Changes in structural and pigmentary colours in response to cold stress in Polyommatus icarus butterflies

Abstract

While numerous papers have investigated the effects of thermal stress on the pigmentary colours of butterfly wings, such studies regarding structural colours are mostly lacking, despite the important role they play in sexual communication. To gain insight into the possible differences between the responses of the two kinds of colouration, we investigated the effects of prolonged cold stress (cooling at 5 °C for up to 62 days) on the pupae of Polyommatus icarus butterflies. The wing surfaces coloured by photonic crystal-type nanoarchitectures (dorsal) and by pigments (ventral) showed markedly different behaviours. The ventral wing surfaces exhibited stress responses proportional in magnitude to the duration of cooling and showed the same trend for all individuals, irrespective of their sex. On the dorsal wing surface of the males, with blue structural colouration, a smaller magnitude response was found with much more pronounced individual variations, possibly revealing hidden genetic variations. Despite the typical, pigmented brown colour of the dorsal wing surface of the females, all cooled females exhibited a certain degree of blue colouration. UV-VIS spectroscopy, optical microscopy, and scanning and transmission electron microscopy were used to evaluate the magnitude and character of the changes induced by the prolonged cold stress.

Figure 1

Wings of wild butterflies and of butterflies eclosed form pupae subjected to prolonged cooling at 5 °C in dark. (a) Dorsal forewing of a wild male; (b) dorsal forewing of a male emerged from a pupa cooled for 62 days; (c) ventral forewing of a wild male; (d) ventral forewing of a male eclosed form a pupa cooled for 62 days; (e) dorsal hindwing of a wild male; (f) dorsal hindwing of a male emerged from a pupa cooled for 62 days; (g) ventral hindwing of a wild male; (h) ventral hindwing of a male eclosed form a pupa cooled for 62 days; (i) dorsal forewing of a wild female; (j) dorsal forewing of a female emerged from a pupa cooled for 40 days; (k) ventral forewing of a wild female; (l) ventral forewing of a female eclosed form a pupa cooled for 40 days; (m) dorsal hindwing of a wild female; (n) dorsal hindwing of a female emerged from a pupa cooled for 40 days; (o) ventral hindwing of a wild female; (p) ventral hindwing of a female eclosed form a pupa cooled for 40 days.

Figure 2

The averaged quantitative aberration of the ventral wing surfaces versus the cooling time of the pupae. For details of the conversion of pattern alteration into numerical values see the Supplementary Table 1. The red line is a linear fit to the numerically evaluated values of aberration (a = 0.24941 ± 0.00556, R² = 0.99653), the error bars correspond to the standard error of the mean (s.e.m.).

Figure 3

Normalized reflectance spectra averaged over all four wings of the exemplars. Wild males, and males emerged from pupa cooled for 10, 30, and 62 days respectively. The inset shows the peak region of the curves in more detail.

Figure 4

Spectral position of the blue reflectance peak for all the investigated exemplars. When possible all four wings of an individual are represented as individual measurement points close to the same vertical line. Horizontal broken lines indicate the range of wild males with centre at 384 nm marked by dotted broken line. The data of pupae cooled for a certain time are separated by thin vertical lines. We used only the wings sufficiently extended after eclosion to allow reliable spectral measurements.

Figure 5

Optical microscopy of the wings. (a) Wild male, (b) male from a pupa cooled for 22 days, (c) wild female, (d) female from a pupa cooled for 20 days. The inset in the lower left corner shows magnified examples of the cover scales, ground scales and the androconia of the males.

Figure 6

SEM images of the wing scales of the wild male and of the male eclosed from a pupa cooled for 22 days. (a) Wild male, overview of the regular rows of scales, the white arrows point to the cover and ground scales and to the androconia (b) wild male, pepper-pot structure in the volume of a blue scale, (c) cooled male, overview of the disordered scales, (d) cooled male, pepper-pot structure in the regular shape blue scale, (e) cooled male, black scale, (f) cooled male, pepper-pot structure in a narrow blue scale.

Figure 7

SEM images of the wing scales of the female eclosed from a pupa cooled for 20 days. (a) Dentate apex blue scale, (b) rounded apex blue scale, (c) brown scale.

Figure 8

TEM images of cross sections through the blue coloured scales of males and females. (a) Wild male, (b) male emerged from a pupa cooled for 22 days, (c) male emerged from a pupa cooled for 62 days, (d) female emerged from a pupa cooled for 20 days, (e) female emerged from a pupa cooled for 40 days.