1) Mass spectrometry analysis.
The β-carotene standard amount (Extrasynthese, cat n° 0303 S) is very low (~ 1 mg). To overcome these limitations, the concentration of the initial solution was determined by measuring its specific absorbance in hexane solution at 451 nm and using the extinction coefficient of β-carotene (ε1%) in hexane (= 2592). The successive dilutions of the initial solution were then prepared in hexane and injected into HPLC. The β-carotene chromatographic profiles were used to standardize the concentrations. The initial β-carotene solution was kept in amber vial under nitrogen atmosphere at -80°C for a maximum of one week.
CRITICAL STEP 1: For each carotenoid analysis, one gram of aphids was hand-milled using liquid nitrogen. Aphids were weighed before milling and then the resulting powder was carefully recovered. We notice that the water condensation on the aphid cuticle after defrosting tends to bias the weighing.
CRITICAL STEP 2: Carotenoids are light sensible. Consequently all the extractions and standard solution preparations are performed under red-light. Furthermore, these compounds being easily oxidized, the extracts are injected immediately into HPLC column.
CRITICAL STEP 3: Carotenoid extracts dissolved in dichloromethane / [MTBE/methanol], v/v) (see below: Step S9) were not filtrated before HPLC analysis. The solvent mixture is extremely volatile and filtration would considerably reduce the volume of extract.
CRITICAL STEP 4: Carotenoids are also highly heat sensitive. The temperature along the full process and particularly the concentration step in the water bath should not exceed 40°C.
CRITICAL STEP 5: Carotenoids being easily oxidized particularly when extracted out of the matrix, we advice to keep these molecules under their native form in insects stored at -80°C instead of freezing the extract.
GENERAL EXTRACTION AND ANALYTICAL PROCEDURE.
The procedure was adapted from the article published by Taungbodhitham et al. (1998), Dhuique-Mayer et al. (2005) and Mertz et al. (2010). Each analysis was made in triplicate.
The major points of carotenoid analysis are the sampling and sample preparation, extraction, partition/solubilization in appropriate solvents depending on their polar/apolar properties, washing, evaporation of solvents, chromatographic separation, and finally at the end of this process, identification and quantification of the carotenoid compounds.
1st Step: extraction and solvent preparation.
• Prepare 15 mL of solvent A: ethanol (20 mL)/hexane (15 mL) containing 35 mg of BHT as antioxidant.
• Prepare 80 mL of 10% sodium chloride.
2nd Step: extraction and purification steps. This entire step was conducted under red light until transfer in an amber vial. Figure 1 presents the main steps of this procedure.
• S1: weight 1 g of aphids (balance precision ± 0.01 mg).
• S2: carefully milled the aphids in a mortar pestle with liquid nitrogen (very low T°C breaks easily the cuticle of the insects).
• S3: in a 50 mL beaker, add 15 mL of solvent A and 80 mg of MgCO3 to neutralize the acidity of the mixture and stir for 5 min.
• S4: filtrate the mixture on a filter funnel n°2 and wash the precipitate successively with 15 mL of solvent A, 15 mL of ethanol, then with 15 mL of hexane to recover most of lipophilic compounds.
• S5: transfer the solvent mixture in a separatory funnel and washed:
o S5-1: once with 40 mL of 10% sodium chloride (salt enhances the ionic strength of the solution. The hydrophilic compounds are concentrated in the water phase).
o S5-2: twice with 40 mL of distilled water to rinse the eventual salty traces.
• S6: recover the hexanic phase in a beaker.
• S7: dry the phase with 1 g of sodium sulphate and filtrate on a cotton glass in a 100 mL conical ball.
• S8: evaporate the dried organic phase using a rotavapor with a T°C of the water bath not exceeding 40°C.
• S9: the residue is recovered with 250 µL of dichloromethane and 250 µL of MTBE/methanol (80:20, v/v). These solvents being very volatile, it is important to pipette immediately in an amber vial.
• S10: the extract is injected quickly on HPLC for analysis purpose.
HPLC-MS ANALYSIS OF CAROTENOIDS.
• Carotenoids separation with a C30 column (250 X 4.6 mm, 5 µm particle size).
• Gradient solvent program is presented Table 1. Injection volume: 10 µL, UV-vis. Detection: 250- 600 nm. Acetate ammonium (20 mM) was added in solvent A and B to favor ionization of the molecules in the ESI chamber.
• In order to get simultaneously UV-visible and MS data, after passing through the flow cell of the diode array detector, the column eluate was split and 0.5 ml was directed to the ion trap of the LCQ mass spectrometer.
• MS experiments in (+) ion mode, scan range: 100–2000 amu, scan rate: 1 scan/s and temperature for dissolving: 250 °C.
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY QUANTIFICATION OF CAROTENOIDS.
• Column and gradient conditions (Table 1, below) were the same as used in mass spectrometry analysis. Ammonium acetate was not added.
• Injection volume: 20 µL, absorbance followed at 450 nm.
• All carotenoids concentrations are expressed in β-carotene equivalent (standard solutions injected at 450 nm).
These data are summarized in figure 1.
2) Raman imaging spectrometry.
Raman spectrometry provides a fingerprint of molecules specific of its global structure and/or some of its motifs (Roman et al. (2010). The intensity of the peaks is proportional to the quantity of molecules which generate them and can be determined in cell context without destructive extraction process. Raman imaging spectrometry can be also used for a spatial visualization of molecules in tissue allowing the analysis of molecular dynamics. Briefly, υ(C=C) 1500 - 1900 cm-1 and υ(C≅C) 2100 - 2250 cm-1 and υ(C−Η) 2800 - 3000 cm-1 give strong Raman signal. Three peaks specify carotenoid molecules in Raman analysis: C-CH3 (1000 cm-1), C-C (1150 cm-1), C=C(1500 cm-1). Different ratios were determined: the ratio C-C/C=C; C=C (torulene/carotene); C-C / C=C; C-CH3 / ½ (C-C + C=C; ); C-CH3/C-C and C-CH3/C=C. Considering that the Raman peaks result from inelastic scattering involving vibrational levels of molecules, the baseline is related to radiative relaxation processes giving rise to fluorescence. The latter phenomenon strongly depends on the excitation wavelength and the interaction of molecules with its environment (Figure 3).
3) ATP dosage of individual aphid extract in a complex population.
The dosage was conducted according to the recommendation of the company and is based on the visible light production obtained with an equal amount of protein in tested samples (Figure 4).
4) Maintenance and propagation of aphids: selection of aphid variants synthesizing carotenoid molecules.
The pea aphid Acyrthosiphon pisum (Homoptera order, Aphididae family) are raised on the Vicia faba plant. Aphids were maintained in cages in a Sayo incubator at about 22˚C ± 3˚C and/or at 8°C ± 1°C, humidity 60% and with a photoperiodicity of 16/8 hours light/dark. Three colored
phenotypes are the orange, the white and the green. Basically the white phenotype emerged when the plants are declining and food resources are rare. The orange phenotype is dominant in optimal temperature and food resources. Finally the green phenotype was selected over 5 months by placing 10 orange adults each day at 8°C ± 1°C. The orange phenotype was not viable in these conditions (orange larvae died at the stage 3 or 4), but the selected green variant turned out to be robust at this temperature. Placing back the green variant at room temperature results in the fast fading of the green pigment in each individual and its disappearance in approximately two days (aphids become orange again).
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