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Proc Natl Acad Sci U S A. Jul 31, 2012; 109(31): 12626–12631.
Published online Jul 9, 2012. doi:  10.1073/pnas.1207114109
PMCID: PMC3412017
Evolution

Redox alters yellow dragonflies into red

Abstract

Body color change associated with sexual maturation—so-called nuptial coloration—is commonly found in diverse vertebrates and invertebrates, and plays important roles for their reproductive success. In some dragonflies, whereas females and young males are yellowish in color, aged males turn vivid red upon sexual maturation. The male-specific coloration plays pivotal roles in, for example, mating and territoriality, but molecular basis of the sex-related transition in body coloration of the dragonflies has been poorly understood. Here we demonstrate that yellow/red color changes in the dragonflies are regulated by redox states of epidermal ommochrome pigments. Ratios of reduced-form pigments to oxidized-form pigments were significantly higher in red mature males than yellow females and immature males. The ommochrome pigments extracted from the dragonflies changed color according to redox conditions in vitro: from red to yellow in the presence of oxidant and from yellow to red in the presence of reductant. By injecting the reductant solution into live insects, the yellow-to-red color change was experimentally reproduced in vivo in immature males and mature females. Discontinuous yellow/red mosaicism was observed in body coloration of gynandromorphic dragonflies, suggesting a cell-autonomous regulation over the redox states of the ommochrome pigments. Our finding extends the mechanical repertoire of pigment-based body color change in animals, and highlights an impressively simple molecular mechanism that regulates an ecologically important color trait.

Keywords: red dragonflies, nuptial color change, redox-dependent color change, Sympetrum, Crocothemis

Nuptial color change, the body color transition associated with sexual maturation, is commonly found in diverse animals such as mammals, birds, reptiles, amphibians, fish, and insects (14). The distinct color patterns reflecting their reproductive mode must have been shaped by sexual selection, and generally play important roles for their reproductive success. Among insects, many dragonflies display remarkable nuptial coloration (5). The sex-specific color patterns are generally important for partner recognition before mating (5, 6). Territorial males visually recognize vividly colored invader males and aggressively attack them, whereas they permit invasion of dull-colored females as potential mates (7). However, the molecular mechanisms of the sex-related transition in body coloration have been poorly understood.

In many red dragonflies of the genera Crocothemis and Sympetrum, young males are yellow in color and turn red upon sexual maturation, whereas females remain yellowish throughout their lifetime (Fig. 1). Here we report that the yellow-to-red color change in the dragonflies is regulated by redox states of epidermal ommochrome pigments, which extends the mechanical repertoire of pigment-based body color change in animals and highlights an impressively simple molecular mechanism that regulates the ecologically important external trait.

Fig. 1.
Stage- and sex-specific adult color change in dragonflies. (A) C. servilia immature male (Left), mature male (Center), and mature female (Right). (B) Adult males and females of three red dragonflies C. servilia, S. darwinianum, and S. frequens.

Results and Discussion

Identification of Red-Yellow Pigments of Dragonflies.

We attempted to extract the red-yellow pigments from abdominal epidermis of the red dragonflies Crocothemis servilia, Sympetrum darwinianum, and Sympetrum frequens. The pigments were efficiently recovered by 0.5% hydrochloric acid in methanol, which has been often used as solvent for ommochrome pigments (8, 9). By using HPLC and MS, we analyzed the epidermal pigments extracted from sexually mature males and females of C. servilia, S. darwinianum, and S. frequens, and identified two ommochrome pigments, xanthommatin and decarboxylated xanthommatin, irrespective of sex and species of the dragonflies (Fig. 2 and Fig. S1). Cinnabar red-colored males of S. darwinianum and S. frequens, and yellow-colored females of all three species, contained decarboxylated xanthommatin as the major pigment (Fig. 2 BF). Meanwhile, crimson red-colored males of C. servilia contained two major pigments, xanthommatin in addition to decarboxylated xanthommatin (Fig. 2A).

Fig. 2.
Identification of ommochrome pigments from dragonflies. (AF) Chromatograms of ommochrome pigments from males and females of three red dragonflies. Blue lines denote the acetonitrile gradient. (G and H) Electrospray ionization mass spectra of ...

Redox-Dependent Color Change of Ommochrome Pigments of Dragonflies.

Ommochrome pigments generally change their color under oxidative/reductive conditions (Fig. S2A) (8). We confirmed redox-dependent color changes of the pigments extracted from the dragonflies in vitro: the red pigment extracts from sexually mature males turned yellow by an addition of oxidant (NaNO2; Fig. 3A), whereas the yellow pigment extracts from immature males turned red by an addition of reductant (ascorbic acid; Fig. 3B). Chemically synthesized decarboxylated xanthommatin and xanthommatin exhibited similar redox-dependent color changes: red under the reductive condition and yellow under the oxidative condition (Fig. 3 C and D). The dull red color of reduced decarboxylated xanthommatin agrees with the cinnabar-red color of mature males of S. darwinianum and S. frequens, whereas the vivid red color of reduced xanthommatin accounts for the crimson-red color of mature males of C. servilia (Figs. 2 and 3 C and D).

Fig. 3.
Redox-dependent color change of the ommochrome pigments. (A and B) Pigment extract from a mature male and an immature male of C. servilia. (C and D) Reduced and oxidized forms of synthetic decarboxylated xanthommatin and xanthommatin. (E and F) Reductant-induced ...

Redox-Dependent Color Change of Dragonflies in Vivo.

By injecting ascorbic acid solution into the abdomen of the dragonflies, we confirmed a similar color change in vivo: not only yellow immature males but also yellow mature females changed their color to red within several hours after the reductant injection (Fig. 3 E and F). On the contrary, by injecting an oxidant solution into the abdomen of mature males, a red-to-yellow color change was observed but only very subtly and locally, and the insects were severely damaged and usually died soon (Fig. S2B).

Direct Measurement of Redox Status of Ommochrome Pigments in Dragonflies.

Redox conditions of a soluble substance can be measured by using an electrochemical method with a rotating working electrode by applying lower and higher voltages than the redox potential of the soluble substance (Fig. S2C). By using this technique (10), we electrochemically evaluated the relative abundance of the oxidized and reduced forms of the ommochromes in the pigment extracts from the dragonflies. In all three species, the levels of the red reduced form were significantly higher in the mature males than in the immature males and females: the reduced form ratios were from 90% to 100% in mature males, whereas the values were approximately 55% to 75% in mature females and also in immature males and females (Fig. 3 GI).

Analysis of Water-Soluble Reductants.

We examined reduction activities in water extracts of the male and female dragonflies. Although ommochrome pigments are poorly water-soluble, most ommochromes are assumed to be associated with specific binding proteins in vivo (11), and some of the complexes are water-soluble to some extent (12). Accordingly, water extracts of mature male dragonflies were more reddish in color than those of female dragonflies (Fig. 4 A and B), and exhibited stronger reduction activities than those of female dragonflies (Fig. 4C). Addition of ascorbate oxidase did not affect the reduction activities in the water extracts of the dragonflies (Fig. 4 D and E), indicating that the reduction activities are not attributable to ascorbic acid inherently present in the dragonflies. HPLC analysis of the water extracts from the male dragonflies revealed that, of five peak fractions collected (Fig. 4F), the reduction activities were recovered in fraction 4, representing decarboxylated xanthommatin, and fraction 5, corresponding to xanthommatin (Fig. 4G).

Fig. 4.
Analysis of water-soluble reductants in red dragonflies. (A) Abdominal epidermis of a male and a female of C. servilia used in the analysis. (B) Water extracts of a male and a female of C. servilia. (C) Linear sweep voltammograms of the water extracts ...

Possible Mechanisms Underlying Sex-Specific Redox Change of Ommochrome Pigments.

Conceptually, male-specific accumulation of natural reductants such as ascorbic acid may account for the male-specific presence of the reduced ommochrome pigments (13). However, our experimental data refuted this possibility: we could not detect any natural reductants other than the pigments themselves in the water extracts from the male dragonflies (Fig. 4). The eyes of the fruit fly Drosophila melanogaster contain a high amount of reduced form of xanthommatin, in which a significant activity of xanthommatin reductase was detected (14), although reductase genes responsible for the activity have not yet been characterized. In the wings of Heliconius butterflies, ommochrome-binding proteins have been hypothesized to stabilize the redox status of the pigments (15). It is conceivable, although speculative, that male-specific enrichment of reductases and/or pigment-binding proteins may be involved in the redox-dependent color differences in the male and female dragonflies.

Insight from Gynandromorphic Dragonflies.

In the abdomen of the dragonflies, the red/yellow pigments are localized within epidermal cells as pigment granules (8, 16) (Fig. 5 A and B). Gynandromorphic dragonflies have been recorded occasionally (17, 18), and they consistently exhibit discontinuous yellow/red mosaicism in their body coloration (Fig. 5 C and D). These observations suggest the possibility that a cell-autonomous mechanism is involved in the regulation of the dragonfly body color, which may entail, for example, a sex-specific cellular response to hormonal signals.

Fig. 5.
Localization of the ommochrome pigments in dragonflies. (A and B) Intracellular localization of the ommochrome pigments in the abdominal epidermis. (A) Tissue section of a mature male of S. darwinianum. (B) Tissue section of a mature female of S. darwinianum ...

Conclusion and Perspective.

In conclusion, the sex-specific maturation color change in dragonflies is regulated by redox states of the ommochrome pigments. Pigment-based color changes are known from diverse animals, which have been attributed to various mechanisms such as de novo pigment synthesis, pigment degradation, change in pigment localization, and accumulation of food pigments (19). Our finding may extend the mechanical repertoire of the pigment-based body color change, which may be operating not only in dragonflies but also in butterflies and other organisms (15). In Japan, appearance of red dragonflies has been regarded as a symbol of seasonal change, giving a natural feeling of summer/autumn transition adopted by numerous versicles, arts, songs, and paintings (5, 20, 21) (Fig. S3). This study highlights a simple molecular mechanism underlying the ecologically important, as well as aesthetically impressive, biological phenomenon.

Materials and Methods

Experimental Animals and Chemical Treatment.

C. servilia, S. darwinianum, and S. frequens were collected in Tsukuba, Ibaraki Prefecture, Japan. S. darwinianum and S. frequens start to emerge from June to July, and mature males become reddish at the beginning of September and disappear in December. C. servilia emerges from May to October, and adult males turn red several days after emergence. For pigment extraction, abdominal epidermis was dissected from a single adult insect and extracted with 1 mL of 0.5% hydrochloric acid in methanol. In acidic methanol, the redox states of the ommochrome pigments were stable, and the color was maintained at least for 6 mo when stored at −30 °C. The pigments were reduced or oxidized by adding a few drops of 1% ascorbic acid (Sigma) or 1% NaNO2 (Wako), respectively. For the redox-dependent color change analyses in vivo, ascorbic acid (Sigma) and NaNO2 was dissolved in distilled water at 0.1 mg/mL. The reductant/oxidant solution (5 µL each) was microinjected into each live insect abdomen (Fig. 3 E and F and Fig. S2B, arrows) using a glass capillary. For histological observation, adult abdominal epidermis was fixed overnight in freshly prepared 4% (wt/vol) paraformaldehyde in PBS solution at 4 °C. After dehydration by acetone, the tissue was embedded in paraffin, processed into 5-μm serial tissue sections, mounted on glass slides, and observed under a light microscope without staining.

Liquid Chromatography and MS.

Abdominal epidermis was dissected from a single adult insect, and pigments were extracted with 1 mL of 0.5% hydrochloric acid in methanol. After centrifugation, the supernatant was subjected to reversed-phase HPLC directly. HPLC analysis was conducted by using a Waters Alliance 2695 HPLC system fitted with a Gemini-NX C18 column (150 × 4.6 mm, 3 μm particle size; Phenomenex). The absorbance data were collected with a Waters 2487 dual-wavelength absorbance detector. The elution gradient was based on a ternary solvent system [time in min [% solvent A/% solvent B/% solvent C]: 0 min (80/10/10); 5 min (30/60/10); 6 min (80/10/10); and 20 min (80/10/10)]. Solvent A consisted of 0.1% trifluoroacetic acid (TFA) in water, solvent B consisted of 0.1% TFA in acetonitrile, and solvent C consisted of 10 mM ascorbic acid in 0.1% TFA water. For HPLC analysis of water extracts, ascorbic acid was not used in mobile phase [0 min (90/10/0); 5 min (40/60/0); 6 min (90/10/0); and 20 min (90/10/0)]. The column temperature was set at 30 °C, and the flow rate was constant at 1 mL/min. The peak fractions were monitored based on 450 nm (for pigment extract) or 250 nm (for water extract) absorbance and collected manually. The collected pigment fractions were subjected to MALDI-TOF MS. The mass spectra were recorded on a Reflex III system (Bruker Daltonik) by using α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid as matrices. Standard samples of xanthommatin and decarboxylated xanthommatin were chemically synthesized as described previously (22). Standard samples were verified by mass measurements using MALDI-TOF MS and electrospray ionization MS (23). Electrospray ionization mass spectra were recorded with an LCQ Finnigan spectrometer (LCQ Duo; Thermo Finnigan).

Measurement of Oxidation/Reduction Current.

Abdominal epidermis was dissected from a single adult insect and pigments were extracted with 1 mL of 0.5% hydrochloric acid in methanol or 1 mL of distilled water. After centrifugation, the supernatant was collected and 1 mL of distilled water was added just before measurement (within 1 min). The diluted sample was electrochemically analyzed (10) as follows. A three-electrode electrochemical cell with a glassy carbon disk (3 mm in diameter) as a working electrode, a platinum wire as a counter electrode, and Ag-AgCl (3 M NaCl) as a reference electrode were used. Before electroanalysis, the glassy carbon electrode was polished in Al2O3/water slurry, and then voltammetry was conducted in blank buffer (0.25% hydrochloric acid in 50% methanol) between −0.2 V and 0.6 V until a stable voltammetric response was obtained. The working electrode was rotated at 1,500 rpm by a rotating electrode system (model RRDE-3A; ALS), and a linear sweep voltammogram was obtained by scanning of electrode potential from −0.2 V to 0.6 V at a scan rate of 5 mV/s by using a potentiostat (model 900; CH Instruments). The reduction current and oxidation current were calculated at 0.1 V and 0.45 V by subtracting reduction/oxidation currents of blank buffer from those of the sample, respectively.

Measurement of Reduction Activity.

Reduction activity was measured by using an Ascorbic Acid Assay Kit II (Bio-Vision) according to the manufacturer’s instructions. Abdominal epidermis was dissected from a single adult insect and extracted in 0.1 mL, 0.2 mL (Fig. 4E, “1/2”), and 1 mL (Fig. 4E, “1/10”) of distilled water. Each extract was divided into two samples, and background value was calculated by adding ascorbate oxidase to one sample. Standard ascorbic acid solutions (0, 2, 4, 6, and 8 nmol/µL) were used as positive control. Fe+3 is reduced to Fe+2 by any antioxidants present, and the resultant Fe+2 was chelated with a colorimetric probe, thereby exhibiting an intense blue color, which can be monitored by measuring the change in absorption at 593 nm.

Supplementary Material

Supporting Information:

Acknowledgments

We thank M. Umemura, Y. Makino, H. Sawada, and Y. Oba for technical support; N. Ishizawa and I. Kawashima for photos of gynandromorphic dragonflies; K. Okubo and S. Okubo for photos of dragonfly arts and products; and M. Moriyama and Y. Matsuura for insect samples. This work was supported by Japan Society for the Promotion of ScienceGrant-in-Aid for Scientific Research Grant 23780058 (to R.F.) and Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research Grant 22128007 (to T.F.). This study was carried out under the National Institute for Basic Biology Cooperative Research Program (project no. 11-373).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1207114109/-/DCSupplemental.

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