A Buoyancy-based Method of Determining Fat Levels in Drosophila

Summary

Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

Abstract

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

Introduction

Drosophila melanogaster has been used for over a century in the study of genetics and other basic biological questions. In the last few decades, it has become clear that Drosophila is a powerful tool in the study of many human diseases. As 70 - 80% of genes associated with human diseases have an identified fly ortholog^1-4, Drosophila provides a simplified yet translatable system in which to study complex diseases. Metabolism in particular has benefited from such study. Not only are the genetics of metabolism well conserved between flies and humans, but the relevant organs and cell types are also very similar<....

Protocol

1. Collect Eggs from Flies with Genotypes of Interest

NOTE: Ideal crosses consist of 150 virgin flies and at least 75 males. Stock collections should consist of at least 200 flies.

1. Prepare grape plates.
   1. Add 37.5 g agar to 1.5 L distilled water in a 4 L flask.
   2. Autoclave water/agar mix for 50 min at 121 ºC (250 ºF).
   3. While water/agar mix is autoclaving, add 50 g sucrose to 500 ml grape juice and stir with a stir bar on a heated stir plate until sucrose is dissolved.
   4. Turn off the heat and continue stirring to cool the grape juice/sucrose mixture to below 70 ºC. Test the internal temperature wit....

Results

Figure 1 represents an example of how the buoyancy-based assay can detect both higher and lower fat stores in genetically manipulated larvae. Figure 1A shows that excitation or silencing of a certain subset of neurons (E347) in the larval brain induces lower and higher fat stores respectively. The same genetic background control was used in both cases. Figure 1B provides an example of how this phenotype obtained by the density assay is co.......

Discussion

There are a multitude of techniques that have been developed to measure lipid levels^8-10. However, each of these methods comes with some major drawbacks that are addressed by the buoyancy-based assay outlined above. First, this assay is extremely quick. Testing a full concentration gradient takes no more than 30 - 60 min. This is a huge improvement on most of the techniques currently in use. For example, lipid quantitation by MS takes 7 - 9 hr to isolate the lipids and another several hou.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacto Agar	VWR	214030
Welch's Original 100% Grape Juice
Sucrose	Fisher	S512
Tegosept	Genesee Scientific	20-259
Ethanol			200 proof
Baker's yeast	Fleichmann's
Yellow Corn Meal	Quaker		Enriched degerminated
Drosophila Agar Type II	Apex	66-104
Soy Flour	ADM Specialty Ingredients	062-100
Malt Extract	Breiss	5748
Light Corn Syrup	Karo
Propionic Acid	VWR	U330-09
Sodium chloride	Fisher	50147491
Potassium phosphate monobasic	Sigma	P5655-100G
Sodium phosphate anhydrous	Fisher	S3933
Potassium chloride	Fisher	P330-500
35 x 10 mm petri plate	Fisher	08-757-100A
50 ml Conical	Fisher	50-869-569