Research ArticleORGANISMAL BIOLOGY

Genetic switch in UV response of mimicry-related pale-yellow colors in Batesian mimic butterfly, Papilio polytes

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Science Advances  08 Jan 2021:
Vol. 7, no. 2, eabd6475
DOI: 10.1126/sciadv.abd6475
  • Fig. 1 UV response and ultrastructure of dorsal pale-yellow wing patterns in P. polytes and P. aristolochiae.

    (A to D) Pale-yellow patterns photographed under (A) bright-field and (B and C) UV (375 nm) illumination, without a UV transmitting filter [(B); UV fluorescence] and with the filter [(C); UV reflectance]. Merged images of UV fluorescence and UV reflectance are shown in (D). Pale-yellow spots are numbered starting from the inside. (E) Scales from the pale-yellow pattern across morphs photographed under bright-field (top) and UV (375 nm) illumination (bottom; UV fluorescence). (F) Scanning electron micrographs (SEM) of scales [asterisks in (E)] from pale-yellow patterns. Scale bars, 1 cm (A to D), 500 μm (E), and 5 μm (F). Photo credit: Yûsuke KonDo and Tasuku Kitamura, The University of Tokyo.

  • Fig. 2 Changes in UV response of pale-yellow spots on hind wings after ethanol treatment.

    (A) A hind wing dipped in 70% ethanol for 1 day (left) and an untreated hind wing (right). In mimetic female and P. aristolochiae, UV fluorescence and UV reflection were not changed. In the nonmimetic female, however, UV fluorescence was attenuated and UV reflection was enhanced in ethanol-treated wings. (B) Ultrastructure of ethanol-treated pale-yellow scale. The scales were collected from the fourth spot on the hind wing (see Fig. 1). No structural changes were observed in the mimetic female and P. aristolochiae. In the nonmimetic, however, the clogged structure seen in the intact ethanol-untreated scales (see and compare with Fig. 1F) disappeared almost completely, resulting in the mimetic-like ultrastructure. Scale bars, 5 μm. Photo credit: Shinichi Yoda, The University of Tokyo.

  • Fig. 3 Relative reflectance spectra of pale-yellow spots in P. polytes and P. aristolochiae.

    Reflectance spectra correspond to males (black), nonmimetic females (green), mimetic females (red), si–dsx (H) mosaic pale-yellow patch of mimetic females (see the asterisk in Fig. 4B) (blue), and P. aristolochiae (yellow). As the optical spectrometer detected all wavelengths when a light with a specific wavelength was irradiated, even when the fluorescence intensity in the UV range (around 380 nm) was included in the reflectance data, we made a modification to raw spectra corresponding to male (broken black) and nonmimetic females (broken green) to take this into account and generate corrected spectra (black and green) (see text and Materials and Methods). K.D., knockdown.

  • Fig. 4 UV response and ultrastructure of mosaic RNAi phenotype of dsx (H) in mimetic females.

    (A) siRNA targeting dsx (H) was introduced into the left dorsal hind wing by electroporation. The detailed phenotype is represented in (B) and (C). (B to E) Pale-yellow patterns photographed under (B) bright-field and (C) UV (375 nm) illumination, without a UV transmitting filter [(C); UV fluorescence] and with the filter [(D); UV reflectance]. Merged images of UV fluorescence and UV reflectance are represented in (E). Similar to the previous results in (24), nonmimetic-like pale-yellow mosaic patches appeared (blue arrowheads). Pale-yellow spots are numbered starting from the inside. The asterisk in (B) indicates the ectopic pale-yellow patch sampled for the reflectance spectra measurement (Fig. 3). (F) Scales from pale-yellow pattern between the RNAi side (left) and the control side (right) photographed under bright-field (top) and UV (375 nm) illumination (bottom; UV fluorescence). (G) SEM of scales [asterisks in (F)]. Scale bars, 1 cm (A), 2 mm (B to E), and 25 μm (F). Photo credit: Yûsuke KonDo, Tasuku Kitamura, and Shinichi Yoda, The University of Tokyo.

  • Fig. 5 Relative expression levels of the genes involved in NBAD and kynurenine pathway in the dsx (H) knockdown wing of mimetic females at P10.

    We estimated the gene expression level by real-time PCR using RpL3 as an internal control. Error bars show the SD of 15 biological replicates. P values are given by paired one-sided Student’s t test or Welch’s t test. n.s., not significant.

  • Fig. 6 UV response and ultrastructure of mosaic RNAi phenotype of ebony in nonmimetic forms.

    (A) siRNA targeting ebony was introduced into the left dorsal hind wing through electroporation. The detailed phenotype is represented in (B) and (C). (B to E) Pale-yellow patterns photographed under (B) bright-field and (C) UV (375 nm) illumination, without a UV transmitting filter [(C); UV fluorescence] and with the filter [(D); UV reflectance]. Merged images of UV fluorescence and UV reflectance are represented in (E). Pale-yellow spots are numbered starting from the inside. (F) Scales from the pale-yellow pattern between the RNAi side (left) and the control side (right) photographed under bright-field (top) and UV (375 nm) illumination (bottom; UV fluorescence). (G) SEM of scales [asterisks in (F)]. Scale bars, 1 cm (A), 2 mm (B to E), and 25 μm (F). Photo credit: Yûsuke KonDo, Tasuku Kitamura, and Shinichi Yoda, The University of Tokyo.

  • Fig. 7 Photoreceptor sensitivity in human, blue tit, and swallowtail butterfly.

    Bar graphs of the relative excitation of the set of photoreceptors for pale-yellow coloration across P. polytes morphs and P. aristolochiae are shown. The reflectance spectra of Fig. 3 were convoluted with a CIE D65 standard illuminant spectrum and each of the photoreceptor spectral sensitivities. The obtained values were subsequently normalized to the highest excitation per morphs. Each bar represents the calculation for an individual photoreceptor. The broken lines and light blue bars indicate the relative excitations by considering the effect of UV absorbance and UV fluorescence, respectively. Human (Homo sapiens: top), blue tit (Cyanistes caeruleus: middle), and Asian swallowtail butterfly (P. xuthus: bottom) closely related to P. polytes.

  • Fig. 8 Hypothetical model for genetic regulation of UV response and scale ultrastructure in the pale-yellow region across P. polytes morphs and P. aristolochiae.

    (A) Gene expression patterns of dsx (H) and genes involved in the kynurenine/NBAD pathway across P. polytes morphs during the pupal stage. Dsx (H) is up-regulated during early pupal stage in mimetic females (Hh, pink line) but not in males with the same genotype (Hh, blue line). The expression pattern of nonmimetic females (hh) is not shown because of the lack of dsx (H). Expression patterns of the genes involved in the kynurenine/NBAD pathway (asterisk) during late pupal stage in nonmimetic (green) and mimetic females (pink) are from (9). (B) Genes involved in kynurenine/NBAD pathway are repressed by dsx (H) in the mimetic females (Hh), similar to the scales observed in P. aristolochiae, with numerous pores and higher UV reflectance than in the nonmimetic. In contrast to the mimetic females, genes involved in the kynurenine/NBAD pathway are probably up-regulated because of the lack of dsx (H) in the nonmimetic females or repression of the dsx expression in the males, resulting in the induction of kynurenine/NBAD pathway gene expression, leading to the formation of papiliochrome II associated with UV fluorescence under UV light. The scales of the nonmimetic have clogged ultrastructure with papiliochrome II.

Supplementary Materials

  • Supplementary Materials

    Genetic switch in UV response of mimicry-related pale-yellow colors in Batesian mimic butterfly, Papilio polytes

    Shinichi Yoda, Kousuke Sakakura, Tasuku Kitamura, Yûsuke KonDo, Kazuki Sato, Ryosuke Ohnuki, Itsuki Someya, Shinya Komata, Tetsuya Kojima, Shinya Yoshioka, Haruhiko Fujiwara

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