Supplementary Material for: Plasticity of the Worker Bumblebee Brain in Relation to Age and Rearing Environment

The environment experienced during development can dramatically affect the brain, with possible implications for sensory processing, learning, and memory. Although the effects of single sensory modalities on brain development have been repeatedly explored, the additive or interactive effects of multiple modalities have been less thoroughly investigated. We asked how experience with multisensory stimuli affected brain development in the bumblebee <i>Bombus impatiens</i>. First, to establish the timeline of brain development during early adulthood, we estimated regional brain volumes across a range of ages. We discovered significant age-related volume changes in nearly every region of the brain. Next, to determine whether these changes were dependent upon certain environmental stimuli, we manipulated the visual and olfactory stimuli available to newly emerged bumblebee workers in a factorial manner. Newly emerged bumblebees were maintained in the presence or absence of supplemental visual and/or olfactory stimuli for 7 days, after which the volumes of several brain regions were estimated. We found that the volumes of the mushroom body lobes and calyces were larger in the absence of visual stimuli. Additionally, visual deprivation was associated with the expression of larger antennal lobes, the primary olfactory processing regions of the brain. In contrast, exposure to plant-derived olfactory stimuli did not have a significant effect on brain region volumes. This study is the first to explore the separate and interactive effects of visual and olfactory stimuli on bee brain development. Assessing the timing and sensitivity of brain development is a first step toward understanding how different rearing environments differentially affect regional brain volumes in this species. Our findings suggest that environmental factors experienced during the first week of adulthood can modify bumblebee brain development in many subtle ways.