Foraging Path-length Protocol for Drosophila melanogaster Larvae

Summary

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Abstract

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Introduction

Since the discovery of the white gene in Thomas Hunt Morgan's laboratory in 1910, the fruit fly, Drosophila melanogaster (D. melanogaster), has been used as a model for the study of the molecular and physiological underpinnings of various biological processes. The popularity of D. melanogaster largely stems from the considerable quantity and variety of genetic tools. Drosophila's small size, relative ease of handling and short generation time render it an ideal model for genetics studies. Equally important is Drosophila's capacity to demonstrate many of the phenotypes expressed by more complex ....

Protocol

1. Prepare Grape Plates and Holding Bottles for Collection of Larvae

1. To make holding bottles, cut holes in one side of 6 oz fly culture bottles, large enough to fit a fly vial plug for air supply (Fig. 1D).
2. To make grape plates, prepare 250 ml of grape juice medium (1.8% agar, 45% grape juice, 2.5% acetic acid, 2.5% ethanol) by boiling the agar, grape juice and most of the water, cool down to 70 ºC (stir while cooling), then add acetic acid and ethanol and bring up to volume with water.
3. Use a syringe (without needle) to fill 35 x 10 mm Petri dish lids until the surface of the grape juice medium makes a do....

Results

Differences in path-length of the rover and sitter for strains and the effect of food deprivation on path-length are illustrated in Fig. 3. Data collected over three consecutive days of testing showed a significant strain effect (F_(1,421) = 351.89, p < 2.20 x 10^-16; Fig. 3A), with rovers traveling farther than sitters. In addition to the strain effect, there was also a significant food treatment effect (F_{(1, 421)}

Discussion

The path-length assay outlined here offers a robust and simple measure of foraging behavior of Drosophila larvae. The protocol follows the general methodology described in Sokolowski², but has since been improved in regards to efficiency and experimental controls. To the best of our knowledge this method is the only available method for measuring larval path-length. The original version of the path-length protocol^{2, 3, 15, 16} tested larvae on Petri dishes with a thin layer of yeast paste ap.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6 oz fly culture bottles	Fisher Scientific	AS355
Fly vial plugs	Droso-Plugs	59-201
35 x 10 mm Petri dishes	Falcon	351008
100 x 15 mm Petri dishes	Fisher	875712
60 x 15 mm Petri dishes	VWR	25384-168
Dissecting probes	Almedic	2325-58-5300
Yeast	Lab Scientific	FLY-8040-20F