Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Summary

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.

Abstract

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.

Introduction

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 done^1-7. However, most of these efforts have been focused on relatively complex social behaviors, such as aggressive interactions^3,6, various aspects of courtship^3,8-12, and how social experience affects other behaviors such as learning, or circadian rhythm^13-17. 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’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 — neurobeachin^21,22 — 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 humans^25,26. 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.

Protocol

1. Equipment and Reagents Created In-house (See List of Material for Others)

1. Prepare a Drosophila cold anesthesia apparatus to perform fly work.
   1. 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).
   2. Fill the box with crushed ice, cover with the porous polyethylene sheet. 
2. Prepare a T-maze apparatus; adapt the apparatus previously described in details ^28-31.
3. Prepare a fly aspirator as described ³².
4. Prepare a phototaxis response apparatus to displace flies in and out of test vials without stress:
   1. Use a countercurrent apparatus, as described by^29,33.

NOTE: The T-maze can also be used for other purposes, as described by²⁸.

5. 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.

2. Preparing the Flies before the Experiment

1-2 days before performing the experiment: 

1. Maintain flies on standard cornmeal-agar-molasses-yeast medium at 25 °C on a 12 hr light/dark cycle.
2. Collect the responder flies under cold anesthesia 1-2 days prior to performing the experiment
   1. Transfer the flies from the bottles in which they have been raised into 50 ml Falcon tubes, using a funnel.
   2. Place the falcon tube with the flies under ice level, into an insulated ice bucket.
   3. Place the cold anesthesia apparatus with crushed ice at -4 °C to chill.
   4. Wait 5 min for the flies to enter a chill coma.
   5. Transfer the flies onto the porous polyethylene sheet of the cold anesthesia apparatus.
   6. 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.
3. Collect the emitter flies 1-2 days prior to performing the experiment.
   1. Gently collect Canton-S mixed-sex flies from their bottles, using a mouth aspirator, controlling for their number.
   2. Maintain these flies in group of 60-100, as they will be the emitters.
   3. Prepare as many samples of emitters as data points needed.
4. 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.
5. 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.

2 hr before performing the experiment:

6. Ensure that the temperature in the room in which the experiment is performed is around 23-25 °C, with even light, and humidity above 30%.
7. Transfer the responders and emitters fruit flies into fresh food vials to ensure that these flies are not starved.
8. Let the fruit flies adjust to the environment for 2 hr, on the bench on which the experiment will be performed.

3. Performing the Social Avoidance Experiment

1. Perform each experiment at the same time of the day, in a range of 3–4 hr in the afternoon between Zeitgeber time ZT5 and ZT9.
2. Place the T-maze on the pounding pad, and tighten the screw clip so the elevator is stable.
3. Transfer the responder flies into a fresh test vial. Snap this vial containing the responder flies onto the top part of the T-maze.
4. Slant the T-maze apparatus and tap it on the pounding mat so the fruit flies fall in the elevator.
5. 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 – to prevent the flies from escaping.
6. Obtain vials with dSO (see Section 5 to generate dSO-free vials that were previously occupied by flies).
   1. While the responder flies are adjusting to this new environment, place the emitter flies in a test vial.
   2. Place a piece of cotton to close the vial so the fruit flies do not escape.
   3. 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.
   4. Remove the emitter flies out of the fresh vial — by transferring in a food vial — and quickly place this dSO filled vial into one of the two sides of the T-maze apparatus.
   5. Alternate the side of placement for the dSO filled vial in each new run.
7. Place a fresh test vial on the other side of the T-maze.
8. 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.
9. Let the flies choose between the fresh vial and the dSO vial, for 1 min, unless noted otherwise.
10. After 1 min, move the elevator up to separate the flies in the fresh vial, dSO vial, and those stuck in the elevator.
11. Count the number of flies in each vial, and in the elevator.
12. Repeat this for each genotype/condition.

4. Analyzing the Social Avoidance Data

1. Count the number of fruit flies in each vial for each genotype, and transfer the data to a spreadsheet.
2. 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.
3. Compare the PIs’ means using statistical analysis software for analysis of the results.
   1. Ideally, prepare 2-3 internal repeats for each experiment, and 3 independent repeats, performed on different days, with different bottles or crosses³⁰.
   2. Use Gaussian distribution statistical tests, as the data follow a normal distribution.
   3. When testing one effect (i.e., density — 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.
   4. When group experiments are performed, test both conditions and genders (empty vials, stressed and un-stressed flies; and gender effect — Figure 1), using a two-way ANOVA.

5. Generate Vials Previously Occupied by Unstressed Flies — Perform Instead of 3.5 to 3.6

1. Use phototaxis to prepare vials occupied by flies that have not been stressed, as a control.
2. Transfer the flies in a test vial, and snap it into the countercurrent apparatus, with fresh empty vials in the opposite location.
3. 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.
4. 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.
5. 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.
6. At that time, initiate the choice experiment as in 3.2 to 3.5. Skip 3.6 and 3.7.
7. Move the slider of the apparatus, to separate the flies that vacated the test vial from the other side.
8. Use the vacated test vial in the T-maze right away for the choice experiment, as in 3.7.

Results

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 — left or right^19,28-31. In this case, the efficiency at which the flies avoid the dSO is quantified by calculating and comparin...

Discussion

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...

Materials

List of materials used in this article

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