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. × Modulation of feeding behavior and peripheral taste response by aversive molecules in D. melanogaster Marie-Jeanne Sellier 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.
Moreover, as the molecules were presented in solution, evaporation became an issue and the tests had to be conducted at a high humidity rate (~70 %). Limiting evaporation in MultiCAFE experiments is particularly important, for three reasons. First, if one wants to measure consumption accurately, evaporation should be kept to a minimum in order to decrease statistical errors. During the pilot tests, we experienced conditions where evaporation was four or five times higher than the flies’ consumption. Reducing evaporation allowed us to reduce variability between tests. Secondly, the controls have to be carefully chosen so that they truly represent the evaporation present in the test tubes. In our dose-response curves, some points are negative, especially at high doses of alkaloids where no ingestion occurs. The most likely explanation is that evaporation in tubes containing flies is reduced as compared to tubes which are empty. Thirdly, evaporation may alter the actual concentration of antifeedants experienced by the flies. Since the liquid column is enclosed into a tube limiting passive diffusion and convection, the surface of the liquid is probably more concentrated in antifeedant (and sugar) than the rest of the tube. So far, the best way to limit this concentration seems to reduce evaporation as much as possible. To limit evaporation, the outer side of each capillary was dipped into mineral oil and the excess of oil was wiped with a paper towel. For each test and for each condition, a control vial without flies was placed into the experimental chamber to monitor evaporation of the capillaries.
Second generation of the assay (boxes)
To further reduce evaporation, we modified the setup, using boxes instead of vials. In this system, the capillaries were inside the box and were consequently less exposed to airstreams (Figure 9). The six capillaries were disposed on a microscope slide with double faced tape and equally spaced (~ 5 mm). The slide was then placed in a plastic box (95 x 76 x 15 mm, Caubère, Increasingconcentration of antifeedant France) with repositionable adhesive pads (Patafix, UHU). The flies were transferred into the box without anesthesia.
Influence of fly density on intake in the MultiCAFE
The dose-response curves obtained with the MultiCAFE may combine the taste discrimination capacities of the flies with memory performances (Motosaka et al., 2007) and a number of social interactions like competition (Dierick and Greenspan, 2006; Vrontou et al., 2006) or social facilitation (Shimada et al., 1987; Tinette et al., 2004; Tinette et al., 2007). The fly density in the test chambers was likely to modulate the agonist and antagonist interactions between animals and thus, to have an influence on the results of the MultiCAFE. In order to establish the impact of the number of flies on MultiCAFE tests, we compared the responses to a series of dilutions of quinine using densities of 10, 20, 40 or 60 flies. Each test condition (density × sex) was replicated 10 times in vials.
Influence of the arrangement of the series of concentration of quinine
Raffa et al. (2002) used a multiple-choice assay to test the effect of isopimaric acid on Lymantria dispar larvae. They coated leaf disks with various concentrations of isopimaric acid and provided the caterpillars with up to 5 different concentrations at a time. The leaf discs were disposed in circle and the various doses were presented in 6 different configurations. They found that the caterpillars could discriminate between the concentrations of isopimaric acid and that the configuration had an effect on the intake of the larvae. Indeed, the dose response profile had the same shape for each configuration but some arrangements seemed to elicit an increase in the consumption of the lower doses and thus a higher probability to find significant differences of intake between doses. To assess if the order of presentation of the capillaries had an impact on the dose-response curves in the MultiCAFE, we tested two conditions (a) capillaries disposed in a row of increasing concentrations and (b) capillaries disposed in random order, using groups of 40 flies and 10 replicates per condition and per sex in vials. The 10 randomized order corresponding to the 10 replicates were obtained using the random function as a macro under Excel. No difference was observed between the curves for males (p = 0.1843, MANOVA, Figure 11).
Number of replicates needed to build a dose response-curve
This first set of data led us to consider that 10 repetitions for each experimental condition could be considered as a reasonable number to get a good estimate of the dose response curves obtained with quinine. In order to go beyond this rule of thumb, we ran a statistical estimate of the reduction of variability obtained when using increasing numbers of repetitions. We used all experiments performed with the fructose control and randomly selected subsets of these data to estimate the variability. As shown on Figure 13, we observed that the standard deviation reached a plateau at about 15 repetitions.
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
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
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