This study investigates the role of copper cells in the insect midgut, specifically their function in gut acidification. By utilizing Drosophila as a model organism, the research outlines a method to monitor gut acidification through a pH-sensing indicator dye.
The insect midgut contains a copper cell region - CCR - which is responsible for gut acidification. This region contains specialized secretory cells - copper cells - that produce acid, which passes through the narrow channel formed by the neighboring interstitial cells. The acid releases into the intestinal lumen to aid digestion.
To monitor gut acidification in Drosophila, take adult flies starved for an adequate duration. Place the flies in a Petri plate containing a single drop of fly food supplemented with bromophenol blue, or BPB - a pH-sensing indicator dye. Allow the starved flies to forage for a fixed period.
After food ingestion, acid produced by the copper cells reaches the intestinal lumen and mixes with the food containing BPB. At an acidic pH, the BPB molecules get protonated, which changes their color from blue to yellow - indicating acidification.
On completion of the experimental duration, place the Petri plate on ice. A low temperature ceases neuromuscular activity in the flies and anesthetizes them. Place an individual fly under a stereomicroscope and surgically isolate the intact digestive tract.
Examine the CCR color to determine the status of gut acidification. A yellow coloration indicates acidification, while a blue coloration indicates the absence of acidification.
Inspect the digestive tract of all the flies, and calculate the percentage of individuals displaying gut acidification.
Start arranging for the gut acidification monitoring assay with the preparation of the fly food with pH-sensing bromophenol blue or BPB dye. To do so, melt the fly food in a microwave, and let it cool until lukewarm. Add 1 milliliter of 4% BPB to 1 milliliter of the lukewarm food with a mixing well. Using a pipette, add the fly food containing BPB into a single dot of approximately 200 microliters in the center of a Petri dish.
Next, collect 0 to 2-days-old non-virgin, female, Drosophila melanogaster flies, and allow the flies to recover on standard cornmeal food. Before the experiment, starve the flies for 24 hours at room temperature in a vial containing a laboratory wipe tissue soaked with approximately 2 milliliters of deionized water.
Transfer starved flies into a Petri dish containing single dots of the freshly prepared fly food supplemented with 2% BPB to allow the flies to forage for four hours at room temperature, while exposed to light.
After four hours, collect the flies. Then, proceed to surgically isolate the guts of the anesthetized flies in 1x PBS with forceps under a stereomicroscope. Count the number of the flies that show robust BPB staining in the guts, and calculate the percentage using the equation.
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