Developmental studies of brain volumes can reveal which portions of neural circuits are sensitive to environmental inputs. In social insects, differences in relative investment across brain regions emerge as behavioral repertoires change during ontogeny or as a result of experience. Here, we test the effects of maturation and social experience on morphological brain development in Polistes fuscatus paper wasps, focusing on brain regions involved in visual and olfactory processing. We find that mature wasps regardless of social experience have relatively larger brains than newly emerged wasps and this difference is driven by changes to mushroom body calyx and visual regions but not olfactory processing neuropils. Notably, social wasps invest more in the anterior optic tubercle (AOT), a visual glomerulus involved in color and object processing in other taxa, relative to other visual integration centers the mushroom body calyces compared with aged socially naïve wasps. Differences in developmental plasticity between visual and olfactory neuropil volumes is discussed in light of behavioral maturation in paper wasps, especially as it relates to social recognition. Previous research has shown that P. fuscatus need social experience to develop specialized visual processing of faces, which is used to individually recognize conspecifics. The present study suggests that the AOT is a candidate brain region that could mediate facial processing in this species.

(a) Animal collection and housing

Nests with reproductive pupa (i.e. gynes) were collected in August 2019 in Brandon, VT, USA and August 2020 in Ithaca, NY, USA. All adults on the nest were removed and nests were affixed to the top of a clear plastic container and provided ad libitum sugar and water as well as construction paper. Each morning and late afternoon nests were checked for new adult females which were split into one of three categories. (i) Newly emerged wasps had their brains immediately dissected and fixed for immunolabeling and imaging (N= 12, figure 1). (ii) Isolated wasps were removed from the nest upon eclosion and isolated in individual plastic containers also containing construction paper and ad libitum sugar and water (N=14, figure 1). (iii) The social rearing group were paint marked according to the date they emerged and allowed to remain on the nest (N=14, figure 1). Maturing wasps were kept in identical housing conditions in the same humidified rearing room kept at 29°C with a 14:10 light- dark cycle and allowed to develop within each treatment for 58 to 71 days (figure 1).

(b) Brain volume and body size measurements

Brains were dissected whole mount in 4% Paraformaldehyde solution and stored over night at 4°C. Brains were stained using previously established methods in honey bees [32]. In brief, they were washed with 0.01M PBS with 3% Triton X (6x20 min., PBSTX, Sigma-Aldrich P4417; X100) and incubated for 3 days with 1:800 anti-synapsin (3C11, DSHB). Following incubation brains were again washed with 0.01M PBSTX (6x20 min.) and incubated for 3 days with 1:320 donkey anti-mouse antibodies conjugated with Alexa 488 (Jackson Immuno, 715-546-151) and 1:80 Phalloidin-TRITC (Milipore Sigma, P1951). Brains were dehydrated in methanol series to 100% methanol and cleared with methyl salicylate (Sigma-Aldrich, M6752). One half of each brain was then imaged on a confocal microscope (Zeiss LSM710 Inverted Axio Observer) with a C-Apochromat10x/0.45 water-immersion objective and a 0.6x digital zoom using the appropriate filter and laser setting for each fluorescent molecule and 8x line averaging with 12 mm optical sections. Brain scans were then manually reconstructed using Amira Software (Thermo Fisher Scientific) and volumes of neuropils were extracted. Each body was photographed using a dissection microscope with a scale in frame. The length of hind tibia, thorax width, and log transformed length of wing was calculated in ImageJ [33]. A PC1 score of body size was calculated for each individual.

We manually reconstructed the lobula, medulla, anterior optic tubercle, antennal lobe, mushroom body lip, mushroom body collar, mushroom body basal ring. The lip, collar, and basal ring have 3 values. L=Lateral calyx, M=Medial calyx, T=combined total of both Lateral and Medial Calyx volumes. Volumes are in cubic microns. Each wasp also has body size measures including wing length, thorax width, and hind tibial length in milimeters. A PC1 for body size was created using thorax width, hind tibial length and log transformed wing length. Note column explains why a brain region may be missing for a given individual, as some preps had partial damage making the neuropil volume incomplete.