Sequencing Data analysis

The mRNA-seq data was treated using the cufflinks v2.2.1 workflow³⁶. First, we aligned the reads with TopHat to the Apis mellifera genome, assembly Amel_4.5. Second, aligned transcripts were assembled with Cufflinks, then the transcripts were merged to the final transcriptome assembly using the Official Gene Set OGSv3.2 as a reference genome annotation, and finally cuffdiff was performed to calculate the FPKM (fragments per kilobase per million mapped reads).

As to potential miRNAs, we removed the adaptors as the first step. Reads of 18–30 nt length were kept and compared with the microRNA data of Apis mellifera in the mirBASE (http://www.mirbase.org) by blast. The RPM (reads per million) were calculated as a measure of the expression levels of the microRNAs.