Reproducible phenotype alteration due to prolonged cooling of the pupae of Polyommatus icarus butterflies.

The phenotypic changes induced by prolonged cooling (2-12 weeks at 5 °C in the dark) of freshly formed Polyommatus icarus pupae were investigated. Cooling halted the imaginal development of pupae collected shortly after transformation from the larval stage. After cooling, the pupae were allowed to continue their developmental cycle. The wings of the eclosed specimens were investigated by optical microscopy, scanning and cross-sectional transmission electron microscopy, UV-VIS spectroscopy and microspectroscopy. The eclosed adults presented phenotypic alterations that reproduced results that we published previously for smaller groups of individuals remarkably well; these changes included i) a linear increase in the magnitude of quantified deviation from normal ventral wing patterns with increasing cooling time; ii) slight alteration of the blue coloration of males; and iii) an increasing number of blue scales on the dorsal wing surface of females with increasing cooling time. Several independent factors, including disordering of regular scale rows in males, the number of blue scales in females, eclosion probability and the probability of defect-free eclosion, showed that the cooling time can be divided into three periods: 0-4 weeks, 4-8 weeks, and 8-12 weeks, each of which is characterized by specific changes. The shift from brown female scales to first blue scales with a female-specific shape and then to blue scales with a male-specific shape with longer cooling times suggests slow decomposition of a substance governing scale formation.