Name: methods for staging pupal periods and measurement of wing pigmentation of drosophila guttifera
Uploaded: 2018-01-24T15:00:00
Description: Watch this Scientific Journal Video about Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera at JoVE.com

Vi takker Sean B. Carroll og Thomas Werner for at levere flyve bestande, Naoyuki Fuse til udstyr, Byung Seok Jin for hans hjælp til at filme, Kiyokazu Agata til mentorordninger og Elizabeth Nakajima for Dansk redigering. Dette arbejde blev støttet af KAKENHI 17K 19427 og Takeda Science Foundation.

1. flyve lager
<ol><li>Bruge Drosophila guttifera for alle de følgende protokoller.</li>
	<li>Bruge plastik hætteglas (diameter 25 mm x højde 96 mm) og cellulose stik (diameter 23 mm x højde 26 mm) for stock vedligeholdelse. Brug en standard cornmeal/sukker/gær/agar mad og følge en publikation beskrives tre andre alternative opskrifter for denne art2. Bemærk: D. guttifera (stock antallet 15130-1971.10) er fastsat af Drosophila arter Stock Center på 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. 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> The Drosophilidae of the southwest. 4313, 7-216 (1943).</li><li>Setoguchi, S., Takamori, H., Aotsuka, T., Sese, J., Ishikawa, Y., Matsuo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+and+courtship+behavior+in+Drosophila+prolongata.">Sexual dimorphism and courtship behavior in Drosophila prolongata.</a> J Ethol. 32 (2), 91-102 (2014).</li><li>Werner, T., Jaenike, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophilids of the Midwest and Northeast. , (2017).</li><li>Arnoult, L., 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=Emergence+and+diversification+of+fly+pigmentation+through+evolution+of+a+gene+regulatory+module.">Emergence and diversification of fly pigmentation through evolution of a gene regulatory module.</a> Science. 339 (6126), 1423-1426 (2013).</li><li>Camino, E. M., Butts, J. C., Ordway, A., Vellky, J. E., Rebeiz, M., Williams, T. M. <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+origination+and+diversification+of+a+dimorphic+gene+regulatory+network+through+parallel+innovations+in+cis+and+trans.">The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans.</a> PLoS Genet. 11 (4), e1005136 (2015).</li><li>Gompel, N., Prud'homme, B., Wittkopp, P. J., Kassner, V. A., 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=Chance+caught+on+the+wing:+cis-regulatory+evolution+and+the+origin+of+pigment+patterns+in+Drosophila.">Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila.</a> Nature. 433 (7025), 481-487 (2005).</li><li>Glassford, W. J., 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=Co-option+of+an+ancestral+Hox-regulated+network+underlies+a+recently+evolved+morphological+novelty.">Co-option of an ancestral Hox-regulated network underlies a recently evolved morphological novelty.</a> Dev. 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. P., 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=Morphological+evolution+through+multiple+cis-regulatory+mutations+at+a+single+gene.">Morphological evolution through multiple cis-regulatory mutations at a single gene.</a> Nature. 448 (7153), 587-590 (2007).</li><li>Tanaka, K., Barmina, O., Kopp, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+mechanisms+underlie+the+evolutionary+diversification+of+Drosophila+sex+combs.">Distinct developmental mechanisms underlie the evolutionary diversification of Drosophila sex combs.</a> Proc Natl Acad Sci U S A. 106 (12), 4764-4769 (2009).</li><li>Werner, T., Koshikawa, S., Williams, T. M., 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=Generation+of+a+novel+wing+colour+pattern+by+the+Wingless+morphogen.">Generation of a novel wing colour pattern by the Wingless morphogen.</a> Nature. 464 (7292), 1143-1148 (2010).</li><li>Wittkopp, P. J., 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=Intraspecific+polymorphism+to+interspecific+divergence:+genetics+of+pigmentation+in+Drosophila.">Intraspecific polymorphism to interspecific divergence: genetics of pigmentation in Drosophila.</a> Science. 326 (5952), 540-544 (2009).</li><li>Lawrence, P. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+pixels+to+picograms:+a+beginners'+guide+to+genome+quantification+by+Feulgen+image+analysis+densitometry.">From pixels to picograms: a beginners' guide to genome quantification by Feulgen image analysis densitometry.</a> J Histochem Cytochem. 50 (6), 735-749 (2002).</li><li>Koshikawa, S., Miyazaki, S., Cornette, R., Matsumoto, T., Miura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+size+of+termites+(Insecta,+Dictyoptera,+Isoptera)+and+wood+roaches+(Insecta,+Dictyoptera,+Cryptocercidae).">Genome size of termites (Insecta, Dictyoptera, Isoptera) and wood roaches (Insecta, Dictyoptera, Cryptocercidae).</a> Naturwissenschaften. 95 (9), 859-867 (2008).</li></ol>

Vi beskriver her protokoller til definition af puppe stadier, at fjerne puparium for detaljeret observation, måling varigheder af puppe faser, og måling af intensiteten af sorte pletter på wing i D. guttifera. Disse protokoller kan anvendes til mange Drosophila og relaterede flyve arter, navnlig arter med wing pigmentering.
Grundig observation og beskrivelse af mere detaljerede udviklingsmæssige begivenheder ville give yderligere underopdeling af faser. I mange tilfælde ...

Nogle af de morfologiske træk af Drosophila er diversificeret blandt arter1,2,3,4,5. Vi kan nærme sig spørgsmålet om hvordan morfologiske diversitet opstår ved at sammenligne mekanismerne af generation af disse morfologier. Eksempler på sådanne morfologier er larver trichomes, adult sex kamme, ydre kønsorganer apparater, abdominal pigmentering og fløj pigmentering6,7,8,9, 10 , 11 , 12 , 13 , 14 , 15. for at studere morfologiske forskelle blandt voksne, observation og analyse af de puppe stadier er vigtige, fordi voksne træk skæbne bestemmes i de sene larve stadier og efterfølgende morfogenese provenuet puppe i perioden.
Udviklingsmæssige biologi studier af Drosophila melanogaster, "timer APF" (timer efter puppe dannelsen) er den fælles metode til at angive en puppe stadie16. Dette system beskæftiger absolutte tid efter puppe dannelse og er meget bekvemt for rutinemæssig eksperimenter. Men udviklingsmæssige hastighed kan variere blandt pupper, og kan blive påvirket af små genetiske, epigenetiske eller microenvironmental forskelle, og derfor har den samme absolutte tid efter puppe dannelse garanterer ikke at pupper er på samme udviklingsstadiet. I mange tilfælde er stadier defineret af morfologiske træk at foretrække frem for at sammenligne flere individer. Især, kræver en sammenligning mellem arter præcise iscenesættelse og sammenligning mellem tilsvarende (homolog) faser.
Bainbridge og Bownes17 anerkendt 20 puppe etaper (P1 til P15(ii)) baseret på morfologiske egenskaber i Drosophila melanogaster pupper. Denne iscenesættelse er det mest udbredte system af morfologiske udviklingsmæssige midlertidige18. I en tidligere undersøgelse udført vi puppe iscenesættelse af Drosophila guttifera for at skabe et grundlag for wing pigmentering undersøgelser19. D. guttifera har en sort prikkede mønster på sine vinger og er en af model arter for wing pigmentering dannelse20. Selv om vi omtales de morfologiske kriterier, der er beskrevet i Bainbridge og Bownes' forskning17, vi målte direkte fase varigheder af serie observationer19, istedet for benytter Bainbridge og Bownes' skøn over scenen varigheder fra observerede frekvens. Her beskriver vi metode til puppe iscenesættelse og måling af varigheder af puppe faser i Drosophila bruges i Fukutomi mfl19.
For at undersøge den udviklingsmæssige mekanisme af wing pigmentering, vi har brug at kende når i puppe eller voksen etaper pigmentering opstår. Fukutomi mfl. 19 kvantificeret optisk tætheder (ODs) af pigmentering i puppe og voksen faser ved billedanalyse af wing billeder. Pigmentering af Drosophila vinger menes at være forårsaget af ophobning af sort melanin21. Til kvantificering af ODs, blev grå-skala billeder og ImageJ software (https://imagej.nih.gov/ij/)22 brugt. For at genkende og kvantificere spot-specifikke pigmentering (ΔOD), trække vi OD uden for en plet fra OD inde i en spot. For at gøre denne metode reproducerbare og objektive, skal steder af OD måling bestemmes ved hjælp af wing vener som vartegn. I denne artikel beskriver vi i detaljer denne metode til kvantificering af wing pigmentering i 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

Diversificeret arter af Drosophila (bananfluen) giver mulighed for at studere mekanismer bag udvikling og genetiske ændringer ansvarlig for evolutionære forandringer. Især er den voksne fase en rig kilde af morfologiske træk for artskrydsede sammenligning, herunder wing pigmentering sammenligning. For at studere udviklingsmæssige forskelle mellem arterne, er detaljeret observation og passende iscenesættelse påkrævet til nøje sammenligning. Her beskriver vi protokoller for iscenesættelse af puppe perioder og kvantificering af wing pigmentering i en polka-prikket frugtflue, Drosophila guttifera. Først, vi beskrive metoden til detaljeret morfologiske observation og definition af puppe faser baseret på morfologier. Denne metode omfatter en teknik til at fjerne puparium, som er den ydre chitinous tilfælde af puppe, aktivere detaljeret observation af puppe morfologier. Andet, vi beskrive metoden til at måle varigheden af definerede puppe faser. Endelig vil beskrive vi metode til kvantificering af wing pigmentering baseret på billedanalyse ved hjælp af digitale billeder og ImageJ software. Med disse metoder, kan vi etablere et solidt grundlag for at sammenligne udviklingsprocesser af voksne træk puppe gennemførelsesstadier.

Den puppe periode af D. guttifera er inddelt i 17 faser (P1 - P15(ii), billeder af tre repræsentant faser (P1, P5 - 6, P10) er vist figur 3, og alle 17 trin er illustreret i figur 4). Selvom Bainbridge og Bownes17 anerkendt 20 etaper i D. melanogaster, kunne nogle af disse faser ikke anvendes til D. guttifera. Rækkefølgen af to udviklingsmæssige begivenheder, udseendet af det...

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

Protokoller for iscenesættelse puppe perioder og måling af wing pigmentering af Drosophila guttifera er beskrevet. Iscenesættelse og kvantificering af pigmentering giver et solidt grundlag for at studere udviklingsmæssige mekanismer af voksne træk og aktiverer artskrydsede sammenligning af trait udvikling.

Metoder for iscenesættelse puppe perioder og måling af Wing pigmentering af Drosophila guttifera

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

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Biology

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

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During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...