# Protocol to measure temporal consumption and feeding choices of Drosophila adults using capillaries

Perrine Colombi, Béatrice Denis, Violette Wallart, Chloé M.C. Richard, David Renault, Frédéric Marion-Poll

► To cite this version:

Perrine Colombi, Béatrice Denis, Violette Wallart, Chloé M.C. Richard, David Renault, et al. Protocol to measure temporal consumption and feeding choices of Drosophila adults using capillaries. STAR Protocols, 2025, 6 (3), pp.103931. 10.1016/j.xpro.2025.103931. hal-05232947

HAL Id: hal-05232947

https://hal.science/hal-05232947v1

Submitted on 6 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Protocol

Protocol to measure temporal consumption
and feeding choices of *Drosophila* adults using
capillaries

<figure><img src="image_2.png" /></figure>

Here, we present a high-throughput assay for continuously measuring the consumption and feeding choices of individual *Drosophila* adults over periods of 30 min to several hours. We describe steps for starving flies, introducing flies, filling capillaries, and monitoring consumption from capillary tubes. We then detail procedures for the automated collection of observations and the analysis of the results. This simple, low-cost protocol enables high-precision recording of the consumption and feeding choices of adult flies.

Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Perrine Colombi,
Béatrice Denis,
Violette Wallart,
Chloé M.C. Richard,
David Renault,
Frédéric Marion-Poll

frederic.marion-poll@
universite-paris-saclay.fr

Highlights

Build a *Drosophila* feeding recording system with 3D-printed feeding chambers and webcam

Steps to prepare an
experiment and
capture individual
flies feeding for
several hours

Procedures for
analysis of food
selection and
consumption across
time

Colombi et al., STAR Protocols
6, 103931

September 19, 2025 © 2025
The Author(s). Published by
Elsevier Inc.

https://doi.org/10.1016/j.
xpro.2025.103931
Protocol

Protocol to measure temporal consumption and
feeding choices of *Drosophila* adults using capillaries

Perrine Colombi,<fnref n="1" /> Béatrice Denis,<fnref n="1" />,<fnref n="4" /> Violette Wallart,<fnref n="1" /> Chloé M.C. Richard,<fnref n="3" /> David Renault,<fnref n="3" /> and Frédéric Marion-Poll<fnref n="1" />,<fnref n="2" />,<fnref n="4" />,<fnref n="5" />,*

SUMMARY

Here, we present a high-throughput assay for continuously measuring the con-
sumption and feeding choices of individual *Drosophila* adults over periods of
30 min to several hours. We describe steps for starving flies, introducing flies,
filling capillaries, and monitoring consumption from capillary tubes. We then
detail procedures for the automated collection of observations and the analysis
of the results. This simple, low-cost protocol enables high-precision recording of
the consumption and feeding choices of adult flies.

For complete details on the use and execution of this protocol, please refer to
Colombi<fnref n="1" /> and Richard et al.<fnref n="2" />

BEFORE YOU BEGIN

The insects taste system reflects adaptation to their feeding strategies, notably in the number of gustatory receptors, which is much higher in polyphagous species than in specialists.<fnref n="3" /> At the physiological level, this system is relatively plastic as its sensitivity can change following exposure to different chemicals, such as pollutants or toxicants,<fnref n="2" /> or following starvation that decreases the aversiveness of bitter substances.<fnref n="4" /> Changes also occur according to internal needs, for example in females, which need more protein after mating.<fnref n="5" />

Many approaches have been designed to evaluate feeding preferences and to measure how much food is ingested in the model insect *Drosophila melanogaster*.⁶,⁷ When screening for qualitative feeding discrimination defects, the most popular approach is to monitor 50-70 flies given a choice between two colored food substrates. After 1 h 30 min, the operator scores the number of flies with a colored abdomen of either color and computes a feeding preference index.⁸⁻¹⁰ To measure feeding quantitatively, Seymour Benzer's group introduced the CApillary FEeder (CAFE) assay¹¹ that is now widely used to manually measure the consumption of flies.¹²⁻¹⁵ At least two designs derived from CAFE have been developed to follow the consumption of individual flies: Expresso¹⁶ that requires a special enclosure around each capillary and ARC, a combined feeding and activity monitor assay.¹⁷

Here, we describe a CAFE protocol, named as multiple capillaries feeding (multiCAFE), routinely
used in our laboratory for several years, to evaluate the sensitivity of flies to different bitter sub-
stances<sup>1,12,18</sup> or to different treatments such as solvents.<sup>2</sup> It is able to monitor feeding from individ-
ual flies across time, using off-the-shelf components such as a webcam, microcapillary tubes and

<figure><img src="image_3.png" /></figure>

<footnote n="1">¹Laboratoire Évolution, Génomes, Comportement et Écologie, CNRS, IRD, Université Paris-Saclay, Institut Diversité, Ecologie et Evolution du Vivant (IDEEV), 91190 Gif-sur-Yvette, France</footnote>

<footnote n="2">²Université Paris-Saclay, AgroParisTech, 91120 Palaiseau, France</footnote>

<footnote n="3">³Université de Rennes, CNRS, ECOBIO (Ecosystèmes, biodiversité, évolution) - UMR 6553, 263 Avenue du G al Leclerc, CS 74205, 35042 Rennes Cedex, France</footnote>

<footnote n="4">⁴Technical contact</footnote>

<footnote n="5">⁵Lead contact</footnote>

<footnote>*Correspondence: frederic.marion-poll@universite-paris-saclay.fr
https://doi.org/10.1016/j.xpro.2025.103931</footnote>
3D-printed cages with 10 cells to enclose individual flies. It records the levels of colored liquids within 5 µL calibrated capillaries, using images sampled at regular intervals (usually 1 image per minute). As evaporation rate is often a critical factor, we devote two cells of each cage to monitor evaporation of liquids in the absence of flies. With a 1280 x 768 pixel webcam image centered on the multiCAFE arena, one capillary fits into 300-500 pixels. This corresponds to 10-16 nL which is enough to resolve individual feeding bouts of a single fly. With this approach, a trained operator is able to run a batch of 32 cages in a morning, i.e. 256 flies recorded during 2 h. The stacks of images are analyzed offline using a custom plugin, multiCAFE, running under an image analysis program, ICY.¹⁹

**Institutional permissions**

Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. This protocol ensures minimal handling of the insects tested and is nondestructive.

**multiCAFE setup**

© Timing: Only once (1–2 days)

1. Print MultiCAFE cages, template and comb (provided in the DataVerse).

**Note:** These cages are designed with an open-source 3D-editor software, OpenSCAD. Two microscope glass slides (75 x 25 x 1 mm)) form the top and bottom of 10 fly cells (6 x 24 x 2.4 mm). A third microscope slide supporting an array of 20 capillaries is inserted in the multi-CAFE device just before the experiment so that each fly can access two capillaries. The capillaries are aligned beforehand on a 3D-printed template with parallel grooves and stuck to a slide. We also designed a transversal barrier (or comb) to prevent flies from escaping the cells in the absence of capillaries. The cage and the microscope slides are reusable and washed between experiments.

**Note:** We currently use a 3D resin printer (Form3+, FormLabs) at maximal resolution using white general-purpose resin SLA. This can be done also by a third-party company.

2. Prepare a recording box. The MultiCAFE cage is placed in the cover of a food container (15 x 10.8 x 18.5 cm, 1.8 l, Lock&Lock).

a. Drill one hole at the bottom of the box to let the webcam cable and the USB connector pass through it.

b. Drill another hole on one of its sides to secure the webcam holder to the box with a screw. Alternatively, 3D-print a webcam support to fit and glue it into the container. This support is provided in the DataVerse.

3. Place a light panel vertically against the wall or inside a climatic chamber.

**Note:** We use light panels made with a central diffusing material and 2 rows of LEDs (A3 or A2, Display Concept, Belgium). These panels are connected to a 12 V DC source.

4. Place a horizontal shelf in front of the panel to support recording boxes that will be placed facing the light panel.

**Note:** Using an A2 panel, we can fit 16 recording boxes on 4 shelves.

5. Place the shelves and the recording boxes in front of the light panel.
**Note:** We use a temperature and humidity-controlled chamber (DR-36VL, Plant Climatics, Germany) to maintain the environment at 25°C and 70-80% RH. In this case, pass the webcam wires and the alimentation of the panel through a lateral hole.

**Video recording setup**

© Timing: Only once (1 h)

6. Install a computer next to the recording station and connect the USB cameras to the computer.

**Note:** Since the sampling rate is low (1 image per minute), even a regular office computer can be used. However, in order to connect up to 16 cameras to one computer, we used 4 PCIe extension cards with 4 independent USB controllers (P5Q4A-USB-CARD, Startech) and a computer board with 4 16x PCIe slots.

7. Install iSpy (or any suitable program capable of sampling images at regular intervals).

8. Connect the webcam(s) to iSpy. For each camera added, adjust the recording parameters, sampling resolution, sampling rate and destination of the stored images.

**Note:** Using iSpy is not mandatory. Any program capable of capturing images at regular intervals would be suitable, provided that the jpg files generated are named as follow. The multiCAFE plugin has been designed to read files named according to the format: "camID-DD. MM.YY_hh.mm.ss.jpg", with ID= camera number, DD= 2 digits of day number, MM= 2 digits of month number, YY= 2 digits of year number, hh= 2 digits of hour number (24 h cycle), mm= 2 digits of month number, ss= 2 digits of seconds.

**multiCAFE analysis setup**

© Timing: Only once (1 h)

9. Install Java JDK 11 on a computer (tested on Windows 10 or Windows 11).

**Note:** By default, the version of Java installed is version 8. The current version of multiCAFE plugin does require version 11 (or higher) to detect properly the feeding activities.

10. Install ICY on a computer.¹⁹

**Note:** This open-source program is a superset the general-purpose image analysis program ImageJ.²⁰

11. Download the MultiCAFE plugin from Icy website and install it.

**Note:** Icy and the plugin multiCAFE works also on a MacIntosh or on a computer under Linux.

**Preparation of stock solutions**

© Timing: 30 min

12. Prepare stock solution of erioglaucine at 125 mg/ml in distilled water (solution 1).

13. Prepare 2 ml of a 30 mM sucrose solution with erioglaucine by adding 20.536 mg of sucrose in 1.8 ml distilled water to which 0.2 ml of solution 1 is added (solution 2 and control).

14. Prepare 1 ml of 10 mM caffeine. To do so, add 1.942 mg caffeine to 1 ml of solution 2 (solution 3).
15. Prepare test solution of 1 mM caffeine with 30 mM sucrose. Add 0.9 ml of solution 2 with 0.1 ml of solution 3 (test solution).

Optional: Freeze the solutions at -20 °C in 2-5 ml aliquots for long-term storage.

Δ CRITICAL: Do not keep sucrose solutions more than one week in the fridge.

Note: Before experiments, bring the solutions at 20°C-25°C.

KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>Agar-agar type 1</td><td>Any (Sigma-Aldrich)</td><td>Cat#70143</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>CAS 57-50-1</td></tr><tr><td>Caffeine</td><td>Sigma-Aldrich</td><td>CAS 58-08-2</td></tr><tr><td>Erioglaucine</td><td>Sigma-Aldrich</td><td>CAS 3844-45-9</td></tr><tr><td colspan="3">Experimental models: Organisms/strains</td></tr><tr><td>Drosophila melanogaster/ Canton S</td><td>Bloomington Stock Center</td><td># 64349; males, 3- to 5-day old</td></tr><tr><td>D. melanogaster/W1118</td><td>Kristin Scott's laboratory (UC Berkeley)</td><td>Marella et al.<sup>21</sup>; males, 3- to 5-day old</td></tr><tr><td>D. melanogaster/W1118</td><td>Yves Grau's laboratory strain (U Montpellier)</td><td>Mitri et al.<sup>22</sup>; males, 3- to 5-day old</td></tr><tr><td colspan="3">Software and algorithms</td></tr><tr><td>3D-cages models</td><td>DataVerse</td><td>https://data.indores.fr/dataset.xhtml? persistentId=doi:10.48579/PRO/4THWV2</td></tr><tr><td>Camera surveillance program</td><td>iSpy 64 bits - Windows</td><td>https://www.ispyconnect.com/</td></tr><tr><td>Java 11</td><td>Oracle</td><td>https://www.oracle.com/fr/java/ technologies/javase/jdk11-archive- downloads.html</td></tr><tr><td>Image analysis program ICY</td><td>https://icy.bioimageanalysis.org/</td><td>N/A</td></tr><tr><td>multiCAFE plugin running under ICY</td><td>https://icy.bioimageanalysis.org/ plugin/multicafe/</td><td>N/A</td></tr><tr><td>R version 4.5.0</td><td>www.r-project.org</td><td>N/A</td></tr><tr><td>RStudio</td><td>www.rstudio.com</td><td>N/A</td></tr><tr><td>Data sample</td><td>DataVerse</td><td>https://data.indores.fr/dataset.xhtml? persistentId=doi:10.48579/PRO/4THWV2</td></tr><tr><td colspan="3">Other</td></tr><tr><td>Standard Drosophila rearing tubes</td><td>Any (Greiner Bio-One)</td><td>CAT#205101</td></tr><tr><td>Fly aspirator</td><td>N/A</td><td>N/A</td></tr><tr><td>32 mm 5 µL glass capillaries</td><td>Hirschmann-Geräte, Germany</td><td>Hirschmann 9000105</td></tr><tr><td>multiCAFE 3D models (cage, template, stopper, camera holder)</td><td>3D model available to download from DataVerse</td><td>https://data.indores.fr/dataset.xhtml? persistentId=doi:10.48579/PRO/4THWV2</td></tr><tr><td>Resin 3D printer</td><td>Any (FormLabs)</td><td>Form3+</td></tr><tr><td>Plastic food box</td><td>LocknLock</td><td>15 x 10.8 x 18.5 cm; volume: 1.8 L</td></tr><tr><td>Web camera</td><td>Logitech</td><td>HC920</td></tr><tr><td>Light panel (white)</td><td>Any (Display Concept, Belgium)</td><td>A2 or A3</td></tr><tr><td>High-speed USB card extension (optional)</td><td>StarTech</td><td>P5Q4A-USB-CARD</td></tr><tr><td>Incubator</td><td>Any (Plant Climatics, Germany)</td><td>(DR-36VL)</td></tr></tbody></table>

STEP-BY-STEP METHOD DETAILS

Virgin fly collection

Timing: 2-3 days
<figure><img src="image_4.png" /><figcaption>**Figure 1. Fly preparation**</figcaption></figure>

(A) To control the age of the flies, adults should be collected from the rearing tubes just after eclosion.

(B) If needed, separate males (or females).

(C) Transfer selected flies on fresh food medium during 2-3 days.

(D) Transfer flies into a tube with no food but with a humid filter paper during 18-22 h. If no starvation is necessary, flies can remain on the food medium until the experiment.

1. Empty rearing tubes and collect flies the next day.

2. Sort flies by sex after narcotizing them on CO₂ (Figure 1B).

3. Transfer chosen flies to a rearing tube with new food medium for 2-3 days (Figure 1C).

△ CRITICAL: Flies should be healthy. Contamination of the food medium with bacteria, mites or fungi have a negative impact on feeding although flies look perfectly healthy and active.

**Note:** We usually use males unless comparing feeding between sexes is required. However, the same protocol can be used for females.

**Note:** Drosophila males can mate with females as soon as their cuticle hardens, therefore make sure to sort sexes soon after emergence.

## Starve flies

### Timing: 18-22 h

4. Prepare rearing tubes with a piece of filter paper (8 x 10 cm) at the bottom humidified with 1 ml of water.

5. Transfer flies of the proper age (usually 2-3 days old) of a given physiological status (mated/non-mated) to the rearing tube with the humidified filter paper.

6. Place the tubes at the experimental temperature (usually 25°C) for the time needed for starvation (usually 18-22 h) (Figure 1D).

**Note:** In order to motivate flies to feed, it is often necessary to deprive them from food (Figure 1). The starvation time depends on the particular strain and species tested. This step is not mandatory but if flies are not starved, they will start feeding later and their aversive response to bitter substances will be higher than after starvation.

## Make test solutions ready to be used and prepare the recording box

### Timing: 1 min

7. Bring the test solutions from the freezer or from the fridge at 20°C-25°C.

**Note:** If solutions were stored in bottles, transfer 0.8 ml in 1.5 ml Eppendorf tubes.
<figure><img src="image_5.png" /><figcaption>**Figure 2. Introduce flies into multiCAFE cage**</figcaption></figure>

(A) Material needed: (1) a multiCAFE cage, (2) two microscope slides, (3) a comb, (4) a mouth aspirator, and (5) at least 8 flies as needed.

(B) Introduce a microscope slide between the vertical branches of the multiCAFE cage, by sliding it in the lateral grooves up to the end.

(C) Add a "comb" to prevent the slide from moving up and later to prevent flies from escaping. Flip the cage upside down so that the slide is now at the bottom of the cage.

(D) Catch a fly with your aspirator.

(E) Slide in a microscope slide over the first cage (that will remain empty) and with a mouth aspirator, blow out one fly in the next cage. Push quickly the microscope slide over the cage to trap the fly. Repeat the process 8 times. Note: usually, we leave empty the cells 1 and 10 (see numbers below the row of cells on the figure).

(F) At the end of the process, center the slide and fix it in position with tape on each side. Eventually label each tape to remember how to place the capillaries (here "suc/caf" on the left for "sucrose versus caffeine" and "CS" on the right tab for "Canton S").

8. Place the tubes in a holder and open the caps.

9. Place a humidified filter paper in the recording box.

**Transfer flies into the cage**

Timing: 2-5 min (for 1 cage)

10. For this step, you will need one cage, one stopper (or comb), two microscope glass slides and one custom-made fly aspirator as well as 8-10 flies (Figure 2A).

11. Insert one microscope slide vertically down to the cells area. This glass slide will form the bottom of each cell (Figure 2B).

12. Insert a "comb" on that side to close the notches.
**Note:** The notches are meant to introduce the capillaries in the cells in a later step. The comb will prevent the microscope slide from moving up (Figure 2C) and males to escape.

13. Flip up the cage (Figure 2D).

14. Introduce the second microscope slide horizontally to cover cell 1 (which will remain empty).

**Note:** This slide forms the top of each cell (Figure 2F).

15. Collect one fly with a fly aspirator without anesthetizing it from the starvation tube (Figure 2E) and blow it gently into cell 2 (Figure 2F).

16. Move the slide over cell 2 to trap the fly.

17. Repeat steps 4-5 until the 8 cells contain one fly.

18. Center the top microscope slide over the cages and place a tape or putty over the two ends of the microscope slide to prevent it to slide away.

**Note:** It is handy to add visual marks on the pieces of tape describing the arrangements of the capillaries (for example C 10 on the left and S 10 on the right). These notes will then appear on each image recorded.

△ CRITICAL: Transferring flies without anesthetizing them is mandatory as CO₂ or cold-anesthetized flies need time to recover from the anesthesia. Although they seem active and seem to recover quickly, they feed much less than un-anesthetized flies.

**Note:** Transferring flies in the cells requires patience and gentleness. Mechanical shock and stress do not help flies to enjoy drinking.

## Transfer test solutions in the capillaries and fix them on a glass slide

◎ Timing: 2-5 min (for one cage)

19. For this step, you will need one cage template, one microscope slide, double sided sticky tape, forceps, 5 µL capillary tubes (n=20), and two 1.5 Eppendorf tubes with your solutions (Figure 3A).

20. Insert 4-10 microcapillaries in 1.5 ml Eppendorf tubes containing 0.8 ml of the test solutions. The capillaries will fill up by capillary action (Figure 3B).

21. Collect each capillary with fine tweezers and dry its outer surface by rolling it on a sheet of paper (Figure 3B).

22. Lay down each capillary on its correct place on the template.

**Note:** Since we use two capillaries per cage, it is easier to put in place the first solution in notches 1, 3, ..., 19 and then the second test solution in notches 2, 4, ..., 20 (Figure 3B).

23. Once all capillaries are parallel to each other on the template, carefully align them vertically with the edge of a microscope slide for example.

24. Take a microscope slide, cut a piece of double-sided sticky transparent tape of the length of the slide and place it approximately at the middle of the slide (Figure 3C).

25. Center this slide over the template (with the tape side facing the capillaries) and gently press the slide over it.

**Note:** Make sure to adjust the position of the glass slide so that the capillaries openings will be accessible to the flies (2-3 mm is sufficient) (Figure 3D).
<figure><img src="image_6.png" /><figcaption>**Figure 3. Fill the capillaries and align them on a microscope glass slide**</figcaption></figure>

The capillaries are filled by capillarity when plunged into the solution within an Eppendorf. Take each capillary out of the Eppendorf and dry the outer surface by rolling it on a sheet of paper. The capillaries can be handled with forceps. Align the capillaries parallel to each other on a 3D-printed template to transfer them onto a microscope slide.

(A) Material needed: (1) a multiCAFE template, (2) one microscope slide, (3) double sided sticky tape, (4) fine forceps to handle capillaries, (5) capillaries, (6) test solution 1 and (7) test solution 2.

(B) Insert capillaries in the Eppendorf tubes. Catch one capillary at a time and roll it over a sheet of paper to dry it externally. Then position it in one of the grooves of the template. Do it sequentially so that all capillaries filled with solution 1 are on the left of each cage and all capillaries of solution 2 are on the right.

(C) Place double-sided sticky tape over the length of the microscope slide and cut the sides.

(D) Turn the slide over and position it over the capillaries that have been aligned previously. Press gently the glass slide to make the capillaries stick to the tape and then remove the slide. If some capillaries are slightly mis-oriented realign them with forceps or repeat the operation.

## Insert the capillaries and transfer the cage into a recording box

Timing: 1 min (for one cage)

26. Insert the microscope slide with the capillaries array into the multiCAFE device, with the capillaries facing the notches and with the glass slide on top (Figure 4A).

27. Remove the fly stopper (or comb) and push gently the slide inside the cage and against the other glass slide already in position, making sure that flies can access the tip of the capillaries.

**Note:** The capillaries need to be well aligned with the notches. Use forceps to re-position the ones that do not fit in.
<figure><img src="image_7.png" /><figcaption>**Figure 4. Prepare the cage for recording**</figcaption></figure>

(A) Insert the microscope slide with the capillaries into the multiCAFE cage, remove the comb and push the slide so that the capillaries protrude inside of each cell. If some capillaries are misaligned, gently realign them with forceps.

(B) Place the multiCAFE cage in the cap of the recording box. The cap has lateral flaps and a central groove. The dimensions of the multiCAFE cage are adapted to fit inside of the central groove. Secure the cage to the cap with putty.

(C) Place the cap with the cage over the recording box and close the flaps. Note the undulated blue line that represents a humidified filter paper placed next to the multiCAFE cage. Position the recording box in front of the light panel. It is fine to start recording even before the cap is in place, to ensure no image is lost.

(D) Sample of the first image of an experiment. The camera zooms on a single cage.

28. Place the multiCAFE in the plastic cover of the recording box; eventually secure it in the cap with putty or tape (Figure 4B).

29. Close the recording box with the cover (and the multiCAFE) and place it in front of a light panel at a distance of about 1 cm to avoid heat generated by the light panel (Figure 4C).

30. Start recording from the corresponding camera using iSpy (Figure 4D).

**Δ CRITICAL:** Performing this step quickly to avoid flies to feed before the recording starts.

**Note:** If this issue is critical for the experiment, modified multiCAFE cages can be printed to make use of a thin rigid plastic sheet preventing flies to access the capillaries.
Record images

Timing: 2 h to several days

31. Edit images label from within iSpy to describe the experiment and the stimuli tested, for example, "cam01_male fly strain choice sucrose_30mM caffeine 10 mM date time".

32. If the experiment spans several days, it might be necessary to replace the capillaries.

a. Prepare a slide with fresh capillaries.

b. Remove the cage from the box.

c. Remove the slide with the old capillaries.

d. Insert a new slide with fresh capillaries.

e. Insert the cage in the recording box.

33. After the recording, place multiCAFE cages in a freezer to euthanize the flies.

34. Wash multiCAFE cages and slides and dry them for further use.

35. Save a copy of your image data at another place, for example on a USB disk or on a network disk, for later analysis and archive.

Note: Once the flies and the multiCAFE cage are placed into the box and the recording started, the operator is not required to do anything.

**Note:** Organize your data logically so that the data can be re-analyzed eventually after some time and understood by other people. We usually save data in a directory with a name related to the experimental question and into subdirectories with names starting with the date, and including the different cameras. It is useful to label images or to add comments on a paper tag with information, otherwise recordings are useless if the lab book is lost.

▲ CRITICAL: Acquire good quality images, with enough contrast, focus and making sure that the camera and the cage are stable. Images that the human eye can easily interpret are sometimes difficult to analyze with the algorithms implemented.

Analyze images with the multiCAFE plugin

Timing: About 5 min per cage

36. Load Icy and the plugin multiCAFE.

37. Load the stack of files from the disk.

38. Describe the parameters of the experiment performed by filling text fields with information such as experiment, fly strain, stimulus, concentration, camera, sex.

39. Place a ROI line over each capillary and store the length in pixels of the first capillary.

40. Generate a kymograph for each capillary – this will generate an image showing the evolution of the liquid within each capillary.

41. Detection of the liquid level from each kymograph.

a. Apply an image filter over the kymograph.

b. Run threshold algorithm to detect the transition between the air and the colored liquid.

Note: Sometimes, one of the solutions does not have the same color than the other, which depends on the product and its concentration. In that case, a different filter and detection threshold must be used for each solution.

42. Once each stack has been analyzed, export the data as an Excel file.
**Note:** Each line of the Excel file describes measures made from one capillary along time (as separate columns t1... tn) and descriptors entered by the user concerning the experiment and the content of each capillary and cell.

43. This file can be treated as a database file and analyzed by Excel using pivot tables, or analyzed under R.

Δ CRITICAL: Draw the line carefully without overdoing it. Given the precision of the camera, the jitter is about 2-4 pixels. If a 5 µL capillary spans 300 pixels, this represents only about 1% error.

**Note:** The multiCAFE analysis software detects the position of the liquid in each capillary along time (Figure 5). The liquid level decreases with time because of the combination of flies feeding and evaporation. While evaporation is continuous (more pronounced at the beginning of the experiment), flies feeding is visible as discrete events. We correct the consumption of each fly by subtracting the average evaporation observed in two control cages without flies.

**Note:** The raw data presented in this paper can be downloaded from the DataVerse listed in the key resource table.

## EXPECTED OUTCOMES

Following this protocol, we compared the responses of commonly used strains of *D. melanogaster* to 0.1 mM caffeine (mixed with 30 mM sucrose) versus 30 mM sucrose to examine the feeding responses of individuals offered this 2-choice situation (Figure 5). The objective of this experiment was to make sure that no major difference of feeding was to be expected between fly strains originating from different laboratories and that a mutation affecting the gene "white" did not have a major impact of these feeding activities.

After collecting data from the cages, each stack of images was analyzed with the multiCAFE plugin, first by annotating each record with proper descriptors and by marking the position of each capillary on the images with a line ROI. Once these ROI lines were set and properly annotated, kymographs were generated to follow the evolution of the liquid during each experiment. The upper limit of liquid in each capillary was then detected using a manually defined threshold applied to images transformed with one of the filters available. This upper limit was transformed into a curve, which was subtracted with the curve observed on capillaries recording evaporation. Eventually, individual curves were curated if a glitch happened during the experiment. Once all recordings were analyzed with the multiCAFE plugin, these data were exported to an Excel file, showing the descriptors and the measures of each capillary on individual rows.

## QUANTIFICATION AND STATISTICAL ANALYSIS

Data from the MultiCAFE plugin are exported within three tables: raw data, data corrected for evaporation within each cage, and sum (L+R) and ratio (L-R)/(L+R) of the left (L) and the right (R) capillaries. Each line of the table describes the measures and the parameters related to a single capillary. The first 20 columns are descriptors such as the directory name of the stack of files, camera number, experiment descriptor, stimulus, concentration, strain of fly, sex, left or right capillary, etc. These columns are followed by the measures done on each picture or on each time interval. Typically, one table regroups all measures related to one experiment, i.e. different cages, and at different dates.

In order to ease plotting the data and analyzing time dependent parameters, these tables typically need to be reformatted, by "unpivoting" the N columns with the measures (value at ti, i = 1 to N) into
A

<figure><img src="image_8.png" /></figure>

B

<figure><img src="image_9.png" /></figure>

C

<figure><img src="image_10.png" /></figure>

D

<figure><img src="image_11.png" /><figcaption>**Figure 5. Analyze feeding with multiCAFE plugin**</figcaption></figure>

(A) MultiCAFE plugin has several sections starting with the description of the experiment (one recording or stack of jpg files), which can be loaded one by one ("Open") or as a group of files ("Search"). Here, the user has loaded the 10 experiments analyzed in Figure 6. Each experiment is associated with 8 text fields with values entered by the operator. These text fields play the role of database descriptors that can be selected to display a subset of the experiments. These data fields can be edited and are saved along with the results in an xml file, stored in a directory "results" along with the stack of images.

(B) The first step of the analysis is to define where the capillaries are, by placing ROIs (the green lines) over each capillary. The user needs also to define which cells contain a fly. Those with no fly are used to evaluate the evaporation.

(C) When the ROIs are placed over the capillaries, kymographs for each capillary can be generated. A kymograph is simply a reconstructed image obtained by collating the column of pixels under one capillary a long time. The y axis summarizes the color of the pixels from the top to the bottom of one capillary. The x axis corresponds to the time. The section "levels" of multiCAFE regroup tools to analyze the upper limit of the liquid in each capillary. On the figure, the notches on the green curve correspond to large feeding events.

(D) When limits are detected, the corresponding curves are displayed (left capillary: red; right capillary: blue). Cells with no flies (n flies = 0) are displayed with a gray background and will be used to evaluate the evaporation, otherwise the background is white (n flies = 1 or more; n < 0 is used to indicate that this measure should be discarded). The curve values and the associated descriptors are then exported to an Excel file using procedures regrouped in the "Export" section. In the example shown, the Excel file will include the measures done on all experiments. MultiCAFE also allows to edit the curves and to measure additional parameters such as gulps ("Levels" section) and flies position (in the "Cages" section) - these procedures are not described here.

N rows, including two columns with time (t_i) and the measure (value_j). We provide procedures to do it with Microsoft software Excel (using PowerQuery and PowerPivot) and with R routines. It allows users to quickly visualize the data and scan them for artefacts (for example one capillary results
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 6. Analyze an experiment after exporting data**</figcaption></figure>

Example of curves generated with Excel from the 10 recordings obtained on groups of flies given the choice between 30 mM sucrose (orange curve) and 0.1 mM caffeine mixed with 30 mM sucrose (blue curves).

(A) Volume ingested from each capillary after compensating for evaporation in two cages with 8 Canton S flies. 2 cameras were used (cam09 and cam10). In each section, the curves correspond to the evolution across time (0 to 120 min) of the liquid level in one cell (A0 to A9; B0 to B9). Cells A0 and A9, (resp. B0 and B9) were empty and their average value was subtracted to the raw liquid level of the other cells containing a fly in the same cage. This representation is useful to check if data are correctly analyzed.

(B) The average consumption a long time (120 min) of the three categories of flies tested. The mean value is represented here along with the standard error to the mean (SEM) and correspond to measures made on 16 flies (CS), 32 flies (W1118 strain1) or 31 flies (W1118 strain 2).

(C) Average consumption ± SEM. at t=120 min in the three strains tested. For CS, n=16 flies, for each W1118 strain, n=32 and n=31.

diverging notably from the others), display the desired parameters, for example the consumption of
each individual fly, the average (or median) value of the consumption with time (Figure 6).

LIMITATIONS

The multiCAFE test is used to quantitatively compare the consumption of flies from two capillaries. It is robust and flexible. For example, the number of choices given to flies can be increased by changing the configuration of the cages, and the number of flies per cage can be increased.

When the focus of the experiment is to evaluate the flies' choices, the analysis should stop when either solution is exhausted or no longer accessible to the flies. In our current setup, we routinely
used this test for 2-4 h runs. For longer periods, a larger capillary can be used (e.g. 10 µL) and/or the capillaries need to be changed regularly.

Evaporation in capillaries must be kept as low as possible. Although our experimental protocol compensates for evaporation (by subtracting the amount evaporated in capillaries placed into an empty cage), if evaporation is too high, it could induce significant changes in the concentration of the solutions between the beginning and end of the experiment. In our hands, evaporation during a 2 h session is between 0.05-0.2 µL. To limit evaporation, we place the cages in a temperature- and humidity-regulated chamber and add a damp piece of filter paper inside the recording box.

## TROUBLESHOOTING

Two types of problems may arise when using this protocol. The first category mostly concerns capillaries and how to ensure that changes of the liquid level reflect fly feeding and not other factors. The second category is related to data analysis using the current version of the multiCAFE plugin. Most of these issues can be addressed relatively easily.

### Problem 1: Evaporation too high

Evaporation is a major concern in multiCAFE experiments, especially when the consumption of flies is low. A high evaporation rate affects the precision of the measurements and the concentration of the chemicals. Because evaporation occurs at the air/liquid interface, a small surface layer of liquid contains more molecules than in the rest of the solution. Besides, flies do not perform well in a dry environment as they are sensitive to desiccation.

#### Potential solution

* Increase the humidity inside of the observation box by placing a piece of filter paper humidified inside of the box.

* Place the observation box inside of a cabinet with a high humidity and control the temperature. Usually, we perform experiments at 25°C and 75% R.H.

* Prepare the capillaries in a room where the ambient air is not too dry.

### Problem 2: Liquid flowing out of the capillaries

In some cases, the capillaries do not hold liquid when placed vertically. This may be due to a combination of physical factors, such as the viscosity of the liquid being too low or the use capillaries with an excessively large diameter. In our experiment with nonionic surfactants, the liquid did not hold in the capillaries when the cage was placed vertically.

#### Potential solution

* The simplest solution is to place the box in a tilted position at an angle at which the liquid does not flow out. The tilting angle can be large so that the capillaries may rest almost horizontally instead of vertically.

* An alternative is to increase the viscosity of the solution by adding yeast extract.¹¹

* Another approach is to dip the tip of the capillary into a thin layer of 0.5% agar, to form a plug at the end of the capillary.

### Problem 3: Liquid forming a drop outside of the capillary

In some cases, a drop forms outside of the capillary. As a consequence, reading the volume of liquid ingested by the fly is incorrect. This situation also tends to worsen with time, with the liquid flowing outside of the capillary rapidly.
**Potential solution**

* Once a drop is formed, discard the capillary and fly.

* Prevent this by carefully drying out the outside of the capillary before putting it in place.

* Avoid strong shocks to the cage or to the microscope slide holding the capillaries.

**Problem 4: Liquid level not at the bottom of the capillaries**

Occasionally, the bottom of the liquid is not at level with the bottom end of the capillary. If the distance is large, flies may be unable to access the liquid.

**Potential solution**

* Avoid transporting the capillaries upside down.

* Ensure that capillaries are prepared in a room with high humidity to reduce evaporation during preparation.

* Gently tap the cage to allow the liquid descend to the tip. This needs to be done cautiously to prevent drops from forming outside the capillaries (see problem 3).

**Problem 5: Bubble in the capillary**

During preparation, capillaries are filled by dipping them into a solution of liquid, either entirely or partly. Usually, this is sufficient for the liquid to flow in by capillary action. In rare cases, bubbles form in the capillary.

**Potential solution**

* A single bubble inside the capillary has a negligible influence on the reading. The bubble usually remains in place and does not migrate to the top or bottom of the capillary.

* If the bubble is noticed before the capillaries are in place in the multiCAFE, the easiest way to remove it is to remove the faulty capillary and replace it with a new one.

**Problem 6: Liquid going up in the capillaries instead of down**

In some cases, especially during long recording session (4 h and more), the liquid in a few capillaries goes up against gravity and centers in the middle section. As a result, flies can no longer feed on these capillaries. In our experience, this occurs mainly in capillaries containing the control solution (30 mM sucrose) and never in capillaries containing sucrose mixed with a bitter molecule. We presume that when flies actively feed on one capillary, they release saliva, which may change the viscosity and surface tension of the liquid. We did not find a completely satisfactory solution to this problem, which is relatively rare.

**Potential solution**

* Once this occurs, if the experiment involves testing whether flies prefer one capillary over the other, the only solution is to discard the measures when the liquid is no longer accessible to the fly.

* One possibility is to increase the volume of the capillary used (10 µL instead of 5 µL). This will delay the occurrence of this phenomenon.

* Another approach is to dip the tip of the capillary into a thin layer of 0.5% agar so that it forms a plug at the end of the capillary. The plug tends to prevent the lower level of the liquid to go back inside of the capillary.

**Problem 7: Position of the capillaries shifting on the images**

In the current design of our experimental setup, the multiCAFE cage is inserted inside the top cover of the plastic container and the camera is placed at the bottom. In some cases, the camera or the
cage move slowly during the recording session. Another cause of disturbance comes from the user
introducing a new cage and inadvertently pushing other cages near it.

Potential solution

* Place the camera within a frame and secure its position inside the box. We designed a 3D printed support to encase the camera and fit it to the bottom of the box. This encasing can be permanently secured to the box using glue.

* Secure the multiCAFE cage to the cover of the box. We use putty (UHU Stick), but magnetic tape could also be used.

* Most movements can be corrected during the analysis of the results, either by dividing the recording into successive phases (with the user redefining the position of the capillaries during each phase) or by using a registration program.

* Alternatively, make use of a registration program to align all images of your experiment and perform the analysis of the modified stack.

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead con-
tact, Frédéric Marion-Poll (fr Frederic.marion-poll@universite-paris-saclay.fr).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contacts, Frédéric Marion-Poll (frédéric.marion-poll@universite-paris-saclay.fr) and Béatrice Denis (beatrice.denis@university-paris-saclay.fr).

Materials availability

All materials can be purchased from the mentioned (or other) companies. The 3D files to print the multiCAFE device are
available on DataVerse. This study did not generate new unique reagents.

Data and code availability

The plugin MultiCAFE is available on the website of the *Icy* program. The raw data and analysis files as well as the 3D models are available on *DataVerse*.

ACKNOWLEDGMENTS

The work was supported by ANR BITTERTOX, program ANR-19-CE34-0011, and by running support from UMR EGCE and AgroParisTech.

AUTHOR CONTRIBUTIONS

Conceptualization, F.M.-P. and C.M.C.R.; experiment, P.C., B.D., V.W., and C.M.C.R.; methodology, P.C., B.D., and F.M.-P.; software and 3D design, F.M.-P.; formal analysis, P.C., B.D., F.M.-P., and C.M.C.R.; writing - review and editing, F.M.-P., D.R., B.D., P.C., and C.M.C.R.; funding acquisition, F.M.-P. and D.R.; supervision, F.M.-P. and D.R. All authors have approved the manuscript.

DECLARATION OF INTERESTS

The authors declare no competing interests.

REFERENCES

1. Colombi, P. (2021). Modulation de la prise de nourriture par des molécules amères chez *Drosophila melanogaster* (Université Paris-Saclay). PhD Thèse de doctorat.

2. Richard, C.M.C., Renault, D., Wallart, V., Denis, B., Tarapacki, P., Marion-Poll, F., and Colinet, H. (2025). Effects of nonionic surfactants on life history traits of *Drosophila melanogaster*. *Environ. Sci. Pollut. Res. Int.* 32, 3809–3821. https://doi.org/10.1007/s11356-025-35932-4.

3. French, A., Ali Agha, M., Mitra, A., Yanagawa, A., Sellier, M.-J., and Marion-Poll, F. (2015). *Drosophila* bitter taste(s). Front. Integr. Neurosci. 9, 58. https://doi.org/10.3389/fnint. 2015.00058.

4. LeDue, E.E., Mann, K., Koch, E., Chu, B., Dakin, R., and Gordon, M.D. (2016). Starvation-induced depotentiation of bitter taste in *Drosophila*. Curr. Biol. 26, 2854-2861. https://doi.org/10.1016/j.cub.2016.08.028.

5. Ganguly, A., Dey, M., Scott, C., Duong, V.K., and Arun Dahanukar, A. (2021). Dietary macronutrient imbalances lead to compensatory changes in peripheral taste via independent signaling pathways. J. Neurosci. 41, 10222-10246. https://doi.org/10.1523/ jneurosci.2154-20.2021.

6. Deshpande, S.A., Carvalho, G.B., Amador, A., Phillips, A.M., Hoxha, S., Lizotte, K.J., and Ja, W.W. (2014). Quantifying *Drosophila* food
intake: comparative analysis of current methodology. Nat. Methods 11, 535–540. https://doi.org/10.1038/nmeth.2899.

7. Marx, V. (2015). *Metabolism: feeding fruit flies.* Nat. Methods 12, 609–612.

8. Aryal, B., Dhakal, S., Shrestha, B., Sang, J., Nhuchhen Pradhan, R., and Lee, Y. (2022). Protocol for binary food choice assays using *Drosophila melanogaster*. STAR Protoc. 3, 101410. https://doi.org/10.1016/j.xpro.2022.101410.

9. Tanimura, T., Isono, K., Takamura, T., and Shimada, I. (1982). Genetic dimorphism in the taste sensitivity to trehalose in *Drosophila melanogaster*. J. Comp. Physiol. 147, 433–437. https://doi.org/10.1007/BF00612007.

10. Meunier, N., Marion-Poll, F., Rospars, J.P., and Tanimura, T. (2003). Peripheral coding of bitter taste in *Drosophila*. J. Neurobiol. 56, 139–152. https://doi.org/10.1002/neu.10235.

11. Ja, W.W., Carvalho, G.B., Mak, E.M., de la Rosa, N.N., Fang, A.Y., Liong, J.C., Brummel, T., and Benzer, S. (2007). Prandiology of *Drosophila* and the CAFE assay. Proc. Natl. Acad. Sci. USA 104, 8253–8256. https://doi.org/10.1073/pnas.0702726104.

12. Sellier, M.-J., Reeb, P., and Marion-Poll, F. (2011). Consumption of bitter alkaloids in *Drosophila melanogaster* in multiple-choice

test conditions. Chem. Senses 36, 323–334. https://doi.org/10.1093/chemse/bjq133.

13. Diegelmann, S., Jansen, A., Jois, S., Kastenholz, K., Velo Escarcena, L., Strudthoff, N., and Scholz, H. (2017). The CApillary FEeder assay measures food intake in *Drosophila melanogaster*. J. Vis. Exp. 14, e55024. https://doi.org/10.3791/55024.

14. Segu, A., Radhakrishnan, D., and Kannan, N.N. (2023). Modified Capillary Feeder assay using micro-tips to measure real-time feeding in *Drosophila*. STAR Protoc. 4, 102393. https://doi.org/10.1016/j.xpro.2023.102393.

15. Qi, W., Yang, Z., Lin, Z., Park, J.Y., Suh, G.S.B., and Wang, L. (2015). A quantitative feeding assay in adult *Drosophila* reveals rapid modulation of food ingestion by its nutritional value. Mol. Brain 8, 87. https://doi.org/10.1186/s13041-015-0179-x.

16. Yapici, N., Cohn, R., Schusterreiter, C., Ruta, V., and Vosshall, L.B. (2016). A taste circuit that regulates ingestion by integrating food and hunger signals. Cell 165, 715–729. https://doi.org/10.1016/j.cell.2016.02.061.

17. Murphy, K.R., Park, J.H., Huber, R., and Ja, W. W. (2017). Simultaneous measurement of sleep and feeding in individual *Drosophila*. Nat. Protoc. 12, 2355–2366. https://doi.org/10.1038/nprot.2017.096.

18. French, A.S., Sellier, M.-J., Ali Agha, M., Guigue, A., Chabaud, M.-A., Reeb, P.D., Mitra, A., Grau, Y., Soustelle, L., and Marion-Poll, F. (2015). Dual mechanism for bitter avoidance in *Drosophila*. J. Neurosci. 35, 3990–4004. https://doi.org/10.1523/jneurosci.1312-14.2015.

19. de Chaumont, F., Dallongeville, S., Chenouard, N., Hervé, N., Pop, S., Provoost, T., Meas-Yedid, V., Pankajakshan, P., Lecomte, T., Le Montagner, Y., et al. (2012). Icy: an open bioimage informatics platform for extended reproducible research. Nat. Methods 9, 690–696. https://doi.org/10.1038/nmeth.2075.

20. Abramoff, M.D., Magelhaes, P.J., and Ram, S.J. (2004). Image processing with ImageJ. Biophot. Int. 11, 36–42.

21. Marella, S., Fischler, W., Kong, P., Asgarian, S., Rueckert, E., and Scott, K. (2006). Imaging taste responses in the fly brain reveals a functional map of taste category and behavior. Neuron 49, 285–295. https://doi.org/10.1016/j.neuron.2005.11.037.

22. Mitri, C., Soustelle, L., Framery, B., Bockaert, J., Parmentier, M.-L., and Grau, Y. (2009). Plant insecticide L-canavanine repels *Drosophila* via the insect orphan GPCR DmX. PLoS Biol. 7, e1000147. https://doi.org/10.1371/journal.pbio.1000147.