tbody><tr><td>1/16 inch clear acrylic</td><td>McMaster-Carr</td><td>8589K12</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>1/8 inch clear acrylic</td><td>McMaster-Carr</td><td>8589K42</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>3/16 inch clear acrylic</td><td>McMaster-Carr</td><td>8560K219</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>1/32 inch black delrin</td><td>McMaster-Carr</td><td>8575K132</td><td>Used to make arenas; see Supplemental Material 1 for designs.</td></tr><tr><td>Hex screws, 1 inch long (50x)</td><td>McMaster-Carr</td><td>92314A115&amp;nbsp;</td><td>Used to make arenas. Can be replaced by 3/4 inch screws (92314A113, McMaster-Carr) for 32-chamber arenas.</td></tr><tr><td>Thumb nuts (25x)</td><td>McMaster-Carr</td><td>92741A100</td><td>Used to make arenas. Can be replaced by regular hex nuts (90480A005, McMaster-Carr).</td></tr><tr><td>Camcorder</td><td>Canon</td><td>Vixia HF R700</td><td>Can be replaced by any consumer comcorder.</td></tr></tbody></table>

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...

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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

Research

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Neuroscience

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

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are also a useful scientific model to study motion vision and flight dynamics in a controlled laboratory setting. As the larvae develop in organically polluted water, they are useful models for investigating investment in microbial immunity. While large scale commercial breeding for agriculture already occurs, there are no standardized protocols for maintaining captive populations for scientific studies. This is important as commercial captive breeding programs focusing on mass output during peak pollination periods may fail to provide a population that is consistent, stable and robust throughout the year, as is often needed for other research purposes. Therefore, a method to establish, maintain and refresh a captive research population is required. Here, we describe the utilization of an artificial hibernation cycle, in addition to the nutritional and housing requirements, for long term maintenance of Eristalis tenax. Using these methods, we have significantly increased the health and longevity of captive populations of E. tenax compared to previous reports. We furthermore discuss small scale rearing methods and options for optimizing yields and manipulating population demographics.

Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

The overall goal of these procedures is to establish, maintain and refresh a captive population of Eristalis tenax in a research setting.

With an estimated 6000 species worldwide, hoverflies are ecologically important as alternative pollinators to domesticated honeybees. However, they are ...

Environment

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

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

Methods to Assay Drosophila Behavior

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

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

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 New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...

Measuring and Altering Mating Drive in Male Drosophila melanogaster

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Rearing and Long-Term Maintenance of Eristalis tenax Hoverflies for Research Studies

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

Methods to Assay Drosophila Behavior

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

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

Conditions Affecting Social Space in Drosophila melanogaster

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Assessing Differences in Sperm Competitive Ability in Drosophila