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 ^....

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 t....

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