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Summary

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

Abstract

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.

Introduction

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 < 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.

Protocol

1. Maintaining and preparing experimental flies

1. Maintain flies in vials containing 10 mL of freshly made food (1% agar, 2.4% brewer’s yeast, 3% sucrose, 5% cornmeal) in an incubator at 25 °C with 60% humidity. Set the light cycle of the incubator to 12-h light:12-h dark.
2. To ensure that a large number of the desired genotype flies ecloses simultaneously, culture young flies (1−3 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.
3. 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 3−5 days.

2. Counting crop contractions

1. Anesthetize the flies with CO₂ and take one fly into a dissecting plate well containing 200 μL 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.
2. 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.
3. 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.
4. Repeat step 2.3 for 5x between 30 s intervals.
5. 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.

3. Preparing dyed food

1. Weigh and dissolve the blue dye (Table of Materials) in PBS at a concentration of 20% (w/v).
2. 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.

4. Feeding flies with dyed food

1. Transfer groups of same-aged flies to the vials with starvation food (1% agar in distilled water) for 4 h to ensure food intake.
2. 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.

5. Dissecting flies and collecting dye samples in crop and gut

1. Anesthetize the flies with CO₂ and take one fly into a dissecting plate well containing 200 μL of 1x PBS.
2. 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 μL of 1x PBS.
3. Wash the body 2x by gently shaking it in 200 μL of 1x PBS using a pair of tweezers to clean the dye attached to the fly body.
4. Gently and smoothly open the abdomen using two pairs of tweezers and carefully separate the whole gut from the body.
5. Carefully take off the crop from the whole gut and put it in a tube with 100 µL of 1x PBS.
6. Lastly, put the whole gut without crop (hereafter referred to as gut) in another tube with 100 µL of 1x PBS.
7. Grind the crop and gut respectively in tubes using pipette tips to make the dye dissolve in the PBS.
8. Repeat steps 5.1−5.7 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.

6. Calculating dye amounts in crop and gut

1. Centrifuge the sample tubes at the highest speed for 1 min and transfer 90 µL of supernatant to the wells of a 96 well plate.
2. 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.
3. Add a series of 90 µL standards to the wells of the 96 well plate.
4. Measure the absorbance of the samples and standards at 630 nm with a plate spectrophotometer.
5. 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.
6. Calculate the amount of dye by multiplying the sample concentration by 0.1 mL.

Representative Results

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 Drosophila. As an inhibitor of TORC1, the nprl2 mutant fly displays hyperactivation of TORC1 and GI digestion defects^8,9. The crop size in a nprl2 mutant is normal at 3 days old and enlarged at 15 days old, compared with its genetic background control (yw)⁸. To evaluate the crop motility assay, 3-day-old nprl2 mutant flies and yw controls were used. Each crop was counted five times and the average value was used (Supplemental Table 1). The numbers of the crop contraction during the five repeats were similar, which suggests that PBS might not affect the crop physiology in short feedings and is suitable for crop contraction counting. The nprl2 mutation significantly decreased the rate of crop contraction (Figure 1).

Similar to the mammalian stomach, the Drosophila crop keeps contracting to move the food into the gut. To further confirm the function of Nprl2 on the crop, food movement was detected. The flies were fed with dyed food for 30 min and dissected immediately to detect the amount of dye in the crop and the gut using spectrophotometry. As shown in Figure 2A, the blue dye amounts in the crops of control and nprl2 mutants were similar, consistent with the previously reported comparable crop size⁸. The nprl2 mutants had less dye in the gut, which may be associated with the decreased contraction rate (Figure 2B).

Figure 1: Crop contraction difference between control and nprl2 mutant males. The 3-day-old male flies were dissected, and the crop contraction rate was counted. The crop contraction frequency of each fly is displayed as a data point. Error bars represent SD from the indicated data point. ***, P < 0.001. Please click here to view a larger version of this figure.

Figure 2: Food distribution difference between control and nprl2 mutant males after a short feeding time. The 3-day-old male flies were fed with food containing 0.5% blue dye for 30 min and then dissected immediately to detect the dye amount. (A) The dye amount in the crop. (B) The dye amount in the gut without crop. **, P < 0.01; NS, not significant. Please click here to view a larger version of this figure.

Supplemental Table 1: The original data of crop contraction used in Figure 1. Please click here to download this table.

Discussion

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 ingestion^10,11. The method of feces number counting can be used to estimate the amount of feces creation¹². However, the food movement in Drosophila body is under the control of many factors, including crop motility. Crop function cannot be easily evaluated using CAFE and feces counting methods. This protocol can quantitatively evaluate crop motility and food passaging from crop to gut in Drosophila.

Crop motility is essential for food passaging and Drosophila survival. Some gene mutations or virus infections that affect crop function result in decreased lifespan^7,13,14. This protocol can be used to screen and evaluate the mutants and drugs that affect crop motility. The crop contraction counting is used to detect crop motility frequency and the spectrophotometry measuring food distribution in crop and gut is used to predict crop motility efficiency. These two methods are easy to perform and highly sensitive. Furthermore, the spectrophotometry method can be modified to detect food usage in Drosophila. For example, food ingestion within a short time can be evaluated by detecting the dye amount in the whole gut. The continuous food distribution status between crop and gut can be assessed by detecting the dye amounts in the flies fed with dye food for few days.

There are a few technical considerations in this protocol. For the crop contraction counting method, it is essential to dissect and take out the crop carefully in PBS buffer. Saline solution is not the physiological environment of the crop. The crop must remain connected to the body, and the fly must be alive and awake; otherwise, the crop loses the ability to contract. It is suggested to count crop contraction in intact flies as well¹³. For the spectrophotometry method, the separation of the crops from the dissected GI and their transfer into a 96 well plate after food dye feeding should be done carefully and quickly. The dye is used to indicate the amount of food in the crop and gut. During the dissection process, the crop and gut should be in contact. If dye leaks into the dissection media, the sample cannot be used. With practice, a skilled technician can finish dissection and crop separation within 30 s.

Previously, these methods were used to evaluate the crop motility in 15-day-old nprl2 mutant flies that had enlarged crops⁸. In this case, the crop contraction and food distribution in 3-day-old nprl2 mutants with normal crop size were quantified. Consistent with the decreased crop contraction rate, the nprl2 mutants displayed less food dye in the gut. These results suggest that the nprl2 mutants have some defects in crop motility even at a young age. The yw background was used as a wild type control because it is the genetic background of the nprl2 mutant. For other experiments, strains like Canton S and w¹¹⁸, might be used as controls. Other groups use a different dissecting solution (123 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 8 mM MgCl₂, 35.5 mM sucrose, pH = 7.1) for detecting crop contraction^13,14,15. The crop contraction rate found in the control flies in this study is lower than that reported by Solari et al.¹⁵, but higher than Chtarbanova et al.¹⁴ and Peller et al.¹³. This difference may be caused by the different genetic backgrounds or dissection media.

In all, blue dye spectrophotometry together with crop contraction can be efficiently used to evaluate crop motility. The protocol presented here helps to make Drosophila a good model for GI tract physiology study.

Materials

96-well plate	Thermo fisher	269620
Brillant Blue FCF	Solarbio	E8500	also called FD&C Blue No. 1
Centrifuge	Thermo fisher	Heraeus Pico 17
Spectrophotometer	Spectra Max	cMax plus
Tweezers	Dumont	11252-30