Name: measuring crop motility and food passaging in drosophila
Uploaded: 2020-05-09T14:30:00
Description: Watch this Scientific Journal Video about Measuring Crop Motility and Food Passaging in Drosophila at JoVE.com

This work was supported by National Natural Science Foundation of China (No. 31872287), Natural Science Foundation of Jiangsu Province (NO. BK20181456) and Six talent peaks project in Jiangsu Province (No. SWYY-146).

1. Maintaining and preparing experimental flies
<ol>
	<li>Maintain flies in vials containing 10 mL of freshly made food (1% agar, 2.4% brewer&#8217;s yeast, 3% sucrose, 5% cornmeal) in an incubator at 25 &#176;C with 60% humidity. Set the light cycle of the incubator to 12-h light:12-h dark.</li>
	<li>To ensure that a large number of the desired genotype flies ecloses simultaneously, culture young flies (1&#8722;3 days old) in standard food with dry yeast on the surface for 3 days. Transfer the adults to a new food via...

<ol>
	<li>Kusano, M., 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=Gastrointestinal+motility+and+functional+gastrointestinal+diseases.">Gastrointestinal motility and functional gastrointestinal diseases.</a> Current Pharmaceutical Design. 20 (16), 2775-2782 (2014).</li><li>Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+analysis+of+human+disease-associated+gene+sequences+in+Drosophila+melanogaster.">A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.</a> Genome Research. 11 (6), 1114-1125 (2001).</li><li>Apidianakis, Y., Rahme, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+for+human+intestinal+infection+and+pathology.">Drosophila melanogaster as a model for human intestinal infection and pathology.</a> Disease Models &amp; Mechanisms. 4 (1), 21-30 (2011).</li><li>Lemaitre, B., Miguel-Aliaga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Digestive+Tract+of+Drosophila+melanogaster.">The Digestive Tract of Drosophila melanogaster.</a> Annual Review of Genetics. 47, 377-404 (2013).</li><li>Miguel-Aliaga, I., Jasper, H., Lemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+Physiology+of+the+Digestive+Tract+of+Drosophila+melanogaster.">Anatomy and Physiology of the Digestive Tract of Drosophila melanogaster.</a> Genetics. 210 (2), 357-396 (2018).</li><li>Strand, M., Micchelli, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quiescent+gastric+stem+cells+maintain+the+adult+Drosophila+stomach.">Quiescent gastric stem cells maintain the adult Drosophila stomach.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (43), 17696-17701 (2011).</li><li>Ren, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beadex+affects+gastric+emptying+in+Drosophila.">Beadex affects gastric emptying in Drosophila.</a> Cell Research. 24 (5), 636-639 (2014).</li><li>Xi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TORC1+inhibitor+Nprl2+protects+age-related+digestive+function+in+Drosophila.">The TORC1 inhibitor Nprl2 protects age-related digestive function in Drosophila.</a> Aging. 11 (21), 9811-9828 (2019).</li><li>Wei, Y., Reveal, B., Cai, W., Lilly, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GATOR1+Complex+Regulates+Metabolic+Homeostasis+and+the+Response+to+Nutrient+Stress+in+Drosophila+melanogaster.">The GATOR1 Complex Regulates Metabolic Homeostasis and the Response to Nutrient Stress in Drosophila melanogaster.</a> G3. 6 (12), 3859-3867 (2016).</li><li>Ja, W. W., 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=Prandiology+of+Drosophila+and+the+CAFE+assay.">Prandiology of Drosophila and the CAFE assay.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (20), 8253-8256 (2007).</li><li>Diegelmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CApillary+FEeder+Assay+Measures+Food+Intake+in+Drosophila+melanogaster.">The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster.</a> Journal of Visualized Experiments. (121), e55024 (2017).</li><li>Edgecomb, R. S., Harth, C. E., Schneiderman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+feeding+behavior+in+adult+Drosophila+melanogaster+varies+with+feeding+regime+and+nutritional+state.">Regulation of feeding behavior in adult Drosophila melanogaster varies with feeding regime and nutritional state.</a> Journal of Experimental Biology. 197, 215-235 (1994).</li><li>Peller, C. R., Bacon, E. M., Bucheger, J. A., Blumenthal, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+gut+function+in+drop-dead+mutant+Drosophila.">Defective gut function in drop-dead mutant Drosophila.</a> Journal of Insect Physiology. 55 (9), 834-839 (2009).</li><li>Chtarbanova, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+C+virus+systemic+infection+leads+to+intestinal+obstruction.">Drosophila C virus systemic infection leads to intestinal obstruction.</a> Journal of Virology. 88 (24), 14057-14069 (2014).</li><li>Solari, 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=Opposite+effects+of+5-HT/AKH+and+octopamine+on+the+crop+contractions+in+adult+Drosophila+melanogaster:+Evidence+of+a+double+brain-gut+serotonergic+circuitry.">Opposite effects of 5-HT/AKH and octopamine on the crop contractions in adult Drosophila melanogaster: Evidence of a double brain-gut serotonergic circuitry.</a> PLoS One. 12 (3), 0174172 (2017).</li></ol>

In Drosophila ingested food moves from the crop to the gut for digestion. During this process, the nutrients are absorbed, and the waste is expelled out of the body as feces. Thus, comparing food ingestion together with feces ejection can be used to roughly assess the speed of food movement in the body. The method of capillary feeder (CAFE) is widely used to measure food ingestion10,11. The method of feces number counting can be used to estimate the amou...

Most animals have a digestive tube called the gastrointestinal (GI) tract to absorb energy and nutrients from the environment. The human GI tract is composed of four parts: the esophagus, stomach, small intestine, and large intestine (colon). Food passage from the stomach to the intestine is essential for nutrient absorption. Some effectors, such as aging, toxic drugs, and infection, cause disordered GI tract motility and gastric emptying, which is related to some diseases and their symptoms such as dyspepsia, gastroesophageal reflux disease, and constipation1.
The fruit fly (Drosophila melanogaster) is a widely used model animal in biomedical research due to its easy genetic manipulation. Importantly, about 77% of genes associated with human disease have a homolog in Drosophila2. Research using Drosophila has made enormous advances in our understanding of many disease mechanisms. As a powerful genetic model organism, Drosophila is widely used in GI tract research3. Drosophila has a simpler digestive tract, which is divided into three discrete domains: foregut, midgut, and hindgut4. The crop, a part of the foregut, is a bag-like structure that serves as a site for ingested food storage. The midgut is a long tube and functions as the site for food digestion and nutrient absorption through the epithelial layer, which consists of absorptive enterocytes (ECs) and secretory enteroendocrine (EE) cells5. Interestingly, the stomach function in Drosophila is divided into two parts: the crop functions as food storage and the copper cell region (CCR) is a highly acidic region with a pH &#60; 36. In Drosophila, the ingested food is initially moved to the crop and subsequently pumped into the midgut7. Thus, the crop plays a critical role in food passaging. Enveloped by visceral muscles and consisting of a complex array of valves and sphincters, the crop keeps contracting and moving food into the midgut for further digestion.
This protocol allows for the detection of food movement from the crop to the midgut in Drosophila. Crop contraction is evaluated by counting crop contraction frequency. In addition, the effect of the crop on food passaging is investigated by detecting the food distribution between crop and gut. Furthermore, the food distribution can be used to reflect immediate food movement or basic food status using different feeding periods. Taken together, this protocol provides methods to rapidly evaluate crop motility and food passaging in Drosophila.

<table>
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>Thermo fisher</td>
			<td>269620</td>
			<td></td>
		</tr>
		<tr>
			<td>Brillant Blue FCF</td>
			<td>Solarbio</td>
			<td>E8500</td>
			<td>also called FD&#38;C Blue No. 1</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo fisher</td>
			<td>Heraeus Pico 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Spectra Max</td>
			<td>cMax plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>11252-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring crop motility and food passaging in drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. Disordered GI motility and gastric emptying cause multiple diseases and symptoms. As a powerful genetic model organism, Drosophila can be used in GI motility research. The Drosophila crop is an organ that contracts and moves food into the midgut for further digestion, functionally similar to a mammalian stomach. Presented is a protocol to study Drosophila crop motility using simple measurement tools. A method for counting crop contractions to evaluate crop motility and a method for detecting the distribution of food dyed blue between the crop and gut using a spectrophotometer to investigate the effect of the crop on food passaging is described. The method was used to detect the difference in crop motility between control and nprl2 mutant flies. This protocol is both cost-efficient and highly sensitive to crop motility.

These methods to count crop contraction rate and detect dyed food distribution can be used to evaluate crop function on food motility. The crop contraction reflects the frequency of pushing food into the gut. The distribution of dye in the fly after a short feeding period indicates immediate food passaging from crop to midgut.
Target of rapamycin complex 1 (TORC1) is a master regulator that mediates nutrient and cell metabolism. TORC1 inhibition extends lifespan in many organisms, including <e...

Watch this Scientific Journal Video about Measuring Crop Motility and Food Passaging in Drosophila at JoVE.com

Measuring Crop Motility and Food Passaging in Drosophila

The goal of this protocol is to measure crop contraction and quantify food distribution in the Drosophila gut.

Measuring Crop Motility and Food Passaging in Drosophila

Most animals use the gastrointestinal (GI) tract to digest food. The movement of the ingested food in the GI tract is essential for nutrient absorption. ...

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