DOI: 10.5061/dryad.wm37pvmnq
title: Behavioral performance and division of labor influence brain mosaicism in the leafcutter ant Atta cephalotes

description: Brain evolution is hypothesized to be driven by behavioral selection on
 neuroarchitecture. We developed a novel metric of relative neuroanatomical
 investments involved in performing tasks varying in sensorimotor and
 processing demands across polymorphic task-specialized workers of the
 leafcutter ant Atta cephalotes and quantified brain size and structure to
 examine their correlation with our computational approximations.
 Investment in multi-sensory and motor integration for task performance was
 estimated to be greatest for media workers, whose highly diverse
 repertoire includes leaf-quality discrimination and leaf-harvesting tasks
 that likely involve demanding sensory and motor processes. Confocal
 imaging revealed that absolute brain volume increased with worker size and
 functionally specialized compartmental scaling differed among workers. The
 mushroom bodies, centers of sensory integration and learning and memory,
 and the antennal lobes, olfactory input sites, were larger in medias than
 in minims (gardeners) and significantly larger than in majors
 (“soldiers”), both of which had lower scores for involvement of olfactory
 processing in the performance of their characteristic tasks. Minims had a
 proportionally larger central complex compared to other workers. These
 results support the hypothesis that variation in task performance
 influences selection for mosaic brain structure, the independent evolution
 of proportions of the brain composed by different neuropils.
description: Mature fully sclerotized
 workers collected from colonies Ac09, Ac16, Ac20, and Ac21 were
 decapitated immediately prior to brain dissection and fixation. Workers
 were sampled from five worker size groups identified by head width (HW):
 minims (0.6mm±0.1mm), medias (1.2mm±0.1mm, 1.8±0.1mm, or 2.4mm±0.1mm), and
 majors (3.0mm or larger). Brains (n=30) from workers sampled from Ac09,
 Ac20, and Ac21 were dissected in ice-cold HEPES Buffered Saline (HBS),
 placed in 16% zinc-formaldehyde (Ott 2008) and fixed overnight at room
 temperature (RT) on a shaker. Whole brains were processed to visualize the
 presynaptic protein synapsin. Fixed brains were washed in HBS six times,
 10 minutes per wash, and fixed in Dent’s Fixative (80% MeOH, 20% DMSO) for
 minimally 1 hour. Brains were then washed in 100% methanol and either
 stored at -17°C or immediately processed. Brains were washed in 0.1M Tris
 buffer (pH=7.4) and blocked in PBSTN (5% neutral goat serum, 0.005% sodium
 azide in 0.2% PBST) at RT for 1 hour before incubation for 3 days at RT in
 primary antibody (1:30 SYNORF 1 in PBSTN; monoclonal antibody anti-synorf
 3C11 obtained from DSHB, University of Iowa, IA, USA; 62). They were
 washed 6x10 minutes in 0.2% PBST and incubated in the secondary antibody
 (1:100 AlexaFluor 488 goat anti-mouse in PBSTN) for 4 days at RT. Brains
 were then washed a final time (6x10 minutes in 0.2% PBST) and dehydrated
 in an ethanol and PBS series (10 minutes per concentration,
 30/50/70/95/100/100% ethanol in 1x PBS), then cleared with and immersed in
 methyl salicylate, and mounted on stainless steel glass windowed slides
 for imaging. Brains were imaged with a Nikon C2
 confocal microscope and images were manually annotated using Amira 6.0
 software to quantify neuropil volumes (not including cell bodies). The
 individual who annotated all brains for the study did not have any
 expectation of specific outcomes and did not have knowledge of predictions
 generated by our model. The annotation process involved using paintbrush-
 or magic wand-style tools to select areas to be included in a given
 neuropil in a given single scan of a 3D stack. The margins of focal
 neuropil regions were identified visually (or automatically when using the
 magic wand tool) based on the presence of synapsin staining. The magic
 wand-style tool was used primarily to annotate the antennal lobe
 glomeruli. Every third frame was annotated manually (or every other frame
 in the case of the antennal lobes) and intervening frames were filled in
 using the interpolation function of Amira. Interpolated frames were also
 checked and edited for accuracy. Annotated slices were then used to
 calculate the 3D volume of each neuropil using Amira and these data were
 exported for analysis. We recorded the volumes of OL, AL, MB, CX, SEZ, and
 ROCB. We use the term ROCB for simplicity and to correspond with our
 ability to associate specific compartments with sensorimotor functions to
 describe the tissue composed of the superior neuropils, lateral horn,
 ventrolateral neuropils, inferior neuropils, and ventromedial neuropils,
 as designated in a fruit fly brain (Ito et al. 2014). For the ALs, only
 glomerular tissue was included (excluding aglomerular neuropil and all
 soma layers). For the OLs, we measured only the medulla and lobula
 neuropils, excluding surrounding cell bodies. Similarly, measurements of
 the SEZ did not include somata. We also measured and separately examined
 substructures of the MB: the medial calyces (MB medial calyces), lateral
 calyces (MB lateral calyces), and peduncle and lobes (MB peduncle). Our
 peduncle measurements incorporated vertical and medial lobes; these
 metrics are included in all discussions of the peduncle. The volumes of
 these components were combined to quantify total MB size (total MB) across
 worker size groups. For bilateral structures, one hemisphere was measured,
 and for compartments located along the brain midline (SEZ and CX), the
 whole structure was measured (Supplementary Table 1; Supplementary Table
 2). When calculating total brain volume, we excluded all soma layers and
 used only neuropil volumes.