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Two-choice test using food dyes
The most commonly used test consists in allowing flies to feed in the dark on two food substrates mixed either with a blue or a red food dye (Tanimura et al., 1982) (Figure 5). After exposure to the food, the abdomen color of each fly is checked (red, blue or purple when they fed on both sources, empty when they did not feed) and a preference index is computed. If the tested substance was associated with the blue dye, the index will be: I = (Nb blue flies + ½ Nb purple flies) / (Nb blue flies + Nb purple flies + Nb red flies). If the index is comprised between 0 and 0.5, the tested molecule is attractive, if the index is between 0.5 and 1, the substance is considered as deterrent. An index of 0.5 shows neutrality.
This test has a good sensitivity and relies on the actual consumption of the flies and not only their presence. Nevertheless, it is limited to the study of binary choices and requires an experienced observer to assess the color of the flies’ abdomen. The amount consumed by the flies can be estimated with a spectrophotometer (Tanimura et al., 1982) under the assumption that the content of the flies’ abdomen reflects what has been ingested.
As we previously mentioned, the test based on food dyes, which has already been used in many studies and has a good sensitivity, relies on the actual consumption of the flies and not only their presence. Nevertheless, the major drawbacks of this test are its inability to perform Modulation of feeding behavior and peripheral taste response by aversive molecules in D. melanogaster Marie-Jeanne Sellier
more than two-choice assays and the relative difficulty in assessing the color of the abdomen. The amount consumed by the flies can be estimated but a spectrophotometer is required. Moreover, the consumption of the flies cannot be monitored through time as the flies must be sacrificed in order to get the results of the test.
Left: 96 -microwell plate filled with the two agar solutions tested (Isono and Morita, 2010). Right: After the test, the number of flies having a blue, red or purple abdomen is determined.
Capillary feeder (CAFE)
In rats and mice, “self-service bottles” are commonly used to study feeding behaviors (Glendinning et al., 2005; Pittman et al., 2006; Inoue et al., 2007; Tordoff et al., 2008). The same principle has been used in insects, such as ad hoc capillary feeders for houseflies (Dethier, 1976) or 100 µl capillaries for the flesh-fly Sarcophaga bullata (Cheung and Smith, 1998). More recently, Ja et al. (2007) studied the feeding behavior of D. melanogaster adults with 5 µl micro-capillary tubes. With this system, called Capillary Feeder (CAFE), they analyzed the prandial behavior of flies, the influence of population density or humidity and the impact of ethanol or paraquat on food intake (Figure 6). The quantity of liquid ingested by the flies can be recorded in real time by monitoring the level of the liquid within the capillaries. This test has been used successfully as a no choice or two-choice assay on D. melanogaster to study the regulation of feeding by peripheral clocks (Xu et al., 2008; Chatterjee et al., 2010), the effect of leucokinin on meal size regulation (Al-Anzi et al., 2010) or how the food content in protein and carbohydrate affects lifespan and fecundity (Lee et al., 2008) or sleep-wake behavior (Catterson et al., 2010) for example.
The level of liquid in the capillary tubes is monitored and consumption can be measured through time (Ja et al., 2007).
Introduction to a quantitative multiple-choice assay
We have described various behavioral tests available to measure feeding preferences in D. melanogaster. All these tests have proven to give results but they also have disadvantages. Most of them are not directly related to the consumption, or the quantitative data are not readily accessible. The CAFE assay seemed to be the only available test to fulfill this condition. Moreover, in order to build dose-response profiles quickly, we chose to develop a multiple-choice test. Given the limitations of existing assays, we tried to design another approach to evaluate flies selectivity and absolute consumption. We adapted the CAFE assay and evaluated the use of a system to test feeding preferences in flies by providing them access to a series of 6 capillary tubes filled with solutions containing different concentrations of an antifeedant. This approach, that we called MultiCAFE, gives the possibility to build dose-response profiles directly.
However, some theoretical problems arise from this setup. One of the potential limitation of the MultiCAFE is that it may not make it easy for flies to discriminate among the different capillary feeders because of the multiplicity of choices available (Prince et al., 2004) (Figure 7). The consumption of two substances or two concentrations can differ greatly whether they are presented alone or simultaneously (Shimada et al., 1987; Akhtar and Isman, 2004). This could influence the apparent antifeedant potency of a given concentration of a bitter substance in the MultiCAFE. Binary choices might be easier to deal with for the flies. Indeed, memorization and comparison of the options should be quicker when only two choices are provided, rather than when many different types of food are available, even if fruit flies seem to be capable of visual learning (Schnaitmann et al., 2010). On the other hand, the multiplicity of options may introduce such complexity that the flies’ choice might involve instant decisions, related to hunger and taste detection, more than memory. In this way, we can wonder if multiple-choice tests can be considered as equivalent to multiple no-choice tests. If this is the case or if, at least, the sensitivity of the multiple-choice test is close to the sensitivity of no-choice or two-choice assays, then the MultiCAFE would give the possibility to compare the antifeedant activity of different substances or to describe mutants’ impairments.
The fact that multiple substances (or concentrations) presented at the same time can be more difficult to discriminate, as compared to two-choice assays, might increase the number of repetitions required in order to decrease variability (Raffa et al., 2002). Moreover, we can wonder if providing the flies with both palatable and non-palatable food sources might elicit “compensative” feeding. As the flies do not eat the deterrent food, they may eat more of the appetitive food to compensate and keep a constant total consumption. Some protocol issues may arise as well from multiple-choice assays. Indeed, the way to present the different food sources is likely to have an effect on the choice or the intake of the flies. Moreover, high fly densities might trigger competition for the most palatable food sources.
Using the MultiCAFE, we try to answer some of these questions. First, we show that the fly density as well as the order of presentation of the concentrations has an influence on the quinine dose-response profile, while the spacing between the capillary tubes does not seem to modify the flies’ feeding behavior. Secondly, we evaluate the variability generated by the MultiCAFE according to the number of repetitions. We also compare the sensitivity of the MultiCAFE assay used as a no-choice, two-choice or multiple-choice test. Then, we build dose-response profiles for 8 alkaloids and rank them according to their antifeedant potency in the MultiCAFE. Finally, we test a mutant supposed to have caffeine-detection impairments and show that, in addition to the lower caffeine discrimination, this mutant also seems to have a general intake defect.
Description of the MultiCAFE setup
First generation of the assay (vials)
Unless otherwise specified, the flies used in these experiments are Canton-S flies, graciously given to our laboratory by Pr. Teiichi Tanimura. Emerged flies (~1 day old) were transferred to a freshly prepared food medium for 2 to 3 days and maintained in a rearing chamber at 25 °C. The flies were first sexed (after numbing them on ice), transferred to plastic tubes provided with humidified filter paper and starved for 20 – 22 hours. Just before the experiment, these flies were numbed on ice and transferred into experimental vials (23.5 dia. × 40 mm, SARSTEDT). All experiments were performed at the beginning of the afternoon, to prevent any effect of the circadian rhythm, at 25°C under complete darkness.
Experimental vials were closed by a plug (28.5 mm Buzz-Plugs, Fisherbrand), cut to 0.8 cm height and sliced in two halves (Figure 8). On one half of this modified plug, we disposed a row of six 5 µl micro-capillary tubes (Hirschmann Laborgeräte, Germany) on a strip of double-sided sticky tape. The capillaries were equally spaced (~ 1 mm unless otherwise specified) and protruded inside of the vial by ~ 5 mm. Each row of capillary tubes was filled with serial dilutions (0, 0.001, 0.01, 0.1, 1 and 10 mM) of a test compound mixed with 35 mM fructose and 0.125 mg / ml of blue food dye (brilliant blue, FCF (C37H3409SNa), Tokyo Kasei Co.). According to earlier tests, this dye has no effect on taste sensitivity and is not toxic to flies at the concentration used (Tanimura et al., 1982). As a control, we also tested a row of capillaries with only fructose and the blue dye.
Table of contents :
I. General introduction
1. Mechanisms evolved by the insects to cope with the secondary plant compounds
2. Morphology and physiology of taste in Drosophila melanogaster
II. The MultiCAFE: a quick feeding preference test to build dose-response curves
1. Feeding preference tests in D. melanogaster
A. Test based on the fly density
B. Proboscis extension reflex (PER)
C. Two-choice test using food dyes
D. Capillary feeder (CAFE)
2. Introduction to a quantitative multiple-choice assay
3. Description of the MultiCAFE setup
A. First generation of the assay (vials)
B. Second generation of the assay (boxes)
4. Statistical analysis
5. Influence of fly density on intake in the MultiCAFE
6. Influence of the arrangement of the series of concentration of quinine
7. Effect of the spacing of the capillary tubes
8. Number of replicates needed to build a dose response-curve
9. Comparison of the test used as a no-choice, two-choice or multiple-choice assay
10. Determination of the EC50 of various alkaloids
11. Responses of a ΔGr66a mutant to caffeine with the MultiCAFE
12. Conclusion on the MultiCAFE
13. Screening of some molecules extracted from endemic plants of the Canary Islands ….. 41
A. Identification of pericallone as a potential deterrent molecule
B. Possible inhibitory effect of pericallone on sugar detection
C. Perspectives of this study
III. Mixture interactions: involvement of the bitter cell in the sugar cell inhibition
Modulation of feeding behavior and peripheral taste response by aversive molecules in D. melanogaster Marie-Jeanne Sellier
AgroParisTech / INRA-UPMC UMR PISC 1272 6
2. Electrophysiological recording technique
3. Correlation between the electrophysiological and the behavioral responses
4. Specificity of the inhibition
5. Test for a lateral interaction between the sugar and bitter cells
A. Electrophysiological inhibition of the S cell in L2-lacking flies
B. Inhibition of (sucrose + strychnine) consumption in L2-lacking flies
IV. Experience-induced modulation of feeding
2. Attempt to set up a paradigm of habituation with caffeine
3. Modulation of the P450 activity with metyrapone
4. Conclusion on the habituation experiments
5. Adaptation to sugars
A. Previous results obtained in Linda Kennedy’s laboratory
B. Changes in fructose or glucose consumption following exposure to these sugars
C. Modulation of the electrophysiological response for fructose and glucose
D. Discussion on sugar experience-induced modifications
V. General conclusion on the PhD project and perspectives of the study