The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

Summary

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

Abstract

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

Introduction

Eating is essential; however, deregulation of food intake resulting in eating disorders such as bulimia, anorexia or the general tendency to overeat imposes costs on individuals and society^1,2,3. The goal of the present research is to uncover regulatory mechanisms of food intake and to provide a strategy for circumventing disorder formation. Numerous studies using mammalian model organisms have provided new insights of the circuitry and the role of signaling systems in eating disorders^4,5,6. Nevertheless, our knowledge of the neuronal and molecular bases underlying these disorders remains far from complete. In recent years, the fruit fly Drosophila melanogaster has become a valuable model system for unraveling basic mechanistic insights into the regulation of metabolism^7,8,9. The CApillary FEeder (CAFE) assay for Drosophila melanogaster was established in the lab of Seymour Benzer in 2007 inspired by an earlier work by Dethier in blowfly^{10, 11}. The CAFE assay made it possible to directly measure food intake in Drosophila melanogaster. In this behavioral test system, flies feed on liquid food provided in graded glass capillaries placed inside a vial. The decline of the capillary meniscus indicates loss of food solution via evaporation and food consumption. Determining the evaporation rate by vials without flies allows the accurate quantification of food intake.

The CAFE assay is one of several behavioral paradigms used to measure feeding in Drosophila melanogaster and researchers have to choose the most appropriate one for their specific question. The decision to use a certain assay should consider the following points: the nature of the food provided; the feeding condition; the measurement of intake or uptake of nutrients and investigation food consumption or response to food.

The CAFE assay as described in this report is ideal for following food intake of a liquid food source under an upright feeding condition. Alternatively the food intake can be measured for a fly group on a colored food source in a vial or on a plate. Flies are normally killed or anesthetized after feeding and the amount of ingested dye is determined by spectrometry or visual inspection of the stained abdomen. Flies start to excrete the ingested food only 30 min after intake, therefore this approach is difficult to use for the analysis of continuous longer feeding behaviors^{12, 13}.

In contrast flies are kept intact when absorbable dyes with radioactive tracers are used and their consumption of radioisotope is scored in a scintillation counter^{14, 15}. Absorption of the radiolabel by the fly digestive system makes long-term food uptake measurement possible, but might lead to underestimation of consumption because of non-absorbed and excreted tracer molecules. Another approach to measure response to food in Drosophila melanogaster is the proboscis extension response (PER), which normally occurs for food intake¹⁶. This elegant method measures the initial response to a food stimulus but does not record the quantity of intake. Food intake is dynamically adjusted during feeding using several post-digestive feedback signals that are critical for the regulation of feeding^{17, 18}. Several attempts have been made in recent years to semi-automate data collection in the PER assay^{19, 20}. The PER is detected by an electric pad or a combination of electrodes and counted via computer. Combining the PER assay with radioisotope uptake revealed that this assay is limited by low sensitivity to detecting quantity feeding differences¹⁸. The manual feeding assay (MAFE)²¹, in which a fly is fed manually with a glass capillary, was recently developed to measure food uptake in a single immobilized fly. The MAFE assay eliminates the interferences of foraging and feeding initiation and has a time resolution of seconds, and initiation of PER and food consumption can be monitored independently in the assay. However, the way in which immobilization of the fly affects certain aspects of feeding behavior (e.g. locomotion, motivation) still needs to be investigated. For excellent comparative reviews of different assays for measuring food consumption in Drosophila melanogaster and to help researchers finding the most appropriate one, see reports by Deshpande and Marx ^{13, 22}.

The CAFE assay avoids some of the disadvantages of other assays described above and combines simplicity of use with reliable measurement of food intake. Here, a detailed description of the CAFE assay is provided and we show a simple setup modification to reduce evaporation. Representative results including a two food choice assay (short and long term) and the sucrose uptake of flies is demonstrated. In the discussion we compare our described method with alternative ways to perform the CAFE assay, and highlight potential limitations.

Protocol

1. The CAFE Assay

NOTE: The assay consists of three components: an experimental vial, a specific lid and micro- capillaries. A plastic box with cover is used to transport the prepared vials and to control the humidity more efficiently.

1. Use a Drosophila melanogaster culture plastic vial (optional 8 cm height, 3.3 cm diameter) as a tube for the assay.
2. Seal the vial with a manufactured Plexiglas lid containing an O-ring (Figures 1A, 1B). Load flies by tapping or with a blowpipe through the lid's central opening (0.9 cm diameter), which also allows for air circulation and water supply, and close the hole with a sponge bung. Six smaller conical openings (0.4 cm upper diameter, 0.3 cm inner diameter) surround the central hole and fit the pipette tips of 2 - 20 µL volume to hold the capillaries in place. (see supplementary figures for technical details of the lid.)
   NOTE: The use of a sponge stopper with openings for the capillaries instead of the custom-made lid used in our manuscript is possible. Our customized lid allows safe handling of the prepared vials minimizing the risk of capillaries falling down.
3. To present the liquid food, use 5 µL microcapillaries with 1 µL marks. Position the capillaries in the conical openings in the lid by cutting off the top of a 2 - 20 µL pipette tip and inserting the tip into the hole (Figure 1B, marked with red edge). To prevent flies from escaping, insert an uncut 2 - 20 µL pipette tip into the same opening.
4. To safely handle multiple prepared vials, place them into a plastic box with a gridded inlay (Figure 2A).

2. Preparation of Flies

1. Keep flies on standard food at 25 °C, 60% relative humidity and a 12 h/12 h light-dark cycle.
2. To control breeding conditions, introduce 35 virgin females and 15 males for each experimental group into a plastic culture vial (9.8 cm height, 4.8 cm diameter) containing 50 mL fly food. Allow flies to lay eggs for the first 3 days, then transfer adult flies to fresh food vials and let them lay eggs for two more days. After this repeat the transfer again. Discard adult flies after 2 more days.
3. As food intake is dependent on fly size, determine the weight of a group of 100 flies by anesthetizing 2- to 3-day-old adult flies using a CO₂ fly pad and collect them into a 1.5 mL plastic tube and measuring with a standard laboratory scale. Determine the wet weight of at least four independent fly groups sorted by sex (Table 1); use the weight to calculate µL food consumption per mg fly. Use the value to determine the amount of food that a single fly feeds per experiment and adjust the number of food-filled capillaries accordingly to avoid emptying of the capillaries by feeding.
   1. For a 3 h assay, use 20 flies and two filled capillaries. For a long-term experiment (> 3 h and up to 9 days), use a group of eight flies with a supply of four filled capillaries (reliable results cannot be obtained with less than eight flies under the described conditions).
4. Separate flies into groups (8 or 20 flies) after measuring weight under CO₂ exposure. Transfer the group to a new food vial (containing 15 mL standard food) to allow recovery from CO₂ sedation for 48 h prior to the experiment. Use 4- to 6-day-old flies for the CAFE assay.
5. As non-starved wild-type flies feed only marginally^{19, 21}, pre-starve flies for 3 h feeding experiments. No fasting is required when food consumption is monitored over several days. For fasting, transfer flies 16 to 20 h prior to testing by gently tapping them into a vial containing only a 45-mm diameter folded filter paper moistened with ~0.5 mL ddH₂O (double-distilled water), and close with a plugged CAFE assay lid.

3. Preparation of Liquid Food

1. Prepare a 3 M (10%, w/v) sucrose stock solution by filling 102.6 g sucrose (C₁₂H₂₂O₁₁) to 100 mL ddH₂O. Pipette 3 µL, 33 µL, 333 µL, 3.3 mL and 6.6 mL of the stock solution into a 15 mL plastic tube; add 2 mL of food color (for red: Cochineal [E124]; for blue: Indigo carmine [E132]) and fill to 10 mL with ddH₂O. The resulting concentrations are 0.001, 0.01, 0.1, 1, and 2 M sucrose.
   NOTE: The food dye allows visualizing the meniscus more easily. However the dye might have an impact on the food intake. To avoid a bias due to the dye dispense the food dye or randomized the usage of dyes to the food samples during the experiment and groups.
2. To test for alcohol preference pipette 333 µL of the 3 M sucrose stock solution in a 15 mL plastic tube. Add 1.5 mL (2.3 mL) of 100% EtOH (ethanol) and add ddH₂O up to 10 mL to result in 15% (0.25 mM) and a 23% (0.39 mM) working solutions.
3. Keep stock solutions at -20 °C and working solutions at 4 °C; use within 1 week.
4. Fill up to 10 capillaries at the same time with a colored food solution, by capillary force. Insert the ends of the capillaries into the sucrose solution (holding the capillaries at a 45° angle to the solution). Stop if the liquid reaches the top (5 µL) mark of the capillary, and remove excess solution on the outside and inside with tissue paper.

4. Assembly and Performing the CApillary FEeder Assay

1. If fasting is not needed, transfer the experimental flies to the assay by tapping or by blow-pipe. Make sure to include three control vials without flies to quantify evaporation.
2. Carefully remove a pipette tip (2 - 20 µL volume) that is closing one of the outer openings, and insert a filled glass capillary, bottom-end first. Secure the capillary by placing the pipette tip back next to the capillary. If several food solutions are being tested, repeat this procedure accordingly.
3. Place the capillary ends inside all vials at the same level to avoid bias that could occur if the food sources were located at different heights (3 - 4 cm from the lid); keep a distance to the filter paper to prevent the capillary from leaking by accidently touching the filter paper or different viscosities of food sources.
4. Label the upper end of the colored liquid using a marker pen (mark_beginning). To ensure the different capillaries can be identified, label them individually using a color or stripe code.
5. Place multiple prepared CAFE assays inside a plastic box with gridded inlay and transfer the box (Figure 2A) to a secure position under laboratory conditions or in a temperature-, light- and humidity-controlled climate chamber (parameters: 25 °C, 60% relative humidity, 12 h/12 h light-dark cycle) for the experimental period (e.g. 3 h or days).
6. As bottom filter paper dries out if the assay is performed over several days, apply fresh water every 24 h via the sponge bung (100 µL) to keep humidity constant inside the assay. Use four separate vials (8 cm height, 3.3 cm diameter) filled with 30 mL ddH₂O as humidity devices and place them next to the CAFE assays in the plastic box. Use a cover for the plastic box to create humidity controlled environment during the experiment (Figure 2A).
   NOTE: Broader variability occurs under laboratory conditions; however, it is feasible to perform the CAFE assay at room temperature (e.g., in a classroom). The use of a humidification device (filter paper, with or without a wet sponge bung, filled water vials and cover for the plastic box) is highly encouraged to decrease evaporation (Figure 2B).
7. Replace the capillaries with freshly filled ones for long term experiments every 24 h. Make note of dead flies before each 24 h interval and use the number of live flies to calculate consumption per fly for the following period. Discard the old capillaries after measuring the decline of the meniscus (see 5.1).
   NOTE: During a 3 h experiment we hardly see any dead flies. During a 4 days study we usually find 1 - 3 dead flies.
8. At the end of the assay or before replacing the capillary, mark the lower meniscus of the capillary (mark_end) with a marker pen while the CAFE assay is still in the upright position. Discard the data if mark_end is not below the initial mark (mark_beginning). Do not remove the lid, as this might change the meniscus.
9. Carefully remove the capillaries from the assay and store them for data collection. Check if the liquid inside the capillary reached the lower end if not discard the data, as food was not accessible to the flies. Collect all capillaries per vial as a group. Insert uncut pipette tips into all openings to prevent flies from escaping. Dismantle the setup and wash the vials, lids and sponge bungs in a soap bath and dry overnight at room temperature for further use.
   NOTE: Flies can be further analyzed after the assay. Confirm food uptake by eye or under a dissection microscope.
10. Repeat experiments with the same genotypes on at least three different days.

5. Data Collection and Analysis

1. Measure the distance between mark_beginning and mark_end on the capillary using a caliper or a ruler. To transfer data directly to a spreadsheet, use a USB (Universal Serial Bus) connected digital caliper (Figure 1E). Discard the capillaries after the measurement.
2. Account for capillary size to calculate food uptake or evaporation. For example, consider a capillary that is 73 mm long and contains 5 µL of food solution. A 14.6 mm decrease in the meniscus reflects the uptake of 1 µL solution. Calculate food uptake using the following formula:
   Food uptake (µL) = measured distance (mm)/ 14.6 mm
3. To exclude the effect of evaporation on food intake, calculate mean evaporation in the three (at minimum) control vials without flies. Subtract this mean value from the value obtained for food consumption by the flies.
4. Use the following formula to determine total consumption per fly:
   Food consumption (µL) = (Food uptake [µL] - Evaporative loss [µL])/total number of flies in the vial. For long-term experiments use the number of flies alive before the start of the 24 h interval.
5. To account for differences in body size, such as between male and female flies, normalize food consumption to body weight (µL food/mg fly).
6. Use statistical software for data analysis. For normally distributed data, use student's T-tests to determine differences between two fly groups, and use ANOVA (analysis of variance) with post hoc Tukey Cramer tests for more than two groups. In a choice situation, analyze differences from random choice using a nonparametric one-sample sign test.

Results

Flies of the w¹¹¹⁸ genotype are used to demonstrate how the assay is performed. The w¹¹¹⁸ mutants are commonly used to generate transgenic lines and to control the genetic background of transgenes marked with the white gene. Normally, for behavioral experiments, all transgenic lines are backcrossed for five generations to the same w¹¹¹⁸ stock, which is used as an experimental control. We show different experiments: a com...

Discussion

The report describes the CAFE assay in a step-by-step fashion, focusing on the technical setup and its successful performance in the laboratory. Due to its simplicity, this assay could also be used educationally as a school experiment. The examples show that the assay allows investigation of food sensing, preference and consumption in Drosophila melanogaster over short and longer time periods (hours to days). The CAFE assay has been used widely in the field to investigate subjects including food and drug consump...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Vials (breeding)	Greiner Bio-One	960177	www.greinerbioone.com
Vials (CAFE assay)	Greiner Bio-One	217101	www.greinerbioone.com
Lid-CAFE assay	Workshop	–	–
Plastic box, low wall	Plastime	353	www.plastime.it
Cover for the plastic box	Workshop	–	–
Capillaries	BLAUBRAND	REF 7087 07	www.brand.de
Pipette tips	Greiner Bio-One	771290	www.greinerbioone.com
Filter paper circles	Whatman	10 311 804	www.sigmaaldrich.com
D(+)-Sucrose	AppliChem	57-50-1	www.applichem.com
Ethanol absolute	VWR Chemicals	20,821,330	www.vwr.com
Food color (red, E124)	Backfun	10027	www.backfun.de
Food color (blue, E133)	Backfun	10030	www.backfun.de
Soap solution (CVK 8)	CVH	103220	www.cvh.de
Digital caliper	GARANT	412,616	www.hoffmann-group.com
Vials (breeding)			Height 9.8 cm, diameter 4.8 cm
Vials (CAFE assay)			Height 8 cm, diameter 3.3 cm
Lid-CAFE assay			Produced in university workshop, technical drawing supplied Please click here to download this file.
Plastic box, low wall			A plastic grid inlay was custom-made for 8 x 10 vial positions
Cover for the plastic box			Dimensions (37 x 29 x 18 cm)
Capillaries			DIN ISO 7550 norm,  IVD-guideline 98/79 EG, ends polished
Pipette tips			Pipettes for the outer circle are cut according to the lid
Filter paper circles			45 mm diameter works nicely if folded for the vials used
D(+)-Sucrose			Not harmful
Ethanol absolute			Highly flammable liquid and vapor
Food color (red, E124)			Not stated
Food color (blue, E133)			Not stated
Soap solution (CVK 8)			Odor neutral soap
Digital caliper
Standard fly food			(for 20 L)
Agar			160 g
Brewer's Yeast			299.33 g
Cornmeal			1,200 g
Molasses			1.6 L
Propionic acid			57.3 mL
Nipagin 30%			160 mL