Quantifying Abdominal Pigmentation in Drosophila melanogaster

Summary

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Abstract

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Introduction

Pigmentation shows enormous phenotypic variation between species, populations, and individuals, and even within individuals during ontogeny^1,2,3,4,5,6. Although there are myriad studies of pigmentation in a wide variety of animals, pigmentation has perhaps been best studied in Drosophila melanogaster, where the full power of molecular genetics has been used to elucidate the developmental and physiological mechanisms that regulate pigmentation and how these mechani....

Protocol

1. Specimen Mounting

NOTE: Store dead flies in 70% ethanol in water prior to imaging.

1. Pour 10 mL of 1.25% agar dissolved in boiling water in a 60 mm x 15 mm Petri dish and allow it to set.
2. Under a dissecting microscope, use a pair of fine-point forceps to make a ~20 mm ong, 2 mm wide, 1 mm deep groove in the surface of the gel. Using fine forceps, embed the ventral side of an adult fly in the groove, with the dorsal side of the fly projecting above the gel.
   NOTE: The looseness of the gel allows for easy repositioning without damaging the specimen. The same groove can be used for multiple specimens, although it w....

Results

The protocol was used to explore the effect of rearing temperature on abdominal pigmentation. Previous studies have shown that an increase in developmental temperature results in a decrease in the spread of abdominal pigmentation in several species of Drosophila, including D. melanogaster^30,32. Specifically, in abdominal tergites 3 and 4, the extent of pigmentation (width of the pigment band) decreases from 17 °C.......

Discussion

This methodology allows for the precise, rapid, and repeatable acquisition of pigmentation data in a quantitative form suitable for multiple downstream analyses. The method has been used to acquire data on the effect of temperature on abdominal pigmentation in an isogenic line of flies. However, the methodology could be used in forward-genetics studies to identify genes that underlie pigmentation differences between individuals, populations, or species, or reverse-genetic studies to explore the effects of specific genes .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dumont #5 Biology Forceps	FST	11252-30
Agar	Sigma-Aldrich	5040
Dissecting Scope	Leica	MZ16FA
Base	Leica	MDG41
Camera	Leica	DFC280
Gooseneck Cold Light Source	Schott	ACE 1
Image Acquisition Control Software	Micro-Manager v1.3.20		https://micro-manager.org/
Image Analysis Software	ImageJ		https://imagej.nih.gov/ij/
Data Analysis Software	R 3.3.2		https://www.r-project.org/
LED	Thor Labs	LEDWE-15
Multimeter	Fluke	Fluke 75 Series II
60 mm x 15 mm Petri dish	Celltreat Scientific Products	229663
Stage micrometer	Klarman Rulings, Inc.	KR-867