DOI: 10.5061/dryad.8w9ghx3s8
title: Data from: Differential neuroanatomical, neurochemical, and behavioral impacts of early-age isolation in a Eusocial insect

description: Social experience early in life appears to be necessary for the
 development of species-typical behavior. Although isolation during
 critical periods of maturation has been shown to impact behavior through
 gene expression and brain development in invertebrates and vertebrates,
 workers of some ant species appear resilient to social deprivation and
 other neurobiological challenges that occur during senescence or due to
 loss of sensory input. It is unclear if and to what degree neuroanatomy,
 neurochemistry, and behavior will show deficiencies if social experience
 in the early adult life of worker ants is compromised. We reared workers
 of Camponotus floridanus from adult eclosion under conditions of
 social isolation for two to 53 days, quantified brain compartment volumes,
 recorded biogenic amine levels in individual brains, and evaluated
 movement and behavioral performance to compare the neuroanatomy,
 neurochemistry, brood-care behavior, and foraging (predatory behavior) of
 isolated workers with that of workers experiencing natural social contact
 after adult eclosion.  We found that the volume of the antennal
 lobe, which processes olfactory inputs, was significantly reduced in
 workers isolated for an average 40 days, whereas the size of the mushroom
 bodies, centers of higher-order sensory processing, increased after
 eclosion and was not significantly different from controls. Titers of the
 neuromodulators serotonin, dopamine, and octopamine remained stable and
 were not significantly different in isolation treatments and controls.
 Brood care, predation, and overall movement were reduced in workers
 lacking social contact early in life. These results suggest that the
 behavioral development of isolated workers of C. floridanus is
 specifically impacted by a reduction in the size of the antennal lobe.
 Task performance and locomotor ability therefore appear to be sensitive to
 a loss of social contact through a reduction of olfactory processing
 ability rather than change in the size of the mushroom bodies, which serve
 important functions in learning and memory, or the central complex, which
 controls movement.
description: <strong>Colony collection and
 culture</strong> Three queenright colonies of
 <em>C. floridanus</em> were collected in or near Gainesville,
 Florida in 2012 and reared in Harris environmental chambers at 25°C and
 40-55% relative humidity on a 12:12 light cycle.  Parent colonies from
 which workers were sampled had a minimum size of 100, equally distributed
 between minors and majors.  Artificial nests were made from test tubes 75%
 filled with water and fitted with a tight cotton plug and placed in
 Fluon®-lined (Bioquip, Rancho Dominguez, CA) plastic nest boxes 16x11x6 cm
 to 32x22x6 cm depending on colony size.  Colonies were fed 1M sucrose on
 saturated cotton balls and varied protein sources (mealworms, scrambled
 eggs, and fruit flies) every other day in rotation.
 <strong>Worker social isolation</strong>
 <em>C. floridanus</em> has a dimorphic worker caste.
  We used minor workers because of their diverse behavioral repertoire,
 which includes brood care and foraging, rather than majors, which are
 limited to defense.  Four groups of callow minors were collected from each
 of three parent colonies.  Randomly selected callows were isolated from
 nestmates for 2-3 days (n=29), 5-6 days (n=36), 20-22 days (n=44), and 40
 days (n=29, mean = 40.08 days, range = 30 – 53 days; herein 40 days) in
 separate Fluon®-lined boxes (16 x 11 x 6 cm).  Socially isolated workers
 (“isolates”) were provided similar conditions in smaller nest boxes and
 the same diet as parent colonies.  After the prescribed isolation period,
 isolates were assayed for behavioral performance and subsequently
 sacrificed for immunohistochemical analysis of brain structure, or for
 quantification of brain biogenic amine titers.  Callow (n = 29) and mature
 minor workers (“matures”, n = 46) were collected and assayed using the
 same methods.  Callow and mature controls served as comparative groups for
 2-day isolates and 40-day isolates, respectively, and we report primarily
 on differences in behavior, neuroanatomy, and neurochemistry between these
 groups.  We compared brain volumes of callow controls estimated to be 2
 days old and 2--3-day isolates to ensure there were no intrinsic
 differences in brain size post eclosion.  We used 5- and 20-day isolates
 lacking age-matched controls to estimate the trajectory of behavioral and
 neural development from 2 days to 53 days of isolation (Supp Fig 1-4).
  Isolated workers had a survival rate of 63.7%.  Because of the relatively
 high mortality of workers experiencing longer periods of isolation, sample
 sizes for 40-day isolates are generally lower.  Head width, measured as
 the widest distance across the eyes, served as a proxy for body size.  To
 control for the effect of body size on brain size, we compared the head
 widths of workers across isolation periods and control groups and found no
 significant differences (p=0.40, R2=0.098, F=1.18, Z=0.25, SS type 3, Supp
 Fig 1a).  Our sampling of workers for assignment to isolation or control
 treatments was thus unlikely to influence brain volume.
 <strong>Behavioral performance
 assays</strong> Isolated and control workers used
 for behavior (n = 16 [2 day], 19 [5 day], 20 [20 day], 11 [40 day], 8
 [callow] and 13 [mature]) were exposed to a randomized series of assays to
 determine foraging ability and ability to recognize and care for brood,
 tasks that affect colony fitness.  Simplified illustrations of the assays
 can be found in Fig 1a.  <strong>Predatory
 Behavior</strong> A single worker was carefully
 placed in a Fluon®-lined Petri dish (9cm) and allowed to acclimate for 2
 minutes prior to assay initiation.  Using Dumont No. 5 fine forceps
 sanitized with ethanol and dried, a live fruit fly was offered to the
 worker and the response was observed for 2 minutes.  The degree of
 predatory behavior was scored on a 5-point scale; higher scores indicate
 greater predatory behavior.  1. no response or avoidance; 2. olfactory
 (antennal) investigation; 3. mandibular flaring; 4. latent attack (delayed
 and intermittent); and 5. immediate attack (persistent predatory attack
 from first encounter with prey).  The assay arena was also sanitized with
 ethanol between trials to minimize influence from social signals or prey
 odors. <strong>Brood recognition and
 care</strong> To determine if isolates differ in
 the ability to recognize and care for immature siblings with similar
 efficacy as control matures and callows, we evaluated their social
 interactions with pupae.  Each focal worker was carefully placed in a 14
 cm diameter Petri dish test arena where a 30 x 10 mm red plastic hollow
 tube provided a darkened area into which workers could move pupae.  Three
 pupae were added at three different locations equidistant from the
 darkened chamber before a worker was introduced.  Workers could relocate
 pupae either inside or outside of the dark chamber or have no interaction.
  Pupae were recorded as aggregated (“piled”) if workers moved two or more
 together at any location.  Workers showing this level of response began
 pupal care immediately.  We did not assign lower scores to workers that
 clustered pupae without moving them into the darkened area.  The darkened
 tube provided the opportunity for a level of pupal care that would emulate
 natural placement of immatures within a colony.  However, no worker moved
 pupae into the tube and we therefore did not include this behavior in the
 calculation of brood-care scores.  Some workers did not move any pupae,
 but they themselves moved into the darkened tube.  This was scored as
 avoidance. Worker interactions with pupae were observed
 directly for 5 minutes; after an additional 15 minutes , the final
 placement of pupae was recorded (total trial length = 20 minutes).  Brood
 recognition and care was measured on a 7-point scale: 1. no interaction or
 avoidance throughout the duration of the trial; 2. olfactory (antennal)
 investigation; 3. mouthpart contact; 4. one pupa moved during the last
 15-minute period; 5. one pupa moved in the first five-minute period; 6.
 two pupae moved beginning in the first five-minute period; 7. three pupae
 moved beginning in the first five-minute period.  Levels 4 and 5
 distinguish the rate of response to pupae, which we interpret as sensory
 processing ability that could be sensitive to social isolation.  Higher
 scores indicate greater brood-recognition ability (likely olfactory),
 restoration of brood clusters through pupal transport, and sustained
 engagement with brood.  Workers received scores of 5-7 independent of
 where pupae were transported.
 <strong>Locomotion</strong> To
 examine if isolation affected physiological and/or biomechanical
 capability to move pupae or attack prey, we used a simple and robust proxy
 to broadly assess neuromuscular impairment.  Focal workers were placed in
 the center of a 10 cm diameter Fluon®-lined Petri dish divided into four
 equal  quadrants by a crosshair pattern drawn beneath.  After a two-minute
 acclimation period, the number of times a worker moved among the four
 quadrants was recorded during a five-minute period using a digital hand
 counter.   Movement between quadrants was only scored when the worker
 passed a division line by at least one body length.
 <strong>Neuroanatomical and neurochemical
 measurements</strong><br>Immunohistochemistry and confocal
 microscopy Intact brains (n = 8 [2 day], n = 11 [5
 day], n = 15 [20 day], n = 14 [40 day], n = 5 [matures], and n = 7
 [callows]) were dissected in ice-cold Ringer’s solution, and then
 immediately placed in ice-cold zinc-formaldehyde fixative with shaking
 overnight.  Fixed brains were then washed in HEPES buffered saline 6 times
 (10 minutes/wash) and fixed in Dent’s Fixative (80% MeOH, 20% DMSO) for 1
 hour.  Brains were placed in 100% methanol and stored at -17 C° for 1-3
 weeks until processed.  Brains were next rinsed 6 times (10 minutes/rinse)
 with 3% Triton X-100 in 1X PBS (3% PBST).  Brains were stored overnight in
 5% normal goat serum/0.2% PBST/0.005% sodium azide for blocking.  After
 blocking, brains were incubated for 4 days RT with an anti-synapsin
 antibody to stain neuropils (1:30, Development Studies Hybridoma Bank,
 Iowa City, Iowa) in constant nutation.  Brains were washed with 0.2% PBST
 (6x10 minutes), then incubated in secondary antibody (1:100 AlexaFluor
 goat ɑ-mouse) in 5% normal goat serum/0.2%PBST/0.005% sodium azide for 4
 days at RT with constant nutation.  Brains were washed in 0.2% PBST (6x10
 minutes), then dehydrated through an ethanol and PBS series (30, 50, 70,
 95, 100% ethanol in 1XPBS).  Brains were cleared and immersed in methyl
 salicylate and mounted on stainless steel glass-windowed slides for
 confocal imaging. Brains were imaged with a Nikon C2 +
 Si spectral laser scanning confocal microscope (Nikon, Melville, NY, USA)
 with sections at approximately 1.5 um throughout the entire brain.  Images
 were manually annotated using Amira 6.0 software to quantify neuropil
 volumes.  The individual who annotated brains was blind to brain sample
 category, thus minimizing human bias.  The margins of neuropils were
 identified visually or with magic wand tools in Amira.  Every eighth frame
 was annotated manually.  Biomedisa interpolation [48] was used on frames
 without labels, then evaluated and edited by the annotator for accuracy.
  We labeled volumes of the optic lobe (OL; vision), antennal lobe (AL;
 olfaction), mushroom body (MB; higher-order processing, learning, and
 memory), lateral calyx (MB-LC), and medial calyx (MB-MC), peduncles
 (MB-P), central complex (CX; movement and navigation), suboesophageal zone
 (SEZ; mouthpart control), and rest of the central brain (ROCB).  ROCB
 describes tissue composed of the superior neuropils, ventromedial
 neuropils, ventrolateral neuropils, and the lateral horn.  For each brain
 compartment, volumes of only one intact hemisphere were recorded.  If both
 hemispheres were intact, the labeler chose one randomly.
 A detailed description of brain anatomy and development in
 relation to age and experience in C. floridanus is presented in Gronenberg
 et al.  We compared the relative volumes of brains across all groups and
 scaling relationships among brain compartments.  Relative volumes were
 calculated by dividing the volume of each focal compartment by the ROCB
 volume.  Damaged compartments were excluded from analysis.  If one or more
 compartments or the ROCB was damaged, the sample was excluded.  The MB-LC
 and MB-MC exhibited similar volumetric trends and thus were combined to
 one metric (MB-C). <strong>Quantification of
 brain biogenic amines</strong> Isolated workers
 were quickly dissected (n = 11 [2 days], n = 10 [5 days], n = 12 [20
 days], n = 3 [40 days], along with control mature workers (n = 11) to
 quantify levels of serotonin (5HT), dopamine (DA), and octopamine (OA)
 using isocratic, reversed-phase high-performance liquid chromatography
 with electrochemical detection (HPLC-ED).  Monoamine quantification was
 performed blind to treatment.  The HPLC-ED system (formerly ESA, Inc.,
 Chelmsford, MA, USA) included a model 584 pump, a model MD-150 (3 x 150
 mm) reversed-phase analytical column, a model 5011A dual-channel
 coulometric analytical cell, and a Coulochem III electrochemical detector.
  Brains were dissected in less than 5 minutes in ice-cold Ringer’s
 solution, homogenized in mobile phase manually with a plastic
 polypropylene pestle in a centrifuge tube, and centrifuged.  The
 supernatant was then injected into the HPLC column, and monoamine levels
 were quantified with reference to external standards.  Mobile phase was
 formulated as 50 mM citrate/acetate buffer, 1.5 mM sodium dodecyl sulfate,
 0.01% triethylamine, and 25% acetonitrile in MilliQ water.  Multiple daily
 runs of the external standards accounted for any changes in ambient
 conditions. <strong>Statistical analysis and data
 visualization</strong> All statistics analyses
 were performed in RStudio version 4.2.3.  For generalized linear mixed
 models, we used the package ‘RRPP’.  We tested for statistical
 significance using ANOVA with residual randomization in a permutation
 procedure of 1000 iterations and the estimation me­­­­­­­­thod of ordinary
 least squares, given combined sample size of 25 or more.  We included
 colony and head width as random effects in our behavior, brain volume, and
 biogenic amine modeling; neither had a significant effect.  Isolation
 significantly correlated with head width only when head width was nested
 with the microscope and experimenter, rather than head width alone,
 meaning that slight variation between ocular micrometers of the
 microscopes, rather than treatment, influenced the effect of head width.
  ANOVA and modeling details can be found in Supp Tables Set 1 and 2.
  Following ANOVA, post-hoc tests were done on pairwise comparisons using
 Wilcoxon rank sum exact tests with a Benjamini-Hochberg (BH) procedure to
 correct for multiple tests.  Depending on the sample size of 40-day
 isolates, we included type III error to account for sample size bias.
  Additional nonparametric statistical analyses including Kruskal-Wallis H
 tests and post-hoc pairwise comparisons using t tests with a Bonferroni
 correction, which agreed with our modeling, can be found in Supp Tables
 Set 1 and 2.  We then ran studentized Breusch-Pagan (BP) tests from the
 ‘lmtest’ package to confirm homoscedasticity in our model.  If
 heteroscedasticity was found in raw values, we log transformed our data
 and reran the model and confirmed homoscedasticity. Heteroscedasticity
 could not be removed from only one model, which was our mixed model
 analysis of MB-P scaled volumes in Supplementary Tables, Set 1. However,
 our model nonetheless agrees with our additional nonparametric statistical
 tests.  We confirmed that log-transformed data resulted in the same
 pairwise comparison results as raw data, confirming that log
 transformation for model fitness did not create false pairwise findings.
  Because 5-day isolates and 20-day isolates did not have a socially
 typical, age-matched control and were instead meant to provide insight
 into the trajectory of neurodevelopment and behavior, we excluded these
 groups from the analysis reported in the manuscript.  Our analyses
 including these excluded groups can be found in Supp Figs 1-4 and in Supp
 Tables, Set 2.   Figures were created using the package ‘ggplot2’.  Figure
 assembly was done in Adobe Illustrator (Adobe Illustrator 2023).
  Illustrations of ants in Visual Abstract, Fig 1a, and Supp Fig 1b are
 courtesy of BioRender.com.
description: Excel, RStudio.   
description: # Behavioral and Neural Resilience to Isolation in a Eusocial Insect
 Coauthors Billie C. Goolsby, E. Jordan Smith, Isabella B. Muratore, Zach
 N. Coto, Mario L. Muscedere, James F. A. Traniello Last updated June 29,
 2024 #### Uploaded by Billie Goolsby at Stanford University
 ([bgoolsby@stanford.edu](mailto:bgoolsby@stanford.edu)) --- This dataset
 contains our raw data collected from three titular experiments:
 immunohistochemistry (IHC), behavior assays, and high performance liquid
 chromatography (HPLC). Feel free to peruse our data, code, and
 supplementary data. #### Description of the data and file structure The
 supplementary tables are uploaded in Excel format. The tab sheets are as
 such: BehaviorAnalysis, HeadwidthandBrainMassAnalysis, Compartment
 Analysis, AbsoluteTiterAnalysis, RelativetoTotalTiterAnalysis, and
 RatioAnalysis. #### **Supplementary Tables Set 1**: Statistics excluding
 5- day and 20- day isolate groups The supplementary tables are uploaded in
 Excel format. The tab sheets are as such: BehaviorAnalysis,
 HeadwidthandBrainMassAnalysis, Compartment Analysis,
 AbsoluteTiterAnalysis, RelativetoTotalTiterAnalysis, and RatioAnalysis.
 **BehaviorAnalysis** is a sheet within **Supplementary Tables Set 1**
 which shows the detailed ANOVA and pairwise tests for locomotion,
 predation, and brood care, the behavior studied in the manuscript Table
 1*:* Analysis of Variance of Activity, using three variables: days in
 isolation, original ant colony, and individual head width. Permutation
 Procedure: Randomization of Null Model Residuals. Number of Permutations:
 1000. Estimation Method: Ordinary Least Squares. Sums of Squares and
 Cross-products: Type I. Headwidth and Colony size are random effects.
 Effect sizes (Z) based on F distributions. Signif codes: 0 ‘***’ 0.001
 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1  Studentized Breusch-Pagan test is in the
 table legend. Table 2*:* Pairwise Comparisons for Activity. Pairwise
 comparisons using Wilcoxon rank sum test with continuity correction and
 Benjamini-Hochberg procedure. The comparison groups are callows, 2-day
 isolates, and 40-day isolates. Table 3*:* Analysis of Variance of
 Predation, using Residual Randomization. Permutation Procedure:
 Randomization of Null Model Residuals. Number of Permutations: 1000.
 Estimation Method: Ordinary Least Squares. Sums of Squares and
 Cross-products: Type I. Headwidth and Colony size are random effects.
 Effect sizes (Z) based on F distributions. Signif codes: *0 ‘’ 0.001 ‘’
 0.01 ‘’ 0.05 ‘.’ 0.1 ‘ ‘ 1* Studentized Breusch-Pagan test is in the table
 legend. Table 4*:* Pairwise Comparisons for Predation. Pairwise
 comparisons using Wilcoxon rank sum test with continuity correction and
 Benjamini-Hochberg procedure. Table 5*:* Analysis of Variance of Brood
 Care, using Residual Randomization. Permutation Procedure: Randomization
 of Null Model Residuals. Number of Permutations: 1000. Estimation Method:
 Ordinary Least Squares. Sums of Squares and Cross-products: Type I.
 Headwidth and Colony size are random effects. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1 Studentized Breusch-Pagan test: BP = 2.836, df = 6, p-value = 0.8291
 Table 6*:* Pairwise Comparisons for Brood Care. Pairwise comparisons using
 Wilcoxon rank sum test with continuity correction and Benjamini-Hochberg
 procedure. **BehaviorAnalysis_KW** is a sheet within **Supplementary
 Tables Set 1** which shows the Kruskal-Wallis H test with pairwise student
 t tests for locomotion, predation, and brood care Table 7*:* Pairwise
 Comparisons for Activity and Kruskal Wallis H test. Kruskal-Wallis χ2 =
 22.837, df = 3, p-value = 4.366e-05. Pairwise comparisons using t tests
 with pooled SD and bonferroni adjustment. Table *8:* Pairwise Comparisons
 for Predation and Kruskal Wallis H test. Kruskal-Wallis χ2 = 18.352, df =
 3, p-value = 0.0003721. Pairwise comparisons using t tests with pooled SD
 and bonferroni adjustment. Table 9*:* Pairwise Comparisons for Brood Care
 and Kruskal Wallis H test. Kruskal-Wallis χ2 = 15.195, df = 3, p-value =
 0.001658. Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. **Headwidth** is a sheet within is a sheet within
 **Supplementary Tables Set 1** which shows the detailed ANOVA for
 headwidth and microscope interactions. Table 10*:* Analysis of Variance of
 Headwidth and Microscope Interactions, using Residual Randomization.
 Permutation Procedure: Randomization of Null Model Residuals. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Isolation nested in Microscope and
 Colony size are random effects. Effect sizes (Z) based on F distributions.
 Signif codes: 0 ‘** *’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. studentized
 Breusch-Pagan test: BP = 19.444, df = 9, p-value = 0.02167. We reason this
 is likely due to nested values of headwidth being dependent on microscope
 rather than isolation. **CompartmentAnalysis** is a sheet within
 **Supplementary Tables Set 1** which shows the detailed ANOVA and pairwise
 tests for brain volumes. Table 11*:* Analysis of Variance of AL volume,
 using Residual Randomization. Permutation Procedure: Randomization of Null
 Model Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 4.245, df = 6, p-value = 0.6436
 Table 12*:* Pairwise Comparisons for AL. Pairwise comparisons using
 Wilcoxon rank sum exact test using Benjamini-Hochberg p-value adjustment.
 Table 13*:* Analysis of Variance of CX volume, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 3.4994, df = 5, p-value = 0.744
 Table 14: Analysis of Variance of MB-C volume, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 7.9901, df = 6, p-value = 0.2388
 Table 15*:* Pairwise Comparisons for MB-C. Pairwise comparisons using
 Wilcoxon rank sum exact test using Benjamini-Hochberg p-value adjustment.
 Table 16*:* Analysis of Variance of MB-P volume, using Residual
 Randomization.Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 15.505, df = 6, p-value = 0.01667.
 This heteroscedasticity could not be improved by transformations. However,
 we reason that this has to do with reduced sample size, as the model with
 5- and 20- day isolates is homoscedastic. Pairwise interactions do not
 show significance. Table 17*:* Pairwise Comparisons for MB-P. Pairwise
 comparisons using Wilcoxon rank sum exact test using Benjamini-Hochberg
 p-value adjustment. Table 18*:* Analysis of Variance of OL volume, using
 Residual Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 10.052, df = 6, p-value = 0.1225
 Table 19*:* Analysis of Variance of SEZ volume, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 10.908, df = 6, p-value = 0.09127
 **CompartmentAnalysis_KW** is a sheet within **Supplementary Tables Set
 1** which shows the Kruskal-Wallis H test with pairwise student t tests
 for brain volumes. Table 20*:* Pairwise Comparisons for AL volume and
 Kruskal Wallis H test. Kruskal-Wallis χ2 = 12.988, df = 3, p-value =
 0.004662. Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 21*:* Pairwise Comparisons for CX volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 2.0944, df = 3, p-value = 0.553.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 22*:* Pairwise Comparisons for MBCC volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 16.874, df = 3, p-value = 0.0007502.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 23*:* Pairwise Comparisons for MBP volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 9.0266, df = 3, p-value = 0.02894.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 24*:* Pairwise Comparisons for OL volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 1.8614, df = 3, p-value = 0.6017.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 25*:* Pairwise Comparisons for SEZ volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 2.5928, df = 3, p-value = 0.4588.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. **AbsoluteTiterAnalysis** is a sheet within **Supplementary
 Tables Set 1** which shows the detailed ANOVA and pairwise tests for
 absolute biogenic amine titers. Table 26*:* Analysis of Variance of
 Absolute Dopamine Titers, using Residual Randomization. Permutation
 Procedure: Randomization of Null Model Residuals. Headwidth and Colony
 size are random effects. Number of Permutations: 1000. Estimation Method:
 Ordinary Least Squares. Sums of Squares and Cross-products: Type III.
 Effect sizes (Z) based on F distributions. Signif codes: 0 ‘***’ 0.001
 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized Breusch-Pagan test: BP =
 3.9058, df = 5, p-value = 0.563 Table 27*:* Pairwise Comparisons for
 Isolation and Absolute Dopamine Titers. Pairwise comparisons using
 Wilcoxon rank sum exact test using Benjamini-Hochberg p-value adjustment.
 Table 28*:* Analysis of Variance of Absolute Octopamine Titers, using
 Residual Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 3.8885, df = 5, p-value =0.5656
 Table 29*:* Pairwise Comparisons for Isolation and Absolute Octopamine
 Titers. Pairwise comparisons using Wilcoxon rank sum exact test using
 Benjamini-Hochberg p-value adjustment. Table 30*:* Analysis of Variance of
 Absolute Serotonin Titers, using Residual Randomization. Permutation
 Procedure: Randomization of Null Model Residuals. Headwidth and Colony
 size are random effects. Number of Permutations: 1000. Estimation Method:
 Ordinary Least Squares. Sums of Squares and Cross-products: Type III.
 Effect sizes (Z) based on F distributions. Signif codes: 0 ‘***’ 0.001
 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized Breusch-Pagan test: BP =
 9.9201, df = 5, p-value = 0.07753 Table 31*:* Pairwise Comparisons for
 Absolute Serotonin Titers. Pairwise comparisons using Wilcoxon rank sum
 exact test using Benjamini-Hochberg p-value adjustment.
 **AbsoluteTiterAnalysis_KW** is a sheet within **Supplementary Tables Set
 1** which shows the Kruskal-Wallis H test with pairwise student t tests
 for absolute biogenic amine titers. Table 32*:* Pairwise Comparisons for
 absolute dopamine and Kruskal Wallis H test. Kruskal-Wallis χ2 = 28.867,
 df =4, p-value = 8.321E-06. Pairwise comparisons using t tests with pooled
 SD and bonferroni adjustment. Table 33*:* Pairwise Comparisons for
 absolute octopamine and Kruskal Wallis H test. Kruskal-Wallis χ2 = 19.902,
 df = 2, p-value = 4.769E-05. Pairwise comparisons using t tests with
 pooled SD and bonferroni adjustment. Table 34*:* Pairwise Comparisons for
 d absolute serotonin and Kruskal Wallis H test. Kruskal-Wallis χ2 =
 19.902, df = 2, p-value = 4.769E-05. Pairwise comparisons using t tests
 with pooled SD and bonferroni adjustment. **RelativetoTotalTiterAnalysis**
 is a sheet in **Supplementary Tables Set 1** which shows the the detailed
 ANOVA and pairwise tests for a specific biogenic amine (5HT for example)
 relative to the total quantified biogenic amines (5HT+DA+OA). Table 36*:*
 Analysis of Variance of Relative Octopamine to Total Biogenic Amines,
 using Residual Randomization. Permutation Procedure: Randomization of Null
 Model Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 7.7007, df = 5, p-value = 0.1735
 Table 37*:* Pairwise Comparisons for Isolation and Relative Octopamine to
 Total Biogenic Amines. Pairwise comparisons using Wilcoxon rank sum exact
 test using Benjamini-Hochberg p-value adjustment. Table 38*:* Analysis of
 Variance of Relative Serotonin to Total Biogenic Amines, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 6.1353, df = 5, p-value = 0.2933
 Table 39*:* Pairwise Comparisons for Isolation and Relative Serotonin to
 Total Biogenic Amines. Pairwise comparisons using Wilcoxon rank sum exact
 test using Benjamini-Hochberg p-value adjustment.
 **RelativetoTotalTiterAnalysis_KW** is a sheet within **Supplementary
 Tables Set 1** which shows the Kruskal-Wallis H test with pairwise student
 t tests for a specific biogenic amine (5HT for example) relative to the
 total quantified biogenic amines (5HT+DA+OA). Table 40*:* Pairwise
 Comparisons for Relative Dopamine to Total Biogenic Amines and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 4.8873, df =2, p-value = 0.08684.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 41*:* Pairwise Comparisons for Relative Octopamine to
 Total Biogenic Amines and Kruskal Wallis H test. Kruskal-Wallis χ2 =
 9.9771, df = 2, p-value = 0.006816. Pairwise comparisons using t tests
 with pooled SD and bonferroni adjustment. Table 42*:* Pairwise Comparisons
 for Relative Serotonin to Total Biogenic Amines and Kruskal Wallis H test.
 Kruskal-Wallis χ2 = 9.4064, df = 2, p-value = 0.009066. Pairwise
 comparisons using t tests with pooled SD and bonferroni adjustment.
 **RatioAnalysis** is a sheet within **SUpplementary Tables Set 1** which
 shows the detailed ANOVA and pairwise tests for biogenic amine titers
 relative to each for (e.g. OA:5HT or OA:DA). Table 43: Analysis of
 Variance of OA:5HT Ratios, using Residual Randomization. Permutation
 Procedure: Randomization of Null Model Residuals. Residuals. Headwidth and
 Colony size are random effects. Number of Permutations: 1000. Estimation
 Method: Ordinary Least Squares. Sums of Squares and Cross-products: Type
 III. Effect sizes (Z) based on F distributions. Signif codes: 0 ‘***’
 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized Breusch-Pagan test: BP
 = 3.795, df = 5, p-value = 0.5793 Table 44*:* Pairwise Comparisons for
 Isolation and OA:5HT Ratio. Pairwise comparisons using Wilcoxon rank sum
 exact test using Benjamini-Hochberg p-value adjustment. Table 45*:*
 Analysis of Variance of OA:DA Ratios, using Residual Randomization.
 Permutation Procedure: Randomization of Null Model Residuals. Headwidth
 and Colony size are random effects. Number of Permutations: 1000.
 Estimation Method: Ordinary Least Squares. Sums of Squares and
 Cross-products: Type III. Effect sizes (Z) based on F distributions.
 Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized
 Breusch-Pagan test: BP = 5.3165, df = 5, p-value = 0.3785 Table 46*:*
 Analysis of Variance of 5HT:DA Ratios, using Residual Randomization.
 Permutation Procedure: Randomization of Null Model Residuals. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 6.4161, df = 5, p-value = 0.2678
 Table 47*:* Pairwise Comparisons for Isolation and 5HT:DA Ratio. Pairwise
 comparisons using Wilcoxon rank sum exact test using Benjamini-Hochberg
 p-value adjustment. **RatioAnalysis_KW** is the Kruskal-Wallis H test with
 pairwise student t tests for biogenic amine titers relative to each for
 (e.g. OA:5HT or OA:DA). Table 48*:* Pairwise Comparisons for OA:5HT and
 Kruskal Wallis H test. Kruskal-Wallis χ2 = 13.574, df =2,p-value =
 0.001128. Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 49*:* Pairwise Comparisons for OA:DA to Total Biogenic
 Amines and Kruskal Wallis H test. Kruskal-Wallis χ2 = 1.4579, df = 2,
 p-value = 0.4824. Pairwise comparisons using t tests with pooled SD and
 bonferroni adjustment. Table 50*:* Pairwise Comparisons for 5HT:DA and
 Kruskal Wallis H test. Kruskal-Wallis χ2 = 7.7952, df = 2, p-value =
 0.02029. Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. #### **Supplementary Tables Set 2**: Statistics including 5-
 day and 20- day isolate groups **BehaviorAnalysis** is a sheet within
 **Supplementary Tables Set 2** which shows the detailed ANOVA and pairwise
 tests for locomotion, predation, and brood care, the behavior studied in
 the manuscript. Table *1:* Analysis of Variance of Activity, using
 Residual Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Number of Permutations: 1000. Estimation Method: Ordinary Least
 Squares. Sums of Squares and Cross-products: Type I. Headwidth and Colony
 size are random effects. Effect sizes (Z) based on F distributions. Signif
 codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1 Studentized
 Breusch-Pagan test: BP =6.819, df = 8, p-value = 0.5563 Table 2*:*
 Pairwise Comparisons for Activity. Pairwise comparisons using Wilcoxon
 rank sum test with continuity correction and Benjamini-Hochberg procedure.
 Table 3*:* Analysis of Variance of Log-transformed Predation, using
 Residual Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Number of Permutations: 1000. Estimation Method: Ordinary Least
 Squares. Sums of Squares and Cross-products: Type I. Headwidth and Colony
 size are random effects. Effect sizes (Z) based on F distributions. Signif
 codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1 Studentized
 Breusch-Pagan test: BP = 12.313, df = 8, p-value = 0.1378 Table 4*:*
 Pairwise Comparisons for Log-transformed Predation. Pairwise comparisons
 using Wilcoxon rank sum test with continuity correction and
 Benjamini-Hochberg procedure. Table 5: Analysis of Variance of Brood Care,
 using Residual Randomization. Permutation Procedure: Randomization of Null
 Model Residuals. Number of Permutations: 1000. Estimation Method: Ordinary
 Least Squares. Sums of Squares and Cross-products: Type I. Headwidth and
 Colony size are random effects. Effect sizes (Z) based on F distributions.
 Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1 Studentized
 Breusch-Pagan test: BP = 4.6552, df = 8, p-value = 0.7937 Table 6*:*
 Pairwise Comparisons for Brood Care. Pairwise comparisons using Wilcoxon
 rank sum test with continuity correction and Benjamini-Hochberg procedure.
 **BehaviorAnalysis_KW** is a sheet within **Supplementary Tables Set 2**
 which shows the Kruskal-Wallis H test with pairwise student t tests for
 locomotion, predation, and brood care. Table 7*:* Pairwise Comparisons for
 Activity and Kruskal Wallis H test. Kruskal-Wallis χ2 = 29.077, df = 5,
 p-value = 2.239E-05. Pairwise comparisons using t tests with pooled SD and
 bonferroni adjustment. Table 8*:* Pairwise Comparisons for log-transformed
 Predation and Kruskal Wallis H test. Kruskal-Wallis χ2 = 20.302, df = 5,
 p-value = 0.001097. Pairwise comparisons using t tests with pooled SD and
 bonferroni adjustment. Table 9*:* Pairwise Comparisons for Brood Care and
 Kruskal Wallis H test. Kruskal-Wallis χ2 = 17.047, df = 5, p-value =
 0.004412. Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. **Headwidth** is a sheet within is a sheet within
 **Supplementary Tables Set 2** which shows the detailed ANOVA for
 headwidth and microscope interactions. Table 10*:* Analysis of Variance of
 Headwidth and Microscope Interactions, using Residual Randomization.
 Permutation Procedure: Randomization of Null Model Residuals. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Headwidth and Colony size are random
 effects. Effect sizes (Z) based on F distributions. Signif codes: 0 ‘***’
 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. studentized Breusch-Pagan test: BP
 = 28.762, df = 13, p-value = 0.007073. We reason this is likely due to
 nested values of headwidth being dependent on microscope rather than
 isolation. **CompartmentAnalysis** is a sheet within **Supplementary
 Tables Set 2** which shows the detailed ANOVA and pairwise tests for brain
 volumes. Table 11*:* Analysis of Variance of AL volume, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 6.2279, df = 8, p-value = 0.6217
 Table 12*:* Pairwise Comparisons for AL. Pairwise comparisons using
 Wilcoxon rank sum exact test using Benjamini-Hochberg p-value adjustment.
 Table 13*:* Analysis of Variance of CX volume, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 9.2273, df = 8, p-value = 0.3235
 Table 14*:* Analysis of Variance of MB-C volume, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 12.672, df = 8, p-value = 0.1236
 Table 15*:* Pairwise comparisons using Wilcoxon rank sum exact test using
 Benjamini-Hochberg p-value adjustment. *Table 16:* Analysis of Variance of
 MB-P volume, using Residual Randomization.Permutation Procedure:
 Randomization of Null Model Residuals. Headwidth and Colony size are
 random effects. Number of Permutations: 1000. Estimation Method: Ordinary
 Least Squares. Sums of Squares and Cross-products: Type III. Effect sizes
 (Z) based on F distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’
 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized Breusch-Pagan test: BP = 11.663, df = 8,
 p-value = 0.1669 Table 17: Analysis of Variance of OL volume, using
 Residual Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 7.6094, df = 8, p-value = 0.4725
 Table 18*:* Analysis of Variance of SEZ volume, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 10.625, df = 8, p-value = 0.2239
 **CompartmentAnalysis_KW** is a sheet within **Supplementary Tables Set
 2** which shows the Kruskal-Wallis H test with pairwise student t tests
 for brain volumes. Table 19*:* Pairwise Comparisons for AL volume and
 Kruskal Wallis H test. Kruskal-Wallis χ2 = 19.738, df = 5, p-value =
 0.001399. Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 20*:* Pairwise Comparisons for CX volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 3.3538, df = 5, p-value = 0.6456.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 21*:* Pairwise Comparisons for MBCC volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 27.839, df = 5, p-value = 3.913E-05.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 22*:* Pairwise Comparisons for MBP volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 8.1137, df = 5, p-value = 0.1501.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 23*:* Pairwise Comparisons for OL volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 3.7328, df = 5, p-value = 0.5885.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 24*:* Pairwise Comparisons for SEZ volume and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 5.0543, df = 5, p-value = 0.4093.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. **AbsoluteTiterAnalysis** is a sheet within **Supplementary
 Tables Set 2** which shows the detailed ANOVA and pairwise tests for
 absolute biogenic amine titers. Table 25*:* Analysis of Variance of
 Absolute Dopamine Titers, using Residual Randomization. Permutation
 Procedure: Randomization of Null Model Residuals. Headwidth and Colony
 size are random effects. Number of Permutations: 1000. Estimation Method:
 Ordinary Least Squares. Sums of Squares and Cross-products: Type III.
 Effect sizes (Z) based on F distributions. Signif codes: 0 ‘***’ 0.001
 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized Breusch-Pagan test: BP =
 8.7971, df = 7, p-value = 0.2676 Table 26*:* Pairwise Comparisons for
 Isolation and Absolute Dopamine Titers. Pairwise comparisons using
 Wilcoxon rank sum exact test using Benjamini-Hochberg p-value adjustment.
 Table 27*:* Analysis of Variance of Absolute Octopamine Titers, using
 Residual Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 9.6028, df = 7, p-value = 0.2122
 Table 28*:* Pairwise Comparisons for Isolation and Absolute Octopamine
 Titers. Pairwise comparisons using Wilcoxon rank sum exact test using
 Benjamini-Hochberg p-value adjustment. Table 29*:* Analysis of Variance of
 Log-Transformed Absolute Serotonin Titers, using Residual Randomization.
 Permutation Procedure: Randomization of Null Model Residuals. Headwidth
 and Colony size are random effects. Number of Permutations: 1000.
 Estimation Method: Ordinary Least Squares. Sums of Squares and
 Cross-products: Type III. Effect sizes (Z) based on F distributions.
 Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized
 Breusch-Pagan test: BP = 8.0529, df = 7, p-value = 0.328 Table 30*:*
 Pairwise Comparisons for Log-Transformed Absolute Serotonin Titers.
 Pairwise comparisons using Wilcoxon rank sum exact test using
 Benjamini-Hochberg p-value adjustment. **AbsoluteTiterAnalysis_KW** is a
 sheet within **Supplementary Tables Set 2** which shows the Kruskal-Wallis
 H test with pairwise student t tests for absolute biogenic amine titers.
 Table 31*:* Pairwise Comparisons for absolute dopamine and Kruskal Wallis
 H test. Kruskal-Wallis χ2 = 28.867, df =4, p-value = 8.321E-06. Pairwise
 comparisons using t tests with pooled SD and bonferroni adjustment. Table
 32*:* Pairwise Comparisons for absolute octopamine and Kruskal Wallis H
 test. Kruskal-Wallis χ2 = 35.533, df = 4, p-value = 3.611E-07. Pairwise
 comparisons using t tests with pooled SD and bonferroni adjustment. Table
 33*:* Pairwise Comparisons for log-transformed absolute serotonin and
 Kruskal Wallis H test. Kruskal-Wallis χ2 = 43.62, df = 4, p-value =
 7.695E-09. Pairwise comparisons using t tests with pooled SD and
 bonferroni adjustment. **RelativetoTotalTiterAnalysis** is a sheet in
 **Supplementary Tables Set 2** which shows the the detailed ANOVA and
 pairwise tests for a specific biogenic amine (5HT for example) relative to
 the total quantified biogenic amines (5HT+DA+OA). Table 34*:* Analysis of
 Variance of Relative Dopamine to Total Biogenic Amines, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 11.506, df = 7, p-value = 0.118.
 Table 35*:* Analysis of Variance of Relative Octopamine to Total Biogenic
 Amines, using Residual Randomization. Permutation Procedure: Randomization
 of Null Model Residuals. Headwidth and Colony size are random effects.
 Number of Permutations: 1000. Estimation Method: Ordinary Least Squares.
 Sums of Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 4.8937, df = 7, p-value = 0.6729.
 Table 36*:* Pairwise Comparisons for Isolation and Relative Octopamine to
 Total Biogenic Amines. Pairwise comparisons using Wilcoxon rank sum exact
 test using Benjamini-Hochberg p-value adjustment. Table 37*:* Analysis of
 Variance of Relative Serotonin to Total Biogenic Amines, using Residual
 Randomization. Permutation Procedure: Randomization of Null Model
 Residuals. Headwidth and Colony size are random effects. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 11.344, df = 7, p-value = 0.1243.
 Table 38*:* Pairwise Comparisons for Isolation and Relative Serotonin to
 Total Biogenic Amines. Pairwise comparisons using Wilcoxon rank sum exact
 test using Benjamini-Hochberg p-value adjustment.
 **RelativetoTotalTiterAnalysis_KW** is a sheet within **Supplementary
 Tables Set 2** which shows the Kruskal-Wallis H test with pairwise student
 t tests for a specific biogenic amine (5HT for example) relative to the
 total quantified biogenic amines (5HT+DA+OA). Table 39*:* Pairwise
 Comparisons for Relative Dopamine to Total Biogenic Amines and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 7.266, df =4, p-value = 0.1225.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 40*:* Pairwise Comparisons for Relative Octopamine to
 Total Biogenic Amines and Kruskal Wallis H test. Kruskal-Wallis χ2 =
 13.578, df = 4, p-value = 0.008769. Pairwise comparisons using t tests
 with pooled SD and bonferroni adjustment. Table 41*:* Pairwise Comparisons
 for Relative Serotonin to Total Biogenic Amines and Kruskal Wallis H test.
 Kruskal-Wallis χ2 = 17.264, df = 4, p-value = 0.001717. Pairwise
 comparisons using t tests with pooled SD and bonferroni adjustment.
 **RatioAnalysis** is a sheet within **Supplementary Tables Set 2** which
 shows the detailed ANOVA and pairwise tests for biogenic amine titers
 relative to each for (e.g. OA:5HT or OA:DA). Table 42*:* Analysis of
 Variance of OA:5HT Ratios, using Residual Randomization. Permutation
 Procedure: Randomization of Null Model Residuals. Residuals. Headwidth and
 Colony size are random effects. Number of Permutations: 1000. Estimation
 Method: Ordinary Least Squares. Sums of Squares and Cross-products: Type
 III. Effect sizes (Z) based on F distributions. Signif codes: 0 ‘***’
 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized Breusch-Pagan test: BP
 = 9.0945, df = 7, p-value = 0.2459. Table 43*:* Pairwise Comparisons for
 Isolation and OA:5HT Ratio. Pairwise comparisons using Wilcoxon rank sum
 exact test using Benjamini-Hochberg p-value adjustment. Table 44*:*
 Analysis of Variance of OA:DA Ratios, using Residual Randomization.
 Permutation Procedure: Randomization of Null Model Residuals. Headwidth
 and Colony size are random effects. Number of Permutations: 1000.
 Estimation Method: Ordinary Least Squares. Sums of Squares and
 Cross-products: Type III. Effect sizes (Z) based on F distributions.
 Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘ 1. Studentized
 Breusch-Pagan test: BP = 5.767, df = 7, p-value = 0.4535. Table 45*:*
 Analysis of Variance of 5HT:DA Ratios, using Residual Randomization.
 Permutation Procedure: Randomization of Null Model Residuals. Number of
 Permutations: 1000. Estimation Method: Ordinary Least Squares. Sums of
 Squares and Cross-products: Type III. Effect sizes (Z) based on F
 distributions. Signif codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ‘
 1. Studentized Breusch-Pagan test: BP = 9.8857, df = 7, p-value = 0.1951.
 Table 4*6:* Pairwise Comparisons for Isolation and 5HT:DA Ratio. Pairwise
 comparisons using Wilcoxon rank sum exact test using Benjamini-Hochberg
 p-value adjustment. **RatioAnalysis_KW** is a sheet within Supplementary
 Tables 2 that shows the Kruskal-Wallis H test with pairwise student t
 tests for biogenic amine titers relative to each for (e.g. OA:5HT or
 OA:DA). Table 47*:* Pairwise Comparisons for OA:5HT and Kruskal Wallis H
 test. Kruskal-Wallis χ2 = 24.067, df =4, p-value = 7.745E-05. Pairwise
 comparisons using t tests with pooled SD and bonferroni adjustment. Table
 48*:* Pairwise Comparisons for OA:DA to Total Biogenic Amines and Kruskal
 Wallis H test. Kruskal-Wallis χ2 = 1.6187, df = 4, p-value = 0.8054.
 Pairwise comparisons using t tests with pooled SD and bonferroni
 adjustment. Table 49*:* Pairwise Comparisons for 5HT:DA and Kruskal Wallis
 H test. Kruskal-Wallis χ2 = 13.246, df = 4, p-value = 0.01013. Pairwise
 comparisons using t tests with pooled SD and bonferroni adjustment. ####
 Raw Data Behavior The raw data for behavior is in
 behaviorandheadwidth.xlsx with the more polished behavior in
 behaviorkey_behavioronly.xlsx. There are 4 tabs: behavior_simplified,
 raw_behavior_data, headwidthavg_basedonmicroscope, normalizedheadwidth,
 animalcollection, and animalcollection-byisolation. ###### For
 behaviorkey\_behavioronly.xlsx below: a - *Sample ID* is id given to each
 ant b - *Purpose* is what each animal was used for (brain mass, hplc,
 behavior and ihc, ihc only, behavior only) c - *Colony* is where each ant
 originated from d - *Eclosed* is the date each ant eclosed e -
 *DissectedandBehavior* is the date each animal was assayed for behavior
 and dissected afterwards. f - *ActualDaysDifferent* is the raw number of
 days that an ant was isolated for: column e - column d g - *Isolation* is
 the classified group each animal was put into (ie. a 31-day isolate would
 be grouped into the 30-day\ isolate group) h - *Isolation30and45merged* is
 the new classification where 30-day and 45-day isolate groups were merged
 into a new isolate group ("40-day isolates") i -
 *ZoomonMicroscope* is the microscope zoom that was used for each
 dissection of the ant (always 4x) j - *WhichMicroscope* is which
 microscope in our lab was used to each dissection in order to account for
 variation between microscopes. (such as microscope 1) k - *HeadWidth* is
 the headwidth of each individual measured using the optical ruler on the
 microscope l - *NormalizedHeadwidth* is the head width of each individual
 divided by the average headwidth measured on that specific microscope (the
 average headwidth on microscope 1 is 51.44, so the headwidth would be
 divided by this value). m -Assay1 is the first assay that was done on the
 behavioral trial, which was selected randomly (predatory behavior, brood
 behavior, activity) - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 n - Assay2 *is the second assay that was done on the behavioral trial,
 which was selected randomly (predatory behavior, brood behavior, activity)
 - NONE means the behavioral trials weren't run for this individual
 and to refer to the* Purpose* of the individual which will indicate the
 animal was used for something not related to behavior.  o - Assay3 *is the
 third assay that was done on the behavioral trial, which was selected
 randomly (predatory behavior, brood behavior, activity) - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  p - TimeAssay1 *is the time of the
 first assay that was done on the behavioral trial, which was selected
 randomly (predatory behavior, brood behavior, activity) - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  q - TimeAssay2 *is the time of the
 second assay that was done on the behavioral trial, which was selected
 randomly (predatory behavior, brood behavior, activity) - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  r - TimeAssay3 *is the time of the
 second assay that was done on the behavioral trial, which was selected
 randomly (predatory behavior, brood behavior, activity) - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  s -Predatory is the score given to
 the animal's predatory behavior. - NONE means the behavioral trials
 weren't run for this individual and to refer to the Purpose of the
 individual which will indicate the animal was used for something not
 related to behavior.  t - ActivityQuad1 is the number of instances the
 animal entered quadrant 1 of the activity arena. - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  u -ActivityQuad2 is the number of
 instances the animal entered quadrant 2 of the activity arena. - NONE
 means the behavioral trials weren't run for this individual and to
 refer to the* Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  v - ActivityQuad3 is the
 number of instances the animal entered quadrant 3 of the activity arena. -
 NONE means the behavioral trials weren't run for this individual and
 to refer to the* Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  w - ActivityQuad4 *is the
 number of instances the animal entered quadrant 4 of the activity arena. -
 NONE means the behavioral trials weren't run for this individual and
 to refer to the* Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  x - ActivitySum *is the
 sum of columns t-w. - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 y - Activity *is the non-equation, duplicated format of column x, the
 values are identical. - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 z - Brood  *is the brood score given to each individual. - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  #### For behaviorandheadwidth.xlsx
 below: Within the sheet behavior_simplified, variable name and
 description: a - *Sample ID* is id given to each ant b - *Colony* is where
 each ant originated from c - *Isolation* is the classified group each
 animal was put into (ie. a 31-day isolate would be grouped into the 30-day
 isolate group) d - *Predatory* is the score given to the animal's
 predatory behavior. - NONE means the behavioral trials weren't run
 for this individual and to refer to the *Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 e -* ActivityQuad1* is the number of instances the animal entered quadrant
 1 of the activity arena. - NONE means the behavioral trials weren't
 run for this individual and to refer to the* Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  f -* ActivityQuad2* is the number of instances the animal
 entered quadrant 2 of the activity arena. - NONE means the behavioral
 trials weren't run for this individual and to refer to the* Purpose*
 of the individual which will indicate the animal was used for something
 not related to behavior.  g -* ActivityQuad3* is the number of instances
 the animal entered quadrant 3 of the activity arena. - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  h -* ActivityQuad4 *is the number
 of instances the animal entered quadrant 4 of the activity arena. - NONE
 means the behavioral trials weren't run for this individual and to
 refer to the* Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  i -* ActivitySum *is the
 sum of columns t-w. - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 j -* Activity *is the non-equation, duplicated format of column x, the
 values are identical. - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 k -* Brood  *is the brood score given to each individual. - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  l - *ZoomonMicroscope* is the
 microscope zoom that was used for each dissection of the ant (always 4x) m
 - *WhichMicroscope* is which microscope in our lab was used to each
 dissection in order to account for variation between microscopes. (such as
 microscope 1) n - *HeadWidth* is the headwidth of each individual measured
 using the optical ruler on the microscope o - *NormalizedHeadwidth* is the
 head width of each individual divided by the average headwidth measured on
 that specific microscope (the average headwidth on microscope 1 is 51.44,
 so the headwidth would be divided by this value). ###### Within
 raw\_behavior\_data, variable name and description: a - *Sample ID* is id
 given to each ant b - *Purpose* is what each animal was used for (brain
 mass, hplc, behavior and ihc, ihc only, behavior only) c - *Colony* is
 where each ant originated from d - *Eclosed* is the date each ant eclosed
 e - *DissectedandBehavior* is the date each animal was assayed for
 behavior and dissected afterwards. f - *ActualDaysDifferent* is the raw
 number of days that an ant was isolated for: column e - column d g -
 *Isolation* is the classified group each animal was put into (ie. a 31-day
 isolate would be grouped into the 30-day isolate group) h -
 *ZoomonMicroscope* is the microscope zoom that was used for each
 dissection of the ant (always 4x) i - *WhichMicroscope* is which
 microscope in our lab was used to each dissection in order to account for
 variation between microscopes. (such as microscope 1) j - *HeadWidth* is
 the headwidth of each individual measured using the optical ruler on the
 microscope k - *NormalizedHeadwidth* is the head width of each individual
 divided by the average headwidth measured on that specific microscope (the
 average headwidth on microscope 1 is 51.44, so the headwidth would be
 divided by this value). l - *Assay1* is the first assay that was done on
 the behavioral trial, which was selected randomly (predatory behavior,
 brood behavior, activity) - NONE means the behavioral trials weren't
 run for this individual and to refer to the*Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  m -*Assay2* is the second assay that was done on the behavioral
 trial, which was selected randomly (predatory behavior, brood behavior,
 activity) - NONE means the behavioral trials weren't run for this
 individual and to refer to the *Purpose* of the individual which will
 indicate the animal was used for something not related to behavior.  n
 -*Assay3* is the third assay that was done on the behavioral trial, which
 was selected randomly (predatory behavior, brood behavior, activity) -
 NONE means the behavioral trials weren't run for this individual and
 to refer to the *Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  o -*TimeAssay1* is the
 time of the first assay that was done on the behavioral trial, which was
 selected randomly (predatory behavior, brood behavior, activity) - NONE
 means the behavioral trials weren't run for this individual and to
 refer to the *Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  p -*TimeAssay2* is the
 time of the second assay that was done on the behavioral trial, which was
 selected randomly (predatory behavior, brood behavior, activity) - NONE
 means the behavioral trials weren't run for this individual and to
 refer to the *Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  q -*TimeAssay3* is the
 time of the second assay that was done on the behavioral trial, which was
 selected randomly (predatory behavior, brood behavior, activity) - NONE
 means the behavioral trials weren't run for this individual and to
 refer to the *Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  r -*Predatory* is the
 score given to the animal's predatory behavior. - NONE means the
 behavioral trials weren't run for this individual and to refer to the
 *Purpose* of the individual which will indicate the animal was used for
 something not related to behavior.  s -*ActivityQuad1* is the number of
 instances the animal entered quadrant 1 of the activity arena. - NONE
 means the behavioral trials weren't run for this individual and to
 refer to the *Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  t - *ActivityQuad2* is
 the number of instances the animal entered quadrant 2 of the activity
 arena. - NONE means the behavioral trials weren't run for this
 individual and to refer to the *Purpose* of the individual which will
 indicate the animal was used for something not related to behavior.  u -
 *ActivityQuad3* is the number of instances the animal entered quadrant 3
 of the activity arena. - NONE means the behavioral trials weren't run
 for this individual and to refer to the *Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 v - *ActivityQuad4* is the number of instances the animal entered quadrant
 4 of the activity arena. - NONE means the behavioral trials weren't
 run for this individual and to refer to the *Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  w - *ActivitySum* is the sum of columns t-w. - NONE means the
 behavioral trials weren't run for this individual and to refer to the
 *Purpose* of the individual which will indicate the animal was used for
 something not related to behavior.  x - *Activity* is the non-equation,
 duplicated format of column x, the values are identical. - NONE means the
 behavioral trials weren't run for this individual and to refer to the
 *Purpose* of the individual which will indicate the animal was used for
 something not related to behavior.  y - *Brood* is the brood score given
 to each individual. - NONE means the behavioral trials weren't run
 for this individual and to refer to the *Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 ###### Within headwidthavg\_basedonmicroscope, variable name and
 description: This sheet shows how we averaged the average headwidth (sum
 of all headwidth divided by total number of measurements) per microscope.
 a - *Sample ID* is id given to each ant b - *Purpose* is what each animal
 was used for (brain mass, hplc, behavior and ihc, ihc only, behavior only)
 c - *Colony* is where each ant originated from d - *Eclosed* is the date
 each ant eclosed e - *DissectedandBehavior* is the date each animal was
 assayed for behavior and dissected afterwards f - *ActualDaysDifferent* is
 the raw number of days that an ant was isolated for: column e - column d g
 - *Isolation* is the classified group each animal was put into (ie. a
 31-day isolate would be grouped into the 30-day isolate group) h -
 *ZoomonMicroscope* is the microscope zoom that was used for each
 dissection of the ant (always 4x) i - *WhichMicroscope* is which
 microscope in our lab was used to each dissection in order to account for
 variation between microscopes. (such as microscope 1) j - *HeadWidth* is
 the headwidth of each individual measured using the optical ruler on the
 microscope ***Within normalizedheadwidth, variable name and
 description:*** This sheet shows how we averaged the average headwidth
 (sum of all headwidth divided by total number of measurements) per
 microscope to create the normalized headwidth value in column k. a -
 *Sample ID* is id given to each ant b - *Purpose* is what each animal was
 used for (brain mass, hplc, behavior and ihc, ihc only, behavior only) c -
 *Colony* is where each ant originated from d - *Eclosed* is the date each
 ant eclosed e - *DissectedandBehavior* is the date each animal was assayed
 for behavior and dissected afterwards. f - *ActualDaysDifferent* is the
 raw number of days that an ant was isolated for: column e - column d g -
 *Isolation* is the classified group each animal was put into (ie. a 31-day
 isolate would be grouped into the 30-day isolate group) h -
 *ZoomonMicroscope* is the microscope zoom that was used for each
 dissection of the ant (always 4x) i - *WhichMicroscope* is which
 microscope in our lab was used to each dissection in order to account for
 variation between microscopes. (such as microscope 1) j - *HeadWidth* is
 the headwidth of each individual measured using the optical ruler on the
 microscope k - *NormalizedHeadwidth* is the head width of each individual
 divided by the average headwidth measured on that specific microscope (the
 average headwidth on microscope 1 is 51.44, so the headwidth would be
 divided by this value). ###### Within animalcollection, variable name and
 description: a - *Sample ID* is id given to each ant b - *Purpose* is what
 each animal was used for (brain mass, hplc, behavior and ihc, ihc only,
 behavior only) c - *Colony* is where each ant originated from d -
 *Eclosed* is the date each ant eclosed e - *DissectedandBehavior* is the
 date each animal was assayed for behavior and dissected afterwards. f -
 *ActualDaysDifferent* is the raw number of days that an ant was isolated
 for: column e - column d g - *Isolation* is the classified group each
 animal was put into (ie. a 31-day isolate would be grouped into the 30-day
 isolate group) h - *Isolation30and45merged* is the new classification
 where 30-day and 45-day isolate groups were merged into a new isolate
 group ("40-day isolates") i - *ZoomonMicroscope* is the
 microscope zoom that was used for each dissection of the ant (always 4x) j
 - *WhichMicroscope* is which microscope in our lab was used to each
 dissection in order to account for variation between microscopes. (such as
 microscope 1) k - *HeadWidth* is the headwidth of each individual measured
 using the optical ruler on the microscope l - *NormalizedHeadwidth* is the
 head width of each individual divided by the average headwidth measured on
 that specific microscope (the average headwidth on microscope 1 is 51.44,
 so the headwidth would be divided by this value). m -* Assay1* is the
 first assay that was done on the behavioral trial, which was selected
 randomly (predatory behavior, brood behavior, activity) - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  n -* Assay2 *is the second assay
 that was done on the behavioral trial, which was selected randomly
 (predatory behavior, brood behavior, activity) - NONE means the behavioral
 trials weren't run for this individual and to refer to the* Purpose*
 of the individual which will indicate the animal was used for something
 not related to behavior.  o -* Assay3 *is the third assay that was done on
 the behavioral trial, which was selected randomly (predatory behavior,
 brood behavior, activity) - NONE means the behavioral trials weren't
 run for this individual and to refer to the* Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  p -* TimeAssay1 *is the time of the first assay that was done
 on the behavioral trial, which was selected randomly (predatory behavior,
 brood behavior, activity) - NONE means the behavioral trials weren't
 run for this individual and to refer to the* Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  q -* TimeAssay2 *is the time of the second assay that was done
 on the behavioral trial, which was selected randomly (predatory behavior,
 brood behavior, activity) - NONE means the behavioral trials weren't
 run for this individual and to refer to the* Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  r -* TimeAssay3 *is the time of the second assay that was done
 on the behavioral trial, which was selected randomly (predatory behavior,
 brood behavior, activity) - NONE means the behavioral trials weren't
 run for this individual and to refer to the* Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  s -* Predatory* is the score given to the animal's
 predatory behavior. - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 t -* ActivityQuad1* is the number of instances the animal entered quadrant
 1 of the activity arena. - NONE means the behavioral trials weren't
 run for this individual and to refer to the* Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  u -* ActivityQuad2* is the number of instances the animal
 entered quadrant 2 of the activity arena. - NONE means the behavioral
 trials weren't run for this individual and to refer to the* Purpose*
 of the individual which will indicate the animal was used for something
 not related to behavior.  v -* ActivityQuad3* is the number of instances
 the animal entered quadrant 3 of the activity arena. - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  w -* ActivityQuad4 *is the number
 of instances the animal entered quadrant 4 of the activity arena. - NONE
 means the behavioral trials weren't run for this individual and to
 refer to the* Purpose* of the individual which will indicate the animal
 was used for something not related to behavior.  x -* ActivitySum *is the
 sum of columns t-w. - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 y -* Activity *is the non-equation, duplicated format of column x, the
 values are identical. - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 z -* Brood  *is the brood score given to each individual. - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  ###### Within
 animalcollection-byisolation, variable name and description: This sheet is
 identical to animal collection, just sorted by isolation periods. a -
 *Sample ID* is id given to each ant b - *Purpose* is what each animal was
 used for (brain mass, hplc, behavior and ihc, ihc only, behavior only) c -
 *Colony* is where each ant originated from d - *Eclosed* is the date each
 ant eclosed e - *DissectedandBehavior* is the date each animal was assayed
 for behavior and dissected afterwards. f - *ActualDaysDifferent* is the
 raw number of days that an ant was isolated for: column e - column d g -
 *Isolation* is the classified group each animal was put into (ie. a 31-day
 isolate would be grouped into the 30-day isolate group) h -
 *Isolation30and45merged* is the new classification where 30-day and 45-day
 isolate groups were merged into a new isolate group ("40-day
 isolates") i - *ZoomonMicroscope* is the microscope zoom that was
 used for each dissection of the ant (always 4x) j - *WhichMicroscope* is
 which microscope in our lab was used to each dissection in order to
 account for variation between microscopes. (such as microscope 1) k -
 *HeadWidth* is the headwidth of each individual measured using the optical
 ruler on the microscope l - *NormalizedHeadwidth* is the head width of
 each individual divided by the average headwidth measured on that specific
 microscope (the average headwidth on microscope 1 is 51.44, so the
 headwidth would be divided by this value). m -* Assay1* is the first assay
 that was done on the behavioral trial, which was selected randomly
 (predatory behavior, brood behavior, activity) - NONE means the behavioral
 trials weren't run for this individual and to refer to the* Purpose*
 of the individual which will indicate the animal was used for something
 not related to behavior.  n -* Assay2 *is the second assay that was done
 on the behavioral trial, which was selected randomly (predatory behavior,
 brood behavior, activity) - NONE means the behavioral trials weren't
 run for this individual and to refer to the* Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  o -* Assay3 *is the third assay that was done on the behavioral
 trial, which was selected randomly (predatory behavior, brood behavior,
 activity) - NONE means the behavioral trials weren't run for this
 individual and to refer to the* Purpose* of the individual which will
 indicate the animal was used for something not related to behavior.  p -*
 TimeAssay1 *is the time of the first assay that was done on the behavioral
 trial, which was selected randomly (predatory behavior, brood behavior,
 activity) - NONE means the behavioral trials weren't run for this
 individual and to refer to the* Purpose* of the individual which will
 indicate the animal was used for something not related to behavior.  q -*
 TimeAssay2 *is the time of the second assay that was done on the
 behavioral trial, which was selected randomly (predatory behavior, brood
 behavior, activity) - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 r -* TimeAssay3 *is the time of the second assay that was done on the
 behavioral trial, which was selected randomly (predatory behavior, brood
 behavior, activity) - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 s -* Predatory* is the score given to the animal's predatory
 behavior. - NONE means the behavioral trials weren't run for this
 individual and to refer to the* Purpose* of the individual which will
 indicate the animal was used for something not related to behavior.  t -*
 ActivityQuad1* is the number of instances the animal entered quadrant 1 of
 the activity arena. - NONE means the behavioral trials weren't run
 for this individual and to refer to the* Purpose* of the individual which
 will indicate the animal was used for something not related to behavior. 
 u -* ActivityQuad2* is the number of instances the animal entered quadrant
 2 of the activity arena. - NONE means the behavioral trials weren't
 run for this individual and to refer to the* Purpose* of the individual
 which will indicate the animal was used for something not related to
 behavior.  v -* ActivityQuad3* is the number of instances the animal
 entered quadrant 3 of the activity arena. - NONE means the behavioral
 trials weren't run for this individual and to refer to the* Purpose*
 of the individual which will indicate the animal was used for something
 not related to behavior.  w -* ActivityQuad4 *is the number of instances
 the animal entered quadrant 4 of the activity arena. - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  x -* ActivitySum *is the sum of
 columns t-w. - NONE means the behavioral trials weren't run for this
 individual and to refer to the* Purpose* of the individual which will
 indicate the animal was used for something not related to behavior.  y -*
 Activity *is the non-equation, duplicated format of column x, the values
 are identical. - NONE means the behavioral trials weren't run for
 this individual and to refer to the* Purpose* of the individual which will
 indicate the animal was used for something not related to behavior.  z -*
 Brood  *is the brood score given to each individual. - NONE means the
 behavioral trials weren't run for this individual and to refer to
 the* Purpose* of the individual which will indicate the animal was used
 for something not related to behavior.  ### Biogenic Amines ##### The raw
 data for biogenic amines (HPLC) is biogenicamines.xlsx. ###### Within
 RawValues, variable name and description: a - *HW* is the headwidth of the
 animal used for biogenic amines b - *Colony* is where each ant originated
 from c - *Isolation* is the classified group each animal was put into (ie.
 a 31-day isolate would be grouped into the 30-day isolate group) d - *DA*
 is the raw value of dopamine (pg/ant) from the HPLC e - *5HT* is the raw
 value of serotonin (pg/ant) from the HPLC f - *OA* is the raw value of
 octopamine (pg/ant) from the HPLC g - *relDA* is the amount of dopamine
 relative to the summed value of DA+5HT+OA h - *rel5HT* is the amount of
 5HT relative to the summed value of DA+5HT+OA i - *relOA* is the amount of
 OA relative to the summed value of DA+5HT+OA j - *ratioOA5HT* is the ratio
 of OA to 5HT k - *ratioOADA* is the ratio of OA to DA l - *ratio5HTDA* is
 the ratio of 5HT to DA m - *Isolation30and45merged* is the new
 classification where 30-day and 45-day isolate groups were merged into a
 new isolate group ("40-day isolates") **Brain Volume** The data
 for brain volumes is in brainvolumetricdata.xlsx with 11 tabs. All
 variable names and description are consistent for each brain reagion
 within the tabs: ALonly is only volumes for the antennal lobe, SEZonly is
 subesophageal zone volumes, OLonly is the optic lobe volume only, MBPonly
 is volumes for the mushroom body peduncle, MBMConly is the mushroom
 body-medial calyx volume, MBLConly is the mushroom body-lateral calyx
 volume, CXonly is the central complex volume. We used the last column on
 these tabs, titled RelVolumetoROCB. Because MBMC and MBLC showed similar
 trends, we combined their relative to ROCB values on the MBC_calculation
 tab ###### Within RawIHCData, variable name and description: a - Animal -
 Animal ID per individual b - SimplifiedDay - Isolation group c - Day
 Isolation - Absolute period of isolation d - Pixel - pixels of image e -
 Voxel - voxels of image f - Nr - Numerical label for the material g -
 Material - Brain region h - Label - Numeric label for the material - 1 for
 exterior (all unlabeled material) i - Count - output from Amira, not used
 j - IRV - Imputed Relative volume (not used) k - IAV - Imputed Absolute
 volume (not used) l - Relvolumetototalbrain - relative volume (m of
 ant/sum of all m's for that ant) m - Volume - volume of brain region
 as quantified in Amira and scaled to voxels and pixels n - CenterX -
 output from Amira, not used o - CenterY - output from Amira, not used p -
 CenterZ - output from Amira, not used q - Mean - output from Amira, not
 used r - Deviation - output from Amira, not used s - Variance - output
 from Amira, not used t - Min - output from Amira, not used u - Max -
 output from Amira, not used v - CumulativeSum w - Notes - any comments on
 missing volumes due to damage x - RelvolumetoROCB - relative volume
 (m/ROCB's m for that individual) y - Colony - colony of origin z -
 Isolation30and45merged - isolation group if 30 and 45 day isolates are
 merged to make 40 days **Missing data code: NA** ## Code/Software The R
 code needed to run our data is the following: headwidthforpaper_v5.R
 headwidthforpaper_v5_only2and40.R hplcforpaper_v4.R
 hplcforpaper_v4_only2and40.R ihcorpaper_v5.R ihcorpaper_v5_only2and40.R
 behaviorforpaper_v4_only2and40.R behaviorforpaper_v4.R