DOI: 10.5061/dryad.f7m0cfz51
title: Data from: Effects of PcG gene knockdowns on adult morphology

description: 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.
description: Oncopeltus fasciatus 4th instar nymphs were injected with 1 μg
 (0.5 μL) of <em>ampicillin resistance</em>,
 <em>Polycomb</em> (<em>Pc</em>),
 <em>Enhancer of zeste</em> (<em>E(z)</em>) and
 <em>jing</em> double-stranded RNA using a syringe and a pulled
 borosilicate needle. The nymphs were reared at 20°C, 26.5°C or
 33°C.<strong> </strong>Whole bodies of adult <em>O.
 fasciatus </em>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.
description: # Effects of PcG gene knockdowns on adult morphology
 [https://doi.org/10.5061/dryad.f7m0cfz51](https://doi.org/10.5061/dryad.f7m0cfz51) In this study, the role of epigenetic regulation underlying robustness was explored in the aposematic coloration of the milkweed bug, *Oncopeltus fasciatus*. *Polycomb* (*Pc*), *Enhancer of Zeste* (*E(z)*) and *jing* were knocked down in the fourth instar of *O. fasciatus* and the adult morphology was analyzed. The data provided are measurements made on individual adult bugs that were used to generate the graphs in the manuscript. ## Description of the data and file structure **1. Wing_measurements:** This file includes the area of the forewings, the length of the forewings and the width of the forewings of individual bugs as shown in Supplemental Figure S2. The ratio of length/width was calculated by dividing the length by the width. Measurements were obtained from adults that were injected with *ampr*, *E(z)*, *jing* or *Pc* dsRNA during the 4th instar and reared at 26.5°C. These measurements were used to generate the graphs shown in Supplemental Figure S2. dsRNA injected: Double-stranded RNA that was injected Wing area (mm2): Total area of the forewing in mm^2 Wing length (mm): The length of the wing in mm Wing width (mm): The width of the wing in mm Ratio of length/width: Length of the wing divided by width of the wing **2. Leg_measurements:** This file includes the length of the femur, the length of tibia and the length of the tarsus of individual bugs as shown in Supplemental Figure S5. Measurements were obtained from one foreleg (FL), one middle (ML) and one hindleg (HL) of each adult that was injected with *ampr*, *E(z)*, *jing* or *Pc* dsRNA during the 4th instar and reared at 26.5°C. These measurements were used to generate the data shown in Supplemental Figure S5. null indicates missing value due to absence of structure in the specimen. dsRNA injected: Double-stranded RNA injected Individual#: Identifier FL Femur: Foreleg femur length in mm FL Tibia: Foreleg tibia length in mm FL Tarsi: Foreleg tarsus length in mm ML Femur: Midleg femur length in mm ML Tibia: Midleg tibia length in mm ML Tarsi: Midleg tarsus length in mm HL Femur: Hindleg femur length in mm HL Tibia: Hindleg tibia length in mm HL Tarsi: Hindleg tarsus length in mm **3. Head_melanization_measurements:** This file includes the amount of melanization in the head for each individual bug that was injected with dsRNA and reared at different temperatures. The amount of melanization represents the proportion of the linear distance between the ocelli that is melanized (see Figure 2B). In addition, column F contains the normalized amount of melanization relative to the average melanization at 26.5°C (column E). These measurements were used to generate the graphs shown in Figure 2. dsRNA injected: Double-stranded RNA that was injected Temperature: Rearing temperature of bugs Amount of melanization: Proportion of the linear distance between the ocelli that is melanized % melanization: Percentage of the linear distance between the ocelli that is melanized. 100% = completely black; 0% = no melanization Average melanization at 26.5°C: Average proportion of the linear distance the ocelli that is melanized at 26.5°C Normalized ratio: Amount of melanization at the respective temperature divided by the average melanization at 26.5°C **4. Wing_melanization_measurements:** This file includes the percent melanization of one of the forewings (see Figure 3B) of each individual bug that was injected with dsRNA and reared at different temperatures. In addition, column F contains the normalized amount of melanization relative to the average melanization at 26.5°C (column E). These measurements were used to generate the graphs shown in Figure 3. dsRNA injected: Double-stranded RNA that was injected Temperature: Rearing temperature of bugs Melanized area/total area: Proportion of total wing area that is melanized. 1 = completely black; 0 = no melanization % melanized area: Percentage of the total wing area that is melanized. 100% = completely black; 0% = no melanization Average melanized area at 26.5°C: Average proportion of the wing area that is melanized at 26.5°C Normalized ratio: The proportion of melanized area at the respective temperature divided by the average proportion of melanized area at 26.5°C **5. Scutellum_melanization_measurements:** This file includes the percent melanization of the scutellum (see Figure 4C) of each individual bug that was injected with *ampr* or *jing* dsRNA and reared at different temperatures. In addition, column F contains the normalized amount of melanization relative to the average melanization at 26.5°C (column E). These measurements were used to generate the graphs shown in Figure 4. dsRNA injected: Double-stranded RNA that was injected Temperature: Rearing temperature of bugs Melanized area/total area: Proportion of total scutellum area that is melanized. 1 = completely black; 0 = no melanization % melanized area: Percentage of the total scutellum area that is melanized. 100% = completely black; 0% = no melanization Average melanized area at 26.5°C: Average proportion of the scutellum area that is melanized at 26.5°C Normalized ratio: The proportion of melanized area at the respective temperature divided by the average proportion of melanized area at 26.5°C **6. Abdomen_melanization_measurements:** This file includes the percent melanization of the abdomen (see Figure S7A) of each individual bug that was injected with dsRNA and reared at different temperatures. In addition, column M-O contains the normalized amount of melanization relative to the average melanization at 26.5°C (column J-L). These measurements were used to generate the graphs shown in Figures S7 and S8. Sex: The sex of the adult dsRNA injected: Double-stranded RNA that was injected Temperature: Rearing temperature of bugs A3 melanized area/total area: Proportion of total ventral abdominal segment 3 area that is melanized. 1 = completely black; 0 = no melanization A4 melanized area/total area: Proportion of total ventral abdominal segment 4 area that is melanized. 1 = completely black; 0 = no melanization A5 melanized area/total area: Proportion of total ventral abdominal segment 5 area that is melanized. 1 = completely black; 0 = no melanization A3 % melanized area: Percentage of the total ventral abdominal segment 3 area that is melanized. 100% = completely black; 0% = no melanization A4 % melanized area: Percentage of the total ventral abdominal segment 4 area that is melanized. 100% = completely black; 0% = no melanization A5 % melanized area: Percentage of the total ventral abdominal segment 5 area that is melanized. 100% = completely black; 0% = no melanization A3 Average melanized area at 26.5°C: Average proportion of the ventral abdominal segment 3 area that is melanized at 26.5°C A4 Average melanized area at 26.5°C: Average proportion of the ventral abdominal segment 4 area that is melanized at 26.5°C A5 Average melanized area at 26.5°C: Average proportion of the ventral abdominal segment 5 area that is melanized at 26.5°C A3 normalized ratio: The proportion of melanized ventral abdominal segment 3 area at the respective temperature divided by the average proportion of melanized ventral abdominal segment 3 area at 26.5°C A4 normalized ratio: The proportion of melanized ventral abdominal segment 4 area at the respective temperature divided by the average proportion of melanized ventral abdominal segment 4 area at 26.5°C A5 normalized ratio: The proportion of melanized ventral abdominal segment 5 area at the respective temperature divided by the average proportion of melanized ventral abdominal segment 5 area at 26.5°C