Name: measuring crop motility and food passaging in drosophila
Uploaded: 2020-05-09T14:30:00.000+00:00
Duration: 6 min 13 s
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 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>

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

Video: Measuring Crop Motility and Food Passaging in Drosophila

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

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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

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Methods to Assay Drosophila Behavior

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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

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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

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Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

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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

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The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

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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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