A Single-fly Assay for Foraging Behavior in Drosophila

Summary

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.

Abstract

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.

Introduction

States of hunger promote two types of appetitive behaviors: food search and food consumption¹. This simple behavioral assay is useful for the study of chemotactic behaviors associated with foraging^2,3. Specifically, it tracks fly position, walking speed and latency to locating a food odor target. Latency of food finding serves as metric for measuring changes in the sensitivity of the fly's odor detection system downstream of changes in its internal appetitive state. A manual version of this assay was previously used to show GABA-B receptor signaling is important for odor localization behavior in adult flies³. The current automated ....

Protocol

1. Fly Collection and Starvation

1. Rear the experimental flies under controlled temperature and humidity conditions (e.g. 21 °C, 50-60% relative humidity) on a 12-hr light/dark cycle.
2. Collect female flies on the day of eclosion and place them, along with 4-5 males, into new food vials (maximum 30 per vial). Age flies 2-5 days.
3. Prepare the chambers for fly starvation.
   1. Push a single tissue (4.8 x 8.4 inch) down to the bottom of an empty plastic vial. Completely soak the tissue with distilled water. Use an object to push down on the tissue and gently squeeze out excess water.
   2. Invert the vial to discard th....

Results

The data analysis software and the layout, an example of which can be seen in Figure 1, are used to evaluate each fly's performance during its 10 min trial according to a set of analysis criteria. The following criteria are used to determine whether data from each fly will be used for data analysis and are designed to eliminate those flies that are unable to perform the food search task due to injury, illness, stress, or lack of motivation.

Flies that are inactive for more tha.......

Discussion

In this protocol, we describe a step-by-step procedure for the food search behavior assay. In addition to food related odors, it may also be adapted for the study of the fly's ability to locate other odor objects. For example, it may be applied toward the study of mate localization behavior in male flies³ .There are several additional considerations for this protocol that we will mention here regarding this procedure:

First, the rearing temperature determines how long experiment.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Apple Cider Vinegar	Spectrum		commercially available
Agarose, Type VII	Sigma-Aldrich	A0701	low gelling temperature agarose
Acrylic Testing Plate		custom	Plate contains 6 arenas. Each arena is 60 mm in diameter 6 mm in height. See testing plate diagrams for specific measurements.
LabVIEW V.8.5	National Instruments	776670-09	platform for programs: PositioningTool.vi, FlyTracking--Six Zones.vi NOTE: "elapsed time.vi", "time into file.vi", and "two object detect.vi" are included subroutines that must be available in order for the main data acquisition program "FlyTracking--Six zones.vi" to run.
LabVIEW Vision 8.5
LabVIEW Vision Acquisition Software 8.5
LabVIEW Vision Builder AI 3.5
Igor Pro V.6	Wavemetric, Inc.		platform for macro: Data Analysis for Fly Tracking--Six Zones
Basler scA1390-17fm	National Instruments	779980-01	Digital Camera NOTE: driver for camera available at Baslerweb.com
8 mm lens	National Instruments	780024-01	Lens for Basler Digital Camera
Ground Glass Diffuser Plate	Edmund Optics	custom	Diffuses light, 25 cm x 30 cm
US Std. No. 100	Fischer Scientific	04-881X	Sieve with nominal opening of 150 μm
Lighting Option 1
LED backlight 660 nm (20 cm x 20 cm)	Spectra West	BL47192	a simpler but more expensive lighting option.
Power Supply for LED Backlight	Spectra West
Lighting Option 2
660 nm LEDs	Superbrightleds	RL5R1330	Wavelength 660 nm (approximately 7 x 7 LED array for a 14.7 inch x 9.75 inch panel)
Linear DC Power Supply	GW Instek	GPS-1830D	Power supply for LED Panel
Solderless Breadboard	Digikey	922354-ND	Breadboard for LEDs