We thank Sean B. Carroll and Thomas Werner for providing fly stocks, Naoyuki Fuse for equipment, Byung Seok Jin for his assistance in filming, Kiyokazu Agata for mentoring and Elizabeth Nakajima for English editing. This work was supported by KAKENHI 17K19427 and Takeda Science Foundation.

1. Fly stock
<ol>
	<li>Use Drosophila guttifera for all of the following protocols.</li>
	<li>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 Cali...

<ol>
	<li>Carson, H. L., Hardy, D. E., Spieth, H. T., Stone, W. S., Hecht, M. K., Steere, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+biology+of+the+Hawaiian+Drosophilidae.">The evolutionary biology of the Hawaiian Drosophilidae.</a> Essays in evolution and genetics in honor of Theodosius Dobzhansky. , 437-543 (1970).</li><li>Markow, T. A., O'Grady, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: a guide to species identification and use. , (2006).</li><li>Patterson, J. 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Cell. 34 (5), 520-531 (2015).</li><li>Koshikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancer+modularity+and+the+evolution+of+new+traits.">Enhancer modularity and the evolution of new traits.</a> Fly. 9 (4), 155-159 (2015).</li><li>Koshikawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain+of+cis-regulatory+activities+underlies+novel+domains+of+wingless+gene+expression+in+Drosophila.">Gain of cis-regulatory activities underlies novel domains of wingless gene expression in Drosophila.</a> Proc Natl Acad Sci U S A. 112 (24), 7524-7529 (2015).</li><li>McGregor, A. 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A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+in+the+wing+of+Drosophila:+a+study+of+the+engrailed+gene.">Compartments in the wing of Drosophila: a study of the engrailed gene.</a> Dev Biol. 50 (2), 321-337 (1976).</li><li>Bainbridge, S. P., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> J. Embryol. Exp. Morphol. 66, 57-80 (1981).</li><li>Ashburner, M., Golic, K. G., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Laboratory Handbook. , (2005).</li><li>Fukutomi, Y., Matsumoto, K., Agata, K., Funayama, N., Koshikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pupal+development+and+pigmentation+process+of+a+polka-dotted+fruit+fly,+Drosophila+guttifera+(Insecta,+Diptera).">Pupal development and pigmentation process of a polka-dotted fruit fly, Drosophila guttifera (Insecta, Diptera).</a> Dev Genes Evol. 227 (3), 171-180 (2017).</li><li>Koshikawa, S., Fukutomi, Y., Matsumoto, K., Sekimura, T., Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+guttifera+as+a+model+system+for+unraveling+color+pattern+formation.">Drosophila guttifera as a model system for unraveling color pattern formation.</a> Diversity and evolution of butterfly wing patterns: an integrative approach. , (2017).</li><li>True, J. R., Edwards, K. A., Yamamoto, D., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+wing+melanin+patterns+form+by+vein-dependent+elaboration+of+enzymatic+prepatterns.">Drosophila wing melanin patterns form by vein-dependent elaboration of enzymatic prepatterns.</a> Curr Biol. 9 (23), 1382-1391 (1999).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Izumitani, H. F., Kusaka, Y., Koshikawa, S., Toda, M. J., Katoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeography+of+the+Subgenus+Drosophila+(Diptera:+Drosophilidae):+evolutionary+history+of+faunal+divergence+between+the+Old+and+the+New+Worlds.">Phylogeography of the Subgenus Drosophila (Diptera: Drosophilidae): evolutionary history of faunal divergence between the Old and the New Worlds.</a> PLoS ONE. 11 (7), e0160051 (2016).</li><li>Resh, V. H., Card&#233;, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Insects. , (2009).</li><li>DeLean, A., Munson, P. J., Rodbard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+families+of+sigmoidal+curves:+application+to+bioassay,+radioligand+assay,+and+physiological+dose-response+curves.">Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves.</a> Am J Physiol. 235 (2), E97-E102 (1978).</li><li>Robertson, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metamorphosis+of+Drosophila+melanogaster,+including+an+accurately+timed+account+of+the+principal+morphological+changes.">The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological changes.</a> J Morphol. 59 (2), 351-399 (1936).</li><li>McKinney, M. L., McNamara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Heterochrony: the evolution of ontogeny. , (1991).</li><li>Hardie, D. C., Gregory, T. R., Hebert, P. 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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...

Some of the morphological traits of Drosophila are diversified among species1,2,3,4,5. 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 pigmentation6,7,8,9,10,11,12,13,14,15. To study morphological differences among adults, observation and analysis of the pupal stages are important, because the fate of adult traits is determined in the late larval stages and subsequent morphogenesis proceeds during the pupal period.
In developmental biology studies of Drosophila melanogaster, &#34;hours APF&#34; (hours after pupal formation) is the common method to indicate a pupal stage16. This system employs absolute time after pupal formation and is very convenient for routine experiments. However, developmental speed may differ among pupae, and may be affected by slight genetic, epigenetic or microenvironmental differences, and therefore having the same absolute time after pupal formation does not guarantee that pupae are at the same developmental stage. In many cases, stages defined by morphological features are preferable for comparing multiple individuals. Especially, a comparison between species requires precise staging and comparison among corresponding (homologous) stages.
Bainbridge and Bownes17 recognized 20 pupal stages (P1 to P15(ii)) based on morphological features of Drosophila melanogaster pupae. This staging is the most widely used system of morphological developmental staging18. In a previous study, we performed pupal staging of Drosophila guttifera to establish a basis for wing pigmentation studies19. D. guttifera has a black polka-dot pattern on its wings and is one of the model species for wing pigmentation formation20. Although we referred to the morphological criteria described in the Bainbridge and Bownes&#39; research17, we directly measured stage durations by serial observations19, instead of using Bainbridge and Bownes&#39; estimation of stage durations from observed frequency. Here we describe the method of pupal staging and measurement of durations of pupal stages of Drosophila used in Fukutomi et al19.
To study the developmental mechanism of wing pigmentation, we need to know when in pupal or adult stages the pigmentation occurs. Fukutomi et al.19 quantified optical densities (ODs) of pigmentation during pupal and adult stages by image analysis of wing images. The pigmentation of Drosophila wings is thought to be caused by accumulation of black melanin21. For quantification of ODs, gray-scale images and ImageJ software (https://imagej.nih.gov/ij/)22 were used. To recognize and quantify the spot-specific pigmentation (&#916;OD), we subtract the OD outside of a spot from the OD inside of a spot. To make this method reproducible and objective, the places of OD measurement should be determined using wing veins as landmarks. In this article, we describe in detail this method of quantification of wing pigmentation in Drosophila guttifera.

<table>
	<tbody>
		<tr>
			<td>Drosophila guttifera</td>
			<td>The Drosophila Species Stock Center at the U.C. San Diego</td>
			<td>15130-1971.10</td>
			<td>Drosophila guttifera, a fruit fly species used in this article</td>
		</tr>
		<tr>
			<td>Plastic vial</td>
			<td>Hightech</td>
			<td>MKC-30</td>
			<td>Plastic vial, for fly stock maintenance</td>
		</tr>
		<tr>
			<td>Buzz plugs vial and bottle closures for glass vials</td>
			<td>Fisher Scientific</td>
			<td>AS-271</td>
			<td>Cellulose plug, for fly stock maintenance</td>
		</tr>
		<tr>
			<td>White soft sugar</td>
			<td>Mitsui Sugar</td>
			<td>J-500g</td>
			<td>White soft sugar, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Corn flour</td>
			<td>Nippon Flour Mills</td>
			<td>F</td>
			<td>Corn flour, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Corn grits - C</td>
			<td>Nippon Flour Mills</td>
			<td>GC</td>
			<td>Corn grits - C, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Matsuki Kanten Sangyo</td>
			<td>No.602</td>
			<td>Agar powder, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Dry beer yeast</td>
			<td>Asahi Food &#38; Healthcare</td>
			<td>Y2A</td>
			<td>Dry beer yeast, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Butyl p-hydroxybenzoate</td>
			<td>Nacalai Tesque</td>
			<td>06327-02</td>
			<td>Butyl p-hydroxybenzoate, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Wako</td>
			<td>057-00456</td>
			<td>Ethanol, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Flat bottom microtube</td>
			<td>Ina Optica</td>
			<td>CF-0150</td>
			<td>1.5 mL microtube, for collecting pupae</td>
		</tr>
		<tr>
			<td>CAPSULEFUGE</td>
			<td>Tomy</td>
			<td>PMC-060</td>
			<td>Mini microcentrifuge, for collecting pupae</td>
		</tr>
		<tr>
			<td>Sterilized Schale NB</td>
			<td>Sansei Medical</td>
			<td>01-013</td>
			<td>Plastic Petri dish (diameter 90 mm x height 15 mm)</td>
		</tr>
		<tr>
			<td>Serum tube rack</td>
			<td>Iwaki</td>
			<td>9796-050</td>
			<td>Used as a moist chamber, for observation of pupa</td>
		</tr>
		<tr>
			<td>Corning Falcon Easy-Grip tissue culture dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Plastic Petri dish (diameter 35 mm x height 10 mm)</td>
		</tr>
		<tr>
			<td>Falcon standard tissue culture dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>Plastic Petri dish (diameter 60 mm x height 15 mm)</td>
		</tr>
		<tr>
			<td>Push-pin</td>
			<td>Kokuyo</td>
			<td>51233709</td>
			<td>Push-pin, for making pinholes on the microtube lid</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>Stereomicroscope, for morphological observation</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Olympus</td>
			<td>DSE-330-A</td>
			<td>Digital camera, for imaging</td>
		</tr>
		<tr>
			<td>NICETACK double sided tape</td>
			<td>Nichiban</td>
			<td>NW-15SF</td>
			<td>Double sided tape, for removing puparium</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Forceps, for removing puparium</td>
		</tr>
		<tr>
			<td>Van Gogh VISUAL Paint brush</td>
			<td>Talens Japan</td>
			<td>GWVR-#5/0</td>
			<td>Paint brush, for removing puparium</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR 12 well cell culture plate</td>
			<td>Merck</td>
			<td>665-180</td>
			<td>12-well cell culture plate, for measuring durations of pupal periods</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>NaCl, for PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Nacalai Tesque</td>
			<td>285-14</td>
			<td>KCl, for PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;12H2O</td>
			<td>Wako</td>
			<td>196-02835</td>
			<td>Na2HPO4&#183;12H2O, for PBS</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Nacalai Tesque</td>
			<td>28721-55</td>
			<td>KH2PO4, for PBS</td>
		</tr>
		<tr>
			<td>Stepped Neutral Density (ND) Filter 0.04 - 3.0</td>
			<td>Edmund Optics</td>
			<td>64-384</td>
			<td>Stepped density filter, for calibration of pigmentation measurement</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td>1.8.0-101</td>
			<td>ImageJ software, for measurement of intensity of black spots on a wing (https://imagej.nih.gov)</td>
		</tr>
		<tr>
			<td>FINE FROST glass slide</td>
			<td>Matsunami Glass Ind</td>
			<td>FF-001</td>
			<td>Glass slide, for measurement of intensity of black spots on a wing</td>
		</tr>
		<tr>
			<td>Square microscope cover glass 18 x 18</td>
			<td>Matsunami Glass Ind</td>
			<td>C018181</td>
			<td>Cover slip, for measurement of intensity of black spots on a wing</td>
		</tr>
	</tbody>
</table>

methods for staging pupal periods and measurement of wing pigmentation of drosophila guttifera

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.

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...

Watch this Scientific Journal Video about Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera at JoVE.com

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

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.

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

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

The overall goal of this experiment is to stage pupal periods and measure wing pigmentation of Drosophila guttifera. This method can help answer key questions in the evolutionary developmental biology field, such as how pigmentation patterns are developed in Drosophila. The main advantage of this technique is that we can obtain detailed information about morphological development and pigmentation process.Though this method can provide insight into the morphological development and pigmentation process of Drosophila guttifera, it can be applied to other Drosophila species. To begin puparium removal, affix a piece of double-sided tape on piece of paper towel. Place a pupa on the double-sided tape, ventral side up.Locate the space between the anterior side of the puparium and the internal pupa. Grasp and remove the puparium around this gap, using forceps. And expose the anterior side of the head of the pupa.Insert the tip of a forceps by moving it parallel to the anterior-posterior axis. Lift the tip of the forceps to locally break the puparium. Repeat this action until the breakage reaches the posterior part of the puparium.Ensure that a gap is formed between the puparium and pupal legs. And break the ventral side of the puparium while minimizing damage to the internal pupa. After breaking the puparium as much as possible, take out the pupa using a fine paint brush.Place the pupa on a piece of tissue paper that has been moistened with ddH2O and placed in a plastic Petri dish. Immediately take photographs, as the exposed pupa is vulnerable and dries easily. Proceed with image preparation and remove the anterior portion of the puparium with forceps.Take out the pupa and place it into phosphate-buffered saline, or PBS, in a plastic Petri dish. Next, cut the basal joint of a wing. As the wing is folded, place it into a plastic Petri dish filled with ddH2O to extend it by osmotic pressure.Collect newly eclosed adults once every 10 minutes from a stock file. Anesthetize a fly using a CO2 anesthetizing pad. Confirm anesthetization by immobility and cut the basal joint of a wing.Drop 10 microliters of PBS on a glass slide. Place the wing in the drop. And cover the drop with a cover slip.Next, turn on the light of the stereo microscope and set it to maximum. Set the objective lens to 11.5x and set the diaphragm to the most open state. Then, turn on the camera and set the camera.Focus on the sample by moving the focus knob of the microscope. Push the shutter button of the remote control unit to take an image. Take an image of the wing, centered a campaniform sensillum, positioning the distal part of the wing on the left side and the interior part of the wing on the upper side.Open an image with campaniform sensillum spot at the center. Click Image, Type 1, 8-bit, to convert the image to an 8-bit image. Click rectangle in area selection tools and draw a rectangle.Set the right side of the rectangle so that it is located to the right of the campaniform sensillum spot. Click Edit, Selection, Add to Manager, then check the Show All column. Next, click angle tool in the line selection tools.Draw the first line on the posterior line of the third longitudinal vein. Set the left endpoints of the line on the vertex of the previously drawn rectangle. Draw the second line on the upper side of the rectangle.Press the M key to measure the angle between the two lines drawn in this step. Click the window of the image on the screen of the computer. Click Edit, Selection, Add to Manager.Click rectangle in area selection tools and draw a rectangle approximately 1/9th the size of the image window. Click Edit, Selection, Rotate. Write the minus degrees of the previously measured angle in the Angle column and click OK to rotate the rectangle drawn in this step.Move the rectangle drawn by using the arrow keys and set the endpoint of the posterior line of the second longitudinal vein on the left side of the rectangle. And the lower side of the rectangle, attach to the posterior line of the third longitudinal vein. Confirm a perpendicular line from the endpoint of the second longitudinal vein to the posterior line of the third longitudinal vein was drawn.Define the foot of the perpendicular line as point A.Record the X coordinate and the Y coordinate of point A, indicated below area selection tools when placing the cursor on point A.Open the image with point A in the center. Click Image, Type, 8-bit, to convert the image to an 8-bit image. Click rectangle in area selection tools and draw a rectangle of approximately 1/9th the size of the image window.Click Edit, Selection, Specify. Check Oval and Centered column. Write 100 pixels in the width and height columns.Write the X coordinates of point A in the X coordinate column, and write the Y coordinates of point A in the Y coordinate column, then click OK.Next, click Analyze and Measure. If the calibration has already been finished, ODs are indicated in Mean column. Comparing the data of multiple stages revealed that stage P12 is the timing of onset of pigmentation and that pigmentation is completed by 12 hours after eclosion.After its development, this technique paves a way for researchers in the field of evolutionary developmental biology to explore differences in pupal development and pigmentation process among Drosophila species.

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JoVE Journal

Biology

11.4K vistas.  Kyoto University. The overall goal of this experiment is to stage pupal periods and measure wing pigmentation of Drosophila guttifera. This method can help answer key questions in the evolutionary developmental biology field, such as how pigmentation patterns are developed in Drosophila. The main advantage of this technique is that we can obtain detailed information about morphological development and pigmentation process.Though this method can provide insight into the morphological development and pigm...