Name: the capillary feeder assay measures food intake in drosophila melanogaster
Uploaded: 2017-03-17T13:00:00
Description: Watch this Scientific Journal Video about The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster at JoVE.com

We thank the past and present members of the Scholz lab for discussion and Helga D&#246;ring for excellent technical support. We especially thank the members of the Biocenter workshop of the University of Cologne for their support and creativity. The work is supported by SFB 1340, SysMedAlc, and DAAD-Siemens.

1. Il test CAFE NOTA: Il saggio è costituito da tre componenti: un flaconcino sperimentale, un coperchio specifico e capillari micro. Una scatola di plastica con coperchio viene utilizzato per trasportare i flaconi preparati e per controllare l&#39;umidità in modo più efficiente. <ol><li> Utilizzare un Drosophila melanogaster fiala di cultura plastica (opzionale altezza 8 cm, 3,3 centimetri di diametro) come un tubo per il saggio. </li><li> Sigillare la fiala con un coperchio i...

<ol>
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PwC Available from: <a target="_blank" href="https://www.beat.co.uk/assets/000/000/302/The_costs_of_eating_disorders_Final_original.pdf">https://www.beat.co.uk/assets/000/000/302/The_costs_of_eating_disorders_Final_original.pdf</a> (2015)</li><li>Lenard, N. R., Berthoud, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+and+peripheral+regulation+of+food+intake+and+physical+activity:+pathways+and+genes.">Central and peripheral regulation of food intake and physical activity: pathways and genes.</a> Obesity. 16, S11-S22 (2008).</li><li>Magni, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+behavior+in+mammals+including+humans.">Feeding behavior in mammals including humans.</a> Trends in Comp. Endocrinology and Neurobiology. 1163, 221-232 (2009).</li><li>Morton, G. J., Meek, T. 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R., Knapp, J., Chung, P., Heberlein, U., Simpson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+serotonergic+neurons+evokes+hunger+in+adult+Drosophila.">A subset of serotonergic neurons evokes hunger in adult Drosophila.</a> Curr. Biol. 25, 2435-2440 (2015).</li><li>Deshpande, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Drosophila+food+intake:+comparative+analysis+of+current+methodology.">Quantifying Drosophila food intake: comparative analysis of current methodology.</a> Nat. Methods. 11 (5), 535-540 (2014).</li><li>Geer, B. W., Olander, R. M., Sharp, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+dietary+choline+utilization+in+adult+Drosophila+melanogaster+by+radioisotope+methods.">Quantification of dietary choline utilization in adult Drosophila melanogaster by radioisotope methods.</a> J. Insect Physiol. 16, 33-43 (1970).</li><li>Thompson, E. D., Reeder, B. A., Bruce, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+method+for+quantitating+food+consumption+for+mutation+assays+in+Drosophila.">Characterization of a method for quantitating food consumption for mutation assays in Drosophila.</a> Environ. Mol. Mutagen. 18, 14-21 (1991).</li><li>Wong, R., Piper, M. D., Wertheim, B., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+food+intake+in+Drosophila.">Quantification of food intake in Drosophila.</a> PLoS One. 4 (6), e6063 (2009).</li><li>Scheiner, R., Steinbach, A., Classen, G., Strudthoff, N., Scholz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octopamine+indirectly+affects+proboscis+extension+response+habituation+in+Drosophila+melanogaster+by+controlling+sucrose+responsiveness.">Octopamine indirectly affects proboscis extension response habituation in Drosophila melanogaster by controlling sucrose responsiveness.</a> J. Insect Physiol. 69, 107-117 (2014).</li><li>Liu, Y., Luo, J., Carlsson, M. K., N&#228;ssel, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+and+insulin-like+peptides+modulate+leucokinin-producing+neurons+that+affect+feeding+and+water+homeostasis+in+Drosophila.">Serotonin and insulin-like peptides modulate leucokinin-producing neurons that affect feeding and water homeostasis in Drosophila.</a> J. Comp. Neurol. 523, 1840-1863 (2015).</li><li>Ro, J., Harvanek, Z. M., Pletcher, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIC:+high-throughput,+continuous+analysis+of+feeding+behaviors+in+Drosophila.">FLIC: high-throughput, continuous analysis of feeding behaviors in Drosophila.</a> PLoS One. 9 (6), e101107 (2014).</li><li>Itskov, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+monitoring+and+quantitative+analysis+of+feeding+behavior+in+Drosophila.">Automated monitoring and quantitative analysis of feeding behavior in Drosophila.</a> Nat. Commun. 5, 4560 (2014).</li><li>Qi, W., Yang, Z., Lin, Z., Park, J. Y., Suh, G. S. B., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+feeding+assay+in+adult+Drosophila+reveals+rapid+modulation+of+food+ingestion+by+its+nutritional+value.">A quantitative feeding assay in adult Drosophila reveals rapid modulation of food ingestion by its nutritional value.</a> Mol. Brain. 8, 87 (2015).</li><li>Marx, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism:+feeding+fruit+flies.">Metabolism: feeding fruit flies.</a> Nat. Methods. 12 (7), 609-612 (2015).</li><li>Spieth, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Courtship+behavior+in+Drosophila.">Courtship behavior in Drosophila.</a> Annu. Rev. Entomol. 19, 385-405 (1974).</li><li>Devineni, A. V., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+ethanol+consumption+in+Drosophila+models+features+of+addiction.">Preferential ethanol consumption in Drosophila models features of addiction.</a> Curr. Biol. 19 (24), 2126-2132 (2009).</li><li>Lee, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+and+reproduction+in+Drosophila:+New+insights+from+nutritional+geometry.">Lifespan and reproduction in Drosophila: New insights from nutritional geometry.</a> Proc. Natl. Acad. Sci. USA. 105 (7), 2498-2503 (2008).</li><li>Pohl, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+preference+in+Drosophila+melanogaster+is+driven+by+its+caloric+value.">Ethanol preference in Drosophila melanogaster is driven by its caloric value.</a> Alcohol Clin. Exp. Res. 36 (11), 1903-1912 (2012).</li><li>Vargas, M. A., Luo, N., Yamaguchi, A., Kapahi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+S6+kinase+and+serotonin+in+postmating+dietary+switch+and+balance+of+nutrients+in+D.+melanogaster.">A role for S6 kinase and serotonin in postmating dietary switch and balance of nutrients in D. melanogaster.</a> Curr. Biol. 20 (11), 1006-1011 (2010).</li><li>Masek, P., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+taste+discrimination+in+Drosophila.">Limited taste discrimination in Drosophila.</a> Proc. Natl. Acad. Sci. 107 (33), 14833-14838 (2010).</li><li>Pool, A. 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La relazione descrive il saggio CAFE in modo passo-passo, concentrandosi sulla configurazione tecnica e le sue prestazioni di successo in laboratorio. Grazie alla sua semplicità, questo dosaggio potrebbe anche essere usato come educativo esperimento scuola. Gli esempi mostrano che il saggio permette di indagine di cibo rilevamento, privilegiate e di consumo in Drosophila melanogaster per periodi di tempo brevi e più lunghi (ore o giorni). Il saggio CAFE è stato utilizzato ampiamente nel campo di indagare tem...

Mangiare è essenziale; tuttavia, la deregolamentazione di assunzione di cibo con conseguente disturbi alimentari come la bulimia, l&#39;anoressia o la tendenza generale a mangiare troppo impone costi sugli individui e sulla società 1, 2, 3. L&#39;obiettivo della presente ricerca è quello di scoprire meccanismi di regolazione dell&#39;assunzione di cibo e di fornire una strategia per eludere la formazione di disturbo. Numerosi studi che utilizzano organismi modello mammiferi hanno fornito nuove intuizioni del circuito e il ruolo dei sistemi di segnalamento in disturbi alimentari 4, 5, 6. Tuttavia, la nostra conoscenza delle basi neuronali e molecolari alla base di questi disturbi è lungi dall&#39;essere completa. Negli ultimi anni, la mosca della frutta Drosophila melanogaster è diventato un sistema modello valido per svelare intuizioni meccanicistici di base nella regolamentazione dei metabolism 7, 8, 9. Il test capillari alimentatore (CAFE) per Drosophila melanogaster è stato istituito nel laboratorio di Seymour Benzer nel 2007 ispirato da un precedente lavoro di Dethier in blowfly 10, 11. Il saggio CAFE ha permesso di misurare direttamente l&#39;assunzione di cibo in Drosophila melanogaster. In questo sistema di test comportamentali, le mosche si nutrono di alimenti liquidi forniti nei capillari di vetro graduati posti all&#39;interno di un flaconcino. Il declino del menisco capillare indica la perdita della soluzione di alimentare attraverso l&#39;evaporazione e il consumo di cibo. La determinazione del tasso di evaporazione da fiale senza linea consente la quantificazione accurata di assunzione di cibo. Il saggio Café è uno dei diversi paradigmi comportamentali utilizzati per misurare l&#39;alimentazione in Drosophila melanogaster e ricercatori devono scegliere quello più appropriato per la loro specificadomanda. La decisione di utilizzare un certo dosaggio devono considerare i seguenti punti: la natura del cibo fornito; la condizione di alimentazione; la misura di assunzione o l&#39;assorbimento delle sostanze nutritive e consumo di cibo indagini o risposta al cibo. Il saggio CAFE, come descritto in questo rapporto è ideale per seguire l&#39;assunzione di cibo di una fonte di cibo liquido sotto una condizione di alimentazione in posizione verticale. Alternativamente l&#39;assunzione di cibo può essere misurata per un gruppo mosca su una fonte di cibo colorato in una fiala o su una placca. Le mosche sono normalmente uccisi o anestetizzati dopo l&#39;alimentazione e la quantità di colorante ingerita è determinato per spettrofotometria o ispezione visiva dell&#39;addome macchiato. Mosche iniziano a espellere cibo ingerito solo 30 minuti dopo l&#39;assunzione, quindi questo approccio è difficile da usare per l&#39;analisi di alimentazione più continuo comportamenti 12, 13. In contrasto con le mosche sono mantenuti intatti quando colorante assorbibiles con traccianti radioattivi vengono utilizzati e il loro consumo di radioisotopi è segnato in un contatore a scintillazione 14, 15. L&#39;assorbimento di radiomarcato da parte del sistema digestivo fly rende possibile la misurazione alimentare l&#39;assorbimento a lungo termine, ma potrebbe portare a sottovalutare il consumo a causa di molecole non assorbita ed escreta tracciante. Un altro approccio per misurare la risposta al cibo in Drosophila melanogaster è la risposta proboscide estensione (PER), che normalmente si verifica per l&#39;assunzione di cibo 16. Questo metodo elegante misura la prima risposta a uno stimolo cibo, ma non registra la quantità di assunzione. L&#39;assunzione di cibo viene regolato in modo dinamico durante l&#39;alimentazione utilizzando diversi segnali di feedback post-digestive che sono fondamentali per la regolazione di alimentazione 17, 18. Diversi tentativi sono stati fatti negli ultimi anni per la raccolta di dati semi-automatizzare nel saggio PER <sfino class = &quot;xref&quot;&gt; 19, 20. Il PER viene rilevata da un pad elettrico o una combinazione di elettrodi e contato tramite computer. Combinando il saggio PER con radioisotopi assorbimento ha rivelato che questo test è limitata dalla bassa sensibilità per rilevare le differenze di alimentazione quantità 18. Il saggio alimentazione manuale (MAF) 21, in cui una mosca viene alimentato manualmente con un capillare di vetro, è stato recentemente sviluppato per misurare l&#39;assorbimento alimentare in una singola mosca immobilizzato. Il saggio MAFE elimina le interferenze di foraggiamento e l&#39;alimentazione iniziazione e ha una risoluzione temporale di secondi, e l&#39;avvio di PER e il consumo di cibo può essere monitorata in modo indipendente nel saggio. Tuttavia, il modo in cui l&#39;immobilizzazione della mosca colpisce alcuni aspetti del comportamento alimentare (ad es locomozione, la motivazione) deve ancora essere indagato. Per ottime recensioni comparative di diversi test per misurare il consumo di cibo in Drosophila melanogaster e per aiutare i ricercatori trovare quello più adatto, vedi relazioni Deshpande e Marx 13, 22. Il saggio CAFE evita alcuni degli svantaggi di altri saggi descritti sopra e combina semplicità d&#39;uso con misurazione affidabile di assunzione di cibo. Qui, una descrizione dettagliata del saggio CAFE è fornito e mostriamo una semplice modifica di configurazione per ridurre l&#39;evaporazione. I risultati rappresentativi, tra cui un test di due alimenti scelta (a breve e lungo termine) e l&#39;assorbimento di saccarosio di mosche è dimostrata. Nella discussione si confronta il nostro metodo descritto con modi alternativi per eseguire il test CAFE, ed evidenziare potenziali limitazioni.

<table border="0" cellpadding="0" cellspacing="0" width="582">
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:161px;">Vials (breeding)</td>
			<td style="width:117px;">Greiner Bio-One</td>
			<td style="width:123px;">960177</td>
			<td style="width:183px;">www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (CAFE assay)</td>
			<td>Greiner Bio-One</td>
			<td>217101</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Lid-CAFE assay</td>
			<td>Workshop</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Plastic box, low wall</td>
			<td>Plastime</td>
			<td>353</td>
			<td>www.plastime.it</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cover for the plastic box</td>
			<td>Workshop</td>
			<td>&#8211;</td>
			<td>&#8211;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Capillaries</td>
			<td>BLAUBRAND&#160;</td>
			<td>REF 7087 07</td>
			<td>www.brand.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Pipette tips</td>
			<td>Greiner Bio-One</td>
			<td>771290</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Filter paper circles</td>
			<td>Whatman</td>
			<td>10 311 804</td>
			<td>www.sigmaaldrich.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">D(+)-Sucrose</td>
			<td>AppliChem</td>
			<td>57-50-1</td>
			<td>www.applichem.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Ethanol absolute</td>
			<td>VWR Chemicals</td>
			<td>20,821,330</td>
			<td>www.vwr.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (red, E124)</td>
			<td>Backfun</td>
			<td>10027</td>
			<td>www.backfun.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (blue, E133)</td>
			<td>Backfun</td>
			<td>10030</td>
			<td>www.backfun.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Soap solution (CVK 8)</td>
			<td>CVH</td>
			<td>103220</td>
			<td>www.cvh.de</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Digital caliper</td>
			<td>GARANT</td>
			<td>412,616</td>
			<td>www.hoffmann-group.com</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (breeding)</td>
			<td></td>
			<td></td>
			<td>Height 9.8 cm, diameter 4.8 cm&#160;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Vials (CAFE assay)</td>
			<td></td>
			<td></td>
			<td>Height 8 cm, diameter 3.3 cm</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Lid-CAFE assay</td>
			<td></td>
			<td></td>
			<td>Produced in university workshop, technical drawing supplied</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Plastic box, low wall</td>
			<td></td>
			<td></td>
			<td>A plastic grid inlay was custom-made for 8 x 10 vial positions&#160;</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cover for the plastic box</td>
			<td></td>
			<td></td>
			<td>Dimensions (37 x29 x18 cm)</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Capillaries</td>
			<td></td>
			<td></td>
			<td>DIN ISO 7550 norm,&#160; IVD-guideline 98/79 EG, ends polished</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Pipette tips</td>
			<td></td>
			<td></td>
			<td>Pipettes for the outer circle are cut according to the lid</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Filter paper circles</td>
			<td></td>
			<td></td>
			<td>45 mm diameter works nicely if folded for the vials used</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">D(+)-Sucrose</td>
			<td></td>
			<td></td>
			<td>Not harmful</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Ethanol absolute</td>
			<td></td>
			<td></td>
			<td>Highly flammable liquid and vapor</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (red, E124)</td>
			<td></td>
			<td></td>
			<td>Not stated</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Food color (blue, E133)</td>
			<td></td>
			<td></td>
			<td>Not stated</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Soap solution (CVK 8)</td>
			<td></td>
			<td></td>
			<td>Odor neutral soap</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Digital caliper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Standard fly food</td>
			<td></td>
			<td></td>
			<td>(for 20 L)</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Agar</td>
			<td></td>
			<td></td>
			<td>160 g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Brewer`s Yeast</td>
			<td></td>
			<td></td>
			<td>299.33 g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Cornmeal</td>
			<td></td>
			<td></td>
			<td>1200g</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Molasses</td>
			<td></td>
			<td></td>
			<td>1.6 L</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Propionic acid</td>
			<td></td>
			<td></td>
			<td>57.3 mL</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Nipagin 30%</td>
			<td></td>
			<td></td>
			<td>160 mL</td>
		</tr>
	</tbody>
</table>

the capillary feeder assay measures food intake in drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

Le mosche del w 1118 genotipi sono utilizzati per dimostrare come viene eseguito il test. I w 1118 mutanti sono comunemente usati per generare le linee transgeniche e di controllare il background genetico di transgeni contrassegnato con il gene bianco. Normalmente, per esperimenti comportamentali, tutte le linee transgeniche sono reincrociata per cinque generazioni alla stessa w 1118 magazzino, che viene utilizzato come...

Watch this Scientific Journal Video about The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster at JoVE.com

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

Il capillare alimentatore Assay Misure assunzione di cibo in<em&gt; Drosophila melanogaster</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

The overall goal of the CApillary FEeder assay is to observe and measure liquid food intake of adult flies. This method can help to answer key questions in metabolic research such as how actual feeding behavior is initiated and how food consumption is terminated. The main advantages of this technique are that it's easy to use and highly reliable.Visual demonstration of the method is critical as several preparatory steps need to be carefully executed for consistent results. To begin the experiment, obtain a drosophila melanogaster culture plastic vial and seal it with a manufactured Plexiglass lid containing an O-ring. Use five microliter microcapillaries with one microliter marks to present the liquid food.Cut off the top of a two to 20 microliter pipette tip and insert the tip into the whole of the lid to position the capillaries in the conical openings. Insert an uncut two to 20 microliter pipette tip into the same opening to prevent flies from escaping. Load eight or 20 flies by trapping or with a blow pipe through the lid's central opening and close the hole with a sponge bung.Next, place the multiple prepared vials into a plastic box with a gridded inlay to handle them safely. To control breeding conditions, introduce 35 virgin females and 15 males for each experimental group into a plastic culture vial containing 50 milliliter fly food. Allow the flies to lay eggs for the first three days, then transfer adult flies to fresh food vials and let them lay eggs for two more days.After two days, repeat the transfer. On day seven, discard the adult flies. Next, anesthetize two to three day old adult flies using a CO2 pad and collect 100 flies sorted by sex in a 1.5 milliliter plastic tube.Measure their weight with a standard laboratory scale. Determine the wet weight of at least four independent fly groups sorted by sex and use the weight to calculate the microliter food consumption per milligram fly. For a three hour assay, use 20 flies in two filled capillaries.For a long term experiment, use a group of eight flies with a supply of four filled capillaries. Fill up to 10 capillaries simultaneously with previously prepared colored food solution. Insert the capillaries into the food solution and then tilt them to a 45 degree angle to accelerate filling.Stop when the liquid reaches the top five microliter mark of the capillary. Remove excess solution on the outside and inside of the capillary with tissue paper. Carefully remove the pipette tip that is closing one of the outer openings and insert a filled glass capillary, bottom end first.Secure the capillary by placing the pipette tip back next to the capillary. Repeat this step for all food solutions tested. Next, place the capillary ends inside all of the vials at the same level three to four centimeters from the lid.Leave some distance between the filter paper to prevent the capillary from leaking. Use a marker pen to label the upper end of the colored liquid. Label them individually using a stripe code to ensure the different capillaries can be identified.Place prepared CAFE assays into a plastic box with a gridded inlay. Use a sponge bung to apply 100 microliters of fresh water every 24 hours to keep the humidity constant inside the assay. Use four separate vials filled with 30 milliliters of double distilled H2O as humidity devices and place them next to the CAFE assays in the plastic box.Cover the plastic box to create a humidity controlled environment during the experiment. Transfer the box to a secure position in a temperature, light, and humidity controlled climate chamber for the experimental period. Before each 24 hour interval, make note of the dead flies and use the number of live flies to calculate the consumption per fly for the following period.At the end of the assay and before replacing the capillary, keep the CAFE assay in the upright position. Do not remove the lid and mark the lower meniscus of the capillary. Discard the data if the mark end is not below the initial mark.Replace the used capillaries with freshly filled capillaries for long term experiments every 24 hours. Measure the decline of the meniscus, then discard the old capillaries. Then carefully remove the capillaries from the assay and store them for data collection.Check if the liquid inside the capillary reached the lower end. If the liquid inside the capillary did not reach the lower end, discard the data. After the experiment, dismantle the setup and wash the vials, lids, and sponge bungs in a soap bath.Then dry them overnight at room temperature for further use. Repeat experiments with the same genotypes on at least three different days. Using a caliper, measure the distance between mark beginning and mark end on the capillary.Finally, discard the capillaries after the measurements. Two food choices were offered, a 1 molar sucrose solution and a 1 molar sucrose solution with 15%ethanol. Visual examination of the abdomen indicates the flies feed on both solutions.However food consumption per fly is nearly two folds greater for the solution containing ethanol. The ethanol preference was also observed in long term experiments where a group of eight flies had access to similar food sources for four days. The CApillary FEeder assay also reveals that male and female flies prefer higher sucrose concentrations over lower sucrose concentrations.And male flies consume significantly more of the high sucrose concentrations than their female counterparts. While attempting this procedure, it's important to remember to perform the assay in a controlled fashion and to repeat the experiment with the same genotypes on at least three different days. In addition, when testing mutant flies, it's important to consider whether the flies can actually perform the task required to obtain the food, such as sending the food source or climbing up and down the capillary.

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