Comparative RNA-seq of Gall Insect Interactions

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).