We randomly selected eight DE transcripts for validation of differential expression. These transcripts were significantly induced or suppressed in the exposed larvae library relative to the control library to encompass and validate differential expression in both directions (up- and downregulated genes) of the libraries. We also selected four transcripts that were neutral (neither induced nor suppressed in the exposed library relative to the control library) as potential internal reference neutral/loading controls [41]. These reference transcripts consisted of CLIP-domain serine protease, glyceraldehyde 3-phosphate dehydrogenase (gapdh) and two uncharacterized genes (Additional file 1: Table S1) from VectorBase [28]. We ensured that all these transcripts were abundantly expressed in the RNA-seq libraries (based on their RPKM values) to ensure that their expression levels would be within the sensitivity of our Stratagene MX3005P RT-qPCR machine (Agilent Technologies, CA, USA). We obtained DNA sequences of respective genes from VectorBase [28] using the respective gene IDs (Additional file 1: Table S1) and designed primers (Additional file 1: Table S1) from these sequences in silico using primer3 software [42]. In all cases, we ensured that the melting (Tm) and annealing temperatures of the respective forward and reverse primers generated were similar, as determined by pDRAW32 version 1.1.142 software (http://www.acaclone.com) (Additional file 1: Table S1).

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