Monitoring Gut Acidification in the Adult Drosophila Intestine

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 juice^1,2,3,4.

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 exists^2,5,6, 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.

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

1. 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.
2. 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.
3. Prepare the fly food with bromophenol blue (BPB) as follows:
   1. Melt the fly food in a microwave and then let it cool until it is lukewarm.
   2. Add 1 mL of 4% BPB to 1 mL of lukewarm food and mix well.
   3. 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

1. 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.
2. After 4 h, collect the flies and anesthetize them on ice; surgically isolate their guts.
   1. 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.
3. 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).
4. Count only those flies that show robust BPB staining in their guts.
5. 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.

1. Following dissection, mount the samples in PBS onto a glass slide.
2. Acquire the images under a microscope using cellSens Entry software (see the Table of Materials).
   1. Place the prepared slide under the microscope and adjust the sample using the eyepiece.
   2. Shut off the eyepiece to open the shutter for the camera.
   3. Open the software on the connected computer.
   4. Choose the correct objective lenses, click the live button, and select the standard setting with exposer time adjustment.
   5. Focus on the CCR region and take the snapshot.
   6. Right-click on the snapshot image window and save it as a .tif file.
3. Align and process the images further using Fiji software.
   1. Import the .tif file in Fiji software and clear the unrelated background.
   2. Adjust the intensity and contrast to optimize the CCR and other gut regions.
   3. Add the scale bar and save as a .tif file.

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 previously^7,8,9,10,11. 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 yel...

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 sc...

Materials

List of materials used in this article

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