1. Ecology
  2. Evolutionary Biology
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Molecular basis of wax-based color change and UV reflection in dragonflies

  1. Ryo Futahashi  Is a corresponding author
  2. Yumi Yamahama
  3. Migaku Kawaguchi
  4. Naoki Mori
  5. Daisuke Ishii
  6. Genta Okude
  7. Yuji Hirai
  8. Ryouka Kawahara-Miki
  9. Kazutoshi Yoshitake
  10. Shunsuke Yajima
  11. Takahiko Hariyama
  12. Takema Fukatsu
  1. National Institute of Advanced Industrial Science and Technology (AIST), Japan
  2. Hamamatsu University School of Medicine, Japan
  3. Graduate School of Agriculture, Kyoto University, Japan
  4. Graduate School of Engineering, Nagoya Institute of Technology, Japan
  5. Graduate School of Science, The University of Tokyo, Japan
  6. Chitose Institute of Science and Technology, Japan
  7. Tokyo University of Agriculture, Japan
  8. University of Tokyo, Japan
  9. University of Tsukuba, Japan
Research Article
Cite this article as: eLife 2019;8:e43045 doi: 10.7554/eLife.43045
11 figures, 3 videos, 1 data set and 2 additional files

Figures

Stage- and sex-dependent adult color change in O. albistylum as visualized in red, green, blue (RGB) and ultraviolet (UV) light.

(A) Adult males and females of O. albistylum. (B, G) Immature male. (C, H) Mature male. (D, I) Immature female. (E, J) Mature female. (F, K) Mating pair. Images photographed normally (B–F) or through a UV filter (G–K) in the field.

https://doi.org/10.7554/eLife.43045.003
Figure 2 with 1 supplement
Reflectance of the adult body surface at the 5th abdominal segment of O. albistylum.

(A–F) Spectrometry of a round area (6 mm in diameter) in males (A–C) and females (D–F). (A, D) Immature individuals. (B, E) Mature individuals. (C, F) Aged individuals. Solid and dotted lines indicate averaged UV reflectance on the dorsal and ventral sides of the abdomen, respectively. The standard deviation is shaded. (G) Micro-spectrometry of the 5th abdominal segment of a mature male. UV reflectance was measured in eight micro-areas (10 μm x 10 μm each) depicted as red squares in the photos. In the photos, white areas are covered with secreted wax whereas black areas are without wax, presumably because of accidental scratches and cracks on the adult body surface.

https://doi.org/10.7554/eLife.43045.004
Figure 2—source data 1

Spectrometry of a round area (6 mm in diameter) of immature males and females of O. albistylum.

https://doi.org/10.7554/eLife.43045.006
Figure 2—source data 2

Spectrometry of a round area (6 mm in diameter) of mature males and females of O. albistylum.

https://doi.org/10.7554/eLife.43045.007
Figure 2—source data 3

Spectrometry of a round area (6 mm in diameter) of aged males and females of O. albistylum.

https://doi.org/10.7554/eLife.43045.008
Figure 2—source data 4

Micro-spectrometry of the 5th abdominal segment of a mature male of O. albistylum.

https://doi.org/10.7554/eLife.43045.009
Figure 2—source data 5

Micro-spectrometry of immature males and females of O. albistylum.

https://doi.org/10.7554/eLife.43045.010
Figure 2—source data 6

Micro-spectrometry of mature males and females of O. albistylum.

https://doi.org/10.7554/eLife.43045.011
Figure 2—source data 7

Micro-spectrometry of aged males and females of O. albistylum.

https://doi.org/10.7554/eLife.43045.012
Figure 2—figure supplement 1
Micro-spectrometry (of 10 × 10 µm micro-areas) of O. albistylum.

(A–C) Male. (D–F) Female. (A, D) Immature individuals. (B, E) Mature individuals. (C, F) Aged individuals. Three to eight micro-areas were measured for each individual.

https://doi.org/10.7554/eLife.43045.005
Fine structure of the adult body surface at the 5th abdominal segment of O. albistylum.

(A–P) Scanning electron microscopic (SEM) images of the body surface. (Q–X) Transmission electron microscopic (TEM) images of the sectioned cuticle. It should be noted here that the surface wax was dissolved and removed during the processing of the sample for TEM observation. (A–H) Dorsal side of mature male (A–D) and mature female (E–H). Panels B and F are magnified images of panels A and E, as indicated by red rectangles. Likewise, panels C and G are magnified images of panels B and F. (D, H) Cross-sectioned images of cuticle and surface wax. (I–L and Q–T) Dorsal side. (M–P and U–X) Ventral side. (I, M, Q, U) Immature male. (J, N, R, V) Immature female. (K, O, S, W) Mature male. (L, P, T, X) Mature female.

https://doi.org/10.7554/eLife.43045.014
Solubility and wettability of the abdominal wax of O. albistylum.

(A–D) Dorsal side of the 5th abdominal segment of mature males. (A) No treatment. (B) After ethanol treatment. (C) After hexane treatment. (D) After chloroform treatment. (E) Scanning electron microscope images of the dorsal surface of mature male 30 min after hexane treatment. (F) Scanning electron microscope images of dorsal side of a mature male (left) or a mature female (right) after chloroform treatment.

https://doi.org/10.7554/eLife.43045.016
Figure 5 with 1 supplement
Identification and chemical synthesis of surface wax of O. albistylum.

(A) Chromatogram of hexane extract from the dorsal abdomen of a mature male. (B) Chromatogram of chloroform extract from the dorsal abdomen of a mature male. (C) Chromatogram of chloroform extract from the ventral abdomen of a mature male. (D) Chromatogram of chloroform extract from the dorsal abdomen of a mature female. (E) Chromatogram of chloroform extract from the ventral abdomen of a mature female. (F) Chromatogram of chemically synthesized 2-pentacosanone. Asterisks indicate nonspecific peaks that are also detected with the solvent only. The Y-axis shows the abundance of total ion current.

https://doi.org/10.7554/eLife.43045.017
Figure 5—figure supplement 1
Mass spectra and extracted ion chromatogram of dragonfly wax

(A, B, D–H) and of synthetic 2-pentacosanone (C).

https://doi.org/10.7554/eLife.43045.018
Comparison of surface fine structure, reflectance, and wettability between the dorsal abdomen of mature males of O. albistylum and synthetic 2- pentacosanone crystallized on glass plates using three different cooling processes.

(A–H) Scanning electron microscope images. Panels E, F, G and H are magnified images of panels A, B, C and D, respectively, as indicated by red rectangles. (I–L) Micro-spectrometry from a 10 µm x 10 µm area. (M–P) Wettability measured with a 1 nL water droplet. (A, E, I, M) The dorsal abdomen of mature males of O. albistylum. (B, F, J, N) Synthetic wax crystallized by the dropping method. (C, G, K, O) Synthetic wax crystallized by the quenching method. (D, H, L, P) Synthetic wax crystallized by the slow-cooling method.

https://doi.org/10.7554/eLife.43045.019
Figure 6—source data 1

Micro-spectrometry of synthetic 2-pentacosanone crystallized on glass plates using three different cooling processes.

https://doi.org/10.7554/eLife.43045.020
Figure 7 with 1 supplement
UV reflection patterns in O. melania, S. darwinianum and C. servilia.

(A, E) Mating pair of O. melania. (B, F) Mating pair of S. darwinianum. (C, G) Mature male of C. servilia with mature male of O. albistylum. (D, H) Mating pair of C. servilia. Each image photographed normally (RGB) (A–D) or through a UV filter (E–H) in the field. (I–N) Spectrometry of a round area (6 mm in diameter) on the 5th abdominal segment of O. melania (I, L), S. darwinianum (J, M), and C. servilia (K, N). (I–K) Male. (L–N) Female. Solid and dotted lines indicate averaged UV reflectance on the dorsal and ventral sides of the abdomen, respectively. The standard deviation is shaded.

https://doi.org/10.7554/eLife.43045.021
Figure 7—source data 1

Spectrometry of a round area (6 mm in diameter) of O. melania.

https://doi.org/10.7554/eLife.43045.023
Figure 7—source data 2

Spectrometry of a round area (6 mm in diameter) of S. darwinianum.

https://doi.org/10.7554/eLife.43045.024
Figure 7—source data 3

Spectrometry of a round area (6 mm in diameter) of C. servilia.

https://doi.org/10.7554/eLife.43045.025
Figure 7—source data 4

Micro-spectrometry of O. melania.

https://doi.org/10.7554/eLife.43045.026
Figure 7—source data 5

Micro-spectrometry of S. darwinianum.

https://doi.org/10.7554/eLife.43045.027
Figure 7—source data 6

Micro-spectrometry of C. servilia.

https://doi.org/10.7554/eLife.43045.028
Figure 7—figure supplement 1
Micro-spectrometry (of a 10 × 10 µm micro-area) of O. melania (A, D), S. darwinianum (B, E), and C. servilia (C, F).

(A–C) Male. (D–F) Female. Five to ten micro-areas were measured for each individual.

https://doi.org/10.7554/eLife.43045.022
Comparison of wax components on the dorsal and ventral abdomen of O. albistylum, O. melania and S. darwinianum.

(A) Chromatogram of chloroform extract from the dorsal abdomen of a mature male of O. melania. (B) Chromatogram of chloroform extract from the ventral abdomen of a mature female of S. darwinianum. Asterisks indicate nonspecific peaks also detected with the solvent only. (C) Summary of wax components detected from O. albistylum, O. melania and S. darwinianum. Relative amount was judged from the abundance of total ion current. +++, high amount; ++, moderate amount; +, small amount.

https://doi.org/10.7554/eLife.43045.029
Surface wax of Orthetrum species and allied dragonflies.

(A–D) Mature males of O. albistylum (A), O. melania (B), O. luzonicum (C) and O. glaucum (D). (E–F) Gynandromorphic individuals of O. japonimum (E) and Lyriothemis pachygastra (F). Photos courtesy of Mitsutoshi Sugimura (E) and Makoto Machida (F).

https://doi.org/10.7554/eLife.43045.031
Figure 10 with 1 supplement
Genes associated with UV reflective wax.

(A) An androchrome female used for transcriptome analysis. (B) The number of genes that were upregulated in the dorsal abdominal epidermis of males and/or an androchrome female compared to normal females. (C) The list of genes upregulated in the dorsal abdominal epidermis of both males and an androchrome female (max FPKM >1000). Gene expression levels are displayed as a heat map. The numbers indicate FPKM values. Red and blue fills indicate high and low expression levels, respectively. D and V indicate the dorsal and ventral abdominal regions, respectively. (D–F) Expression level of three elongation of very long-chain fatty acids (ELOVL) genes in the dorsal and ventral parts of the epidermis of O. albistylum.

https://doi.org/10.7554/eLife.43045.032
Figure 10—figure supplement 1
List of genes that are upregulated in the dorsal abdominal epidermis of both males and an androchrome female (max FPKM >100).

Gene expression levels are displayed as a heat map. The numbers indicate FPKM values. Red and blue fills indicate high and low expression levels, respectively. D and V indicate the dorsal and ventral abdominal regions, respectively. Andro and Gyno mean androchrome and normal (gynochrome) females, respectively.

https://doi.org/10.7554/eLife.43045.033
Identification of 17 ELOVL genes in dragonflies.

(A) Phylogenetic tree of ELOVL family genes produced on the basis of their amino-acid sequences. A maximum likelihood phylogeny is shown, but neighbor-joining and Bayesian phylogenies exhibit substantially the same topologies. Statistical supporting values are indicated for each node (shown as (bootstrap value of neighbor-joining)/(bootstrap value of maximum likelihood)/(posterior probability of Bayesian)). Asterisks indicate support values < 50%. Blue and gray shading indicates O. albistylum and L. fulva genes, respectively. Accession numbers or annotation identities are shown in parentheses. (B) Alignment of 17 ELOVL genes of O. albistylum. The conserved histidine motif is boxed. (C) Gene expression levels of 17 ELOVL genes in O. albistylum. The numbers indicate FPKM values. Red and blue shading indicates high and low expression levels, respectively. D and V indicate the dorsal and ventral abdominal regions, respectively.

https://doi.org/10.7554/eLife.43045.034

Videos

Video 1
Movie of visible light and UV reflection of O. albistylum in the field.
https://doi.org/10.7554/eLife.43045.013
Video 2
Reversible color change by adding acetone.
https://doi.org/10.7554/eLife.43045.015
Video 3
Movie of visible light and UV reflection of O. melania, S. darwinianum, and C. servilia in the field.
https://doi.org/10.7554/eLife.43045.030

Data availability

The sequences reported in this paper have been deposited in the DNA Data Bank Japan Read Archive, www.ddbj.nig.ac.jp (accession nos. BR001497-BR001513, LC416747-LC416767, DRA001687, DRA001690, DRA001693-DRA001694, DRA001697-DRA001698, DRA001700-DRA001701, DRA001703-DRA001704, DRA001706-DRA001707, DRA001709-DRA001710, DRA001712-DRA001713, DRA001716-DRA001717, DRA007015-DRA007018). All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2,6I-L,7I-N, Figure 2-figure supplement 1, and Figure 7-figure supplement 1.

The following previously published data sets were used
  1. 1

Additional files

Supplementary file 1

Orthetrum albistylum samples and RNA sequencing reads.

https://doi.org/10.7554/eLife.43045.035
Transparent reporting form
https://doi.org/10.7554/eLife.43045.036

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