High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Summary

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Abstract

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the "greater of two goods." However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

Introduction

Drosophila melanogaster is a powerful genetic model organism to study the neural basis of behaviors. The rapid developments of genetic tools to manipulate neurons in a targeted manner and the emergence of sophisticated behavioral analysis tools have significantly improved our ability to dissect the circuit mechanisms that underlie the sensory-motor transformation processes of several innate and learned behaviors^1-3.

Drosophila egg-laying is a suitable model to study the neural basis of simple decision making processes. In particular, Drosophila females have been shown to possess the ability to compare....

Protocol

1. Preparing Flies to be Assayed

1. Culture flies on standard molasses/cornmeal media at incubator set at 25 °C and 65% humidity. Take care to not overcrowd the vials. For example, put 8 females and 6 males into a narrow food vial.
   Note: The "narrow food vial" used here has an inner diameter of 2.3 cm. We typically dispensed about 10 ml of fly food into each vial. The fly food recipe we used is described here: http://flystocks.bio.indiana.edu/Fly_Work/media-recipes/molassesfood.html.
2. 2 - 3 days after females eclosion, prepare vials with fresh yeast paste as shown in Figure 1E. Make fresh yeast paste by mixing 6 g....

Results

The egg-laying chambers are composed of several pieces: a substrate (bottom) piece, a divider (middle) piece, a loading (top) piece, and 2 sliding doors (Figure 1A - D). These pieces are used to independently setup flies and substrates before egg-laying experiments. Figure 1F shows how vials should look when female flies are ready for egg-laying. When flies are given a choice between a plain substrate and a sucrose-containing substrate, females robustly p.......

Discussion

The chambers and protocols described here have several improvements over previous egg-laying assays. First, they increase the throughput of assaying preferences of single animals significantly. Each chamber can assay 30 single females and it takes less than an hour to set up. Second, they increase the consistency of the egg-laying preferences over previous methods. The standardization of the dimensions of the arena, size of the egg-laying substrates, and distance between substrates makes it easier to compare the results .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
UltraPure Agarose	Invitrogen	16500-500
Sucrose	Sigma	S0389
Water bath	Fisher	15-462-6Q
LifeCam Cinema webcam	Microsoft	H5D-00013
Red LEDs	Cree	C503B-RAN-CA0B0AA1
Egg-laying chambers	Custom Built
Camera holders	Custom Built
LED holders	Custom Built
Fly vials (narrow)	Genesee	32-116BC