Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

1. Preparing plant extract

1. Collect Psidium guajava L. leaves³⁰ from an adult tree and process as follows: dry the leaves in an oven at 40 °C for 6 days, then air-dry for 6 days, then dry again in the oven at 40 °C for 4 days, and finally prepare leaf powder by grinding the dry leaves in a grinding mill or a coffee grinder.
2. Macerate 100 g of the dried powder in 1 L of 96% ethanol for 24 h, with continuous stirring using a shaker. Subject the plant residue to the same process once more and evaporate the resulting filtrates to dryness using a vacuum rotary evaporator under reduced pressure (175 mbar) at 40 °C.
3. Dissolve the plant extract in ethanol to obtain the desired concentration. Determine the optimal concentration (using the protocol described here) by testing a series of concentrations.
   1. For plant extracts, test a concentration range of 100 µg/mL, 1 mg/mL, 10 mg/mL, 100 mg/mL and use those that do not affect the survival rate of fly. For pure compounds, test the following range of concentrations: 0.05, 0.5, 5 and 50 mM¹².

2. Preparing food medium

1. Measure 100 mL of distilled water and pour into the beaker with 4 g of sugar and 0.8 g of agar (see Table of Materials). Heat to 100 °C (while stirring) and hold for 10 min.
2. Lower the temperature to 80 °C, add 7.4 g of flour and 2.8 g of yeast while stirring. Heat for at least 20 min, still stirring and controlling the temperature which should be around 80 °C.
3. Add the moldex-propionic acid solution (1 mL of moldex and 0.3 g propionic acid mix well). Wait for the temperature to drop to around 50 °C, add the plant extract solution (1 mg/mL) and 0.5 g of bromophenol blue powder.
   NOTE: Please refer to section 1.3.1 for more details on the other concentrations to be tested.
4. Pour the food into the Petri dishes and stop when the Petri dish is full (Figure 1A). Allow the Petri dishes to cool to room temperature (about 3 h), then close the lid, and store in the refrigerator at 4 °C.
   ​NOTE: Petri dishes should be stored in the fridge for no more than 2 weeks to avoid water evaporation.

Figure 1: Demonstration of the experimental process for the fecal deposit test. (A) Image showing Petri dishes full of food medium. Make sure to have enough food in the Petri dish, so that no gaps will trap the flies and prevent them from moving. However, do not overload the Petri dish with food so that the surface can be covered evenly. (B) Image of the spatula as described in the protocol. (C) Image of the fecal deposit test as described in the protocol. Please click here to view a larger version of this figure.

3. Preparing flies

1. Prepare the CO₂ tanks, CO₂ blow gun with needles, fly pad, paintbrush, and microscope. The fly station supplies CO₂ to both the fly pad and the blow gun. The fly pad is employed for sorting flies, while the CO₂ blow gun is utilized to anesthetize flies in vials, bottles, and Petri dishes.
2. Standardize the age of the flies by selecting vials that contain pupae (at least 10) and discard the adults flies from the tube using the following method. One by one, turn the vials upside down and insert the needle between the cotton plug and the sidewall of the vial. Anesthetize adult flies with the CO₂ blow gun until all flies are asleep on the cotton plug (a few seconds is needed to anesthetize them, and the anesthetic action will last for a few seconds after releasing the blow gun). Open the vial above a glass bottle containing 70% ethanol and drop the flies into it. Close the vial with the cotton plug and keep it in an incubator at 25 °C with 60% humidity. Set the light cycle of the incubator to 12 h light/12 h dark.
3. After incubation (maximum 8 h), sort them into virgin females and males under the microscope and fly pad by turning them on their back and looking at their genitalia.
   1. Female genitalia are pale compared to male genitalia, which are of reddish color. Males can also be identified by the presence of dark bristles, called sex combs, on their front pair of legs. Divide the flies in two fresh tubes (one for males and one for females) and incubate them for 6-8 days at 25 °C.
      ​NOTE: At 25 °C, the females remain virgin for approximately 8 h after hatching.

4. Fecal deposit test

1. Label the Petri dishes with the corresponding strain, sex, and drug to avoid confusion between the Petri dishes. Stack the Petri dishes on top of each other.
2. Get the Petri dishes containing the dyed food and reverse them on the blotting paper to absorb the extra liquid. Using a spatula (Figure 1B), cut the food into 12 equal parts and then use the spatula to put one slice into an empty Petri dish.
   NOTE: Depending on the number of replicates, the number of slices to be cut can be increased up to 20 per Petri dish. Each slice should be of the same size.
3. Anesthetize the flies with CO₂ until all the flies are asleep on the cotton plug. Transfer six healthy flies in each Petri dish (Figure 1C), and close the lid immediately, then place them in the incubator (25°C, 60% humidity, 12 h light/ 12 h dark).
4. To ensure that the flies do not escape from the Petri dish during the experiment, attach the top and bottom covers of the Petri dish with a tape. For each test group, prepare six replicate Petri dishes (at least).
5. After allowing the flies to rear for 24 h, use CO₂ to anesthetize them, transfer the flies to a container filled with 70% ethanol, and dispose of any remaining food.
6. Keep the Petri dishes and proceed to step 5.

5. Quantification of Petri dishes

1. Set a folder on the computer and rename it, by including the experiment strain name, the sex of the flies, and the type of drugs used. Within this folder, make subfolders named Original, Cut, and Analysis.
2. Scan the Petri dishes by using a high-resolution scanner with a 6,400 pixel-per-inch (ppi) optical resolution. Scan the top and bottom covers of each Petri dish separately by placing them individually in the middle of the scanner field.
3. Open the application installed on the computer. A welcome window will open on the computer screen with all the general settings (Figure 2A).
   NOTE: Do not modify the advanced settings. This step is valid only for users who have the same scanner as proposed in the Table of Materials. Please refer to the guidelines if using another software.
   1. Name the Petri dish in the application, by including sequence number, top or bottom cover, sex, and type of food used. Select the subfolder original set in step 5.1.
   2. Preview the Petri dish. Click on Önizleme at the bottom of the window, wait a few seconds for the scanner to pre-scan, then a window appears on the computer screen. Move the square displayed on the screen to surround the Petri dish.
   3. Click on Scan at the bottom right of the window on the computer screen, the scan is automatically saved as an image in the folder of choice.
4. Crop the image using an application (e.g., the open-source Fiji application), so that no artefacts and food residue are considered as deposits.
   NOTE: The following steps are valid only for users who have the Fiji application. Please refer to the guidelines if using another software.
   1. Open the Fiji application, wait a few seconds until a toolbar appears on the screen.
   2. Drag the image to be cropped onto the toolbar. Select the 3rd icon Polygon selections in the toolbar, crop the unwanted part of the photo (the colored pie chart) by clicking on the screen to mark an angle around the pie chart (Figures 2B-1,2).
      NOTE: When delineating the cropping region within an image, it is imperative that the chosen frames are seamlessly connected end-to-end.
   3. Click on Edit at the top left of the bar then on Fill/Clear outside (Figure 2B–3).
   4. To save the photo, click on File at the top left of the bar, then on Save As, and finally on Tiff. Choose the subfolder Cut set in step 5.1.
      NOTE: When saving the cropped image, make sure that no special characters (e.g., !, &, $, #, _,-,…) or too many characters are present in the file name .

Figure 2: Key steps in the process of analyzing the data from the fecal deposit test. (A) Screenshot showing the setting information of the scan application. (B) Images cropped using the Fiji application. Make sure that no artefacts and food residue are considered deposits. (C) Screenshot showing what it looks like when opening the Excel_merge-v4 application. Please click here to view a larger version of this figure.

6. Fecal deposits identification using the ultimate reader of dung open source software

NOTE: The introduction and usage of the ultimate reader of dung software can be found in Supplementary File 1.

1. First, open the T.U.R.D. software (Supplementary Figure 1). Create a new experiment and give a name to the document, then save it in the subfolder Analysis set in step 5.1.
2. Click on Plates and then on Add plate. Select the Petri dish to be processed. A new window appears with the names of the selected plates and new parameters. Set the block size, offset, min size, and max size (Supplementary Figure 2).
3. To verify that the fecal deposits detected are the right ones, click on Plates, then Inspect Selected Plates, then Graphics, and View annotated images (Supplementary Figure 3). Zoom in and look at the counts. If there are only a few deposits that should not be included in the analysis, deselect the deposits to be excluded (Supplementary Figure 4).
4. For each new image to process, restart from step 6.3.
5. After analyzing the plates with the T.U.R.D. software, modify the number of flies by clicking on No. Flies (Supplementary Figure 5).
6. Modify the group name by clicking on Plates > Edit groups > Add, and then on Group column, choose the group name.
7. Export the different replicate data separately by clicking on Analyze > Descriptive Statistics > Select Group (Supplementary Figure 6). Keep all spreadsheet files (.csv) in the same folder.
8. In order to gather all files (obtained in step 6.7) in a unique spreadsheet, open the application Excel_merge-v4 (Supplementary Coding File 1), wait until the following sentence appear Select the path of the folder (with .csv files):, and then paste the above folder address. For example, the path could be C:ExperimentFecal deposit test, then click Enter twice on keyboard (Figure 2C). After that, a new spreadsheet file is created in the same folder. The new spreadsheet file includes all of the exported files in different sheets.
9. In the previous spreadsheet file, add another sheet to gather the mean of each parameter of all replicates (use the VLOOKUP function for processing the data). An example is given in the Supplementary Table 1.
10. Analyze the p-value.