Summary
Here, we present a standardized protocol for monitoring gut acidification in Drosophila melanogaster with optimal output. We first use this protocol for gut acidification monitoring in Drosophila melanogaster and then demonstrate its use in non-model Drosophila species.
Abstract
The fruit fly midgut consists of multiple regions, each of which is composed of cells that carry out unique physiological functions required for the proper functioning of the gut. One such region, the copper cell region (CCR), is localized to the middle midgut and consists, in part, of a group of cells known as copper cells. Copper cells are involved in gastric acid secretion, an evolutionarily conserved process whose precise role is poorly understood. This paper describes improvements in the current protocol used to assay for acidification of the adult Drosophila melanogaster gut and demonstrates that it can be used on other species of flies. In particular, this paper demonstrates that gut acidification is dependent on the fly's nutritional status and presents a protocol based on this new finding. Overall, this protocol demonstrates the potential usefulness of studying Drosophila copper cells to uncover general principles underlying the mechanisms of gut acidification.
Introduction
In the insect gut, copper cells share cellular and functional similarities with the acid-producing gastric parietal cells (also known as oxyntic) of the mammalian stomach. This group of cells releases acid into the intestinal lumen. The function of acid secretion and anatomy is evolutionarily conserved. The major components of the discharged acid are hydrochloric acid and potassium chloride. The chemical mechanism of acid formation in the cells depends on carbonic anhydrase. This enzyme generates a bicarbonate ion from CO2 and water, which liberates a hydroxyl ion that is then discharged into the lumen through a proton pump in exchange for potassium. Chloride and potassium ions are transported into the lumen by conductance channels resulting in the formation of hydrochloric acid and potassium chloride, the main component of gastric juice1,2,3,4.
Although the mechanisms of acid formation are well understood, much less is known about the physiological mechanisms that regulate acid secretion. The goal of developing this method is to help better delineate the cellular pathways that coordinate acid formation and secretion and determine the role of acid in mediating intestinal physiology and homeostasis. The rationale behind the development and use of this technique is to provide a consistent and reliable method to study the process of gut acidification in Drosophila and non-model organisms. Although a standard protocol for determining Drosophila midgut acidification currently exists2,5,6, significant variability was observed in the extent of acidification in wild-type (WT) flies while using this protocol for studying copper cell function. To understand the basis for this observed variability and obtain consistent results, several aspects of the standard protocol were optimized as described below.
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Protocol
NOTE: The standard laboratory line Oregon R was used as a WT control. All flies were reared on standard cornmeal-molasses medium (containing molasses, agar, yeast, cornmeal, tegosept, propionic acid, and water) at room temperature with 12/12 h light/dark circadian rhythm.
1. Preparing for the assay
- Collect female flies (0-2 days old, non-virgin) under CO2 anesthesia and allow them to recover on standard cornmeal food for at least 3 days before experiments.
- Starve the flies for ~24 h at room temperature (~23 °C) in vials containing a laboratory wipe tissue soaked with ~2 mL of deionized water.
- Prepare the fly food with bromophenol blue (BPB) as follows:
- Melt the fly food in a microwave and then let it cool until it is lukewarm.
- Add 1 mL of 4% BPB to 1 mL of lukewarm food and mix well.
- Using a pipet, add the fly food containing BPB into a single dot (~200 µL) in the center of a Petri dish.
2. Gut acidification monitoring assay
- Transfer starved flies into a Petri dish containing single dots (200 µL) of fly food supplemented with 2% bromophenol blue (BPB). Allow the flies to forage for 4 h at room temperature while exposed to light.
- After 4 h, collect the flies and anesthetize them on ice; surgically isolate their guts.
- Perform the surgery in 1x phosphate-buffered saline (PBS) with forceps under a stereomicroscope (see the Table of Materials). Isolate the gut by holding the thorax with a pair of forceps and pulling down the abdomen with a second pair until the CCR of the gut is visible, taking care to ensure that the intestine remains attached at both ends.
- Determine acidification of the gut by examining the color of the CCR of the gut (Figure 1C; yellow indicates acidified, and blue indicates not acidified).
- Count only those flies that show robust BPB staining in their guts.
- Calculate the percentage using the following equation:
Percentage of flies with acidified guts = number of flies acidified × 100 / (number of flies acidified + number of flies non-acidified)
NOTE: A percentage of 0 indicates that no flies acidified their gut, whereas a percentage of 100 indicates all flies acidified their gut.
3. Mounting and image acquisition
NOTE: This step is additional to acquire and process images for the respective conditions for further analyses as the samples cannot be preserved for long. These images are not being used for any gut acidity quantification.
- Following dissection, mount the samples in PBS onto a glass slide.
- Acquire the images under a microscope using cellSens Entry software (see the Table of Materials).
- Place the prepared slide under the microscope and adjust the sample using the eyepiece.
- Shut off the eyepiece to open the shutter for the camera.
- Open the software on the connected computer.
- Choose the correct objective lenses, click the live button, and select the standard setting with exposer time adjustment.
- Focus on the CCR region and take the snapshot.
- Right-click on the snapshot image window and save it as a .tif file.
- Align and process the images further using Fiji software.
- Import the .tif file in Fiji software and clear the unrelated background.
- Adjust the intensity and contrast to optimize the CCR and other gut regions.
- Add the scale bar and save as a .tif file.
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Representative Results
We starved Oregon R female flies for more than 20 h and then fed them food supplemented with BPB (2%) for ~12 h, as described previously7,8,9,10,11. Bromophenol blue (BPB) is a pH-sensing dye. It changes from yellow at pH 3.0 to blue at pH 4.6 and above. Following gut dissection, as previously reported, some flies were found to produce acid as indicated by yellow color in the CCR of the gut (Figure 1B). Surprisingly, in contrast to published results, the intestines of some flies were blue, suggesting that they had failed to acidify their guts. These inconsistent results indicated that the protocol needed to be modified to optimize for consistent and interpretable outcomes.
To optimize the BPB protocol, two new modifications were incorporated. First, to better control the onset of feeding, flies were starved and then placed on spots of food with BPB in the center of a plate (Figure 1A). Second, we began assaying for gut acidification at time points closer to the onset of feeding. Female flies were starved for >20 h, provided fly food with BPB in a small Petri-plate arena (see Figure 1A), and allowed to feed for various time points until 4 h while dissecting guts at 1 h intervals. The number of acidified guts (yellow color) and non-acidified guts (blue color) was determined, and the percentage of flies showing gut acidification was calculated for each time point (Figure 1B). Within 30 min, ~20% of flies had acidified their gut. After an hour, ~40% of guts showed evidence of acidification, while after 2 h and 3 h of feeding, the percentage of acidified guts increased to ~60% and ~70%, respectively (Figure 1B). This indicates that there is an increase in the percentage of flies showing gut acidification with time. Almost 90-95% of guts were acidified when flies were fed for 4 h (Figure 1B). We used this optimized protocol of 4 h feeding for subsequent experiments.
In addition to the effect of feeding, the effect of temperature at which flies were raised on gut acidification was examined. Flies were reared at 23 °C and 30 °C, and female flies were starved for ~20 h. Flies were then fed fly food supplemented with BPB for 4 h, and the percent of gut acidification was determined as described above. We observed no difference in gut acidification for these two temperatures (Figure 1C), suggesting that temperature, unlike feeding, does not affect gut acidification.
Gut acidification protocol demonstration for non-model organisms
Drosophilae species are phylogenetically separated over millions of years (see Figure 2A). Over this vast period, they have adapted to different habitats and diets12, raising the possibility that some species may not acidify their gut in the same manner as D. melanogaster. We used D. melanogaster (fruit), D. sechecllia (morinda fruit), D. erecta (pandanus fruit), D. pseudoosubcura & D. virilis (plant sap), and D. mojavensis (cactus fruits) (Figure 2B). To demonstrate that this protocol could be used for other Drosophila species, these species were fed fly foods supplemented with BPB for 4 h, and the percent of gut acidification was determined as described above. Robust gut acidification was observed for all species tested (Figure 2B). This result suggests that acidification of the gut is evolutionarily conserved among diverse Drosophila species and that this protocol can easily be implemented for other organisms.
Figure 1: Gut acidification monitoring. (A) Schematic drawing of feeding arena. The blue dot represents fly food with bromophenol blue (a pH-indicating dye). Other spots represent fruit flies. (B) Graphical representation of percentage of flies showing gut acidification fed for different durations over 4 h. Representative gut images of an acidified gut and a non-acidified gut. The red arrow indicates acidic release in the copper cell region of the midgut. n = 4 experiments, 25-30 female flies per experiment. Scale bar = 500 µm each. Asterisks indicate significant differences from the control group (one-way ANOVA, followed by a Bonferroni test) *P < 0.05; **P < 0.01; ****P < 0.0001. (C) Flies were fed fly food with BPB for 4 h at 23 °C or 30 °C. Percentage (%) of flies showing gut acidification. n = 4 experiments, 25-30 female flies per experiment (unpaired t-test followed by non-parametric Mann-Whitney U test and Wilcoxon rank-sum test. Abbreviation: ns = not significant. Please click here to view a larger version of this figure.
Figure 2: Phylogeny of gut acidification phenomenon. (A) Phylogenetic relationship of Drosophila species along with their feeding habit and habitat. 1 mm bar indicates 1 million years. (B) Percentage of flies (Drosophila species) showing gut acidification, fed fly food with BPB for 4 h. n = 4 experiments, 25-30 female flies per experiment (one-way ANOVA, followed by a Bonferroni test). Abbreviation: ns = not significant.Please click here to view a larger version of this figure.
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Discussion
A critical step in this protocol is the proper dissection of the gut to visualize the CCR for the acidification phenotype. The acid released from the copper cells is confined to the CCR when the gut is intact. However, during dissection, leakage caused by rupture of the intestine can lead to diffusion of acid from the CCR and result in a gut mistakenly scored as a negative for acidification. In addition, the yellow color indicative of acidification fades within 5-10 min after dissection, underscoring the importance of scoring intestines for the acidification phenotype soon after isolation. Finally, current protocols7,8,9,10,11 that assay the state of acidification in the fly gut rely on supplementation of fly food with BPB, without consideration of the feeding status of the animals. However, during our studies, we found that acidification of the gut was not constitutive but rather dependent on feeding following prior starvation. As such, accurate evaluation of the acid state of the gut using BPB as an indicator of gut pH requires consideration of the fly's nutritional status along with any other variables being considered.
Acidification of the gut is conserved from lower multicellular to higher organisms. However, little is known about its function in most animals and the full extent of the molecular and cellular pathways that regulate it. In humans, lack of gut acidification is associated with the malabsorption of nutrients, while excess acid in the gut can result in intestinal ulcers13. Thus, insights gained from research on gut acidification are likely to provide new insights into the treatment and cure of intestinal diseases caused by defects in the regulation of acid secretion.
Drosophila has recently emerged as a powerful model for the study of gut acidification2,5,6. Genetic studies have identified genes required for the establishment of acid-secreting cells and the machinery involved in the production of acid. Drug studies have also been carried out. For example, acidification of the gut is prevented when flies are fed acetazolamide, a carbonic anhydrase (CAH) inhibitor7, consistent with the central role that CAH plays in the production of protons necessary for acid production. We expect this protocol to help researchers rapidly and cost-effectively discover drug inhibitors or activators of gut acidity. In addition, the application of this method in combination with genetic and biochemical approaches will help uncover the cellular pathways involved in acid secretion and pinpoint the role of gut acidification in intestinal and organismal homeostasis.
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Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgments
The authors acknowledge that support for work in the author's laboratory is provided by an HHMI Faculty Scholar Award and startup funds from the Children's Research Institute at UT Southwestern Medical Center.
Materials
| Name | Company | Catalog Number | Comments |
| Bromophenol blue | Sigma-Aldrich | B0126 | |
| cellSens software | Olympus | Image aqusition (https://www.olympus-lifescience.com/en/software/cellsens) | |
| D. simulans | Drosophila Species Stock Center at the University of California | Riverside California1 (https://www.drosophilaspecies.com/) | |
| D. erecta | Drosophila Species Stock Center at the University of California | Dere cy1(https://www.drosophilaspecies.com/) | |
| D. pseudoobscura | Drosophila Species Stock Center at the University of California | Eugene, Oregon(https://www.drosophilaspecies.com/) | |
| D. mojavensis | Drosophila Species Stock Center at the University of California | Chocolate Mountains, California (https://www.drosophilaspecies.com/) | |
| Forceps | Inox Biology | Catalog# 11252-20 | |
| Fuji | Fuji | Image processing (https://hpc.nih.gov/apps/Fiji.html) | |
| Glass slide | VWR | Catalog#16005-108 | |
| Kim wipes Tissue | Kimtech | ||
| Microscope and camera | Olympus SZ61 microscope equipped with an Olympus D-27 digital camera | Imaging | |
| Oregon R | Bloomington Drosophila Stock | (https://bdsc.indiana.edu/ # 2376) | |
| Petri dishes | Fisher Scientific | Catalog #FB0875713A | |
| Phosphate-buffered Saline (PBS) | HyClone | Catalog # SH30258.01 | |
| Stereomicroscope | Olympus SZ51 | Visual magnification |
References
- Hollander, F. The composition and mechanism of formation of gastric acid secretion. Science. 110 (2846), 57-63 (1949).
- Forte, J. G., Zhu, L. Apical recycling of the gastric parietal cell H, K-ATPase. Annual Review of Physiology. 72, 273-296 (2010).
- Samuelson, L. C., Hinkle, K. L. Insights into the regulation of gastric acid secretion through analysis of genetically engineered mice. Annual Review of Physiology. 65, 383-400 (2003).
- Yao, X., Forte, J. G. Cell biology of acid secretion by the parietal cell. Annual Review of Physiology. 65, 103-131 (2003).
- Driver, I., Ohlstein, B. Specification of regional intestinal stem cell identity during Drosophila metamorphosis. Development. 141 (9), 1848-1856 (2014).
- Overend,, et al. Molecular mechanism and functional significance of acid generation in the Drosophila midgut. Scientific Reports. 6, 27242 (2016).
- Shanbhag, S., Tripathi, S. Epithelial ultrastructure and cellular mechanisms of acid and base transport in the Drosophila midgut. Journal of Experimental Biology. 212, Pt 11 1731-1744 (2009).
- Dubreuil, R. R. Copper cells and stomach acid secretion in the Drosophila midgut. International Journal of Biochemistry and Cell Biology. 36 (5), 745-752 (2004).
- Martorell,, et al. Conserved mechanisms of tumorigenesis in the Drosophila adult midgut. PLoS ONE. 9 (2), 88413 (2014).
- Strand, M., Micchelli, C. A. Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence. PLoS One. 8 (11), 80608 (2013).
- Storelli, G., et al. Drosophila perpetuates nutritional mutualism by promoting the fitness of its intestinal symbiont Lactobacillus plantarum. Cell Metabolism. 27 (2), 362-377 (2018).
- Abu, F., et al. Communicating the nutritional value of sugar in Drosophila. Proceedings of the National Academy of Sciences of the United States of America. 115 (12), 2829-2838 (2018).
- Blecker, U., Gold, B. D. Gastritis and ulcer disease in childhood. European Journal of Pediatrics. 158 (7), 541-546 (1999).
