Agradecemos a Sean B. Carroll e Thomas Werner para fornecer estoques voar, Naoyuki fusível para equipamentos, Byung Seok Jin por sua assistência na filmagem, Kiyokazu Agata para tutoria e Elizabeth Nakajima para edição inglesa. Este trabalho foi financiado pelo KAKENHI 17K 19427 e Takeda Science Foundation.

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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. 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Os autores não têm nenhum conflito de interesses.

Aqui descrevemos os protocolos para definição dos estágios pupal, removendo o casulo para observação detalhada, medindo durações dos estágios pupal e a medida da intensidade das manchas de preto em uma ala em d. guttifera. Esses protocolos podem ser aplicados para muitos Drosophila e relacionados a moscas espécies, especialmente com pigmentação de asa.
Observação em profundidade e descrição de eventos mais detalhados do desenvolvimento permitiria mais subdivisão dos estágios. Em muitos casos, um evento do desenvolvimento, exigindo dissecação ou seccionamento de uma pupa não é adequado para definição de estágio, porque um tem que matar uma pupa para estadiamento e posterior utilização de que o indivíduo é difícil. Para o uso de uma nova espécie de drosófila , um deve empregar distinguíveis de fora o casulo do desenvolvimento eventos como o primeiro passo. Dependendo da finalidade do estudo, um pode então subdividir mais fases baseadas em particular organogênese ou outros eventos do desenvolvimento.
Para comparação interespecífica de múltiplos estágios, uma dificuldade potencial é uma inversão da ordem do desenvolvimento eventos entre espécies (heterocronia27). Por exemplo, em d. melanogaster, o túbulo de Malpighi torna-se verde e em seguida o corpo amarelo se torna visível, Considerando que esta ordem pode ser invertida em algumas pupas de guttifera d.19. Nesse caso, a comparação estrita entre fases homólogas é difícil. Dependendo o fenômeno de interesse, um pode precisar de re-definir ou subdividir um estágio particular com base em um evento do desenvolvimento. Por exemplo, podemos mais ou menos selecione pupas de P5 - 6 e fazer comparação interespecífica de expressões de gene em asas pupal usando morfologia da asa como um indicador de temporização do desenvolvimento14.
Normalmente, demora de 10-30 min para remover o casulo. Se alguém quiser observar uma fase curta, pupas devem ser preparadas tendo em conta o tempo que passa durante a remoção do casulo. Por exemplo, se você quiser observar a P12(i) de d. guttifera, que tem apenas 1,1 h duração, preparando-se pupas no P11 daria um bom resultado.
Em nosso protocolo, papel de tecido umedecido é usado para o fundo das imagens pupal. Dependendo do estágio de observação e de estruturas quer mostrar em uma figura, pode-se usar papel de tecido branco ou preto. Para o P3 para a transição de P4(ii), a posição da bolha em pupa é importante para distinguir fases, e papel de tecido preto ajuda a observar a posição da bolha. Para as fases após P5, papel de tecido branco é melhor porque ajuda a observar o corpo amarelo, a cor dos olhos, cerdas e cor do corpo.
Bainbridge e Bownes17 calcula-se a duração da fase pupal da sua frequência de aparecimento. Sua mesa de preparo é o mais amplamente utilizado por d. melanogaster18. Para o seu método, eles preparados quatro garrafas de alimentos contendo cinco fêmeas adultas e cinco adultos do sexo masculino e os manteve no escuro, e em seguida pupas foram aleatoriamente retiradas uma garrafa a cada 11, 12, 13 e 14 dias após o início da postura de ovos. Eles contado o número de pupas em estágios particulares e calculadas as médias. O comprimento de cada fase de pupa pode ser estimado com base em dados da duração total do período pupal e estes dados de frequência. Um problema com este método é que não pode ser usada para estimar a duração exacta dos estágios pupal se cronometrando do desenvolvimento tende a ser sincronizada entre pupas.
Na verdade, nós tentamos Bainbridge e do Bownes método em d. guttifera, e obtivemos dados tendenciosos devido à sincronização entre pupas. Nós não pôde identificar a causa deste fenômeno, mas algumas possibilidades são pupas 1) manteve o ritmo circadiano de sua fase jovem e/ou 2) reagiram para as exposições à luz que ocorreu em observação. Portanto, decidimos para medir a duração do estágio real pela observação direta. Isso minimiza a polarização causada pelo ritmo circadiano.
O método descrito aqui é um método para quantificar o acúmulo extra de melanina, em manchas, em comparação com seus arredores de controle (ΔOD), subtraindo-se do OD da área de controle do OD da área local. Este método foi inspirado por um método para quantificar o conteúdo de DNA nuclear por coloração de Feulgen coloração e análise de imagem (densitometria28,.29). Como um dos potenciais problemas para a aplicação deste método para pigmentação de asa, ΔOD pode ser um valor negativo, especialmente quando uma pupa é muito jovem e não tem quase nenhuma pigmentação. Em fases mais tardias, pode haver alguma pigmentação na área de controle. O uso do OD simples da área local em si pode ser apropriado em vez de ΔOD dependendo da finalidade do estudo. No caso de d. guttifera, uso do OD simples em vez de ΔOD não alterou a tendência dos dados ou as conclusões do estudo.

Alguns dos traços morfológicos da drosófila são diversificados entre espécies1,2,3,4,5. Vamos aproximar-na questão da diversidade morfológica como surge, comparando os mecanismos de geração destas morfologias. Exemplos de tais morfologias são tricomas larvas, pentes de sexo adulto, aparelho genital externo, pigmentação abdominal e asa pigmentação6,7,8,9, 10 , 11 , 12 , 13 , 14 , 15. para estudar diferenças morfológicas entre os adultos, observação e análise da fase de pupa são importantes, porque o destino dos traços adultos é determinado na fase larval atrasado e subsequente morfogênese continua durante o período pupal.
Em estudos de biologia do desenvolvimento da Drosophila melanogaster, "horas APF" (horas depois da formação de pupa) é o método comum para indicar uma fase de pupa,16. Este sistema emprega o tempo absoluto após formação de pupa e é muito conveniente para experimentos de rotina. No entanto, velocidade do desenvolvimento pode diferir entre pupas e pode ser afectada por pequenas diferenças genéticas, epigenéticas ou microenvironmental, e, portanto, tendo o mesmo tempo absoluto após formação pupal não garante que as pupas são ao mesmo estágio do desenvolvimento. Em muitos casos, estágios definidos por características morfológicas são preferíveis para comparar vários indivíduos. Especialmente, uma comparação entre espécies requer preparo preciso e a comparação entre os estágios (homólogos) correspondentes.
Reconhecido de Bainbridge e Bownes17 20 estágios pupal (P1 a P15(ii)) baseada em características morfológicas das pupas de Drosophila melanogaster . Esta plataforma é o mais utilizado sistema de preparo do desenvolvimento morfológico18. Em um estudo anterior, realizamos encenando pupal de Drosophila guttifera para estabelecer uma base para asa pigmentação estudos19. D. guttifera tem um padrão de bolinhas pretas nas asas e é uma das espécies modelo para asa pigmentação formação20. Embora nós referidos os critérios morfológicos descritos na Bainbridge e 'Bownes pesquisa17, medimos diretamente durações de palco por observações serial19, em vez de usando Bainbridge e 'Bownes estimativa de durações de palco observava a frequência. Aqui descrevemos o método de preparo pupal e medição de durações dos estágios pupal de Drosophila usado em Fukutomi et al.19.
Para estudar o mecanismo do desenvolvimento de pigmentação de asa, precisamos saber quando nas fases de pupa ou adultos a pigmentação ocorre. Fukutomi et al. 19 quantificada densidades ópticas (ODs) de pigmentação durante estágios pupal e adultos por análise de imagem de imagens de asa. A pigmentação da drosófila asas é pensada para ser causado pelo acúmulo de melanina negra21. Para quantificação de ODs, foram utilizadas imagens de escala de cinza e ImageJ software (https://imagej.nih.gov/ij/)22 . Para reconhecer e quantificar a pigmentação local-específicos (ΔOD), subtraímos o OD fora de um local o OD dentro de um lugar. Para tornar este método reprodutível e objetiva, os locais de medição de OD devem ser determinados utilizando as veias da asa como Marcos. Neste artigo, descrevemos detalhadamente este método de quantificação de pigmentação de asa em 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

Diversificadas espécies de Drosophila (mosca de fruta) fornecem oportunidades para estudar os mecanismos de desenvolvimento e alterações genéticas responsáveis por mudanças evolutivas. Em particular, a fase adulta é uma fonte rica de traços morfológicos para comparação interespecífica, incluindo comparação de pigmentação de asa. Para estudar as diferenças de desenvolvimento entre as espécies, a observação detalhada e preparo adequado são necessários para comparação precisa. Aqui descrevemos os protocolos para preparo dos períodos pupal e quantificação de pigmentação da asa de uma mosca de fruta bolinhas, Drosophila guttifera. Primeiro, descrevemos o método por observação morfológica detalhada e definição dos estágios pupal baseado em morfologias. Este método inclui uma técnica para remover o casulo, que é o caso de quitinoso exterior da pupa, para permitir a observação detalhada de morfologias pupal. Em segundo lugar, descrevemos o método para medir a duração da fase pupal definido. Finalmente, descrevemos o método para a quantificação de pigmentação de asa com base na análise de imagem usando imagens digitais e o software ImageJ. Com esses métodos, podemos estabelecer uma base sólida para a comparação de processos de desenvolvimento de traços adultos durante estágios pupal.

O período pupal de d. guttifera é dividido em 17 estágios (P1 - P15(ii); imagens de representante de três estágios (P1, P5 - 6, P10) são mostrados a Figura 3e todas as 17 fases estão ilustradas na Figura 4). Embora Bainbridge e Bownes17 reconhecido 20 fases no d. melanogaster, alguns destes estágios não poderiam ser aplicado a d. guttifera. A ordem dos dois eventos do desenvolvimento, a aparência do corpo amarelo (massa de células de galpão dentro do intestino26) e o timing de túbulos de Malpighi, ficando verde, não são estritamente controlados em guttifera d., e, portanto, nós não pode separar P5 (i ), P5(ii) e P6. Também, ao contrário de d. melanogaster, o timing de enegrecimento das cerdas torácicas e abdominais foi sincronizado, e, portanto, nós não pode separar P11(i) e P11(ii)19.
Nós poderia medir o comprimento dos estágios pupal de d. guttifera (tabela 3, de Fukutomi et al.19). Todo o período pupal é mais do que d. melanogaster em 25 ° C17aproximadamente 20 h. Calculamos o ΔODs das áreas ao redor de um campaniform sensillum, dica de veia longitudinal e posterior crossvein. Aqui, nós mostramos o ODs e ΔODs em adultos 7 dias após a eclosão (tabela 4). Comparando os dados de múltiplos estágios, encontramos que o palco P12(i) é a hora de início da pigmentação, e essa pigmentação é completada por 24 h após a eclosão (Figura 5, as medições original usado em Fukutomi et al. 19).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56935/56935fig1.jpg"> Figura 1. Ilustração de remoção casulo. (A) Coloque um lado ventral crisálidas em um pedaço de fita dupla-face. Remova a parte anterior do casulo. (B) quebre o casulo com pinça do lado ventral. (C) depois de quebrar o casulo, tire a pupa usando um pincel. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56935/56935fig2.jpg"> Figura 2. Definição da área de medição pigmentação. (A) o ponto A para um lugar associado com um sensillum de campaniform. (B) o ponto B por um ponto associado com uma ponta de veia longitudinal. (C) o ponto C para um local associado a uma posterior crossvein. (D) ponto D para uma área de controle. Escala barras indicam 250 µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig2large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56935/56935fig3.jpg"> Figura 3. Exemplos de estágios pupal definidos. (A) pupa do palco coberto de P1 com o casulo. (B) pupa da fase P5 - 6. (C) Pupa do palco P10. Os casulos são removidos antes de observação em (B) e (C). Escala barras indicam 500 µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig3large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56935/56935fig4.jpg"> Figura 4. Ilustrações de 17 estágios pupal identificados em d. guttifera. (A) P1, o casulo é branco. (B) P2, a cor do casulo é marrom claro. (C) P3, uma bolha é observada no lado lateral (seta preta). (D) P4(i), a bolha é maior do que em P3 (seta preta), e a pupa é flutuante em PBS. (E) P4(ii), uma lacuna é observada em parte anterior (seta preta) e a parte posterior (seta cinza). (F) P5 - 6, Malpighi túbulos migram (difícil de ver se coberto por casulo). A forma de pupa é formada por epitélio pupal e cutícula pupal. (G) P7, o corpo amarelo pode ser observado no lado dorsal (seta preta). (H) P8, os olhos são amarelos. (I) P9, os olhos são âmbar. (J) P10, os olhos estão vermelhos. (K) P11, cerdas Orbital e ocellar (seta cinza), vibrissae, macrochaetae torácica (seta preta) e do Tarso cerdas são preto e visível. (L) P12(i), as pontas das asas são cinza. (M) P12(ii), todas as partes das asas são cinza (seta preta). (N) P13, as asas são completamente pretas. (O) P14, a cabeça e as pernas estão completamente escurecidas. (P) P15(i), o mecônio pode ser observado no abdômen dorsal (seta preta). (Q) P15(ii), a mosca é eclosing. Detalhes destes estágios foram descritos em Fukutomi et al. 19 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig4large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56935/56935fig5.jpg"> Figura 5. Desenvolvimento de pigmentação ao redor de um sensillum de companiform numa asa. Círculos indicam ΔODs individuais, e barras horizontais indicam médias. P10: n = 10, P11: n = 10, 12(i): n = 10, P15(i): n = 11, 3 h: n = 8, 24 h: n = 7, 7 dias: n = 10. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig5large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Componente de</td>
			<td></td>
		</tr>
<tr>
<td>Açúcar branco macio</td>
			<td>51,6 g</td>
		</tr>
<tr>
<td>Farinha de milho</td>
			<td>g 172,4</td>
		</tr>
<tr>
<td>Grãos de milho - C</td>
			<td>g 86,4</td>
		</tr>
<tr>
<td>Levedura de cerveja seca</td>
			<td>106 g</td>
		</tr>
<tr>
<td>Pó de ágar-ágar</td>
			<td>g 35,28</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>4000 mL</td>
		</tr>
<tr>
<td colspan="2">Ferva por 30 min e deixe esfriar até 70 ° C.</td>
		</tr>
<tr>
<td colspan="2">Adicione 4 g de p-hidroxibenzoato de butilo dissolvido em 40 mL de etanol.</td>
		</tr>
<tr>
<td colspan="2">Misture bem e despeje 9 mL cada frascos plásticos (diâmetro 25 mm x altura 96 mm).</td>
		</tr>
</tbody></table>Tabela 1. A composição dos alimentos de farinha de milho/açúcar/levedura/ágar padrão.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Componente de</td>
			<td>Quantidade</td>
		</tr>
<tr>
<td>NaCl</td>
			<td>80 g</td>
		</tr>
<tr>
<td>KCl</td>
			<td>2 g</td>
		</tr>
<tr>
<td>Na2HPO4·12H2, O</td>
			<td>29 g</td>
		</tr>
<tr>
<td>KH2PO4
</td>
			<td>2 g</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>até 10 L</td>
		</tr>
<tr>
<td></td>
			<td>definir pH 7,4</td>
		</tr>
</tbody></table>Tabela 2. A composição de PBS (1x).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Estágio</td>
			<td>Quer dizer de duração (h)</td>
			<td>s.d.</td>
			<td>n</td>
		</tr>
<tr>
<td>P1 - 2</td>
			<td>1.7</td>
			<td>0.65</td>
			<td>16</td>
		</tr>
<tr>
<td>P3</td>
			<td>2.1</td>
			<td>0.65</td>
			<td>16</td>
		</tr>
<tr>
<td>P4(i)</td>
			<td>2.1</td>
			<td>1,69</td>
			<td>19</td>
		</tr>
<tr>
<td>P4(II)</td>
			<td>0.3</td>
			<td>0.28</td>
			<td>29</td>
		</tr>
<tr>
<td>P5 - 6</td>
			<td>5.0</td>
			<td>3,07</td>
			<td>30</td>
		</tr>
<tr>
<td>P7</td>
			<td>31,9</td>
			<td>7,22</td>
			<td>46</td>
		</tr>
<tr>
<td>P8</td>
			<td>9.6</td>
			<td>2,81</td>
			<td>57</td>
		</tr>
<tr>
<td>P9</td>
			<td>10.9</td>
			<td>2.66</td>
			<td>55</td>
		</tr>
<tr>
<td>P10</td>
			<td>11,7</td>
			<td>2,96</td>
			<td>39</td>
		</tr>
<tr>
<td>P11</td>
			<td>4.4</td>
			<td>2,81</td>
			<td>44</td>
		</tr>
<tr>
<td>P12(i)</td>
			<td>1.1</td>
			<td>0.76</td>
			<td>44</td>
		</tr>
<tr>
<td>P12(II)</td>
			<td>2.0</td>
			<td>0.70</td>
			<td>43</td>
		</tr>
<tr>
<td>P13</td>
			<td>2.2</td>
			<td>0,68</td>
			<td>10</td>
		</tr>
<tr>
<td>P14 - 15(i)</td>
			<td>28,6</td>
			<td>2.75</td>
			<td>10</td>
		</tr>
<tr>
<td>P15(II)</td>
			<td>1.4</td>
			<td>0.87</td>
			<td>10</td>
		</tr>
<tr>
<td>Total</td>
			<td>121.7</td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>Tabela 3. Medido durações dos estágios pupal de guttifera d..
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td>OD</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>ΔOD</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>Campaniform sensilum</td>
			<td>Dica de veia longitudinal</td>
			<td>Crossvein posterior</td>
			<td>Controle</td>
			<td></td>
			<td>Campaniform sensilum</td>
			<td>Dica de veia longitudinal</td>
			<td>Crossvein posterior</td>
		</tr>
<tr>
<td>Indivíduo</td>
			<td>(Ponto A)</td>
			<td>(Ponto B)</td>
			<td>(Ponto C)</td>
			<td>(Ponto D)</td>
			<td></td>
			<td>(Ponto A - ponto D)</td>
			<td>(Ponto B - ponto D)</td>
			<td>(Ponto C - ponto D)</td>
		</tr>
<tr>
<td>1</td>
			<td>0,549</td>
			<td>0.484</td>
			<td>0.515</td>
			<td>0.256</td>
			<td></td>
			<td>0,293</td>
			<td>0.228</td>
			<td>0,259</td>
		</tr>
<tr>
<td>2</td>
			<td>0.529</td>
			<td>0.489</td>
			<td>0.516</td>
			<td>0.254</td>
			<td></td>
			<td>0.275</td>
			<td>0,235</td>
			<td>0.262</td>
		</tr>
<tr>
<td>3</td>
			<td>0.546</td>
			<td>0,48</td>
			<td>0.533</td>
			<td>0,255</td>
			<td></td>
			<td>0.291</td>
			<td>0,225</td>
			<td>0.278</td>
		</tr>
<tr>
<td>4</td>
			<td>0.583</td>
			<td>0.496</td>
			<td>0.566</td>
			<td>0,255</td>
			<td></td>
			<td>0.328</td>
			<td>0,241</td>
			<td>0,311</td>
		</tr>
<tr>
<td>5</td>
			<td>0.523</td>
			<td>0.479</td>
			<td>0.528</td>
			<td>0,235</td>
			<td></td>
			<td>0,288 ponto</td>
			<td>0.244</td>
			<td>0,293</td>
		</tr>
<tr>
<td>6</td>
			<td>0.572</td>
			<td>0.509</td>
			<td>0.546</td>
			<td>0,265</td>
			<td></td>
			<td>0,307</td>
			<td>0.244</td>
			<td>0,281</td>
		</tr>
<tr>
<td>7</td>
			<td>0.568</td>
			<td>0,511</td>
			<td>0,56</td>
			<td>0.256</td>
			<td></td>
			<td>0,312</td>
			<td>0,255</td>
			<td>0.304</td>
		</tr>
<tr>
<td>8</td>
			<td>0,56</td>
			<td>0.507</td>
			<td>0.562</td>
			<td>0,27</td>
			<td></td>
			<td>0,29</td>
			<td>0,237</td>
			<td>0.292</td>
		</tr>
<tr>
<td>9</td>
			<td>0.551</td>
			<td>0,485</td>
			<td>0.569</td>
			<td>0,259</td>
			<td></td>
			<td>0.292</td>
			<td>0,226</td>
			<td>0.31</td>
		</tr>
</tbody></table>Tabela 4. Medido ODs e ΔODs de d. guttifera adultos 7 dias após a eclosão.

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

Protocolos para encenar o período pupal e medição de pigmentação de asa de Drosophila guttifera são descritos. Preparação e quantificação de pigmentação fornecem uma base sólida para estudar os mecanismos do desenvolvimento dos traços adultos e habilitar interespecífica comparação do desenvolvimento de traço.

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

11.5K visualizações.  Kyoto University. Protocolos para encenar o período pupal e medição de pigmentação de asa de Drosophila guttifera são descritos. Preparação e quantificação de pigmentação fornecem uma base sólida para estudar os mecanismos do desenvolvimento dos traços adultos e habilitar interespecífica comparação do desenvolvimento de traço.

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

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In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Dissecação ao vivo de Drosophila Embriões: Métodos ágeis para Screening Coleções Mutant pela coloração de anticorpos

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Microdissecção a laser Aplicada ao perfil de expressão gênica de subconjunto de células a partir do Drosophila Asa Disco

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila Imunohistoquímica Abdomen Pupal

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medição do Tempo de vida em<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Um método melhorado para rápido e preciso de medição de desempenho em vôo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imagem Através do Pupal Caso de<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

A medição da taxa metabólica em<em&gt; Drosophila</em&gt; Usando respirometria

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Ex vivo Cultura de Drosophila Pupal Testículo e único macho da linha germinal Cistos: Dissection, Imaging, eo tratamento farmacológico

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