Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Summary

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

Abstract

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Introduction

Some of the morphological traits of Drosophila are diversified among species^1,2,3,4,5. We can approach the question of how morphological diversity arises by comparing the mechanisms of generation of these morphologies. Examples of such morphologies are larval trichomes, adult sex combs, external genital apparatus, abdominal pigmentation, and wing pigmentation^6,7,8,9,....

Protocol

1. Fly stock

1. Use Drosophila guttifera for all of the following protocols.
2. Use plastic vials (diameter 25 mm x height 96 mm) and cellulose plugs (diameter 23 mm x height 26 mm) for stock maintenance. Use a standard cornmeal/sugar/yeast/agar food and follow a publication described three other alternative recipes for this species².
   NOTE: D. guttifera (stock number 15130-1971.10) is provided by the Drosophila Species Stock Center at the University of California, San Diego. Although D. guttifera belongs to the immigrans-tripunctata radiation, which is distantly related to ....

Results

The pupal period of D. guttifera is divided into 17 stages (P1 - P15(ii); images of three representative stages (P1, P5 - 6, P10) are shown Figure 3, and all 17 stages are illustrated in Figure 4). Although Bainbridge and Bownes¹⁷ recognized 20 stages in D. melanogaster, some of these stages could not be applied to D. guttifera. The order of two developmental events, the appearan.......

Discussion

We describe here the protocols for definition of pupal stages, removing the puparium for detailed observation, measuring durations of pupal stages, and measurement of intensity of black spots on a wing in D. guttifera. These protocols can be applied for many Drosophila and related fly species, especially species with wing pigmentation.

In-depth observation and description of more detailed developmental events would enable further subdivision of stages. In many cases, a develo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drosophila guttifera	The Drosophila Species Stock Center at the U.C. San Diego	15130-1971.10	Drosophila guttifera, a fruit fly species used in this article
Plastic vial	Hightech	MKC-30	Plastic vial, for fly stock maintenance
Buzz plugs vial and bottle closures for glass vials	Fisher Scientific	AS-271	Cellulose plug, for fly stock maintenance
White soft sugar	Mitsui Sugar	J-500g	White soft sugar, for standard cornmeal/sugar/yeast/agar food
Corn flour	Nippon Flour Mills	F	Corn flour, for standard cornmeal/sugar/yeast/agar food
Corn grits - C	Nippon Flour Mills	GC	Corn grits - C, for standard cornmeal/sugar/yeast/agar food
Agar powder	Matsuki Kanten Sangyo	No.602	Agar powder, for standard cornmeal/sugar/yeast/agar food
Dry beer yeast	Asahi Food & Healthcare	Y2A	Dry beer yeast, for standard cornmeal/sugar/yeast/agar food
Butyl p-hydroxybenzoate	Nacalai Tesque	06327-02	Butyl p-hydroxybenzoate, for standard cornmeal/sugar/yeast/agar food
Ethanol	Wako	057-00456	Ethanol, for standard cornmeal/sugar/yeast/agar food
Flat bottom microtube	Ina Optica	CF-0150	1.5 mL microtube, for collecting pupae
CAPSULEFUGE	Tomy	PMC-060	Mini microcentrifuge, for collecting pupae
Sterilized Schale NB	Sansei Medical	01-013	Plastic Petri dish (diameter 90 mm x height 15 mm)
Serum tube rack	Iwaki	9796-050	Used as a moist chamber, for observation of pupa
Corning Falcon Easy-Grip tissue culture dish	Corning	353001	Plastic Petri dish (diameter 35 mm x height 10 mm)
Falcon standard tissue culture dish	Corning	353002	Plastic Petri dish (diameter 60 mm x height 15 mm)
Push-pin	Kokuyo	51233709	Push-pin, for making pinholes on the microtube lid
Stereomicroscope	Olympus	SZX16	Stereomicroscope, for morphological observation
Digital camera	Olympus	DSE-330-A	Digital camera, for imaging
NICETACK double sided tape	Nichiban	NW-15SF	Double sided tape, for removing puparium
Dumont #5 forceps	Fine Science Tools	11252-20	Forceps, for removing puparium
Van Gogh VISUAL Paint brush	Talens Japan	GWVR-#5/0	Paint brush, for removing puparium
Greiner CELLSTAR 12 well cell culture plate	Merck	665-180	12-well cell culture plate, for measuring durations of pupal periods
NaCl	Wako	191-01665	NaCl, for PBS
KCl	Nacalai Tesque	285-14	KCl, for PBS
Na2HPO4·12H2O	Wako	196-02835	Na2HPO4·12H2O, for PBS
KH2PO4	Nacalai Tesque	28721-55	KH2PO4, for PBS
Stepped Neutral Density (ND) Filter 0.04 - 3.0	Edmund Optics	64-384	Stepped density filter, for calibration of pigmentation measurement
ImageJ software	NIH	1.8.0-101	ImageJ software, for measurement of intensity of black spots on a wing (https://imagej.nih.gov)
FINE FROST glass slide	Matsunami Glass Ind	FF-001	Glass slide, for measurement of intensity of black spots on a wing
Square microscope cover glass 18 x 18	Matsunami Glass Ind	C018181	Cover slip, for measurement of intensity of black spots on a wing