Effects of PcG gene knockdowns on adult morphology

Aposematic coloration offers an opportunity to explore the molecular mechanisms underlying canalization. In this study, the role of epigenetic regulation underlying robustness was explored in the aposematic coloration of the milkweed bug, Oncopeltus fasciatus. Polycomb (Pc) and Enhancer of Zeste (E(z)), which encode components of the Polycomb Repressive Complex 1 (PRC1) and PRC2, respectively, and jing, a component of the PRC2.2 subcomplex, were knocked down in the fourth instar of O. fasciatus. Knockdown of these genes led to alterations in scutellar morphology and melanization. In particular, when Pc was knocked down, the adults developed a highly melanized abdomen, head, and forewings at all temperatures examined. In contrast, the E(z) and jing knockdown led to increased plasticity of the dorsal forewing melanization across different temperatures. Moreover, jing knockdown adults exhibited increased plasticity in the dorsal melanization of the head and the thorax. These observations demonstrate that histone modifiers may play a key role during the process of canalization to confer robustness in the aposematic coloration.

Methods
Oncopeltus fasciatus 4th instar nymphs were injected with 1 μg (0.5 μL) of ampicillin resistance, Polycomb (Pc), Enhancer of zeste (E(z)) and jing double-stranded RNA using a syringe and a pulled borosilicate needle. The nymphs were reared at 20°C, 26.5°C or 33°C. Whole bodies of adult O. fasciatus were kept frozen at -20°C. The ventral abdomen of each bug was fixed in 3.7% formaldehyde and mounted in a 70% glycerol:30% PBS solution or an 80% glycerol:20% water solution and then imaged. Adult bugs were imaged under a Leica M165 FC stereo microscope and images were captured using a Leica DFC310 FX camera. Images were analyzed using the ImageJ software.