Fig. 3. Development of differential gene expression as gyne-queen role differentiation in M. pharaonis proceeds.

(A) PCA of the brain transcriptomes of gynes and queens of different ages, with each point representing the transcriptome from a single brain, colored according to binary reproductive role and with symbols representing different ages. The mean PC values for gynes and queens at days 1, 5, 15, and 30 were connected chronologically by solid lines (arrowed, young to old), showing that transcriptome profiles were similar on day 5 but had diverged on days 15 and 30. The larger scatter among the gyne dots may have contributed to the number of significant DEGs between gynes and queens on day 30 being less than on day 15. (B) The number of DEGs between gynes and queens of the same age increased drastically between days 5 and 15, which is the time period during which gynes started laying unfertilized eggs and queens fertilized (and possibly also some unfertilized) eggs, after which no further increase at day 30 was observed. (C) Multidimensional scaling clustering according to semantic similarities (gene membership) of Gene Ontology (GO) terms (62). Representative gene members were written next to GO clusters, showing that genes with queen-biased expression were involved in hormonal responses on day 15 and in biogenesis and metabolic processes on day 30, whereas genes with gyne-biased expression on day 30 were mainly involved in ion transport functions.