Pgem t easy vector 1

3′-UTRs of target genes were obtained by 3′-RACE using specific primers (Supplementary Table 4) and the SMART RACE cDNA Amplification kit (Clontech, Mountain View, CA, USA). All PCR-amplified DNA fragments were cloned into the pGEM-T Easy Vector I (Promega) and sequenced. The 3′-UTR of Pxy-EcR was cloned into the pMIR-REPORT miRNA Expression Reporter Vector (Invitrogen). Mutations at the miR-281-3p seed sequence binding region were introduced at the miRNA target sites in the 3′-UTR of the P. xylostella ecdysone receptor gene (Pxy-EcR, GenBank accession number NM_001309151.1). The mutant construct was used as the negative control. Constructs were transfected into HEK293 cells using Lipofectamine 3000 (Invitrogen). Mimics of Cve-miR-281-3p, Cve-miR-31-5p-1, and Cve-novel22-5p were also transfected into the HEK293 cells. The miRCURY LNA miRNA mimic negative control (Exiqon 479903-001, Woburn, MA, USA) was used as negative control. The Dual-Luciferase Reporter Assay System (Promega) was used to measure the interaction between miRNAs and Pxy-EcR in each sample selected. At 48 h post transfection, the luciferase activity was measured according to the recommended protocol using a Thermo Scientific Varioskan Flash Microplate Reader (Thermo Fisher Scientific).

To identify the sources of miRNAs, primers were designed based on the miRNAs of C. vestalis that did not cover the last variable nucleotides (Supplementary Table 4). Both Pxem_ZJU cells co-cultured with C. vestalis teratocytes and hemocytes of parasitized P. xylostella were collected for miRNA extraction. PCR-amplified fragments of interest were cloned into the pGEM-T Easy Vector I (Promega, Madison, WI, USA) and sequenced. For each group of miRNA homologs, 100 clones were randomly selected and sequenced. Based on sequence differences, the proportion of each miRNA originated from P. xylostella versus C. vestalis was then determined.