Ringraziamo Sean B. Carroll e Thomas Werner per fornire scorte volare, Naoyuki fusibile per attrezzature, Byung Seok Jin per la sua assistenza nella contaminazione, Kiyokazu Agata per tutoraggio ed Elizabeth Nakajima per l'editing di inglese. Questo lavoro è stato supportato da 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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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. 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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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Gli autori non hanno alcun conflitto di interessi.

Descriviamo qui i protocolli per la definizione delle fasi pupal, rimuovendo il pupario per osservazione dettagliata, misurano la durate delle fasi pupal e misurazione dell'intensità delle macchie nere su un'ala a d. guttifera. Questi protocolli possono essere applicati per molti Drosophila e relative specie volare, soprattutto con la pigmentazione di ala.
Descrizione degli eventi inerenti allo sviluppo più dettagliati e approfondita osservazione consentirebbe ulteriore suddivisione delle fasi. In molti casi, un evento inerente allo sviluppo che richiede la dissezione o sezionamento di una pupa non è adatto per la definizione del piano, perché uno deve uccidere una pupa per la stadiazione e continuare ad utilizzare quell'individuo è difficile. Per l'utilizzo di una nuova specie di Drosophila , uno dovrebbe impiegare distinguibili da fuori il pupario eventi dello sviluppo come il primo passo. A seconda dello scopo dello studio, uno può quindi suddividere ulteriormente le fasi basato sul particolare organogenesi o altri eventi inerenti allo sviluppo.
Per confronto interspecifica delle fasi multiple, una potenziale difficoltà è un'inversione dell'ordine di eventi dello sviluppo tra specie (eterocronia27). Ad esempio, in d. melanogaster, l'anatomista diventa verde e poi il corpo giallo diventa visibile, considerando che questo ordine può essere invertito in alcune pupe di guttifera d.19. In tal caso, è difficile confronto strict tra fasi omologhe. A seconda il fenomeno di interesse, uno potrebbe essere necessario ri-definire o suddividere una fase particolare basata su un evento inerente allo sviluppo. Ad esempio, possiamo approssimativamente selezionare pupe di P5 - 6 e fare interspecifico confronto di espressioni geniche nelle ali pupale utilizzando morfologia alare come indicatore di temporizzazione dello sviluppo14.
In genere, ci vogliono 10-30 min per rimuovere il pupario. Se si vuole osservare una tappa breve, pupe dovrebbero essere preparati tenendo conto del tempo che passa durante la rimozione del pupario. Ad esempio, se si vuole osservare P12(i) di d. guttifera, che ha solo 1,1 h durata, preparazione di pupe a P11 darebbe un buon risultato.
Nel nostro protocollo, inumidito carta velina viene utilizzato per lo sfondo delle immagini pupal. A seconda della fase di osservazione e strutture uno vuole mostrare in una figura, uno può usare la carta velina bianca o nera. Per il P3 per la transizione di P4(ii), la posizione della bolla in pupa è importante per distinguere fasi e carta velina nera aiuta a rispettare la posizione della bolla. Per le fasi dopo P5, carta velina bianca è meglio perché aiuta a osservare il corpo giallo, colore degli occhi, setole e colore del corpo.
Bainbridge e Bownes17 stima la durata delle fasi pupal dalla loro frequenza di comparsa. La tabella di gestione temporanea è quello più ampiamente usato per d. melanogaster18. Per il loro metodo, hanno preparato quattro bottiglie di cibo contenente cinque femmine e cinque maschi adulti e li ha tenuti al buio, e quindi pupe sono state prese a caso da una bottiglia ogni alle 11, 12, 13 e 14 giorni dopo l'inizio della deposizione delle uova. Hanno contato il numero di pupe in fasi particolari e calcolate le medie. La lunghezza di ogni stadio pupale potrebbe essere stimata in base i dati della durata totale del periodo pupa e questi dati di frequenza. Un problema con questo metodo è che non può essere utilizzato per stimare la durata precisa di fasi pupal se la tempistica dello sviluppo tende a essere sincronizzato tra pupe.
In realtà, abbiamo provato il metodo Bainbridge e di Bownes in d. guttifera, e abbiamo ottenuto dati distorto a causa della sincronizzazione tra pupe. Non potremmo identificare la causa di questo fenomeno, ma alcune possibilità sono 1) pupe mantenuto ritmo circadiano dal loro stadio giovane e/o 2) hanno reagito alle esposizioni alla luce che si è verificato in osservazione. Pertanto, abbiamo deciso di misurare la durata reale della fase di osservazione diretta. Questo riduce al minimo il pregiudizio causato da ritmo circadiano.
Il metodo qui descritto è un metodo per quantificare supplementare accumulo di melanina nei punti rispetto alla loro circostante zona di controllo (ΔOD), sottraendo il diametro esterno della zona di controllo dal OD della zona spot. Questo metodo è stato ispirato da un metodo per quantificare il contenuto di DNA nucleare di Feulgen macchiatura e analisi delle immagini (densitometria28,29). Come uno dei potenziali problemi per l'applicazione di questo metodo alla pigmentazione ala, ΔOD può essere un valore negativo, soprattutto quando una pupa è molto giovane e non ha quasi nessuna pigmentazione. Nelle fasi successive, ci potrebbero essere alcuni pigmentazione nell'area di controllo. L'uso del semplice OD della zona spot stesso potrebbe essere appropriato anziché ΔOD a seconda dello scopo dello studio. Nel caso di d. guttifera, uso del semplice OD invece di ΔOD non ha cambiato la tendenza dei dati o le conclusioni dello studio.

Alcune delle caratteristiche morfologiche di Drosophila sono diversificate tra specie1,2,3,4,5. Noi possiamo affrontare la questione della diversità come morfologica si pone confrontando i meccanismi di generazione di queste morfologie. Esempi di tali morfologie sono larvale Tricomi, sesso adulto pettini, apparato genitale esterno, pigmentazione addominale e ala pigmentazione6,7,8,9, 10 , 11 , 12 , 13 , 14 , 15. per studiare le differenze morfologiche tra gli adulti, osservazione e analisi delle fasi pupale sono importanti, perché il destino dell'adulti tratti è determinato nelle ultime fasi larvali e successiva morfogenesi procede durante il periodo pupa.
Negli studi di biologia dello sviluppo di Drosophila melanogaster, "ore APF" (ore dopo formazione pupal) è il metodo comune per indicare una fase pupal16. Questo sistema impiega tempo assoluto dopo formazione pupal e molto comodo per gli esperimenti di routine. Tuttavia, velocità inerente allo sviluppo possono differire tra pupe e può essere influenzata da lievi differenze genetiche, epigenetiche o microambientali, e quindi avere lo stesso tempo assoluto dopo formazione pupal non garantisce che le pupe sono allo stesso Stadio di sviluppo. In molti casi, fasi definite da caratteristiche morfologiche sono preferibili per il confronto di più individui. Soprattutto, un confronto tra specie richiede stadiazione precisa e confronto tra le fasi (omologhi) corrispondente.
Bainbridge e Bownes17 riconosciuto 20 fasi pupal (P1 a P15(ii)) basata su caratteristiche morfologiche di Drosophila melanogaster pupe. Questa messa in scena è il sistema più diffuso di gestione temporanea dello sviluppo morfologico18. In uno studio precedente, abbiamo effettuato pupal messa in scena di Drosophila guttifera per stabilire una base per ala pigmentazione studi19. D. guttifera ha un modello di pois neri sulle ali ed è una delle specie modello per ala pigmentazione formazione20. Anche se abbiamo fatto riferimento ai criteri morfologici descritti nella Bainbridge e 'Bownes ricerca17, abbiamo misurato direttamente le durate fase di osservazioni di serie19, invece di utilizzare Bainbridge e 'Bownes stima delle durate di fase da frequenza osservata. Qui descriviamo il metodo di stadiazione pupal e misurazione delle durate delle fasi pupal di Drosophila utilizzato Fukutomi et al.19.
Per studiare il meccanismo inerente allo sviluppo della pigmentazione di ala, abbiamo bisogno di sapere quando nelle fasi di pupal o adulti la pigmentazione si verifica. Fukutomi et al. 19 quantificato densità ottica (ODs) di pigmentazione, pupa e adulto nella fase di analisi dell'immagine di immagini di ala. La pigmentazione delle ali di Drosophila è pensata per essere causato da accumulo di melanina nera21. Per la quantificazione dell'ODs, immagini in scala di grigio e ImageJ software (https://imagej.nih.gov/ij/)22 sono stati usati. Per riconoscere e quantificare la pigmentazione posto specifico (ΔOD), sottraiamo il OD di fuori di un posto dal OD all'interno di un punto. Per rendere questo metodo riproducibile ed obiettivo, dovrebbero essere determinati i posti di misura OD utilizzando le vene ala come punti di riferimento. In questo articolo, descriviamo in dettaglio questo metodo di quantificazione della pigmentazione di ala 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

Diversificato specie di Drosophila (moscerino della frutta) offrono l'opportunità di studiare i meccanismi di sviluppo e cambiamenti genetici responsabili di cambiamenti evolutivi. In particolare, la fase adulta è una fonte ricca di tratti morfologici per confronto interspecifico, compreso il raffronto di pigmentazione di ala. Per studiare le differenze di sviluppo tra specie, osservazione dettagliata e gestione temporanea appropriata sono necessari per confronto preciso. Qui descriviamo i protocolli per la stadiazione dei periodi pupal e quantificazione della pigmentazione di ala a pois moscerino della frutta, Drosophila guttifera. In primo luogo, descriviamo il metodo di osservazione morfologica dettagliata e definizione di fasi pupal basato sulle morfologie. Questo metodo include una tecnica per la rimozione del pupario, che è il caso di chitinoso esterno della pupa, per consentire l'osservazione dettagliata delle morfologie pupale. In secondo luogo, descriviamo il metodo per misurare la durata delle fasi di pupal definite. Infine, descriviamo il metodo per la quantificazione della pigmentazione di ala sulla base di analisi di immagine utilizzando immagini digitali e software ImageJ. Con questi metodi, possiamo stabilire una solida base per il confronto di processi di sviluppo dei tratti adulti durante fasi pupal.

Il periodo pupa di d. guttifera è diviso in 17 tappe (P1 - P15(ii); immagini del rappresentante tre fasi (P1, P5 - 6, P10) sono mostrati nella figura 3e 17 tutte le fasi sono illustrate nella Figura 4). Anche Bainbridge e Bownes17 riconosciuto 20 tappe in d. melanogaster, alcune di queste fasi non poteva essere applicata a d. guttifera. L'ordine di due eventi inerenti allo sviluppo, l'aspetto del corpo giallo (massa di cellule capannone all'interno del midgut26) e la tempistica di tubuli malpighiani svolta verde, non sono rigorosamente controllati in d. guttifera, e quindi non abbiamo potuto separare P5 (i ), P5(ii) e P6. Inoltre, a differenza di in d. melanogaster, la tempistica di annerimento delle setole toraciche ed addominale è stata sincronizzata, e pertanto non abbiamo potuto separare P11(i) e P11(ii)19.
Abbiamo potuto misurare la lunghezza delle fasi pupal di d. guttifera (tabella 3, da Fukutomi et al.19). L'intero periodo pupa è più lungo di quello di d. melanogaster a 25 ° C17circa 20 h. Abbiamo calcolato ΔODs delle zone intorno a un campaniform sensillum, la punta di vena longitudinale e la nervatura trasversale posteriore. Qui, indichiamo l'ODs e ΔODs negli adulti 7 giorni dopo eclosion (tabella 4). Confrontando i dati di varie tappe, abbiamo trovato che la fase P12(i) è la tempistica di insorgenza della pigmentazione, e quella pigmentazione è completata da 24 h dopo eclosion (Figura 5, dalle misure di originale utilizzato in Fukutomi et al. 19).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56935/56935fig1.jpg"> Figura 1. Illustrazione di rimozione pupario. (A) posto un lato ventrale di pupa fino su un pezzo di nastro biadesivo. Rimuovere la parte anteriore del pupario. (B) rompere il pupario con forcipe dal lato ventrale. (C) dopo la rottura del pupario, estrarre la pupa con un pennello. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56935/56935fig2.jpg"> Figura 2. Definizione della zona per la misurazione della pigmentazione. (A) punto A per un posto connesso con un sensillum campaniform. (B) il punto B per un posto connesso con una punta di vena longitudinale. (C) la lettera C per un posto connesso con una vena trasversale posteriore. (D) lettera D per una zona di controllo. Barre della scala indicano 250 µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56935/56935fig3.jpg"> Figura 3. Esempi di precise fasi pupal. (A) la pupa della fase P1 è ricoperta di pupario. (B) pupa della fase P5 - 6. (C) Pupa della fase P10. Il puparia vengono rimossi prima dell'osservazione in (B) e (C). Barre della scala indicano 500 µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig3large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56935/56935fig4.jpg"> Nella figura 4. Illustrazioni di 17 fasi pupal identificati in d. guttifera. (A) P1, il pupario è bianco. (B) P2, il colore del pupario è marrone chiaro. (C) P3, una bolla è osservata nella parte laterale (freccia nera). (D) P4(i), la bolla è più grande di quello in P3 (freccia nera), e la pupa è capace di galleggiare in PBS. (E) P4(ii), un divario si osserva nella parte anteriore (punta di freccia nera) e la parte posteriore (triangolino grigio). (F) P5 - 6, Malpighian tubuli migrano (difficile da vedere se coperto da pupario). La forma pupal è formata da epitelio pupal e cuticola pupa. (G) P7, il corpo giallo può essere osservato nella parte dorsale (freccia nera). (H) P8, gli occhi sono gialli. (I) P9, gli occhi sono ambra. (J) P10, gli occhi sono di colore rossi. (K) P11, orbitale e ocellare setole (triangolino grigio), vibrisse, macrochaetae toracica (freccia nera) e setole tarsali sono neri e visibili. (L) P12(i), le punte delle ali sono grigie. (M) P12(ii), tutte le parti delle ali sono di colore grigio (freccia nera). (N) P13, le ali sono completamente nere. (O) P14, la testa e le gambe sono completamente oscurabile. (P) P15(i), il meconio può essere osservato sulla parte dorsale dell'addome (freccia nera). (Q) P15(ii), la Mosca è schiusura. Dettagli di queste fasi sono stati descritti in Fukutomi et al. 19 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig4large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56935/56935fig5.jpg"> Figura 5. Sviluppo della pigmentazione intorno un sensillum companiform su un'ala. Cerchi indicano singoli ΔODs e barre orizzontali indicano medie. P10: n = 10, P11: n = 10, 12(i): n = 10, P15(i): n = 11, 3 h: n = 8, 24 h: n = 7, 7 giorni: n = 10. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56935/56935fig5large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Componente</td>
			<td></td>
		</tr>
<tr>
<td>Zucchero bianco morbido</td>
			<td>51,6 g</td>
		</tr>
<tr>
<td>Farina di mais</td>
			<td>172,4 g</td>
		</tr>
<tr>
<td>Granulosità di cereale - C</td>
			<td>86,4 g</td>
		</tr>
<tr>
<td>Lievito di birra secco</td>
			<td>106 g</td>
		</tr>
<tr>
<td>Agar in polvere</td>
			<td>35,28 g</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>4000 mL</td>
		</tr>
<tr>
<td colspan="2">Far bollire per 30 min e lasciar raffreddare fino a 70 ° C.</td>
		</tr>
<tr>
<td colspan="2">Aggiungere 4 g di butile p-idrossibenzoato di sodio disciolto in 40 mL di etanolo.</td>
		</tr>
<tr>
<td colspan="2">Mescolare bene e versare 9 mL ciascuno in fiale di plastica (diametro 25 mm x altezza 96 mm).</td>
		</tr>
</tbody></table>Tabella 1. La composizione del cibo di farina di mais/zucchero/lievito/agar standard.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Componente</td>
			<td>Importo</td>
		</tr>
<tr>
<td>NaCl</td>
			<td>80 g</td>
		</tr>
<tr>
<td>KCl</td>
			<td>0%</td>
		</tr>
<tr>
<td>Na2HPO4·12H2O</td>
			<td>29 g</td>
		</tr>
<tr>
<td>KH2PO4
</td>
			<td>0%</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td>fino a 10 L</td>
		</tr>
<tr>
<td></td>
			<td>impostare pH 7,4</td>
		</tr>
</tbody></table>Tabella 2. La composizione del PBS (1x).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Fase</td>
			<td>Dire di durata (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>Totale</td>
			<td>121.7</td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>Tabella 3. Misurate le durate delle fasi pupal di 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>Suggerimento di vena longitudinale</td>
			<td>Nervatura trasversale posteriore</td>
			<td>Controllo</td>
			<td></td>
			<td>Campaniform sensilum</td>
			<td>Suggerimento di vena longitudinale</td>
			<td>Nervatura trasversale posteriore</td>
		</tr>
<tr>
<td>Individuo</td>
			<td>(Punto A)</td>
			<td>(Punto B)</td>
			<td>(Punto C)</td>
			<td>(Punto D)</td>
			<td></td>
			<td>(Punto A - punto D)</td>
			<td>(Punto B - punto D)</td>
			<td>(Punto C - lettera 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</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>Tabella 4. Misurato ODs e ΔODs di guttifera d. adulti 7 giorni dopo eclosion.

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

Protocolli per la gestione temporanea di pupal periodi e misurazione della pigmentazione ala della Drosophila guttifera sono descritti. Staging e quantificazione della pigmentazione forniscono una solida base per lo studio di meccanismi di tratti di adulti e consentire interspecifico confronto tra lo sviluppo del tratto.

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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.

Dissezione di vivere Drosophila Embrioni: Metodi semplificati per lo screening Collezioni Mutant dalla colorazione anticorpale

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.

Microdissezione laser applicata a profili di espressione genica di sottogruppo di cellule dal Drosophila Ala 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 pupa Addome Immunoistochimica

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.

Misura della durata della vita in<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.

Un metodo migliore per precisa e rapida misurazione delle performance in volo<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.

Imaging attraverso il caso pupa di<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.

Misurazione del tasso metabolico in<em&gt; Drosophila</em&gt; Utilizzando 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 della Drosophila pupa testicolo e singolo maschio cisti germinale: Dissezione, Imaging, e trattamento farmacologico

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