Gene ontology enrichment analysis (Huang et al. 2009) was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID v. 6.8) (Dennis et al. 2003). Unique FlyBase IDs (n = 66) of D. melanogaster orthologs of identified differentially expressed genes served as the query gene list. A list of all unique FlyBase IDs (n = 7,083) representing all B. impatiens transcripts was obtained through BLAST by searching against the D. melanogaster transcriptome (TBlastX program parameters: -evalue 0.000001 -outfmt 6 -max_target_seqs 1 -max_hsps 1) and was amalgamated with a few additional manually curated unique FlyBase IDs from the aforementioned differentially expressed gene set to generate the final background gene list of unique FlyBase IDs (n = 7,092). As we are primarily interested in exploring the functions of the most differentially expressed and most reliable set of genes, we identified GO terms which met the EASE score (Hosack et al. 2003) from the set of genes that passed an adjusted Fisher’s exact test P value significance level threshold of P < 0.05 and an FDR-corrected significance threshold of P < 0.001. To summarize the GO terms, we used ReViGO web-based interactive server (Supek et al. 2011) which uses a multidimensional scaling (MDS) approach to cluster GO terms based on semantic similarity measurements. GO terms related with biological processes (n = 18) and their corresponding P values obtained from the aforementioned procedure (i.e., using DAVID v. 6.8) were provided as the input with default settings in the ReViGO interface (available at: http://revigo.irb.hr/) to generate a scatter plot depicting the major nonredundant biological process clusters.

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