80i microscope system

5–10 μm fresh sections were stained using Vitroview Picro-Sirius Red Stain kit (VitroVivo Biotech) as described in the protocol. The sections are covered with Weigert’s Hematoxylin solution for 8 minutes, followed by Picro-Sirius Red stain for 60 minutes. Slides were washed with acidified water, water, ethanol (95% and 100%), and finally, xylene. Subsequently, slides were covered with a coverslip along with Permount. Slides were imaged under the Nikon 80i microscope system.

The TUNEL reaction was performed following the manufacturer’s introduction by using the riboAPO One-Step TUNEL Apoptosis Kit (RiboBio, C11012-1, Guangzhou, China). In brief, the 5-μm thick sections after deparaffinization and rehydration were treated with proteinase K (20 μg/mL) for 15 min and washed in PBS for 5 min. The sections were then applied and kept on 1x TdT enzyme equilibration buffer for 5–10 min at room temperature. Subsequently, the samples were incubated with the TdT enzyme reaction mixture in a humidified chamber at 37 °C for 2 h. After stopping the reaction with 2x SSC buffer at room temperature for 15 min and washing 3 times in PBS, the sections were stained with DAPI for 30 min at room temperature and were washed in PBS 3 times again. The images were captured using a Nikon 80i microscope system.

Histological sectioning, staining (HE) and immunofluorescence were performed for abnormal (including low-tanning and over-tanning nymphs; Supplementary Figure S11) and control nymphs obtained from the ds-TH and ds-lta treatments, respectively. Five samples of the second thoracic (T2) segments of the third-instar of A. pisum from each treatment were selected for hematoxylin–eosin (HE) staining, and five samples of the cross-sections of the T2 segments of the third-instar nymphs from each treatment were selected for immunofluorescence assays. Transverse longitudinal sections, from the head to the tail, of the treated mother aphids were also collected 48 h after the dsRNA injection for immunofluorescence experiments. The method is detailed in S1.3 (Supplementary Material). Digital images were acquired using a Nikon DS-Ri1 camera (Nikon, Tokyo, Japan), a Nikon 80i microscope system (Nikon, Tokyo, Japan), and Nis-Elements v. 3.22.14 (Build 736, Nikon, Tokyo, Japan). Cuticle thickness was determined from the digital images. All images showing Cy^TM3 fluorescence were captured and filtered using the default settings (brightness, contrast, and saturation) of software for comparison.