Ten orange adult aphids were placed each day at 8°C for five months before we obtained a viable and robust colony of green variants (figure 1). By chance, progenies of the orange adult aphids did not survive at 8°C. Most larvae died from larval stage 1 to stage 4. Cold (8°C) conditions successfully selected a viable and robust green aphid lineage from orange descendants of a 22°C-adapted unique founder mother. Importantly, the switch to the cold adapted green variant never occurred within the actual orange adults, which excludes a direct enzymatic induction. The green phenotype is heritable in the conditions in which it emerged, but its singular pigmentation fades away when it is placed back in optimal conditions at 22°C. This demonstrates that a clonal aphid population under pressure of selection is able to generate complex traits guiding environmental fitness and underlying the recruitment of gene networks11. The scenario precludes allele selection as an explanation (success of the phenotypic adaptation too fast for implying a Darwinian process) and strongly supports the hypothesis of an epigenetic regulation. The mechanism might reside in the extensive DNA methylation as the molecular cue to transmit complex traits in the framework of an unchanged genome11. This selection process is summarized in figure 1. On the other hand, if the pink/orange pigmentation is dominant at 22°C in optimal conditions (low population density and abundant resources), the declining conditions (rarefaction of resources, high population density) trigger the progressive disappearance of the pink/orange phenotype and its replacement by pale/white/yellow colours (figure 1). In such case, the colour plasticity (colour shift orange to pale/white/yellow) is proportionally induced by the increase of population density and the rarefaction of resources. The pale/white/yellow phenotype reflects an unfavourable environment and might be referred as survival forms that have turned down some less essential biochemical processes to minimize energy cost.

Following the intriguing discovery of the carotene synthesis genes in the aphid genome, we undertook an extensive analysis of carotenoid molecules by Raman spectrometry imaging and mass spectrum technology in the framework of this genetic/epigenetic context. We took advantage of the rapid crystallisation of carotene molecules to isolate and to solubilise them in ethanol/acetone. A long centrifugation (9,300 x g for 1 hour) of PBS buffered extract of aphids triggers the formation of a pure orange crystallized precipitate at the top of the aqueous phase. Spectral absorbance properties of this precipitate were analyzed and were found to be in accordance with carotenoid molecules. A comparative absorbance profile between the extracts from the green and pale orange phenotype is presented in figure 2. As expected, the decrease of spectral absorbance of the pale aphid acetone/ethanol extract in the wave lengths of carotenoid molecules absorption was spectacular compared to the green aphid extract (Figure 2).

Figure 2 Absorbance properties of green, orange and pale aphid ethanol extracts. The orange pigments were extracted as indicated in Methods. Briefly, 250 mg of adult aphids were centrifuged in Ringer’s buffer and the orange layer was collected. This precipitate was then solubilized in ethanol/acetone (75/25). A comparative spectrum of absorbance was carried out from 320 to 700 nm (A), at 600 nm, 650 nm and 700 nm (B) and at 425 nm, 450 nm and 480 nm (specific peaks of carotene absorbance) (C). A comparative measure was carried out at 295 nm (peak of absorbance of phytoene, a precursor of carotene) and at 450 nm (peak of carotene absorbance) with the orange crystallized precipitate obtained with the green and orange aphid extracts (D). The means of three independent experiments are shown in (A) and bars in (B), (C), (D) represent the average of three experiments +/− S.E (comparison orange versus green, P< 0.002). More pigment is present in green than in orange aphids. Full size image

Imaging of resonance Raman spectrometry allowing non-destructive molecular motif identification and quantification was performed to detect structural elements of carotene molecules directly on living aphids. A 488 nm laser wavelength corresponding to the maximum wavelength of carotene absorption was used to excite the polyene motifs (Raman conditions: 1.2 to 12 mW/1–3 seconds). The shift values [the shifts of the Stokes (lower energy) and Anti-Stokes (higher energy) Raman light scattering correspond to a vibrational mode of a structural motif in a molecule] are expressed as cm−1. The carotene signature corresponding to the three peaks obtained at 1,520 cm−1 (assigned to the C = C stretching vibration), 1,157 cm−1 (CH-CH) and 1,005 cm−1 (CH-CH3) were always found in living aphids even though these molecules are part of a complex biological matrix (figure 3). The laser beam of the Raman imaging apparatus was also directed on the crystals spontaneously formed after crushing adult orange aphids (figure 3). Moreover a Raman imaging control was carried out using the reddish/brown aphid eyes, known to contain, as any eye in all the taxa, a strong concentration of retinal. Retinal (vitamin A) conserves the structural motifs of carotene by the fact that it is the enzymatic conversion product of carotenoid molecules. The three peaks corresponding to the Raman signature of carotene were unambiguously obtained with living aphids and crystals, which suggests high concentration of these compounds (figures 3 and 4). Interestingly, a stronger intensity of the peaks was consistently found with the green compared to the orange phenotype (40% increase) (figure 4). Moreover, the method was able to follow the carotene synthesis in the developing embryos where the signals were correlated with the apparition of the orange pigmentation (see Supplementary Data, figure S3).

Figure 3 Carotene signature in aphid eyes and spontaneous crystals by Raman imaging. A 488 nm laser excitation of the Raman spectrometry was used. A control with pure β-carotene is shown. Two other spectra are shown: the microscope laser was focused on an eye and on spontaneous crystals obtained after crushing adult orange aphids in PBS solution. The 1,550, 1,150 and 1,005 cm−1 shifts correspond respectively to the C = C, CH-CH and CH-CH3 motifs. Conditions of Raman: 488 nm; 1.2 mW; 3 s. The panels at the top represent spontaneous crystals, an eye and embryos (mature orange embryos in a germaria plus an ovariole stained in green with anti HRP10). β-carotene is present in whole aphids and in aphid eyes. Full size image

Figure 4 Carotene signature in aphid adults corresponding to the green and orange phenotype by Raman imaging. Individual orange (A) and green (B) living aphid phenotypes (see figure 1 for details) were analysed according to the same protocol as in figure 3. Each assay was conducted with individual adult aphids ten days after the birth of the first instar larva. A significant increase of the intensity of the three identified signals (1,550, 1,150 and 1,005 cm−1 shifts) was consistently observed for the green phenotype indicating a stronger carotenoid pigmentation. Each colour represents the measures with different individual adult aphids. Raman conditions: 488 nm; 1.2 mW; 1 s. Full size image

The extensive comparative analysis of these molecules between the green and the orange aphids has been performed by mass spectrometry (after chromatographic isolation), in order to quantify few intermediate components in the cascade of carotenoid synthesis. The major components of the carotene family found in the green and orange aphids is reported in table 1. A substantial increase of concentration of trans-β and trans-γ carotene is observed in the green variant compared to the orange (8.3± 1.2 versus 4.0 ± 1.9 and 12.6± 1.5 versus 6.5± 1.8 µg.100 g−1 respectively). At the opposite, the cis-torulene is drastically increased in the orange phenotype (10.7 ± 3.4 versus 3.0 ± 0.6 µg.100 g−1) whereas the trans-torulene (a precursor metabolite) was roughly unchanged (Table 2). Therefore, the mass spectrum analysis confirmed the trend observed by Raman analysis (see Supplementary Data, figure S4).

Table 1 Comparative quantification of carotenoid molecules in the green and orange aphid phenotype by LC-MS/MS. Characteristics of identified carotenoids of orange and green aphids: the molecules were extracted as indicated in Methods and then submitted to HPLC separation and mass spectrum analysis. Strong differences are observed between the two environmentally selected variants originated from a unique parthenogenetic founder mother. No significative differences were observed for all-trans-torulene and 3,4-dehydrolycopene as they were for the other compounds analysed Full size table

Table 2 Quantification (µg.100 g−1) of carotenoids of orange and green aphids Full size table

One well documented role regarding these compounds are the annihilation of singlet oxygen and radical scavengers in plant photosynthesis along with the light harvesting function of chlorophyll5. Because strong carotenoid concentration was observed especially in the green and orange larval forms (despite a high level of variation between individuals in the same colonies when food resources are declining), we tried to unravel some putative physiological functions beside their canonical anti-oxidant properties. We investigated the hypothesis that the photon energy might excite the π delocalized electrons of the carotene polyene structure and trigger an electron transfer to acceptor molecules. Adult aphids (green, orange and white phenotypes) and larvae from orange mothers were placed in dark or left in light photoperiodicity (18/6 hours). Furthermore, these conditioned aphids were returned under light photoperiodicity (18/6 hours) after a dark episode. The striking data show that ATP synthesis is sensitive to light, but differs among the orange (marked effects), green (little effects likely because a strong lipid load in this variant acts as a metabolic reserve) and white phenotypes (no change). Results are summarized in figure 5 and support the concept of photo-conditioning of ATP synthesis in some environmentally shaped variants.

Figure 5 ATP dosage in dark conditioned aphids. (A). Dark exposure of adult aphids. Aphids were placed in dark, then tested after two days (1 and 2; 5 and 6) or alternatively were kept in light two days more as control (3 and 4; 7 and 8) before the measure of ATP content. 1–4 and 5–8 are the separate ATP determinations obtained with green aphids and orange aphids. a and b: ATP dosage determined with the content of 5 and 1 aphids, respectively. c: ATP determination obtained with the content of one white aphid. The standards roughly represent 50 pmoles (blue) to 250 pmoles (red). (B). Dark exposure of larvae. Larvae from orange aphid were kept two days in light (a) or alternatively two days in dark (b). A control white larva aphid kept two days in light after birth is shown (c). The determinations were done with 5 larvae. (C). Comparative time course of the decline of ATP content in embryos and larvae placed in dark. (see methods for experimental design). (D) Light-induced ATP synthesis after a dark exposure episode of orange adult aphids. 1, 2 are ATP dosages from separate experiments. (a) light-exposure control. (b) and (c) are the dark exposure for two and three days, respectively. 3 and 4 are orange adult aphids placed two days in dark (a, b, c). 5 and 6 are the same aphids than in 3 and 4 placed back in light for one day (a) or kept one day (b) or two days more (c) in dark. 7 and 8 are the light rescue of ATP content three days after a dark exposure of two days (c) versus the light control (a). (E) Comparative determination of ATP between green, orange and white adult aphids in dark or light. Bars represent the mean of three different experiments +/−S.E. (** P<0.005). (F) Light-induced ATP synthesis after a dark exposure episode in larvae. Top: First instar larvae were tested at day 0 (1), at day 2 (2) and day 3 (3). Down: Emerged progenies were tested at day 0 (1), at day 2 (2) and finally at day 3 (3). Full size image

To get more insight about the light-dependent reduction-oxidation (redox) process, orange aphid extracts were used to reduce tetrazolium salts (MTT) in presence or absence of light. Although the effect was moderate, an increase of MTT reduction in presence of light was obtained with the orange, but not with the white extract (figure 6). This trend was also obtained with orange embryos incubated with MTT and exposed to light whereas the white embryos in the same conditions display a weak fluctuation of the basal level (figure 6). The same results were observed when the experiments were conducted with pure molecules. Briefly, 100 µl of MTT solubilized in water were placed on a layer of dry β-carotene and illuminated by a regular electric light. The reduction of MTT in blue precipitated formazan was observed as the result of a capture of free electrons generated by the photoactivated carotene, which suggests that the energy of these free electrons is high enough to pass the barrier of the tetrazolium redox potential (see Supplementary Data, figure S5).

Figure 6 Tetrazolium (MTT) reduction by orange aphid extract. 100 µl of tetrazolium solution (1 mM in water) were placed on a glass slide in which 10 µl of orange aphid extracts were added. The system was irradiated by visible light (A) for 30 min or kept in dark (B). Then, the medium was delicately washed out. Top: The photos show the border of the spots where the formazan precipitation is more intense. Unambiguously, an increase of MTT reduction, measured as formazan precipitation on the glass, is observed under light (A). Middle: higher magnification of the photograph above. Bottom: The light exposure of strongly pigmented ovarioles in presence of MTT (1 mM in water) is compared with white/pale ovarioles in the same conditions as above. Produced formazan by orange or white aphid extract (100 µg protein) under light or kept in dark was measured after solubilization in acid/ethanol (C and D). The representations are the mean of three separate experiments. Full size image

Finally the balance NAD+/NADH was measured in the light versus dark context. A series of experiments shows unambiguously a significant increase of the reduced co-enzyme level in particulate fraction enriched in mitochondria when the orange aphids are exposed to light (figure 7). Intriguingly, a drastic decrease in NAD+ (oxidized) concentration was found in the soluble fraction of the extract when the orange aphids were maintained in dark, suggesting that its synthesis is partly controlled by light. By contrast and as expected, the white aphid variants display weak levels of NAD+/NADH, which seem little affected by light (figure 7). Together these data reinforce the hypothesis that light, through biological membranes enriched in pigments, triggers a reducing power that in fine is captured by co-enzymes like NAD+. This reduced co-enzyme is known to transit inside mitochondria through a shuttle mechanism and deliver electrons for the respiratory chain machinery ending with the H+ inflow-driven ATP synthesis13,14,15. Amazingly, we observe that carotene molecules are disposed as a bilayer under the cuticule from 0 to 40 µM in depth, suggesting that this structure might present an optimal efficiency to harvest light energy (figure 7).