Overview
This video demonstrates an assay to monitor gut acidification in Drosophila flies. After the ingestion of food supplemented with a pH indicator dye, the acidification of the intestinal lumen results in a change in the color of the dye. Visual inspection of the digestive tract of the flies to examine the color change provides evidence of acidification.
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.
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Representative Results
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.
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Materials
Name | Company | Catalog Number | Comments |
Bromophenol blue | Sigma-Aldrich | B0126 | |
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 | |
Kim wipes Tissue | Kimtech | ||
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 |