We thank Rachelle Kanippayoor for her help in identifying the new wild-type strain as being of the melanogaster species. R.W.F, O.F. and A.F.S were responsible for research design; R.W.F, M.N. and O.F. performed the experiments. R.W.F, M.N., O.F. and A.F.S. analyzed the data; R.W.F., I.S.M. and A.F.S. wrote the manuscript. 
This work was supported by PSC-CUNY research awards, jointly funded by The Professional Staff Congress and The City University of New York to A.F.S.; by internal funding from Western University to A.F.S. and I.S.M.; by a training support from the National Alliance for Hispanic Health&#8217;s Alliance/Merck Ciencia (Science) Hispanic Scholars Program and a University Fellowship from the Yale Graduate School of Arts and Sciences to R.W.F.

1. Equipment and Reagents Created In-house (See List of Material for Others)
<ol>
	<li>Prepare a Drosophila cold anesthesia apparatus to perform fly work.
	<ol>
		<li>Cut a porous polyethylene sheet to cover a small plastic box (12.7 cm long, 10.2 cm wide), typically the top cover of a pipette tips box (12.7 cm long, 10.2 cm wide, 3.8 cm deep).</li>
		<li>Fill the box with crushed ice, cover with the porous polyethylene sheet.&#160;</li>
	</ol>
	</li>
	<li>Prepare a T-maze apparatus; adapt the apparatus previously described in details 28-31.</li>
	<li>Prepare a fly aspirator as described 32.</li>
	<li>Prepare a phototaxis response apparatus to displace flies in and out of test vials without stress:
	<ol>
		<li>Use a countercurrent apparatus, as described by29,33.</li>
	</ol>
	</li>
</ol>
NOTE: The T-maze can also be used for other purposes, as described by28.
<ol start="5">
	<li>Ensure homogeneous lighting conditions: perform the experiment on a bench covered with a white bench cover, and in front of a white board. 
	NOTE: For these experiments, the fly strains used were Drosophila melanogaster wild-types: Canton-S, Oregon and Samarkand flies from our laboratory stocks34; Elwood flies were collected in Fall 2011 in the Elwood neighborhood of Huntington, on Long Island, New York, USA.</li>
</ol>
2. Preparing the Flies before the Experiment
1-2 days before performing the experiment:&#160;
<ol>
	<li>Maintain flies on standard cornmeal-agar-molasses-yeast medium at 25 &#176;C on a 12 hr light/dark cycle.</li>
	<li>Collect the responder flies under cold anesthesia 1-2 days prior to performing the experiment
	<ol>
		<li>Transfer the flies from the bottles in which they have been raised into 50 ml Falcon tubes, using a funnel.</li>
		<li>Place the falcon tube with the flies under ice level, into an insulated ice bucket.</li>
		<li>Place the cold anesthesia apparatus with crushed ice at -4 &#176;C to chill.</li>
		<li>Wait 5 min for the flies to enter a chill coma.</li>
		<li>Transfer the flies onto the porous polyethylene sheet of the cold anesthesia apparatus.</li>
		<li>Collect the responder flies on the cold anesthesia apparatus. At RT, under a stereomicroscope, separate 3-7 days old male from female flies and maintain these flies in groups of 40.</li>
	</ol>
	</li>
	<li>Collect the emitter flies 1-2 days prior to performing the experiment.
	<ol>
		<li>Gently collect Canton-S mixed-sex flies from their bottles, using a mouth aspirator, controlling for their number.</li>
		<li>Maintain these flies in group of 60-100, as they will be the emitters.</li>
		<li>Prepare as many samples of emitters as data points needed.</li>
	</ol>
	</li>
	<li>Allow at least a night of recovery after collection of the responders and emitters, to minimize any confounding effect of the cold anesthesia and mouth aspirator collection on behavior.</li>
	<li>Open the bag of test vials, to allow for the stale plastic-smelling air in the bag to be replaced by fresh air, at least 1 day prior to the experiment.</li>
</ol>
2 hr before performing the experiment:
<ol start="6">
	<li>Ensure that the temperature in the room in which the experiment is performed is around 23-25 &#176;C, with even light, and humidity above 30%.</li>
	<li>Transfer the responders and emitters fruit flies into fresh food vials to ensure that these flies are not starved.</li>
	<li>Let the fruit flies adjust to the environment for 2 hr, on the bench on which the experiment will be performed.</li>
</ol>
3. Performing the Social Avoidance Experiment
<ol>
	<li>Perform each experiment at the same time of the day, in a range of 3&#8211;4 hr in the afternoon between Zeitgeber time ZT5 and ZT9.</li>
	<li>Place the T-maze on the pounding pad, and tighten the screw clip so the elevator is stable.</li>
	<li>Transfer the responder flies into a fresh test vial. Snap this vial containing the responder flies onto the top part of the T-maze.</li>
	<li>Slant the T-maze apparatus and tap it on the pounding mat so the fruit flies fall in the elevator.</li>
	<li>Move the top part of the elevator down so the flies are between the top and bottom section of the T-maze, but pay attention to keep the elevator above the choice point &#8211; to prevent the flies from escaping.</li>
	<li>Obtain vials with dSO (see Section 5 to generate dSO-free vials that were previously occupied by flies).
	<ol>
		<li>While the responder flies are adjusting to this new environment, place the emitter flies in a test vial.</li>
		<li>Place a piece of cotton to close the vial so the fruit flies do not escape.</li>
		<li>Mechanically agitate this vial on a mini-vortex as follows: vortex for 15 sec, remove the vial from the vortex for 5 sec. Repeat 3 times for a total of 55 sec.</li>
		<li>Remove the emitter flies out of the fresh vial &#8212; by transferring in a food vial &#8212; and quickly place this dSO filled vial into one of the two sides of the T-maze apparatus.</li>
		<li>Alternate the side of placement for the dSO filled vial in each new run.</li>
	</ol>
	</li>
	<li>Place a fresh test vial on the other side of the T-maze.</li>
	<li>Bring the elevator completely down, so the fruit flies can chose between the fresh test vial and the test vial with dSO, and start the timer.</li>
	<li>Let the flies choose between the fresh vial and the dSO vial, for 1 min, unless noted otherwise.</li>
	<li>After 1 min, move the elevator up to separate the flies in the fresh vial, dSO vial, and those stuck in the elevator.</li>
	<li>Count the number of flies in each vial, and in the elevator.</li>
	<li>Repeat this for each genotype/condition.</li>
</ol>
4. Analyzing the Social Avoidance Data
<ol>
	<li>Count the number of fruit flies in each vial for each genotype, and transfer the data to a spreadsheet.</li>
	<li>Using a spreadsheet program, calculate the Performance Index (PI) for each genotype by subtracting the number of flies in the dSO vial from the number of flies in the dSO-free vial, and then dividing by the total number of flies.</li>
	<li>Compare the PIs&#8217; means using statistical analysis software for analysis of the results.
	<ol>
		<li>Ideally, prepare 2-3 internal repeats for each experiment, and 3 independent repeats, performed on different days, with different bottles or crosses30.</li>
		<li>Use Gaussian distribution statistical tests, as the data follow a normal distribution.</li>
		<li>When testing one effect (i.e., density &#8212; Figure 2), use a one-way ANOVA, with post-test for multiple comparison (column by column), or a simple t-test to compare each result to a control.</li>
		<li>When group experiments are performed, test both conditions and genders (empty vials, stressed and un-stressed flies; and gender effect &#8212; Figure 1), using a two-way ANOVA.</li>
	</ol>
	</li>
</ol>
5. Generate Vials Previously Occupied by Unstressed Flies &#8212; Perform Instead of 3.5 to 3.6
<ol>
	<li>Use phototaxis to prepare vials occupied by flies that have not been stressed, as a control.</li>
	<li>Transfer the flies in a test vial, and snap it into the countercurrent apparatus, with fresh empty vials in the opposite location.</li>
	<li>Lay the apparatus horizontally with the distal vial directly in front of a 15 W fluorescent cool-white light, and cover with a black cloth.</li>
	<li>Let the flies gently move in the fresh vial. This might take several minutes. Once most of the flies are walking toward the light in the distal vial, start the timer for 1 min.</li>
	<li>Gently remove the black cloth, switch the location of the light, recover with the black cloth, and allow the flies to leave the test vial.</li>
	<li>At that time, initiate the choice experiment as in 3.2 to 3.5. Skip 3.6 and 3.7.</li>
	<li>Move the slider of the apparatus, to separate the flies that vacated the test vial from the other side.</li>
	<li>Use the vacated test vial in the T-maze right away for the choice experiment, as in 3.7.</li>
</ol>

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The authors declare that they have no competing financial interests.

This protocol describes a detailed procedure for the social avoidance assay. Canton-S will only avoid a vial in which flies have previously been mechanically stressed, and that sex and number of responders does not affect that social avoidance performance. However, genetic background of the responders has a major influence.
The following are several critical steps for performing this experiment successfully: 1) always transfer the flies 2 hr before the experiment and make sure not to disturb t...

The goal of this method is to easily quantify a new aspect of simple social behavior in Drosophila melanogaster, independent from courtship and aggression.
Social interactions are crucial to the proper development and health of individuals within a society, as well as the functionality of a social group as a whole. The high complexity of these interactions necessitates large sample sizes and a system that allows for simplification of the behavior as the genetic and neural bases of social behavior are still poorly understood. Drosophila melanogaster is a powerful genetic model that can be used to identify the genetic and neural bases of social interactions. Indeed, D. melanogaster has a repertoire of complex social behaviors and some direct measurements of socialization have already been done1-7. However, most of these efforts have been focused on relatively complex social behaviors, such as aggressive interactions3,6, various aspects of courtship3,8-12, and how social experience affects other behaviors such as learning, or circadian rhythm13-17. In addition, many of these assays rely on analyzing complex interaction patterns of groups of flies, using video tracking and computer software to analyze the resulting abundance of data. Such analyses are invaluable, and lead to important new insights such as the dynamic of fly-fly interactions in groups7. One limitation, however, is the inaccessibility of these assays to the community at large, and the limited knowledge of the mechanisms underlying recognition of others. In other words, the basis of the emission of a signal by one individual and its recognition by another is still poorly understood18.
In contrast, flies also exhibit a simple behavior, social avoidance, where individuals move away from a signal emitted by stressed flies: the D. melanogaster Stress Odorant or dSO19. In a high-throughput assay, this behavior can be quantified as the avoidance of a stress signal emitted by other flies, or social avoidance19. Flies are placed in a T-maze apparatus and given the choice to avoid a vial containing dSO. Using this assay, CO2 was shown to be a component of the dSO, and part of the neural circuitry necessary to respond to CO2 was dissected19.
The social avoidance assay presented here is similar conceptually to the simple behavioral assays developed in Seymour Benzer&#8217;s laboratory that enabled generations of researchers to dissect complex behaviours20. Analysis of social avoidance can be carried out cost-effectively using the T-maze assay, allowing for more widespread study of social behavior. For example, using this assay we recently demonstrated that different genetic risks for autism have contrasting effects in social behavior assays. Mutants for a candidate gene for autism &#8212; neurobeachin21,22 &#8212; present deficiencies both in social space (described elsewhere23) and social avoidance24. Abnormal dopaminergic signaling is also proposed to play a role in the etiology of autism in humans25,26. In contrast to the results obtained with neurobeachin, we found that social avoidance performance was unaffected by increased or decreased levels of the Drosophila Vesicular Monoamine Transporter (VMAT) in dopaminergic cells, although social space was directly correlated to these levels of VMAT27. The contrasting results obtained with neurobeachin and VMAT underscore the possibility of identifying various forms of asocial behavior, and thus the different underlying neural circuitries modulating the response to others.

<table><tbody><tr><td>Stereo Zoom Microscope&amp;nbsp;</td><td>Nikon&amp;nbsp;&amp;nbsp;</td><td>SMZ-645</td><td>Any other standard scope for fly handling would work</td></tr><tr><td>Small paint brushes&amp;nbsp;</td><td></td><td></td><td>for pushing flies</td></tr><tr><td>Porous Polyethylene, 12&quot; x 12&quot; Sheet</td><td>Flystuff - Genesee</td><td>46-100</td><td>http://www.flystuff.com/ProductInfo.php?productID=46-100&lt;br/&gt; Porous Plastic sheet for the cold anesthesia box</td></tr><tr><td>Mini-Alarm Timer/Stopwatch</td><td></td><td></td><td></td></tr><tr><td>Sharpie pens</td><td></td><td></td><td></td></tr><tr><td>Adhesive Tape</td><td></td><td></td><td></td></tr><tr><td>Mini vortex</td><td>Fisher</td><td>14-955-151&amp;nbsp;</td><td>http://www.fishersci.com/ecomm/servlet/itemdetail&lt;br/&gt; For mechanical agitation of the flies - any vortex would work.</td></tr><tr><td>Corning Life Sciences DL No.:352017, Falcon test tube; round bottom; disposable; no closure, 14 ml; 17 x 100 mm</td><td>Fisher</td><td>14-959-8 &amp;nbsp;&amp;nbsp;</td><td>http://www.fishersci.com/ecomm/servlet/itemdetail?storeId=10652&amp;amp;langId=-1&amp;amp;catalogId=29104&amp;amp;productId=2771811&amp;amp;distype=0&amp;amp;highlightProductsItemsFlag=Y&amp;amp;fromSearch=1&amp;amp;searchType=PROD&amp;amp;hasPromo=0&lt;br/&gt; These snap in place in the in-house made T-maze and counter-current apparatus (see text)</td></tr><tr><td>cotton balls</td><td></td><td></td><td>to close the vials after the experiment.</td></tr><tr><td>trifold board and white bench cover</td><td></td><td></td><td>to provide a white background, and a homogeneous light.</td></tr><tr><td>white bench cover</td><td></td><td></td><td></td></tr><tr><td>pounding pad</td><td></td><td></td><td>any mouse pad works.</td></tr><tr><td>large black cloth</td><td></td><td></td><td>to cover the counter-current apparatus in phototaxis response.</td></tr><tr><td>cool-white light&amp;nbsp;</td><td>Home Depot</td><td>1000516563</td><td>http://www.homedepot.ca/product/illume-26-fluorescent-plug-in-linear/911423&lt;br/&gt; any similar linear light with fluorescent light bulb cool-white at 13-15 W would work</td></tr></tbody></table>

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.

The social avoidance assay is a robust test quantifying the ability of Drosophila melanogaster to recognize a stress signal (dSO) emitted by other flies, and thus assessing one aspect of social interactions. The assay is performed using an apparatus commonly used in various behavior assays known as a T-maze, which present the flies with a choice between two different options &#8212; left or right19,28-31. In this case, the efficiency at which the flies avoid the dSO is quantified by calculating and co...

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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.

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 ...

straightforward-assay-for-quantification-social-avoidance-drosophila

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.2K Views.  Yale University. 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.

Video: Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

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 ...

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 article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Testing Drosophila Olfaction with a Y-maze Assay

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.

Testing Drosophila Olfaction with a Y-maze Assay

We present a choice test to reveal the influence of odorants on Drosophila behavior using a Y-maze assay.

Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, ...

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits&#160;within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized here. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This work describes a simple behavioral paradigm that allows the analysis of aversive associative learning in adult fruit flies. The method is based on suppressing the innate negative geotaxis behavior due to the association formed between a specific environmental context and an electric shock.

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to ...

Behavior

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

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 ...

Drosophila Models for Genetic and Behavioral Traits of Autism Spectrum Disorder

Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurodevelopmental disorders with common behavioral symptoms including deficits in social interaction and ability for communication, enhanced restricted or repetitive behaviors, and also, in some cases, learning disability and motor deficit. Drosophila has served as an unparalleled model organism for modeling a great number of human diseases. As many genes have been implicated in ASD, fruit flies have emerged as a powerful and efficient way to test the genes putatively involved with the disorder. As hundreds of genes, with varied functional roles, are implicated in ASD, a single genetic fly model of ASD is unfeasible; instead, individual genetic mutants, gene knockdowns, or overexpression-based studies of the fly homologs of ASD-associated genes are the common means for gaining insight regarding molecular pathways underlying these gene products. A host of behavioral techniques are available in Drosophila which provide easy readout of deficits in specific behavioral components. Social space assay and aggression and courtship assays in flies have been shown to be useful in assessing defects in social interaction or communication. Grooming behavior in flies is an excellent readout of repetitive behavior. Habituation assay is used in flies to estimate the ability for habituation learning, which is found to be affected in some ASD patients. A combination of these behavioral paradigms can be utilized to make a thorough assessment of the human ASD-like disease state in flies. Using Fmr1 mutant flies, recapitulating Fragile-X syndrome in humans, and POGZ-homolog row knockdown in fly neurons, we have shown quantifiable deficits in social spacing, aggression, courtship behavior, grooming behavior, and habituation. These behavioral paradigms are demonstrated here in their simplest and straightforward forms with an assumption that it would facilitate their widespread use for research on ASD and other neurodevelopmental disorders in fly models.

Author Spotlight: Exploring Autism Spectrum Disorder Symptoms in Fruit Flies &#8212; Genetic Models and Behavioral Tests

Autism Spectrum Disorder (ASD) is associated with impaired social and communicative behavior and the emergence of repetitive behavior. For studying the interrelation between ASD genes and behavioral deficits in the Drosophila model, five behavioral paradigms are described in this paper for assaying social spacing, aggression, courtship, grooming, and habituation behavior.

Autism Spectrum Disorder (ASD) encompasses a heterogeneous group of neurodevelopmental disorders with common behavioral symptoms including deficits in ...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model to study animal avoidance behavior. This protocol introduces a light-spot assay to investigate larval phototactic behavior. The experimental set-up includes two main parts: a visual stimulation system that generates the light spot, and an infrared light-based imaging system that records the process of larval light avoidance. This assay allows tracking of the behavior of larva before entering, during encountering, and after leaving the light spot. Details of larval movement including deceleration, pause, head casting, and turning can be captured and analyzed using this method.

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging system.

The larvae of Drosophila melanogaster show obvious light-avoiding behavior during the foraging stage. Drosophila larval phototaxis can be used as a model ...

Biology

Quantification of Drosophila melanogaster Grooming Behavior for Evaluation of Excessive Grooming Phenotypes

Observable changes in stereotyped grooming are applied translationally in model organisms. These changes are representative of pathologies that elicit similar deviations in human behavior; for example, excessive grooming acts as a proxy for obsessive and compulsive behaviors present in conditions like Tourette Syndrome or obsessive-compulsive disorder. The grooming assay presented allows for the evaluation of abnormal self-grooming phenotypes in Drosophila melanogaster. Flies are recorded for a period of 10 min, and these recordings are observed and annotated blind for previously defined grooming behaviors. Quantitative measures of both grooming bout frequency and the time spent engaging in self-grooming can be obtained by manually annotating the footage. The assay is relatively inexpensive, requires few materials not already available in laboratory environments, and is easily adaptable to fit the specific needs of any given study aiming to observe grooming. Additionally, the low level of skill needed to perform the assay, as compared to computer science-heavy automated methods, makes the protocol well-suited for small labs and students. We discuss in detail the steps required to perform this assay and its present limitations.

The method presented here involves the manual annotation of footage acquired of Drosophila melanogaster for specific grooming behaviors. It allows for quantification of both the number of grooming bouts and overall time spent grooming to evaluate for atypical self-grooming phenotypes.

Observable changes in stereotyped grooming are applied translationally in model organisms. These changes are representative of pathologies that elicit ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Summary

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Chapters in this video

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Methods to Assay Drosophila Behavior

A Single-fly Assay for Foraging Behavior in Drosophila

Testing Drosophila Olfaction with a Y-maze Assay

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

A Simple Technique to Assay Locomotor Activity in Drosophila

Conditions Affecting Social Space in Drosophila melanogaster

Paradigms for Behavioral Assessment in Drosophila Model of Autism Spectrum Disorder

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

Quantification of Drosophila melanogaster Grooming Behavior for Evaluation of Excessive Grooming Phenotypes