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 vial with standard food including wet yeast, for 2 days to allow egg laying. Leave the eggs in the incubator to develop and transfer adult flies to a new vial to collect more eggs.</li>
	<li>Collect the eclosed male or female flies each day and culture them in new vials with standard food at the maintenance condition to the desired age. 
	NOTE: To get more same-age flies, multiple vials of the desired genotype may be set up simultaneously. The vials for adult fly culture should be changed every 3&#8722;5 days.</li>
</ol>
2. Counting crop contractions
<ol>
	<li>Anesthetize the flies with CO2 and take one fly into a dissecting plate well containing 200 &#956;L of 1x phosphate buffered saline (PBS, pH = 7.4) composed of 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, and 1.76 mM KH2PO4.</li>
	<li>Grasp the fly at its thorax using one pair of tweezers, smoothly open the thorax using another pair of tweezers, and then pull the end in opposite directions to open the abdomen. Take the crop and the gut out from the body carefully.</li>
	<li>Wait for the fly to wake up and then visualize the crop and count the number of times it contracts in 1 min. 
	NOTE: Only a complete wave on the crop lobes is counted as one contraction.</li>
	<li>Repeat step 2.3 for 5x between 30 s intervals.</li>
	<li>Calculate the average number of crop contractions per minute. 
	NOTE: During the contraction counting, the fly should be alive, and the gut should be intact and attached at its anterior and posterior ends after dissection.</li>
</ol>
3. Preparing dyed food
<ol>
	<li>Weigh and dissolve the blue dye (Table of Materials) in PBS at a concentration of 20% (w/v).</li>
	<li>Add the 20% blue dye into the boiled liquid maintenance food (step 1.1) with a 1:40 dilution to a final concentration of 0.5% (w/v) during the food cooling process. 
	NOTE: The blue dye is added before the food cooling down and mixed well with stirring. It is optional to dissolve the blue dye in PBS; distilled water is also suitable.</li>
</ol>
4. Feeding flies with dyed food
<ol>
	<li>Transfer groups of same-aged flies to the vials with starvation food (1% agar in distilled water) for 4 h to ensure food intake.</li>
	<li>Transfer the flies to new vials with food dyed blue and culture the flies for the desired time. 
	NOTE: The feeding time is a critical factor and depends on the research purpose. Short feeding, within the time of food passing through, can be used to evaluate the speed of food motility from crop to gut. At the maintenance conditions, the food passes through in about 2 h. However, the time of passing through might be related to culture conditions. Long feeding, up to a few days, can be used to evaluate persistent food distribution status between crop and gut.</li>
</ol>
5. Dissecting flies and collecting dye samples in crop and gut
<ol>
	<li>Anesthetize the flies with CO2 and take one fly into a dissecting plate well containing 200 &#956;L of 1x PBS.</li>
	<li>Grasp the fly at its thorax using one pair of tweezers and take the head off the body using another pair of tweezers. Move the remaining body to a new well containing 200 &#956;L of 1x PBS.</li>
	<li>Wash the body 2x by gently shaking it in 200 &#956;L of 1x PBS using a pair of tweezers to clean the dye attached to the fly body.</li>
	<li>Gently and smoothly open the abdomen using two pairs of tweezers and carefully separate the whole gut from the body.</li>
	<li>Carefully take off the crop from the whole gut and put it in a tube with 100 &#181;L of 1x PBS.</li>
	<li>Lastly, put the whole gut without crop (hereafter referred to as gut) in another tube with 100 &#181;L of 1x PBS.</li>
	<li>Grind the crop and gut respectively in tubes using pipette tips to make the dye dissolve in the PBS.</li>
	<li>Repeat steps 5.1&#8722;5.7 until enough crops and guts are collected for the experiment designed. 
	NOTE: The crop and gut should be fully homogenized, and all dye should be dissolved in the buffer. For research purposes, one or multiple crops or guts can be collected in one tube.</li>
</ol>
6. Calculating dye amounts in crop and gut
<ol>
	<li>Centrifuge the sample tubes at the highest speed for 1 min and transfer 90 &#181;L of supernatant to the wells of a 96 well plate.</li>
	<li>Make a series of blue dye dilutions at concentrations from 1 x 10-7 g/mL to 1 x 10-4 g/mL as standards.</li>
	<li>Add a series of 90 &#181;L standards to the wells of the 96 well plate.</li>
	<li>Measure the absorbance of the samples and standards at 630 nm with a plate spectrophotometer.</li>
	<li>To create a standard curve, plot a line graph of absorbance vs. concentration for each of the standards. Then draw a line of best fit through the points to get the equation used to calculate the dye concentration in the samples.</li>
	<li>Calculate the amount of dye by multiplying the sample concentration by 0.1 mL.</li>
</ol>

<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. 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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. 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The authors have nothing to disclose.

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&amp;amp;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. ...

measuring-crop-motility-and-food-passaging-in-drosophila

Research

JoVE Journal

Biology

5.5K Views.  Yangzhou University. Deze methode wordt gebruikt om de bemotilheid van het gewas te evalueren door het tellen van gewascontractie en het opsporen van de voedseldistributie in enkel de spijsvertering. Het voordeel van deze techniek is dat het voortdurend evalueert gewas beweeglijkheid. Het is goedkoop en gemakkelijk uit te voeren.Deze methode maakt het mogelijk om alleen filler te gebruiken als een model om de spijsvertering te bestuderen op de voedselpassages in het maag-darmkanaal. Helpen om het proces te...

Video: Measuring Crop Motility and Food Passaging in Drosophila

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Neuroscience

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

The goal of this protocol is to show how to perform an improved assay for mechanical nociception in Drosophila larvae. We use the assay here to demonstrate that mechanical hypersensitivity (allodynia and hyperalgesia) exists in Drosophila larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila ...

Behavior

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

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.

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.

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

Measuring and Altering Mating Drive in Male Drosophila melanogaster

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, reductionist, and scalable system for in-depth investigation of motivation using the mating drive of male Drosophila&#160;melanogaster (Drosophila), the methods for which we detail here. The behavioral paradigm centers on the finding that male mating drive decreases alongside fertility over the course of repeated copulations and recovers over ~3 d. In this system, the powerful neurogenetic tools available in the fly converge with the genetic accessibility and putative wiring diagram available for sexual behavior. This convergence allows rapid isolation and interrogation of small neuronal populations with specific motivational functions. Here we detail the design and execution of the satiety assay that is used to measure and alter courtship motivation in the male fly. Using this assay, we also demonstrate that low male mating drive can be overcome by stimulating dopaminergic neurons. The satiety assay is simple, affordable, and robust to influences of genetic background. We expect the satiety assay to generate many new insights into the neurobiology of motivational states.

Measuring and Altering Mating Drive in Male Drosophila melanogaster

This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation.

Despite decades of investigation, the neuronal and molecular bases of motivational states remain mysterious. We have recently developed a novel, ...

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.

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

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

Measuring Crop Motility and Food Passaging in Drosophila

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