DOI: 10.5061/dryad.pg4f4qrm7
title: Age and social experience induced plasticity across brain regions of the paper wasp Polistes fuscatus

description: 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.
description: <b>(a) Animal collection and housing</b>
 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 <i>ad libitum </i>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
 <i>ad libitum </i>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>(b) Brain volume and body size
 measurements</b> 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.
description: 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.