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mec12626-sup-0001-FigS1.epsimage/eps26905KFig. S1 Experimental set-up for Experiment 2. A) Four mother colonies were split into 2 colony fragments each. All colony fragments were provided with a nesting chamber, a foraging area with a cricket, water and sugar water, and equal amounts of workers and brood. B) Queenless colony fragment. The queen is no longer inside the nesting chamber, but some workers continue to perform in-nest tasks. QR = queenright; QL = queenless; in = nonforaging workers; out = foraging workers.
mec12626-sup-0002-FigS2.epsimage/eps607KFig. S2 Microarray hybridization scheme for Experiment 1. For each group of workers, 6 pools were hybridized in a loop design: 3 pools were labelled with the Cy3 dye and other 3 with the Cy5 dye. We used 6 arrays of a 12-plex array slide with 135 000 probe capacity designed by Roche NimbleGen, Inc. (Madison WI).
mec12626-sup-0003-FigS3.epsimage/eps727KFig. S3 Microarray hybridization scheme for Experiment 2. For each group of workers, 6 pools were hybridized in a loop design: 3 pools were labelled with the Cy3 dye and other 3 with the Cy5 dye. We used a whole 12-plex array slide with 135 000 probe capacity designed by Roche NimbleGen, Inc. (Madison WI).
mec12626-sup-0004-FigS4.epsimage/eps700KFig. S4 Validation of analysis of gene expression with R in samples from Experiment 1. Analysis with R produced almost twice the number of transcripts differentially expressed between foraging and nonforaging workers at FDR<0.05 than analysis with sas (1387 vs. 771, respectively). However, a comparative analysis between the two set of transcripts revealed large overlap confirming that the choice of either analysis would not affect the biological significance of the results.
mec12626-sup-0005-FigS5.epsimage/eps1552KFig. S5 Comparative analysis of sets of transcripts differentially expressed at FDR <0.05 in both Experiments 1 and 2. A pool of 89 transcripts were shared (more than expected by chance, hypergeometric test: representation factor: 8.4, P < 7.16e-55), but these did not produce any significantly enriched GO terms. GO terms indicated in the figure refer to the two sets of transcripts analysed separately.
mec12626-sup-0006-FigS6.epsimage/eps1378KFig. S6 Quantitative real-time PCR validation of expression levels of genes of interest. A) Expression levels of the following genes associated with GO terms of interest were analysed using quantitative real-time PCR (see Table S10 for detailed information about these genes and the primers we used): For (food-related behaviour and polyethism), Hym (antibacterial response), mf (muscle development), mhc (locomotion), oat (neurogenesis), spirit (innate immune response) and syt1 (neurotransmitter secretion). Mean expression levels in QLin and QRout were normalized to levels of expression in QRin workers. We used 9 pools for QLin, 8 for QRin and 5 for QRout, each pool being composed by 10 workers from the same colonies used for Experiment 2 (but different individuals). Statistical analysis was performed with nonparametric Wilcoxon comparisons for each pair of treatments: * = P < 0.05; ** = P < 0.01. #For a better visualization of the results, the bar associated with the gene Hym is not represented in full length in QRout: average relative expression for this gene was 3.95 with S.E. ±0.94; QRin = queenright nonforaging workers; QLin = queenless nonforaging workers; QRout = queenright foraging workers. B) Log2-transformed and normalized expression values for the same genes as above after microarray analysis.
mec12626-sup-0007-TableS1-S10.xlsxapplication/msexcel259K

Table S1 Experiment 1: differentially expressed transcripts between foraging vs. nonforaging workers at FDR <0.05.

Table S2 Experiment 1: significantly enriched GO terms and KEGG pathways (functional annotation chart, P < 0.05).

Table S3 Experiment 2: differentially expressed transcripts between queenright nonforaging (QRin) and foraging (QRout) workers at FDR <0.05.

Table S4 Experiment 2: significantly enriched GO terms and KEGG pathways (functional annotation chart, P < 0.05).

Table S5 Experiment 2: differentially expressed transcripts between queenright and queenless workers at FDR <0.1. The list includes transcripts that were differentially expressed either in the QRin-QLin or in the QRout-QLout comparisons.

Table S6 Differentially expressed transcripts that were shared at FDR <0.05 between Experiment 1 and Experiment 2.

Table S7 Comparisons of significantly enriched GO terms from Experiment 1 and Experiment 2 (P < 0.05).

Table S8 Overlap analysis between 17 960 transcripts upregulated in queenless workers and transcripts that were upregulated in foraging and nonforaging workers.

Table S9 Comparative analysis of the 735 transcripts provided with FlyBase annotations that were differentially expressed in foraging vs. nonforaging workers in Experiment 1 and in other previous transcriptome studies.

Table S10 Quantitative real-time PCR validation of expression levels of genes of interest in workers from Experiment 2: gene lists and primers’ sequences.

mec12626-sup-0008-DataS1.docxWord document28KAppendix S1 Supporting information for online publication including further details (with relevant references) on the following aspects: insect collection, rearing and sampling; sample preparation for molecular analyses; protocols for microarray analysis; validation of differential expression of candidate genes using quantitative real-time PCR; comparative studies across species; discussion of genes of interest.

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