Drosophila Egg-Laying Preference Assay: A Method to Test Decision-Making in Flies

Overview

Drosophila females choose between substrates and select sites that are attractive for egg-laying. This video describes a behavioral assay that tests their decision-making, called the egg-laying preference assay. The featured clip demonstrates the procedure with a custom-built setup, which enables researchers not only to test the behavior of many females at the same time but is also compatible with video recording and optogenetic setups.

Protocol

This protocol is an excerpt from Gou et al., High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster, J. Vis. Exp. (2016).

1. 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.

Representative Results

Figure 1: Egg-laying Chambers and Egg-laying Protocol. (A) Fully assembled egg -laying chamber. (B) Disassembled pieces. a: the loading piece (top), b: the divider piece (middle), c: the substrate (bottom) piece. Engineering drawings of these pieces are shown in Supplementary Figure 1-3. (C) The loading piece of the chamber. a': sliding doors, a": rails. The plastic sheets are inserted into the loading piece to serve as a floor to keep the loaded flies in place. We typically put color tapes onto the edge of the plastic sheets (red arrow). (D) The substrate (bottom) and the divider (middle) pieces of the chamber. Agarose is deposited into individual troughs to serve as egg-laying substrates (arrows). Quadrilateral outlines the egg-laying arena for a single fly. (E) Day 0 of collected females/males in a yeasted vial. (F) Day 4 - 5 of collected females/males in a yeasted vial. Note that larvae and adult females have eaten most of the yeast and the surface of the food has become occupied by larvae. The wet surface food crawling with larvae prevents females from laying more eggs in the vial. (G) Schematic depicting the protocol for setting up egg-laying behavior experiments. Please click here to view a larger version of this figure.

Figure 2: Representative Egg-laying Results for Flies Choosing between Sucrose and Plain Substrates. (A) Side view of egg-laying results of wild type flies when they were given a choice between sucrose (150 mM) and plain substrates. White box outlines one egg-laying arena for a single fly. (B) Top view of egg-laying results. White box outlines one egg-laying arena for a single fly. (C) Preference index (PI) of wild type flies when asked to choose between a sucrose-containing substrate and a plain substrate. PI for each female is calculated as follows: (number of eggs on sucrose substrate - number of eggs on the plain substrates)/total number of eggs. Error bar indicates SEM. Please click here to view a larger version of this figure.

Supplementary Figure 1: Engineering Drawings for the Divider and the Substrate Piece of the Chamber. (A-A") Different views of the substrate piece of the chamber. (B-B') Different views of the middle divider piece of the chamber. Please click here to view a larger version of this figure.

Supplementary Figure 2: Engineering Drawings for the Loading Piece of the Chamber. (A-A') Different views of the upper layer of the loading piece of the chamber. (B-B') Different views of the bottom layer of the loading piece of the chamber. Please click here to view a larger version of this figure.

Supplementary Figure 3: Engineering Drawing for the Sliding Door and the Rail on the Chamber. (A) The rail fixed onto the top piece of the chamber. (B) The sliding door for the chamber. Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
UltraPure Agarose	Invitrogen	16500-500
Sucrose	Sigma	S0389
Water bath	Fisher	15-462-6Q
Egg-laying chambers	Custom Built
Fly vials (narrow)	Genesee	32-116BC