S8ap0 stereomicroscope

The morphology of fungal colonies is determined by their ability to grow on PDA, with varied observations of colony traits, such as color, size, and shape. In this study, three plates were incubated at 28 ± 2 °C, and the diameters of colonies were measured daily until the colonies reached the edge of the plate. The growth rate per day on PDA of fungal isolates was calculated. The growth rate experiment was repeated twice. The general morphological characteristics of the fungal isolates were observed using a Leica S8AP0 stereomicroscope (Leica Microsystems, Wetzlar, Germany) with 10× magnification and a Leica DM750 compound microscope (Leica Microsystems, Wetzlar, Germany) with 40× magnification. Mycelial plugs (0.5 cm) were cut from the edges of 3-day-old colonies of fungal isolates and placed on WA covered with sterile dried Napier grasses as substrates to develop pycnidia structures according to previous studies [13 (link),21 (link)].

The growth rate of fungal mycelia was measured on PDA. The macroscopic characteristics of pycnidia formation were observed [20 (link)] with a Leica S8AP0 stereomicroscope (Leica Microsystems, Wetzlar, Germany), whereas the microscopic characteristics of the conidia were examined with a Leica DM750 light microscope (Leica Microsystems, Wetzlar, Germany). The dimensions of the conidia were measured. The fungal cultures were then deposited in a culture collection, and the accession number was assigned.

Newly emerged females and males (within 12 h after eclosion) were microinjected with dsNF-YA, ds-NF-YB, dsNF-YC, or dsGfp. Then each female was allowed to match with two males in a glass tube. Insects were removed at 5 days after matching, and eggs were counted under a Leica S8AP0 stereomicroscope.

Photographs of K. daigremontiana were taken using a Huawei P smart (FIG-LX1) with an Apexel 10x Macro camera attachment. A GXCAM Eclipse (0654) Wi-Fi camera attached to a S8AP0 Stereo Microscope (Leica, USA) was used to visualize notches. Fiji Image J (http://imagej.net/Fiji/Downloads) was used to calculate plant areas and add scale bars. All graphs and statistical analyses were produced using GraphPad Prism Version 8.41. Two-way analysis of variance (ANOVA) (Repeated Measures) with Sidak’s multiple comparisons tests (95% confidence limits) were performed on the Torin2 treatment data. One-way ANOVA with Dunnett’s multiple comparisons tests were performed on the 35S::KdTORa plantlet data.

Galls on leaves of G. obovatum and E. japonica were originally collected from Tomogashima Island (Kada, Wakayama, Japan) and Kibogaoka Cultural Park (Yasu, Shiga, Japan), respectively, and both have been successfully reared in the laboratory [14 (link),15 (link)]. For G. obovatum, the galls with the third instar larva were collected as young galls, and those with the fourth to fifth larva as mature galls. In both cases, the collected galls were cut in half and the larva removed. The intact leaves from the same tree were collected as control samples. For E. japonica, the gall with the fourth instar inside was collected, cut, and larva removed. Intact leaves from the same tree were collected as control samples. Galls and leaves of A. montana were collected from Kyoto Prefectural University, Seika campus (Seika, Kyoto, Japan). Gall and larva RNA were extracted to avoid physical stress by dissection, since the size of the gall was small. Collection, RNA extraction and RNA–sequencing of galls and leaves from R. javanica were performed by collaborators (Hirano and Sato, in preparation). All samples were frozen in liquid nitrogen and kept at -80°C until required for RNA extraction. Photos were taken with an S8AP0 stereomicroscope mounted with an EC3 digital camera (Leica, Germany).

Females were allowed to lay eggs for 2 h, and the hatched nymphs were then monitored every 12 h. Newly hatched 1^st-instar nymphs (n = 20, 0–12 hAE) were collected, and each individual was raised separately in a glass tube. The developmental times of 1^st-, 2^nd-, 3^rd-, 4^th- and 5^th-instar stages were monitored every 12 h. Glucose and triglyceride levels were measured in pooled 24h-adult females (n = 15) as reported previously [46 (link)]. Glucose levels were measured using glucose oxidase reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Triglyceride contents were quantified by enzymatic hydrolysis using the GPO Trinder method with a tissue triglyceride assay kit (Applygen Technologies), according to the manufacturer’s instructions. The glucose and triglyceride contents were calculated based on three biological replicates. Adult longevity was determined by recording the mortality of newly emerged adult females (0–3 hAE, n = 43 for WT^SW and n = 42 for NlInR2^E4) and males (0–3 hAE, n = 52 for WT^SW and n = 42 for NlInR2^E4) every 12 h. To determine fecundity, newly emerged adult females (0–12 hAE) were collected for paired mating assays. Each female (n = 20) was allowed to match with two males in a glass tube. The insects were removed 10 days later and the laid eggs were counted under a Leica S8AP0 stereomicroscope.