A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

Summary

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

Abstract

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

Introduction

Reproductive behaviors typically have high energy costs, especially for females, who produce larger gametes than males and must carefully chose the conditions to raise their developing offspring. Because of the energy cost, it is not surprising that reproduction is connected to nutritional conditions. This is true in most, if not all, animals including mammals, whose puberty can be delayed by malnutrition, and whose sexual drive can be negatively affected by food-restriction¹.

The reproduction of the genetic model organism Drosophila melanogaster is also affected by nutritional conditions. Males court at hig....

Protocol

1. Environmentally Controlled Mating Box

1. To ensure a controlled and easy to clean test area, setup a stainless-steel kitchen cabinet of 120 cm x 64 cm x 85 cm as illustrated in Figure 1A.
   1. Drill one hole at the back of the cabinet just below the ceiling and four sets of four holes into the sides, each with a diameter of 2 cm. Drill the first two sets of four holes, on each side of the box at a height of 7 cm from the bottom of the box and with 12.5 cm in between holes. Drill the other two sets on each side of the box at a height of 35 cm from the bottom.
      NOTE: The four sets of four holes are used for camera power cables ....

Results

Using this continuous assay, mating behavior, and mating frequency in specific, can be determined under experimental environmental conditions. To control environmental conditions, we transformed a stainless-steel kitchen cabinet into a test area, with its own light source and diffusion, which ensures a high abundance of light and a minimum amount of glare from the top of the mating arenas (Figure 1A). The inner test area is completely encased by stainless steel and glass,.......

Discussion

This protocol describes an assay to test mating behavior over 24 h while continuously controlling the environmental cues that a mating couple is hypothesized to use to determine mating frequency. It is possible to increase the mating frequency in response to yeast air delivered through a pressurized air system when the medium contains yeast as well (Figure 2B). Additionally, a similar response in mating frequency can be observed with a simplified food medium containing only agar, peptone, and acetic acid.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cabinet
Stainless steel kitchen cabinet	Horecaworld	7412.0105
White LEDs	Lucky Light	ll-583wc2c-001	Cold white, 20 mAmp and 2 V
Red LEDs	Lucky Ligt	ll-583vc2c-v1-4da	Wavelength between 625 nm, 20 mAmp and 6 V
Resistor	Royal Ohm	CFR0W4J0561A50	560 ohm, 0.25 W, 250 V and 5 % tolerance
Smartphone light meter app	Patrick Giudicelli		Light/Lux Meter FREE, version 1.1.1
Power timer	Alecto	TS-121
Metal brackets			Sharp angle 5 by 5 mm,  2 x 5450 and 1 x 1100 mm long
Frosted glass plate			1190 x 545 x 5 mm
Filter paper sheets	LEE filters	220	White frost
Small fan	Nanoxia Deep silence	4260285292828	80 mm Ultra-Quiet PC Fan, 1200 RPM
Big fan	Nanoxia Deep silence	4260285292910	120 mm Ultra-Quiet PC Fan, 650-1500 RPM
Webcam camera	Logitech	950270	B910 HD WEBCAM OEM, Angle: 78-degree, resolution: 5-million-pixel
Camera software	DeskShare		Security monitor pro
Name	Company	Catalog Number	Comments
Fly rearing
Fly rearing bottles	Flystuff	32-130	6oz Drosophila stock bottle
Flypad	Flystuff	59-114
Wild-type flies			Canton-S
Fly rearing vials	Dominique Dutscher	789008	Drosophila tubes narrow 25x95 mm
Incubator	Sanyo	MIR-154
Magnetic hot plate	Heidolph	505-20000-00	MR Hei-Standard
Agar	Caldic Ingredients B.V.	010001.26.0
Glucose	Gezond&wel	1019155	Dextrose/Druivensuiker
Sucrose	Van Gilse		Granulated sugar
Cornmeal	Flystuff	62-100
Wheat germ	Gezond&wel	1017683
Soy flour	Flystuff	62-115
Molasses	Flystuff	62-117
Active dry yeast	Red Star
Tegosept	Flystuff	20-258	100%
Peptone (bacto)	BD	211677
Acetic Acid	Merck	1000631000	Glacial, 100%
Small petridish	Greiner bio-one	627102	35 x 10 mm with vents
Paraffin film	Bemis NA		Parafilm
Name	Company	Catalog Number	Comments
Yeast and pressurised air set-up
Big petridish	Gosselin	BP140-01	140 x 20.6 mm
Ultrapure water	Millipore corporation		MiliQ
Yeast extract	BD	212750
Agar (pure)	BD	214530	bacto
Glucose (0(+)-glucose monohydrate)	Merck	18270000004
Open caps	Schott	29 240 28	GL45
Silicone septum	VWR	548-0662
Barbed bulkhead fittings	Nalgene	6149-0002
Large PVC tubing			diameter: outer 1.2 cm and inner 0.9 cm
Small PVC tubing			diameters: outer 0.8 cm and inner 0.5 cm
15 ml tube	Falcon
Aquarium pump	Sera precision		Sera air 110 plus, AC 220-240 V, 50/60 Hz, 3 W and pressure >100 mbar
Activated charcoal	Superfish	A8040400	Norit activated carbon
Disposible filter unit	Whatman	10462100
Serological pipettes	VWR	612-1600
Syringe	BD Plastipak	300013
Hot glue	Pattex
Syringe filter	Whatman		FP 30/pore size 0.45 mm CA-S
Name	Company	Catalog Number	Comments
Analysis
Statistics software	R		lme4 package