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 and rank their options before "committing" to depositing an egg onto a given option^4-8. For example, when given only a plain (sucrose-free) substrate or only a sucrose-containing substrate, females readily accept either option for egg-laying. However, when presented with both options, females robust reject the sucrose substrate in some contexts^7,9,10. Relatively little is known about the neural mechanisms that allow females to "choose the greater of two goods", however. A major obstacle has been the lack of an efficient method to assay egg-laying preferences such that one can use a systematic genetic screening approach to study this problem.

In this report, we describe the protocol we developed that allows egg-laying preferences of females to be assayed at single-animal resolution and with substantially improved throughput and consistency over previous methods. Specifically, we provide the blueprints for constructing the chambers we designed, the protocol for preparing the females so that each is primed to lay many eggs, and the protocol for using the chambers.

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 of active yeast with 10 ml of 0.5 % proprionic acid. Use a spatula to apply the yeast paste onto the sidewall of the food vials. Collect 30 - 35 eclosed females together with 20 - 25 males into the vials.
   1. In general, collect the males from the same vials where the females are collected (so as to save time and effort). However, if there is concern about the fertility of males, use WT males instead. Also, yeast paste is important for stimulating egg production therefore prepare it fresh daily.
3. Maintain the collected females/males at 25 °C, 65% humidity, unless the flies are temperature sensitive.
4. After ~4 - 5 days, check the vials to see if females are ready for egg-laying experiments. They are ready when the surface of the food medium is wet from larvae actively burrowing in the food (see Figure 1E - F).
   Note: Females typically withhold laying eggs once the food surface becomes wet and crawling with larvae. This step ensures that females would be ready to lay many eggs when they encounter substrates with desired texture (e.g., ~1% agarose).

2. Chamber Construction, Assembly, Assay Setup

1. Have a machine shop build acrylic egg-laying chambers (Figure 1A - D). The engineering drawing of different pieces were shown in Supplementary Figure 1 - 3. High resolution pictures can also be found here (http://www.rebeccayang.org/pdf/chamber%20design.pdf).
2. Insert plastic sheets into loading (top) piece of the chamber as shown in Figure 1C. This serves as the bottom surface while loading flies into individual egg-laying arenas.
3. Anesthetize females on a CO₂ pad and load them individually into each egg-laying arena. Allow ~30 min for flies to recover from the CO₂ and to become acclimated to the new environment.
4. Prepare the agarose substrates.
   1. For convenience, keep a premade bottle of melted 1% agarose in a 55 °C water bath.
   2. Add the desired amount of stock sucrose solution (2 M) into a 50 ml conical tube and mixing it with appropriate amount of agarose. For example, to prepare the 150 mM sucrose substrate, place 750 µl of 2 M sucrose solution into the tube and then fill the tube with agarose to the 10 ml mark.
   3. Prepare the plain substrate in the same manner but add distilled water instead of sucrose solution.
      Note: The final concentration of agarose in this protocol is slightly less than 1%. In our experience, the exact concentration of agarose does not matter so long as it is controlled to be within ~0.9 - 1.1% and that the two substrates are of the same agarose concentration.
5. Take the substrate (bottom) piece of the chamber and pipette 1,000 µl of agarose substrate into each trough as seen in Figure 1D.
6. Allow agarose to solidify for ~30 min.
7. Once the agarose substrates and flies are ready, assemble all three pieces of the egg-laying chamber and then take out the plastic sheets.
8. Place the chambers in fly incubators.
   Note: Length of egg-laying experiments can vary depending on experimental needs. We typically run the experiment O/N (14 - 16 hr). Also, no significant impact of circadian timing on egg-laying preferences was observed.
9. Anesthetize females by injecting CO₂ into the chamber. Disassemble the chamber, discard the anesthetized flies into fly morgue (i.e., an empty coffee can filled with some corn oil). Take pictures of the results for recordkeeping (see Figure 2).
10. Count the number of eggs manually and calculate preference indices for analysis. Calculate preference index as (N_a - N_b)/(N_a + N_b) where N_a and N_b represent number of eggs on site a vs. site b, respectively.

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