Behav Ecol Sociobiol (2017) 71: 116 DOI 10.1007/s00265-017-2344-y

ORIGINAL ARTICLE

Caste differences in the mushroom bodies of swarm-founding paper : implications for brain plasticity and brain evolution (, )

Sean O’Donnell1 & Susan J. Bulova1 & Sara DeLeon1 & Meghan Barrett2 & Katherine Fiocca2

Received: 28 February 2017 /Revised: 20 June 2017 /Accepted: 25 June 2017 /Published online: 13 July 2017 # Springer-Verlag GmbH Germany 2017

Abstract a positive association of MB investment with social dom- Eusocial reproductive castes (in , fe- inance is widespread. male reproductive queens and sterile workers) differ dra- matically in behavior. Castes may differ in the cognitive Significance statement demands that affect patterns of brain tissue investment. Social insect castes (reproducing queens and sterile workers) Queens and workers diverge most strongly in the ad- differ strongly in behavior, particularly in swarm-founding spe- vanced eusocial, or swarm-founding species, where cies where queens are largely nest-bound. Caste comparisons queens do not forage and rarely leave their nests. We are a powerful model for understanding brain/behavior relation- asked whether reproductive castes of swarm-founding pa- ships. We measured the relative size of a key insect brain re- per wasps in the tribe Epiponini differed in the relative gion, the mushroom bodies (MB), in 16 swarm-founding sizes of their mushroom bodies (MB), a key brain region species. MB are involved in sensory integration, and in learning involved in sensory integration, and in learning and mem- and memory. Queens had relatively larger MB than workers, ory. We measured brain-size corrected volumes of the MB and the magnitude of the queen-worker differences increased dendritic-field neuropils (calyces) and the MB axonal with species average colony size. We suggest the reproductive bundles (peducles and lobes) for queens and workers from dominance and social contact of nest-bound queens promotes 16 species of 10 genera of the tribe Epiponini. The subject greater mushroom body investment. species spanned much of the epiponine phylogeny, differ- ing in colony size and degree of caste differentiation. Keywords Brain evolution . Mushroom bodies . Paper Queens had significantly higher relative MB investment wasps . than workers, both for the MB in toto and for the MB calyces. The magnitude of queen-worker MB size differ- ences did not covary significantly with body size, but Introduction species with larger colonies had stronger caste differences in MB size. A review of caste differences in MB volume The neuroecology of caste in social across a wide range of social Hymenoptera taxa suggested Neuroecology theory assumes brain tissue is costly and rela- tive investment in different brain regions is therefore Communicated by W. Hughes constrained. Neuroecological models predict differences in ’ sensory environments will lead to differences in * ’ Sean O Donnell brain structure as a result of brain plasticity (at the individual [email protected] level) or brain evolution (when comparing species) (Barton et al. 1995; Hampton and Shettleworth 1996;Nivenand 1 Department of Biodiversity Earth and Environmental Science, Laughlin 2008; but see Kamhi et al. 2016). Relative brain Drexel University, Philadelphia, PA 19104, USA region sizes are expected to correspond to the cognitive de- 2 Department of Biology, Drexel University, Philadelphia, PA, USA mands an confronts. Comparisons of individuals or 116 Page 2 of 9 Behav Ecol Sociobiol (2017) 71: 116 species that differ in behavior or ecology can be used to test the stalk-like MB lobes and peduncles (Strausfeld 2002), which hypotheses about the cognitive forces shaping adaptive differ- comprise the Kenyon cell axons; these regions are involved in ences in brain investment. modifying neural activity in the calyx and long-term memory The distinct behavioral roles of females from different re- formation (Pascual and Preat 2001;Giurfa2003). productive castes—mated, egg-laying queens versus unmated, sterile workers—are characteristics of eusocial Social behavior and brain investment Hymenoptera (Holldobler and Wilson 2009). Queen and worker behavior entail different ecological and social environ- Controversy exists over expected direction of queen vs. worker ments. Queen-worker differences are greatest in the complex caste differences in MB size. Based on the limited task repertoire societies of advanced eusocial, swarm-founding species of queens, reduced MB investment could be characteristic of (Jeanne 2003; Jeanne and Suryanarayanan 2011). queens (Julian and Gronenberg 2002; Gronenberg and Riveros In these species, colonies are initiated by social groups of 2009; Roat and da Cruz Landim 2010). Alternatively, the chal- many workers and one to many queens (Jeanne 1991, 2003). lenges of maintaining social and/or reproductive dominance may Swarm-founding queens are largely nest-bound (except for place strong cognitive demands on queens (O'Donnell et al. mating flights and colony emigrations). In-nest queens engage 2007;Smithetal.2010; Rehan et al. 2015). Brain development in extensive social contact but perform relatively few tasks. In is affected by social experience, both positively (social enrich- contrast, workers perform outside-nest tasks including forag- ment) and negatively (social deprivation), in many animals in- ing, but often act alone (Eberhard 1975; O'Donnell 1996; cluding insects (Heisenberg et al. 1995; Ehmer et al. 2001;Lee Richter 2000;O’Donnelletal.2014). We asked whether and Goto 2013;Saleetal.2014; Seid and Junge 2016). Social queens and workers of eusocial wasps (Vespidae) differed in insect’s perception of rewarding stimuli can be modified by so- brain structure, and whether these differences might reflect cial context, and gene expression profiles in the brain are affected behavioral differences between the castes. by social interactions (Barron et al. 2009; McNeill et al. 2016). Social dominance is associated with greater MB size or greater Mushroom bodies as key brain regions in social insect MB neuropil development in facultatively social bees (Smith neuroecology et al. 2010; Rehan et al. 2015) and in primitively social paper wasps (Molina and O’Donnell 2007, 2008; O'Donnell et al. To test for caste differences in brain structure, we focused on the 2007). Few comparisons of mature adult queen and worker mushroom bodies (MB), paired neuropils in the forebrain of brains are available for advanced eusocial species. We sampled insects and other (Strausfeld et al. 1998;Fahrbach MB investment (MB size relative to brain size and to body size) 2006). The MB function in sensory integration, and in learning in both castes from a diverse set of epiponine wasp species to test and memory (Lozano et al. 2001;Davis2005;Farris2013). for caste differences in MB size. A previous study suggested Evolutionary increases in MB size and structural complexity queens, including some epiponines, had larger MB correspond to greater species-typical ecological complexity relative to their peripheral processing lobes than their workers (Farris 2008, 2013). At the individual level, the MB are devel- (O'Donnelletal.2011). However, this caste comparison did not opmentally plastic, varying in volume with behavioral differ- correct for overall brain size differences and may have been ences (Withers et al. 2008; Riveros and Gronenberg 2010; affected by reduced peripheral lobe size in queens due to their Muscedere and Traniello 2012). Positive associations of MB simplified in-nest environments (O’Donnell et al. 2014). size with foraging behavior occur in diverse eusocial Hymenoptera (Farris et al. 2001;O’Donnell et al. 2004; Behavioral diversity in the Epiponini: variation in social Fahrbach and Dobrin 2009; Riveros and Gronenberg 2010; complexity Muscedere and Traniello 2012). Volume increases in the MB calyces (the neuropils receiving input from the sensory lobes) We used a generic phylogeny of the Neotropical radiation of are associated with greater dendrite length and complexity and swarm-founding Epiponini to select subject taxa that spanned synaptic density of the intrinsic MB neurons (Kenyon cells), much of the evolutionary history of the lineage (Fig. 1; Wenzel suggesting greater neural connectivity (Farris et al. 2001; and Carpenter 1994). We asked whether the magnitude of queen- Ehmer and Gronenberg 2004; Jones et al. 2009; Seid et al. worker brain differences covaried with species differences in 2005; Fahrbach 2006). In most eusocial Hymenoptera, distinct social complexity. We used two indicators of variation in caste regions of the MB neuropil (calyx) receive input from visual specialization: colony size and degree of queen-worker develop- and olfactory modalities. The compound eyes input to the MB mental differentiation. Although all epiponines are swarm foun- calyx collar region (visual processing), and the antennae input ders, epiponine species vary widely in colony size: typical adult to the MB calyx lip region (olfactory processing) (Gronenberg populations in mature nests span over five orders of magnitude 2001). We tested whether caste differences in brain structure (Jeanne 1991, 2003). Strength of division of labor and caste were consistent across both calyx regions. We also quantified specialization generally increase with colony size within and Behav Ecol Sociobiol (2017) 71: 116 Page 3 of 9 116

Fig. 1 Genus-level phylogeny, subgenus-level in the case of Polybia,of subgenera sampled in this study (one species each) are indicated with Neotropical swarm-founding paper wasps (Polistinae: Epiponini; Wenzel single asterisk;twospecies(double asterisks) were sampled in two of Carpenter 1994, modified with subsequent generic synonymies: Wenzel the Polybia subgenera and Carpenter 1989; Carpenter et al. 2000; Carpenter 2004). Genera and among eusocial insect species (Anderson and McShea 2001; aldehyde-based fixative (Prefer fixative, Anatech Ltd.) for at least Jeanne 2003; Holbrook et al. 2011; Feinerman and Traniello two months until histological processing. Species, collection 2016). Epiponine species also vary in degree of queen-worker dates, and locations were Agelaia xanthopus, Polybia caste differentiation. Degrees of queen-worker separation range (Pedothoeca) emaciata: August 2006, Costa Rica, 10°18. 1′N, from species lacking discrete castes (i.e., continuous worker-to- 84°47. 9′W; Nectarinella championi, Polybia (Trichinothorax) queen variation) to species with allometric size and shape differ- raui: August 2006, Costa Rica, 10°14. 4′N, 84°54. 3′W; ences between queens and workers (O'Donnell 1998;Nolletal. pallens, Angiopolybia zischkai, Charterginus fulvus, Leipomeles 2004). We asked whether the magnitude of caste brain differ- dorsata, Parachartergus smithii, Polybia (Cylindroeca) ences covaried with caste differentiation or with colony size. dimidiata, Polybia (Furnariana) richardsi, Protopolybia exigua: Epiponines are highly variable in body size, and our sub- June 2007, Ecuador, 0°40. 3′S, 76°24. 0′W; Polybia jects ranged from some of the smallest-bodied epiponine spe- (Trichinothorax) flavitincta: March 2012, Costa Rica: 10°25. 6′ cies (Leipomeles dorstata, Protopolybia exigua)tomoderate- N, 84°1. 2′W Brachygastra smithii, Polybia (Myrapetra) ly large-bodied species (Polybia dimidiata, Apoica pallens) aequatorialis, Polybia (Myrapetra) plebeja: July 2012, Costa (Richards 1978). We tested whether species differences in Rica 10°16. 3′N, 84°49. 4′W. body size were related to the magnitude of queen-worker brain structure differences (O'Donnell et al. 2011;O’Donnell and Bulova 2017). Assigning subjects to castes

We dissected each subject’s gaster (the terminal abdominal body region), exposing the ovaries and examining them at 10× under a Methods binocular dissecting scope. We selected workers that had fila- mentous ovarioles with no visible opaque oocyte swellings and Subject species, collection dates, and locations queens with at least one fully opaque oocyte more than 2.5× longer than broad per ovariole. All subjects were mature wasps We sampled one species per genus (Fig. 1) except in the case of with fully hardened, deeply colored cuticles. We did not know Polybia which has been resolved at the subgenus level the individual histories of the subjects, and we assumed our (Carpenter et al. 2000); we sampled seven species of Polybia samples were representative of mature females of each caste. with a maximum of two species in each of five of the subgenera. Researchers were blinded to subject wasps’ castes but not to Wasps were field-collected into, and stored in, buffered species when collecting neuroanatomical data. 116 Page 4 of 9 Behav Ecol Sociobiol (2017) 71: 116

Histology and neuroanatomy

We cut the fixed wasps’ head capsules from the thorax and dehydrated through a series of increasing ethanol concentra- tions, acetone, and then through increasing concentrations of plastic resin. Resin composition 5.5 g EMbed 812 (a mixture of bisphenolA/epichchlorohydrin epoxy resin [CAS #25068– 38-6]) and epoxy modifier ([CAS #2425–79-8]), 5.7 g dodecenyl succinic anhydride, 0.65 g dibutyl phthalate, and 0.31 g of 2,4,6-tri(dimethylaminoethyl)phenol. We incubated individual wasp heads in 0.1 ml resin in pyramid molds at 60 °C for 72 h and then glued the hardened resin to 0.5 ml acrylic cylinders with cyanoacrylate adhesive. We cut each head along the frontal plane into 12 to 16-μm-thick sections (depending on species) using a rotary microtome with dispos- able steel histology blades. Sections were mounted on gelatin- coated microscope slides and the tissue was stained with Toluidine blue. We cleared the stained sections in a series of increasing ethanol concentrations and cover slipped under transparent mounting medium. We used a compound microscope-mounted digital camera to photograph the tissue sections at 2560 × 1920 pixel resolution, using 2.5× or 5× microscope objectives (depending on species; Fig. 2). For each wasp, we began photographing every other section at the section where brain tissue first became visible. Fig. 2 Top: thin section of resin embedded head capsule of a Leipomeles ImageJ version 1.46 digital imaging analysis software (http:// dorsata queen in frontal view. Some major brain regions are labeled. rsbweb.nih.gov/ij/) was used to quantify the volumes of brain Bottom: artificially colored 3D reconstruction (from serial thin sections) structures. We outlined the target brain regions and quantified of the brain neuropils from a Polybia dimidiata queen, with all major brain neuropils quantified in this study indicated the number of image pixels in the structure using ImageJ and then converted the pixel counts to area using a photograph of a stage micrometer taken at the same resolution and magnification P. dimidiata n = 12); whenever possible, we sampled even as a size reference. We multiplied the areas by section thickness numbers of queens and workers. We dissected the wasp’s to yield estimated volume in cubic millimeters. head capsules from the body at the foramen (narrow We quantified the volumes of the following brain neuropils attachment point to the alitrunk or Bneck^). We (we did not measure the cell body regions surrounding the photographed each head using a digital camera mounted neuropils): optic lobes (medulla and lobula only), antennal on a dissecting scope and used the ruler tool in ImageJ lobes, MB calyx lip, MB calyx collar, MB lobes + peduncle, and photographs of a stage micrometer to convert pixels and central brain mass (central brain mass included the rest of to mm. Heads were photographed face-on in frontal view the protocerebrum, the central complex, and the with the foramen area facing away from the camera rest- subesophageal ganglion) (Fig. 2; O'Donnell et al. 2011, ing against a horizontal glass surface. We measured head 2013, 2014; Rehan et al. 2015). Because queens have smaller width at the widest point, head height from the distal tip peripheral lobes, possibly related to their in-nest ecology of the clypeus at the midline to the vertex, and we used (O’Donnell et al. 2014), we used the volume of the central one half head width as an approximation of head depth. brain mass as the index of overall brain size in the caste com- We then estimated head capsule volume for each individ- parisons. We used total brain volume, the sum of the volumes ual using the formula for an ellipsoid: of all neuropil regions we measured, when analyzing overall species differences in MB size. 4=3  pi  1=2 head width mm  §headheightmm Body size measurements  § head depth mm We measured head capsule sizes for n = 6 individuals (not the brain anatomy subjects, but taken from the same col- We used species mean estimated head volumes as an index onies) of each subject species (except P. flavitincta n =1; of species-typical body size in the statistical analyses. Behav Ecol Sociobiol (2017) 71: 116 Page 5 of 9 116

Social complexity variables effects of phylogeny. We used general linear models (GLM) to test whether the magnitude of queen-worker differences in Colony size Colony sizes were the mean numbers of adults in relative MB size (total MB and MB calyx) varied among mature colonies; values were obtained from published records species with different mechanisms of caste differentiation or from our own field collections (Richards and Richards (i.e., no castes, physiological, or morphological castes: Noll 1951;Richards1978;Jeanne1991). et al. 2004).

Data availability Caste differentiation We categorized each species’ degree of queen-worker caste differentiation based on published mor- All data generated and analyzed during this study are included phometric analyses and our own observations (Shima et al. in this published article and its supplementary information 1994, 1996;Huntetal.2001;Nolletal.2004). We used the files. caste differentiation categories of Noll et al. (2004)toclassify the social species’ degree of queen-worker caste differences as follows: no castes: continuous morphological and ovary de- velopment variation, physiological castes:onlyqueensshow Results ovary development, size castes: queens are larger but similar in shape, and morphological castes: queens are distinct in Caste differences in relative MB size shape (body allometry). Castes differed in their ratios of MB size relative to brain size; Sampling effort, variables, and statistical analyses queens had significantly greater relative MB volumes (Fig. 3; MB/central brain mass ratio; paired t tests—total MB: We analyzed brain architecture of 131 individual female ves- t =3.65,df=15,p =0.002;MBcalyx:t = 3.90, df = 15, pid wasps from 16 species in 10 epiponine genera (Fig. 1; p = 0.001). The mean percent caste difference in relative MB Wenzel and Carpenter 1994; Carpenter et al. 2000). We col- size was 5.4% for total MB and 4.5% for MB calyx. Queens lected neuroanatomical data on five to 13 wasps per species had greater MB:brain size ratios in 13 of the 16 species we   (X = 8.2), with two to ten individuals from each caste (X sampled for total MB volume (Fig. 3;signtestwithHo =0.5, = 3.5 queens, X = 4.7 workers). We used the Epiponini ge- p = 0.01); queens had relatively larger MB calyces in 15 spe- neric and Polybia subgeneric phylogenies cited above. cies (sign test p = 0.0003). These caste differences in relative Because we did not have data on dates of divergence among MB size were significant after applying phylogenetic correc- — genera, all tree branch lengths were set to one in phylogenetic tion (phylogenetic paired t tests total MB: t =3.77, analyses (Martins and Hansen 1997); unresolved nodes were p =0.002;MBcalyx:t =4.03,p =0.001).Thesignificantly arbitrarily resolved but given relatively small branch lengths of 0.001 as recommended by Martins (2004). Standard parametric analyses were performed using SPSS v. 24 software (IBM corp. 2016). For measures of relative brain region size (volumes of MB calyx, MB lip, MB collar, and total MB [calyx plus lobes and peduncle] divided by volume of the central brain mass), we calculated the mean for each caste within species, and then we used paired t tests to assess whether the castes differed consistently across spe- cies in average brain architecture. We used a paired t test with phylogenetic correction to assess whether the caste differences in relative total MB and MB calyx sizes were significant after accounting for the effects of phylogeny using Phytools v. 0.5- 38 software in R (Revell 2012). We used Pearson correlation to test whether the magnitude of queen-worker caste differences in relative MB size (total

MB and MB calyx) covaried with colony size (log10 trans- formed); phylogenetic generalized least squares (PGLS: Fig. 3 Caste differences in MB size relative to brain size for 16 epiponine Martins and Hansen 1997) analysis was performed using wasp species. Each data point represents a single species; the line of unity (1:1 queen-worker ratio) is indicated as a dashed line for reference. Also Compare v. 4.6b software (Martins 2004)toassesswhether note that queen relative MB size was strongly positively correlated with these correlations were significant after accounting for the worker relative MB size among species 116 Page 6 of 9 Behav Ecol Sociobiol (2017) 71: 116 greater relative size of queen MB held for both subregions of Discussion the MB calyx (MB lip olfactory region: t = 4.60, df = 15, p < 0.001; MB collar visual region: t = 3.34, df = 15, Caste differences in brain structure p = 0.004). Worker brains showed a nonsignificant trend to- ward being larger than queens brains (as indicated by the MB tissue investment, as indicated by MB size relative to volume of the central brain mass: paired t test, t = −2.04, brain size, was significantly greater for epiponine queens than df = 15, p =0.059). for their workers. Caste differences in relative MB size held for the entire MB, the MB calyx neuropil, and separately for the olfactory lip and the visual collar regions. A previous Body size and brain allometry study suggested the ratio of MB calyx volume to peripheral lobe volume was greater for queens than workers, but this For the species where we measured head capsule of both pattern may have been affected by reduced peripheral lobe castes, queens and workers did not differ significantly in head volumes in queens (O’Donnell et al. 2014). The caste differ- capsule volume (F1,30 =2.60,p = 0.12). The magnitude of the ences we documented in this study were robust to statistical queen-worker differences did not covary significantly with tests that accounted for the effects of phylogeny. Our samples species mean body size (head capsule volume: total MB, spanned much of the phylogeny of Epiponini, suggesting r = 0.13, n =16,p = 0.64; MB calyx, r =0.09,n = 16, greater queen MB investment is widespread in this clade. p =0.74). The greater MB investment by epiponine wasp queens was consistent with the caste- and social dominance-associated differences in MB development that have been documented Species differences in social structure in independent-founding paper wasps (Molina and O’Donnell 2007, 2008; O'Donnell et al. 2007). Positive relationships of The magnitude of the caste differences in relative MB size social and reproductive dominance with adult brain develop- was significantly positively correlated with mean colony ment, and particularly with MB investment, appear to be size (Fig. 4;totalMB,r =0.63,n =16,p = 0.009; with widespread in social Hymenoptera (Smith et al. 2010;Rehan PGLS phylogenetic correction r = 0.63, n = 15, p =0.012; et al. 2015). Queens are often required to suppress reproduc- MB calyx, r =0.58,n =16,p = 0.019; with PGLS phy- tion in an array of nest mates while being targets of social logenetic correction r =0.58,n =15,p = 0.023). The challenges. In primitively eusocial paper wasps (e.g., magnitude of the caste differences in relative MB size , Mischocyttarus), aggressive interactions are often did not differ significantly among species with different involved in the establishment and/or maintenance of repro- degrees of caste differentiation (total MB, F2,13 =0.16, ductive dominance (West-Eberhard 1986; Tindo and Dejean p = 0.85; MB calyx, F2,13 = 0.38, p =0.69). 2000; Markiewicz and O'Donnell 2001). Queens of some swarm-founding wasps engage in ritualized displays with workers. These displays affect reproductive division of labor, and queen elimination can result from these interactions (West-Eberhard 1977; Strassmann et al. 1991, 1992;Noll and Zucchi 2002;Nascimentoetal.2004; Platt et al. 2004). Our results suggest swarm-founding wasp queens, like repro- ductive and/or socially dominant females in other species, may face cognitive challenges that are processed by mush- room body neurons (Strausfeld et al. 1998; Fahrbach 2006; Farris 2013). The mechanisms by which social interactions are related to adult brain development are poorly understood. In the independent-founding social wasp Mischocyttarus mastigophorus, MB development (calyx neuropil to cell body volume) was highest for queens, but varied continuously in females; MB development covaried with age and Fig. 4 The magnitude of queen-worker differences in mean relative MB (independently) with social dominance status (O'Donnell size plotted against mean colony size (log10 scale) for 16 epiponine wasp et al. 2007; Molina and O'Donnell 2008), suggesting brain species. Each data point represents a single species; the zero line plasticity during adulthood contributed to caste MB differ- (representing no caste difference) is indicated as a solid line, and the best fit least squares linear regression relationship is indicated as a dashed line ences. Gene expression patterns in the brain differed among for reference Polistes paper wasp castes, and mature queens’ brains differed Behav Ecol Sociobiol (2017) 71: 116 Page 7 of 9 116 most strongly from newly emerged adult female brains, fur- References ther suggesting adult experience influenced brain develop- ment (Toth et al. 2007). In contrast to greater queen MB size Anderson C, McShea DW (2001) Individual versus social complexity, in many flying eusocial insects, some ant queens (and repro- with particular reference to ant colonies. 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