Quantification of Drosophila Grooming Behavior

Summary

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

Abstract

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

Introduction

Grooming in Drosophila melanogaster (D. melanogaster ) is a robust innate behavior that involves the coordination of multiple independent motor programs¹. Fruit flies clean their bodies of dust, microbes, and other pathogens which could inhibit normal physiological function such as vision and flight, or lead to significant immune challenges. In sensing and responding to both mechanical² and immune activation³, flies repetitively rub their legs together or on a targeted body region until it is sufficiently clean and grooming progresses to another part of the body. F....

Protocol

1. Preparation

1. Prepare an aspirator for moving live Drosophila from a culture vial to the grooming chamber. Aspirators allow transfer of conscious animals to the behavioral chambers to ensure that anesthesia does not affect subsequent behavioral observation.
   1. Using scissors cut 1.5 feet of tygon tubing ID ⅛", OD ¼".Cut at least 1 inch off of the tip of a 1 mL disposable micropipette tip. Hold a 1 cm square piece of mesh (opening 0.196 inch) over the cut tip.
      NOTE: Most 1mL tips have a gradation line where the tip sits in the package box, typically cut to that line.
   2. Snugly place a fr....

Results

The grooming assay yields quantitative data to assess behavioral performance based on the relative remainder of accumulated dye left on the bodies of flies after a set time of measurement for grooming (30 min). Sample images of the sliding grooming chamber design and major steps of the assay are highlighted in Figure 1. Flies aggregate a significant amount of dye from immediate dusting by vortexing in the presence of dye (Figure 2d, 2e). Dusted flies can .......

Discussion

The grooming assay is relatively straightforward, but we would caution experimenters to pay special attention to the following issues. Maintaining a tight seal by tightening the screws on the top and bottom plates after introducing flies and dye is essential for reproducible results. The Brilliant Yellow dye is very fine and loose joints will allow losses of dye from the edges of the chamber. The irregularity in dye content for each well could easily throw off grooming quantification as non-uniform dusting will increase .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
High-Flex Tygon PVC Clear Tubing	McMaster-Carr	5229K54	ID 1/8", OD 1/4", used with micropipettor tips and mesh to construct mouth aspirators
Micropipette tips (1 mL and 200 μL)	Genesee Scientific	24-165, 24-150R
Nylon Mesh Screen, 2 x 2.6"	McMaster-Carr	9318T44	Used to construct grooming chamber and mouth aspirators
Dumont #5 Forceps	Roboz Surgical Instrument	RS-5050
Brilliant Yellow Dye	Sigma-Aldrich	201375-25G	we recommend use of nitrile gloves while handling this product
Vortexer	Fisher Scientific	12-812	set to "touch"
Ethanol	Carolina Biological Supply	86-1282
1.5 mL microcentrifuge tubes	VWR International	10025-726
0.65 mL microcentrifuge tubes	VWR International	20170-293	tubes can be reused with successive assays
UV 96-well plate	Corning	26014017
BioTek Synergy HTX Platereader	BioTek	need to download catalog to access product number	http://www.biotek.com/products/microplate_detection/synergy_htx_multimode_microplate_reader.html?tab=overview
Gen5 Microplate Reader and Imager Software	BioTek
Microsoft Excel	Microsoft		https://www.microsoftstore.com/store/msusa/en_US/pdp/Excel-2016/productID.323021400?tduid=(65d098c0e83b86c952bdff5b0719c83f)(256380)(2459594)(SRi0yYDlqd0-LI..ql4M2LoZBEhcBljvIA)()
Drosophila Incubator	Tritech	DT2-CIRC-TK
1/4" acrylic plastic	McMaster-Carr	8473K341
8 - 32 nuts	McMaster-Carr	90257A009
8 - 32 x 1" hex cap screws	McMaster-Carr	92185A199	the bottom plate needs to be tapped for this size screw
8 - 32 x 1/2" hex cap screws	McMaster-Carr	92185A194	the second plate from the top needs to be tapped
2 - 56 3/8" flat head phillips machine screws	McMaster-Carr	91500A088	these hold the two middle plates together
0.175" ID, 1/4" OD, 0.34" aluminum pipe	McMaster-Carr	92510A044	Manufactured in-house; product listed is approximately the same dimensions and should work for size 8 screws.  These act as sheaths for the 1" screws and set the hex cap up slightly from the surface of the top plate