Conditions Affecting Social Space in Drosophila melanogaster

Summary

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.

Abstract

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster — or other small insects — 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.

Introduction

Social interactions are crucial to the proper development and health of individuals within a group as a whole, and can be observed across numerous species, from humans (Homo sapiens) to simpler organisms such as fruit flies (Drosophila melanogaster) ^5,6. An individual fruit fly or human share common means to process sensory information during these interactions, whether it be: auditory, visual, olfactory, tactile, or gustation. We and others hypothesize that there is a potentially shared neurocircuitry underlying behavioral responses to social interactions and that the neuronal cells and genes involved might be evolutionary conserved ⁷. Once the initial interaction has occurred, social space between the interacting individuals will either increase (social avoidance ⁸) or decrease (group formation/aggregation ⁵). More complicated interactions, like aggression or courtship, can then take place.

Neither sophisticated tools and methods, nor large investments in time and training are required to quantify this simple form of social behavior, making it a powerful analytical tool. Here, we explain a straightforward protocol that quantifies inter-fly distance, or social space, to assess social interaction in stable groups of Drosophila melanogaster, as used in the following studies ^1-4,9. Social space refers to a measure of the distance between a fly and its closest neighbor ¹⁰. Social space is consistent for a given population of D. melanogaster when experimental conditions are preserved (averaging approximately within 1-2 body lengths), and varies with respect to the social experience of the flies, increasing if the individual has been kept in isolation ¹. Proper vision is necessary to maintain normal social distance, but not classical odorants or cVA perception ¹. Measure of social space can thus be used as a diagnostic tool to analyze social interactions and quantify social behavior in D. melanogaster ¹. We describe here in details how to perform this quantification, and to what extent common experimental variables affect this behavior.

We show that the orientation of the chamber in which the assay is performed, as well as the number of flies — to an extent — do affects social space. It was previously shown that chamber geometry affects spontaneous exploratory movement of flies ^11,12, and this phenomena may ultimately impact where they decide to settle. However, as long as the fly density (fly / cm²) and chamber orientation is kept the same, the social space of the flies also remains constant. The robustness of this assay is illustrated by the fact that independent laboratories using different chamber sizes, shape, and orientation can replicate the result displayed by mutants of the white gene (affecting eye pigmentation), which is an increase social space (vertical triangle or horizontal circle in ¹, horizontal square with airflow in ³).

Our results also indicate that maintaining the time at which the social space experiment is performed is crucial to the consistency of the results, as we show that males, but not females, are further apart in the evenings. However, the differences seen between daytime and evening hours are not due to activity differences of the flies, and we discuss arguments indicating that activity levels are not correlated with social space.

Finally, there are genetic underpinnings to the determination of social space, as indicated by the white mutant already described ^1,3, and the differences between various inbred and wild-caught strains of flies that we present here.

Therefore, this assay makes an excellent tool for studying the effects of genetic as well as environmental factors.

Protocol

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

1. Prepare a Drosophila cold anesthesia apparatus as previously described ⁸.
2. Prepare a fly aspirator as previously described ¹³.
3. Prepare social space chambers and holders.
   1. Order or make glass panes and acrylic spacers to create the social space chambers. Each social space chamber consists of two square glass panes (17.6 cm by 17.6 cm, with a thickness of 0.3 cm), two right triangle acrylic spacers (with a height of 16.5 cm, a base of 8.9 cm, with a thickness of 0.3 cm), and two rectangular spaces (9 cm by 1.5 cm with a thickness of 0.3 cm) (Figure 1A).
   2. Assemble the glass and acrylic social space chamber such that it is identical to the chamber in Figure 1B. To do so, start by placing one triangular spacer flat on top of one square pane of glass such that the right angle of the triangular spacer is aligned with one of the corners of the square pane. Next, place the second triangular spacer flat on top of the square pane, mirroring the first triangular spacer.
      NOTE: Make sure to wear gloves so as not to contaminate the pieces with oils and scents from your hands.
   3. Place two small rectangular spacers flat on top of the square pane, along the side of the pane that is not covered. The acrylic spacers on top of the square plane should now form a triangular arena.
   4. Place a second square pane of glass on top of the acrylic spacers such that it is aligned above the original pane of glass below.
   5. Use four binder clips to secure the panes and spacers together to form the social space chamber. Place one clip near the corner, over the long side of each of the triangular spacers, securing them inside the glass panes. On these same sides, place a clip in the adjacent corner, over each of the rectangular spacers, securing them inside the glass panes.
   6. Assemble a support stand and a flask clamp such that the clamp will maintain the social space chamber in an upright position, as the social space chamber rests on its edge on top of the work surface.
   7. For each repeat in the experiment, assemble a social space chamber and support stand.
4. Prepare a climbing assay response apparatus to check the climbing ability of the flies tested.
   1. Use a countercurrent apparatus, as previously described ^14,15.
5. Ensure homogeneous lighting conditions by performing the experiment on a work surface covered with a white bench cover and in front of a white background.

2. Preparing the Flies before the Experiment

1. Maintain flies in bottles containing standard Drosophila fly food. Keep them in an incubation chamber at 25 °C on a 12 hr light/dark cycle.
2. One to two days prior to the experiment, collect and sex flies under cold anesthesia, as previously described ⁸.
   1. Chill the cold anesthesia apparatus at -4 °C. Place flies into 50 ml plastic tubes. Fully submerge the plastic tubes containing flies into ice in an insulated ice bucket, wait at least 5 min for flies to become immobile.
   2. Place the flies on the polyethylene sheet of the cold anesthesia apparatus. At RT, use a stereomicroscope to separate males from females. Maintain flies, in groups, in vials containing standard Drosophila fly food, not exceeding 40 flies per vial.
3. Two hours before the beginning of the experiment, ensure that the temperature of the room where the experiment will be performed is between 24–25 °C and the humidity is approximately 50%.
4. Transfer the flies into new vials containing food, and place them for 2 hr on the work surface where the experiment will be performed.
   NOTE: For these experiments, the fly strains used were Drosophila melanogaster strains: Canton-Special or Canton-S (CS), w¹¹¹⁸Cs₁₀ (or W - w¹¹¹⁸ outcrossed 10 times to Canton-S), Oregon and Samarkand flies were all from our laboratory stocks ¹⁶; Elwood flies were collected in Fall 2011 in the Elwood neighborhood of Huntington, on Long Island, New York, USA ⁸. Apart when specified otherwise, the results presented were obtained with Canton-S flies (in shades of red).

3. Performing the Experiment

1. Perform the experiment between 12 p.m. and 3 p.m. (Zeitgeber time — the time in hours after the onset of light — of ZT 4 to 7).
2. Prepare the social space chamber for the transfer of flies.
   1. Place a social space chamber flat on a work surface, with the side containing rectangular spacers closest to your body.
   2. Remove one clip nearest your body and slide one rectangular spacer outwards, creating a gap of approximately 1 cm between the rectangular spacers.
   3. Using tape and a marker, label the social space chamber in an upper corner with sex, strain, and repeat number. Take care to ensure the tape does not cover part of the inner triangular arena.
3. Transfer the flies into the social space chamber.
   1. Transfer flies from their vial containing food into a new, empty vial. Aspirate flies from the empty vial and transfer them into the social space chamber.
      1. Inhale to draw the flies into the tip of the aspirator. Place the tip into the 1 cm gap between the rectangular spacers of the social space chamber and exhale at a consistent rate to force the flies into the inner triangular arena.
      2. Immediately slide the rectangular spacer back into place, closing the base of the inner triangular arena, and place the binder clip back on.
4. On a pounding pad, which is located on different bench than where the experimental work takes place, hold the social space chamber upright such that the rectangular spacers are on the bottom side. Pound three times to ensure all flies have fallen to the bottom of the arena.
   NOTE: Due to the acrylic spacer sticking out of the smallest chamber size (Figure 1D), bang elbows on work surface while maintaining a secure hold on apparatus to ensure the flies have fallen to the bottom.
5. Start a timer.
6. Place the social space chamber on the work surface and use the support stand and flask clamp to keep it upright. Place a ruler or a sticker of known length flat against the social space chamber, but not covering any part of the inner triangular arena.
   1. For horizontally positioned experiments, do not place the social space chamber in the support stand; instead lay the social space chamber flat on the work surface.
7. When the flies have settled, usually after around 30 min, take a picture of the social space chamber. Ensure that the frame contains the entire inner triangular arena, the ruler, and the label.
8. Repeat the experiment for each genotype and condition (ideally 3 internal replicates and 3 independent repeats).

4. Analyzing the Social Space Data

1. Import the following Macros in ImageJ ¹⁷
   1. ImageJ is available here: http://rsbweb.nih.gov/ij/.
   2. You can create a 'Measure Distances' command in the Plugins menu by saving the macros below in a file named 'Measure_Distances.txt' in the ImageJ/plugins folder, in the macro subfolder, and using the Help>Update Menus command.'
   3. Copy in a .txt file the macros that are provided in supplementary data and were originally published in ³.
2. Upload the pictures to a computer, and open one picture using ImageJ.
   1. Create a scale by drawing a line from the 0 cm to 1 cm marks on the ruler in the picture, and under the Analyze tab, choose the Set Scale feature to set a scale of 1 cm for that distance (same approach for the sticker of known length). Choose the Global option before applying the scale to the picture.
   2. Crop the picture using the Crop feature under the Image tab such that it includes all of the flies, while removing as much of the rest of the picture as possible.
   3. Make the picture black and white by choosing 8-bit from the Type option under the Image tab.
   4. Remove all background noise from the picture by going to the Threshold feature under the Adjust option of the Image tab. Drag the sliders to enhance or remove the contrast so that the body of each fly can be clearly seen without any other markings in the image. If there are markings that are not flies, capture them with the rectangular tool and delete them.
   5. Set the measurements by using the Set Measurements tool under the Analyze tab. Select Area, Center of Mass, Centroid, and Display Label. Next, choose the Analyze Particles feature under the Analyze tab to create a numbered list of all the black spots representing flies.
      1. When using Analyze particles; set size 0.01-0.1 (do NOT choose pixel unit); circularity 0.00-1.00; show outlines; display results, and add to manager.
      2. Ensure that each black spot (each particle) is an accurate representation of the flies that are in the original picture, by comparing the numbered list to the original picture.
         NOTE: If some flies that are very close together being counted as one particle, manually draw a white line to separate the two flies on the binary image.
   6. While the list is selected, use the Nearest Neighbor — List Distances macro under the second Macros option in the Plugins tab to create a new list containing the distances, in cm, from each fly to their nearest neighbor.
3. Copy the nearest neighbor distances from ImageJ and paste into a column of a spreadsheet program.
   1. Compile all of the data from each repeat onto the same spreadsheet and organize by genotype and condition.
4. Perform data analysis using statistical software. In this experiment, Graph Pad Prism (version 6 for MacOSX, GraphPad Software, San Diego California USA, www.graphpad.com) was used to conduct one-way ANOVA, and Kruskal-Wallis Tests, Tukey’s and Dunnet’s Post-Hoc Tests.
5. The distribution of the distances follows a non parametric distribution ¹, and data are represented as box and Tukey’s whiskers (to eliminate outliers).

5. Climbing Assay (Control Behavior)

1. Use a climbing assay as a control to test flies climbing ability ^14,15,18.
   1. Use the countercurrent apparatus such that three trials can be run in parallel.
2. Transfer 50 to 100 naïve flies (flies that have not been tested before) in three different test vials, and snap them into the 1^st, 3^rd and last slots at the bottom of countercurrent apparatus, with fresh empty vials in the opposite locations.
3. Place fresh, empty vials in the empty slots at the bottom of the apparatus.
4. Tap down the apparatus three times, such that all the flies are at the bottom of each tube, to initiate the experiment.
5. Start the timer for 15 sec (time sufficient for ~100% of 3–7 days old Canton-S to reach the top vial — see Figure 3F).
6. Slide the upper portion of the counter-current apparatus, to displace the top vials by one slot.
7. Collect flies by tapping the apparatus down, to bring all flies in the bottom vials.
   1. 1^st and 2^nd tubes (or 3^rd and 4^th, 5^th and 6^th) correspond respectively to the flies that did not reach to top and the flies that reached the top vial.
8. Calculate the Performance Index (PI).
   1. Count the number of flies in each vial.
   2. The PI is the percentage of flies able to climb on the upper tube.
   3. The means of the PI follow a normal distribution¹, and data are represented as column graphs of mean plus or minus standard error to the mean (SEM).
      NOTE: Alternatively, the PI can be calculated as the percentage of flies able to reach the upper part of the lower vial (for older flies, 5 sec is the time sufficient to quantify a difference in climbing ability between infected and non infected 12 days old Canton-S quantified from a still picture: see results). Others have used different measures of performance in startle-induced negative geotaxis, that are also appropriate to quantify climbing ability ^19-22.

Results

The social space chamber can be used as a tool to quantify the social behavior of Drosophila melanogaster. Acrylic spacers and glass panes are clipped together to form an inner triangular arena that provides a two-dimensional area in which flies can form stable groups without the presence of many potentially confounding cues. When flies are transferred to the vertical arena, they are startled by being tapped down, and they respond by an escape behavior: negative geotaxis. They climb to the vertex of the upright ...

Discussion

In this protocol, a detailed procedure for the quantification of social space was described. Some crucial steps to ensure the experiment is successful are: 1) always use gloves when cleaning and setting up the apparatus, to keep your own oils and scents off the inner chamber of the apparatus, 2) ensure flies are collected at least one day before the experiment to reduce any potential effects of cold anesthesia, 3) 2 hr before the experiment provide new vials containing fresh food to ensure the flies are not starved, and ...

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
Jazz-mix	Fisher	33545	any other standard drosophila food would work
Mini-Alarm Timer/Stopwatch
Sharpie pens
Adhesive Tape
Medium size binder clips	Staples		to hold the chambers together: 1-1/4" (32mm) medium clips with 5/8" capacity
small SupportStands	Carolina	707161	to hold the chambers in a vertical orientation
Buret clamps	Carolina	707362
Digital Camera	Nikon	Coolpix S8000	to take the still pictures
small ruler			to be able to scale the picture
trifold board and white bench cover			to provide a white background, and a homogeneous light.
pounding pad			any mouse pad works.
Prism 6	GraphPad Software Inc.	Prism 6 for Mac OS X	Any statistical analysis software with t-test, one-way and two ANOVA would work