Screening of some molecules extracted from endemic plants of the Canary Islands

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Morphology and physiology of taste in Drosophila melanogaster

Fruit flies react to taste molecules in a way which is quite similar to humans (sometimes more than rodents, see: Gordesky-Gold et al., 2008) and within the detection range of mammals. They are attracted to sugars, avoid bitter and toxic molecules and adapt their consumption of acids and salts to their internal needs (Amrein and Thorne, 2005; Gerber and Stocker, 2007). Although the taste system of mammals and fruit flies is anatomically different, the numerous functional similarities between them, the relative simplicity of the insects’ gustatory system and the molecular tools available make Drosophila melanogaster a good model to study taste perception (Scott, 2005). In drosophila, taste is mediated through taste hairs, called sensilla, located on mouthparts, the legs, the wings margin and the ovipositor (Figure 1, Left). Taste sensilla directly influence feeding activities, especially those located on the mouthparts, i.e. on the labellum or proboscis.
All taste sensilla have a pore at their tip that let chemicals penetrate the hair shaft and contact the dendrites of four gustatory receptor neurons (GRNs). The sensilla on the labellum are classified into three types according to their length (L: long, S: small and I: intermediate) (Shanbhag et al., 2001) (Figure 1, Right). L- and S-type sensilla house four GRNs responding mainly to water (W-cell), sugars (S-cell), low (L1-cell) and high (L2-cell) concentrations of salts respectively (Rodrigues and Siddiqi, 1981; Fujishiro et al., 1984; Hiroi et al., 2002). In S-type sensilla, the L2 cell also responds to bitter compounds. I-type sensilla only contain two GRNs (Shanbhag et al., 2001), one combining the functions of the S and L1 cells and the other being the L2 cell and responding to aversive molecules (Hiroi et al., 2004). The axons of the labellar GRNs directly project to interneurons in the subesophageal ganglion (Ishimoto and Tanimura, 2004; Wang et al., 2004; Amrein and Thorne, 2005; Miyazaki and Ito, 2010).

Feeding preference tests in D. melanogaster

In our work, we needed a behavioral assay to underline potential differences of feeding preferences with or without exposure to an antifeedant compound, in order to highlight habituation. Several behavior tests have been developed to assess feeding preferences in the fruit fly. Here, we are describing the most commonly used.

Test based on the fly density

The simplest approach consists in recording how many flies wander on a treated surface as compared to a control surface (Marella et al., 2006) (Figure 3). This measure is not directly linked to consumption but relies on the fact that flies use taste receptors of their legs and of their mouthparts to check the substrate on which they stand. A preference index is built by counting the number of flies on the two media at different time intervals: I = (Nb of flies on Test medium – Nb of flies on Control medium) / (Nb of flies on Test medium + Nb of flies on Control medium). An index comprised between 0 and 1 shows an attraction towards the test medium, while an index between -1 and 0 shows a deterrence (0 represents neutrality).
Although this test works well when the aversion or the attraction towards one of the media is high, its sensitivity decreases quickly as the two media get closer in taste (personal observation). Flies may feed more on one of the media but they do not seem to spend more time on the preferred medium in this case, leading to an index biased towards indifference. Thus, the results of this test for fine discrimination cannot be trusted.

Screening of some molecules extracted from endemic plants of the Canary Islands

We applied our system to evaluate the antifeedant activity of new chemicals on the fruit fly. This study was done in the frame of a collaboration with Azucena Gonzalez Coloma and Adriana Gonzalez Portero (Instituto de Ciencias Medioambientales, CSIC, Serrano 117, 28006-Madrid, Spain / Instituto de Productos Naturales y Agrobiología, PO Box 195, La Laguna, 38206-Tenerife, Canary Islands, Spain).
Azucena Gonzalez Coloma’s research aims at finding natural compounds with agronomical interest and applications as biopesticides. She is particularly interested in the compounds present in endemic plants of the Canary Islands. About 27 % of the approximately Modulation of feeding behavior and peripheral taste response by aversive molecules in D. melanogaster Marie-Jeanne Sellier 1000 native vascular plant species of the Canary Islands are endemic (Juan et al., 2000) and among them, 70 % are endemic of only one of the 7 islands of the archipelago (Carine and Schaefer, 2010). Some of the compounds produced by these plants seem to have a deterrent or a toxic effect on insects (Gonzalez-Coloma et al., 1999; Fraga et al., 2001; Dominguez et al., 2008) and their bioactivity makes them good candidates to be used as biopesticides. Adriana Gonzalez Portero is doing her PhD partly in the laboratory of Azucena Gonzalez-Coloma and partly in another laboratory situated in Tenerife. She used the MultiCAFE to assess the effect on D. melanogaster of some molecules (euparine, euparone, pericallone and 6-hydroxytremetone) extracted from Pericallis echinata (Asteracea), an endemic plant of the Canary Islands. These compounds are currently tested on caterpillars and aphids in Azucena Gonzalez-Coloma’s laboratory. If some of them had a deterrent effect of drosophila then the multiple tools available in the fruit fly would help understand the modes of action of these molecules.

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Identification of pericallone as a potential deterrent molecule

The main difficulty in this study was the non-solubility of the tested compounds in pure water. This implies to use organic solvents to dissolve them first and then to dissolve this solution in water. There are three conditions to fill: 1) the tested molecule must be soluble in the solvent, 2) the solvent must be soluble in water and 3) the final concentration of solvent in the water solution should be low enough not to be toxic or deterrent for the flies. Preliminary assays were done in order to find the appropriate solvent and concentrations. Unfortunately, euparone could not be tested because neither of the solvents used for this compound was soluble in water.

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
1. Introduction
Modulation of feeding behavior and peripheral taste response by aversive molecules in D. melanogaster Marie-Jeanne Sellier
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
6. Conclusion
IV. Experience-induced modulation of feeding
1. Introduction
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
1. Conclusion
2. Perspectives
References 

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