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.

本研究针对新生伊卡洛斯灰蝶（Polyommatus icarus）蛹在黑暗环境中5℃条件下持续低温处理2~12周所诱导的表型变化展开了探究。低温处理可阻断刚完成幼虫-蛹变态的蛹的成虫发育进程；待低温处理结束后，蛹可继续完成其发育周期。研究人员通过光学显微镜、扫描电子显微镜、横截面透射电子显微镜、紫外-可见分光光度法以及显微分光光度法，对羽化个体的翅膀进行了检测分析。羽化后的成虫呈现出表型变异，该结果与我们此前针对少量个体发表的研究结论高度一致；此类变异具体包括：① 随着低温处理时长增加，翅膀腹面图案与正常个体的量化偏差程度呈线性升高；② 雄性个体的蓝色翅色出现轻微改变；③ 随着低温处理时长增加，雌性个体翅膀背面的蓝色鳞片数量逐渐增多。多项独立评价指标——包括雄性个体鳞片规则排列的紊乱程度、雌性个体蓝色鳞片数量、羽化成功率以及无缺陷羽化概率——均证实，低温处理时长可划分为三个特征阶段：0~4周、4~8周及8~12周，每个阶段均具有特异性的变化特征。随着低温处理时长延长，雌性个体的鳞片从棕色逐步转变为具有雌性特异性形态的蓝色鳞片，进而进一步转变为具有雄性特异性形态的蓝色鳞片，这一现象表明，调控鳞片形成的相关物质发生了缓慢降解。