This work was funded by National Science Foundation grants IOS-1256565 and IOS-1557638 to AWS. We thank Patricia Wittkopp and three anonymous reviewers for their helpful comments on an earlier version of this paper.

1. Specimen Mounting
NOTE: Store dead flies in 70% ethanol in water prior to imaging.
<ol>
	<li>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.</li>
	<li>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: T...

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 ...

Pigmentation shows enormous phenotypic variation between species, populations, and individuals, and even within individuals during ontogeny1,2,3,4,5,6. 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 mechanisms evolve1,6. Much is known about the genes that regulate the biochemical synthesis of pigments in D. melanogaster7,8 and the genes that control the temporal and spatial distribution of this biosynthesis9,10,11,12,13. Furthermore, genetic mapping has identified the genetic loci underlying intra- and interspecific differences in pigmentation in D. melanogaster14,15,16,17. The relationships between pigmentation and pleiotropic traits, such as behavior18,19 and immunity19,20, have also been explored, as has the adaptive significance of pigmentation patterns15,21,22. As such, pigmentation in D. melanogaster has emerged as a powerful yet simple model for the development and evolution of complex phenotypes.
Pigmentation in adult D. melanogaster is characterized by distinct patterns of melanization across the body, particularly on the wings and dorsal thorax and abdomen. It is the pigmentation of each cuticular plate (tergite) on the dorsal abdomen, however, that has received the most research attention. There is considerable variation in this pigmentation (Figure 1A-F), because of both genetic17,23 and environmental24,25 factors. The cuticle of an abdominal tergite is made up of anterior and posterior developmental compartments (Figure 1G), each of which can be further subdivided depending based on pigmentation and ornamentation26. The anterior compartment includes six cuticle types (a1-a6), and the posterior compartment includes three (p1-p3) (Figure 1G). Of these, the p1, p2, and a1 cuticle are typically folded under the tergite in un-stretched abdomens so that they are hidden. The reliably visible cuticle is characterized by a band of heavy pigmentation, here referred to as a &#34;pigment band,&#34; comprised of cuticle types a4 (hairy with moderate bristles) and a5 (hairy with large bristles), with the posterior edge of the band more intensely pigmented than the anterior edge (Figure 1G). Anterior to this band is a region of lightly pigmented hairy cuticle, which has bristles posteriorly (a3) but not anteriorly (a2). Variation in pigmentation between flies is observed in both the intensity of pigmentation and in the width of the pigment band. In general, variation is greatest in the most posterior segments (abdominal segments 5, 6, and 7) and is lower in the more anterior segments (abdominal segments 3 and 4)24. Furthermore, there is a sexual dimorphism in D. melanogaster pigmentation, with males generally having wholly pigmented fifth and sixth abdominal tergites (Figure 4C).
In most studies of abdominal pigmentation in D. melanogaster, pigmentation has been treated as a categorical or ordinal trait, with the pattern measured qualitatively27,28,29 or semi-quantitatively on a scale14,15,16,17,24,30,31,32,33,34,35,36,37. These methods inevitably suffer from a lack of precision, and because they rely on the subjective assessment of pigmentation, it is difficult to compare the data across studies. Several authors have quantified the spatial dimensions of pigmentation38,39, the intensity of pigmentation of a particular cuticle type23,25,39,40, or the average intensity of pigmentation across the abdominal tergite as a whole41,42,43. Nevertheless, these quantification methods do not measure both the intensity and the spatial distribution of abdominal pigmentation simultaneously and therefore do not capture the nuances of how pigmentation varies across the abdominal tergite. Furthermore, several of these quantification methods38,41,42,43 require the dissection and mounting of the abdominal cuticle. This is both time consuming and destroys the sample, making it unavailable for additional morphological analyses. As the understanding of the development and evolution of abdominal pigmentation deepens, more sophisticated tools to quickly and precisely measure both the spatial distribution and the intensity of pigmentation will be required.
The overall goal of this method is to utilize digital image analysis to obtain a replicable and more precise measure of the abdominal pigmentation in D. melanogaster. The methodology includes three stages. First, the adult fly is non-destructively mounted, and a digital image of the dorsal abdomen is taken. Second, using an ImageJ macro, the user defines an anterior-posterior strip of pixels that extends from the anterior of the a2 cuticle to the posterior of the a5 cuticle (green box, Figure 1G) on both the third and fourth abdominal segments. The average pixel value across the width of this strip is then extracted along its long axis, generating a profile that captures the spatial distribution and intensity of pigmentation as it changes from the anterior to the posterior of the tergite. Third, an R script is used to describe the pigmentation profile mathematically using a cubic spline. The R script then uses the spline and its first and second derivative to extract the width of the a2-a5 cuticle, the width of the pigment band, and the maximum and minimum levels of pigmentation. The method therefore quantifies both the spatial characteristics and the depth of abdominal pigmentation.
This methodology quantifies the pigmentation of the third and fourth abdominal tergites, which have been the focus of numerous previous studies1,15,23,24,25,28,33,39,42, either exclusively or in combination with more posterior tergites. Although less variable than the fifth and sixth abdominal tergites, the third and fourth tergites are not completely pigmented in males, so this protocol can be applied to both males and females. Nevertheless, as shown here, the protocol can be used to measure pigmentation in the fifth and sixth abdominal tergites in females. Furthermore, minor modifications of the scripts used to extract the characteristics of the pigmentation profile should allow for the method to be used to quantify the variation in pigmentation in a wide variety of other organisms.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dumont #5 Biology Forceps</td>
			<td>FST</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scope</td>
			<td>Leica</td>
			<td>MZ16FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Leica</td>
			<td>MDG41</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Leica</td>
			<td>DFC280</td>
			<td></td>
		</tr>
		<tr>
			<td>Gooseneck Cold Light Source</td>
			<td>Schott</td>
			<td>ACE 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Acquisition Control Software</td>
			<td>Micro-Manager v1.3.20</td>
			<td></td>
			<td>https://micro-manager.org/</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>ImageJ</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Data Analysis Software</td>
			<td>R 3.3.2</td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>Thor Labs</td>
			<td>LEDWE-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimeter</td>
			<td>Fluke</td>
			<td>Fluke 75 Series II</td>
			<td></td>
		</tr>
		<tr>
			<td>60 x 15 mm Petri dish</td>
			<td>Celltreat Scientific Products</td>
			<td>229663</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage micrometer</td>
			<td>Klarman Rulings, Inc.</td>
			<td>KR-867</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying abdominal pigmentation in drosophila melanogaster

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.

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. melanogaster30,32. Specifically, in abdominal tergites 3 and 4, the extent of pigmentation (width of the pigment band) decreases from 17 &#...

Watch this Scientific Journal Video about Quantifying Abdominal Pigmentation in Drosophila melanogaster at JoVE.com

Quantifying Abdominal Pigmentation in Drosophila melanogaster

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.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

The overall goal of this methodology is to rapidly quantify abdominal pigmentation in the fruit fly Drosophila melanogaster. This method can help answer key questions in the field of evolutionary biology such as identifying the genetic loci that underlie in turn in trust-specific variation morphology and understanding the development and evolution of morphological phenotypes. The main advantage of this technique is that it allows users to non-destructively measure various aspects of abdominal pigmentation including the spread and depth of pigmentation.Though this method can provide insight into Drosophila melanogaster's abdominal pigmentation, it can also be applied to other Drosophila species and easily adapted to measure pigmentation patterns in other insects and animal taxa. Prior to imaging, the flies can be stored in 70%ethanol to maintain their pigmentation. Visualization requires a mounting pad.Load a 60 millimeter Petri dish with about 10 milliliters of 1.25%agar in water. Under a microscope, use fine forceps to dig a 20 millimeter long, two millimeter wide, one millimeter deep trench into the gel. Several trenches can be made in one dish.Then embed flies into the trench with the surface for analysis sitting just above the gel surface. In this case, the dorsal side is of interest. Now, fill the dish with 70%ethanol until the flies are completely submerged.This reduces reflections off the cuticle that can obscure the pigmentation pattern. Now, start the imaging software and digital camera connected to the microscope. For a light source, cold light from a double gooseneck device is ample.In the image analysis software, set the image type to 8-bit. Then in the image capture software, toggle the Live setting to open a view window of the camera. Set the image type to Grayscale.Now, set the light source to its maximum intensity and position the lights about 120 millimeters from the stage. Set the microscope to 60 times magnification so the field of view is three millimeters wide. Then place the two millimeter stage micrometer into the field of view and focus on it.Returning to the software, adjust the exposure time to view the micrometer using the field found in the exposure box. Then set the scale. Open the straight line tool and draw the length of two millimeters along the stage micrometer.Then input that length under the Set Scale option. The window should then display the scale in pixels per micron. Take note of the scale and OK the adjustments.Now, return to the image capture software and use the Stop and Snap commands to capture an image of the stage micrometer. Then return to the image analysis software and save the image as a Tiff file. Now, focus on the dorsal midline of a mounted fly.The midline should be positioned straight up for the best imaging results. Push aside the wings and any other obtruding structures and adjust the lighting to best illuminate the cuticle. Next, set the exposure in the image capture software using the pixel value histogram as a guide to capture the full pixel range of the image without saturation, thereby maximizing contrast.Retain this exposure and lighting throughout the experiment. Now, before capturing an image, ensure that the abdominal segments of interest are in view. Then capture an image and save the image with the sample ID number in the filename.Use the format described in the written protocol. This analysis makes use of a macro provided by the authors. The image series to analysis should all be contained within the same file folder.Now, start the macro and follow the prompted commands. First, identify the location of the stored files. Then identify the location for where the analysis data should be stored.Next, input the number of characters that were used in the filenames so the software reads the image series correctly. The next prompt is for the width of the region of interest. Here, input the number of pixels along the anterior-posterior strip that needs to be analyzed.Now, at the prompt, make a choice to analyze the first image of the series. The other options are to analyze the next image in the series or to exit the analysis. The line tool gets automatically selected.Use it to identify the midline on the image by drawing a line along it in the anterior to posterior direction. Next, follow the macro prompts to identify the posterior edge of Tergite4 using the line tool. The line tool is made available to draw the line from the posterior midline edge to the right lateral edge so that the center of the line is just posterior to the posterior edge of the pigment band.Then define the anterior edge of Tergite4 using the line tool. Draw a line from the anterior midline edge to the right lateral edge. The middle of the line defined by a white square should sit at the anterior edge of the lightly pigmented cuticle.With this information, the macro now generates a pigmentation profile. However, the profile might be inaccurate if bristles or other blemishes cross the strip along which the profile is measured. To correct for this, select the Live preview on the histogram window and adjust the line such that the profile is smooth.For the image analysis strip to accurately capture the various aspects of pigmentation, it is vital to identify an analysis region that will have minimal noise caused by image or specimen irregularities. Now proceed to analyze Tergite3 in the same image and then move on to the next image. The described procedure was used to explore a known change in abdominal pigmentation caused by rearing temperature.The third and fourth tergites were examined several times under various lightings and exposures. Various aspects of the pigmentation were explored. And specifically, it was found that the pigmentation bands were wider when flies were reared at cooler temperatures.The calculated values were then compared to general subjective measures made on the same flies by five different observers. In the assessment of 45 female flies, the average observed ranking of the width of the fourth abdominal pigmentation band was tightly correlated with the macro's automated measurement. The same pigmentation band was also manually measured using imaging software.These results were also strongly correlated with measurements generated using the macro. The software was then tested to measure pigmentation in female ebony mutants which are well-known to have increased pigmentation in the third and fourth pigment bands. The method was also used to measure the fifth and sixth abdominal bands of wild type females.The automated technique always provided useful measurements of the pigment density and band size. After watching this video, you should have a good understanding of how to quickly and precisely quantify abdominal pigmentation in Drosophila. Mastering the technique mostly requires the depth use of forceps.Once mastered, the imaging portion of this technique can be done at a rate of 50 specimens per hour. Extracting the pigmentation measures from these images can be done at a pace of 70 images per hour.

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8.8K visualizações.  Lake Forest College. The overall goal of this methodology is to rapidly quantify abdominal pigmentation in the fruit fly Drosophila melanogaster. This method can help answer key questions in the field of evolutionary biology such as identifying the genetic loci that underlie in turn in trust-specific variation morphology and understanding the development and evolution of morphological phenotypes. The main advantage of this technique is that it allows users to non-destructively measure various aspects of abdominal...