Coevolution of generalist feeding ecologies and gyrencephalic in insects

Sarah M. Farris*† and Nathan S. Roberts‡

*Department of Biology, West Virginia University, P.O. Box 6057, Morgantown, WV 26506; and ‡Program in Biochemistry, Washington and Jefferson College, 51 Lincoln Hall, Washington, PA 15301

Communicated by Gene E. Robinson, University of Illinois at Urbana–Champaign, Urbana, IL, September 27, 2005 (received for review June 22, 2005) Here we demonstrate the independent acquisition of strikingly panded to accommodate the processing needs of highly special- similar brain architectures across divergent insect taxa and even ized somatosensory processing structures on the snout (13). across phyla under similar adaptive pressures. Convoluted cortical Distribution of anatomical features across more phylogeneti- gyri-like structures characterize the mushroom body calyces in the cally diverse insect taxa suggests independent origins, perhaps in brains of certain species of insects; we have investigated in detail response to similar adaptive pressures, in these lineages. This is the cellular and ecological correlates of this morphology in the dramatically illustrated by the distribution of single and double Scarabaeidae (scarab beetles). ‘‘Gyrencephalic’’ mushroom bodies primary calyx . Single ovoid calyces are found in the with increased surface area and volume of calycal synaptic neuro- firebrats, representative of the most basal extant insect order, pils and increased intrinsic number characterize only those and the flies, one of the most derived groups (14–17). Doubled, species belonging to generalist plant-feeding subfamilies, whereas deeply cup-shaped calyces are observed in widely divergent significantly smaller ‘‘lissencephalic’’ mushroom bodies are found groups, such as the hemimetabolous cockroaches and the holo- in more specialist dung-feeding scarab beetles. Such changes are metabolous social Hymenoptera (9). not unique to scarabs or herbivores, because the mushroom bodies What are some potential causal mechanisms and adaptive of predatory beetles display similar morphological disparities in significances of calyx doubling? At the cellular level, mushroom generalists vs. specialists. We also show that gyrencephalic mush- bodies of cockroaches and social Hymenoptera are the largest room bodies in generalist scarabs are not associated with an known, with Ϸ170,000–175,000 Kenyon cells per hemisphere increase in the size of their primary input , the antennal (18, 19). Calyx volume is likely to be large relative to the rest of lobe, or in the number of glomeruli but rather with the brain in these insects, compared with those possessing fewer an apparent increase in the density of calycal microglomeruli and Kenyon cells. Spreading of the calyx into two separate neuropils the acquisition of calycal subpartitions. These differences suggest and convolution of the synaptic neuropil may be a consequence changes in calyx circuitry facilitating the increased demands on of maintaining optimal surface area to volume ratios in large processing capability and flexibility imposed by the evolution of a mushroom bodies. Such a mechanism has been proposed for the generalist feeding ecology. formation of cortical gyri and sulci (gyrification) in mammals, which is pronounced in groups with relatively large cortices such afferent ͉ behavior ͉ carnivore ͉ sociality as primates and cetaceans (20, 21). Developmental studies provide additional support for a positive correlation among cortex volume, neuron number, and gyrencephaly in mammals. he insect mushroom bodies, paired neuropils that act as For example, decreased neuronal number is observed in human centers for sensory discrimination, integration, and associa- T patients and mouse mutants with lissencephalic (smooth cortex) tive and flexible behaviors, are easily recognized due to their mutations (22), whereas increasing the number of neural pre- characteristic cellular architecture (1–5). Mushroom bodies are cursors can generate an abnormally large number of gyri and composed of thousands of tightly packed intrinsic called sulci in the cortex of the mouse (23). Kenyon cells. Kenyon cell are grouped into dorsally We tested the hypothesis that double mushroom body calyces, positioned neuropils termed calyces, which serve as dedicated like cortical gyri and sulci, are associated with an increase in afferent input regions and receive a preponderance of olfactory ͞ mushroom body size relative to single calyces in terms of Kenyon afferents from the antennal lobe and, in some taxa, visual and or cell number, calyx neuropil surface area and calyx volume. We gustatory afferents as well (6–8). Kenyon cell project accomplished this by quantifying mushroom body architecture in ventro-anteriorly to the pedunculus and typically bifurcate into scarab beetles (Coleoptera: Scarabaeidae), species of which may ͞ combined input output structures, the medial and vertical lobes. possess either single or double calyces (24), as illustrated in Fig. Although all insect mushroom bodies share a basic ground- 1 A and B. We also asked whether calyx morphology in scarab plan, substantial morphological and presumably functional mod- beetles was associated with changes in its most prominent source ifications are readily observed (9). Morphological variability of of synaptic input, the antennal lobes (25). Finally, we mapped mushroom bodies often reflects species-specific behavioral ecol- calyx structure according to phylogeny and feeding ecology in ogy. In the social Hymenoptera (including the ants, bees, and other Coleoptera to determine potential behavioral correlates of wasps), the calyx is partitioned into afferent zones characterized calyx diversity in insects. by the dendrites of Kenyon cell subpopulations and by sensory input modality (6, 10, 11). Two of these calyx zones, the lip and Materials and Methods the collar, receive olfactory and visual input, respectively. In Insects and Histology. A total of 48 individual beetles representing ants, the relative size of the lip and collar vary with the size of six scarabaeid subfamilies and 11 different species (see Table 1, their primary sensory input neuropil, the antennal and optic which is published as supporting information on the PNAS web lobes, and also with the importance of each sensory modality in site, for summary information on species identity, feeding ecol- the life of the animal (12). Similar correlations between the periphery and higher processing centers have been observed in mammals. In the star-nosed mole, deemphasis of vision as a Conflict of interest statement: No conflicts declared. primary sensory modality has led to reduction of the eyes and †To whom correspondence should be addressed. E-mail: [email protected]. visual cortex, whereas somatosensory cortex has greatly ex- © 2005 by The National Academy of Sciences of the USA

17394–17399 ͉ PNAS ͉ November 29, 2005 ͉ vol. 102 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508430102 Downloaded by guest on September 30, 2021 Fig. 1. Single and double calyces of scarabaeid mushroom bodies. (A) Single calyx of Onthophagus hecate (Scarabaeinae). (B) Double calyx of Maladera castanea (Melolonthinae) (Scale bars: 20 ␮m.) (C–F) Average brain measurement ratios for beetles with single calyces (black bars) vs. double calyces (white bars). (C) Calyx volume relative to central brain volume. (D) Kenyon cell number relative to central brain volume. (E) Measured calyx surface area relative to predicted calyx surface area. (F) Calyx surface area relative to calyx volume. Brackets, calyces; white arrowheads, sensory input to the calyx from the antennal lobe; black arrowheads, calycal subcompartments. *, Significant difference (P Յ 0.05) as determined by one-way ANOVA. Error bars in C–F represent standard errors of the mean. EVOLUTION ogy, calyx morphology, and sample sizes) were collected as adults bedded brains were sectioned at 10 ␮m on a rotary microtome in the Morgantown, WV, area or ordered from Hatari Inverte- and stained by using Cason’s stain according to the protocol of brates (Portal, AZ). The nonscarabaeids Geotrupes sp. Kiernan (26) to reveal brain architecture. Brains of both male (Geotrupidae), Trox sp. (Trogidae), Odontotaenius disjunctus and female beetles were analyzed, with the exception of antennal (Passalidae), Sphaeridium sp. (Hydrophilidae) and Silpha sp. lobe glomerulus counts in which only male brains were used. The (Silphidae), Scarites subterraneus (Carabidae), Scaphinotus el- age of beetles sampled was not known, but adult mushroom body evatus (Carabidae), and Harmonia axyridis (Coccinellidae) were neurogenesis appears to be negligible or absent for these species also collected in or around Morgantown or obtained from (S.M.F. and J. Miller, unpublished data) and is thus unlikely to Carolina Biological Supply Company (Burlington, NC). Insects be a source of significant variation. were anesthetized with cold and brains dissected in physiological saline, followed by fixation in Carnoy’s fixative at 20° C. Fixation Volume and Cell Number Estimates. Measurements were made in one was allowed to proceed for a duration ranging from 1 h 15 min randomly selected hemisphere of each stained brain. Brain region to1h45mintokeep tissue shrinkage between brains relatively volumes were calculated according to the Cavalieri method (27–29), constant. After fixation, brains were stored in 70% ethanol at 4°C which has been demonstrated to provide accurate estimations of overnight. The next day, dehydration through a graded series of volume from sectioned material by taking into account sampling ethanols (10 min each) was followed by clearing in xylene (2 ϫ frequency and section thickness. First, areal measurements of brain 10 min) and paraffin embedding in a frontal orientation. Em- regions were directly measured from photomicrographs by using

Farris and Roberts PNAS ͉ November 29, 2005 ͉ vol. 102 ͉ no. 48 ͉ 17395 Downloaded by guest on September 30, 2021 Fig. 2. Distribution of calyx morphologies and feeding ecologies across the Coleoptera. The phylogenetic tree of the Scarabaeoidea is after ref. 30; the remainder of the coleopteran tree is after ref. 49. The family Scarabaeidae is considered a monophyletic grouping. (Scale bars, located at the bottom right of each photomicrograph, represent 20 ␮m for brains of Silphidae, Hydrophilidae, Trogidae, and Geotrupidae and 50 ␮m for Passalidae and Scarabaeidae.)

Zeiss AXIOVISION 4 software (Zeiss, Oberkochen, Germany). Sam- Results pling frequency for these measurements was determined for each Double (‘‘gyrencephalic’’) calyces in scarab beetles were posi- brain region [Kenyon cell body region, mushroom body calyx tively associated with a significant increase in calyx volume neuropil (excluding the pedunculus), antennal lobe neuropil, and relative to total central brain size when compared with single total central brain] for each species to provide a volume estimate (‘‘lissencephalic’’) calyces (Fig. 1C; one-way ANOVA F ϭ within 5% of that generated by measurements by using every 10-␮m 124.999, P Ͻ 0.0001). Kenyon cell number was also dramatically section. Typical sampling frequencies ranged from every second increased in gyrencephalic mushroom bodies (Fig. 1D; one-way section (calyx neuropil surface areas and volumes in the smallest ANOVA F ϭ 79.520, P Ͻ 0.0001). The measured surface areas species) to every sixth section (total central brain volume in the of lissencephalic calyces were nearly identical to those predicted largest species). Once sampling frequency was determined, the first for spheres of the same volume, resulting in a measured to section to be sampled in each brain was chosen by tossing a number predicted surface area ratio of Ϸ1 (black bar, Fig. 1E). In of coins equal to the sampling frequency; the number of ‘‘heads’’ contrast, the ratio of measured calyx surface area to predicted was the first section to be sampled after the appearance of the calyx surface area was significantly larger for gyrencephalic structure of interest. calyces (white bar, Fig. 1E; one-way ANOVA F ϭ 46.684, P Ͻ Perimeter measurements of the calyx neuropil and diameters 0.0001). Surface area to volume ratios, however, were signifi- of individual Kenyon cell bodies were also measured from cantly higher in lissencephalic calyces (Fig. 1F; one-way ANOVA ϭ Ͻ photomicrographs by using the Zeiss AXIOVISION 4 software. F 51.556; P 0.0001). This suggests that, although gyren- Surface area of the synaptic neuropil of the calyx was calculated cephaly increases the surface area of the calyx neuropil, it has not from perimeter measurements multiplied by section thickness. kept pace with the expansion in calyx volume in scarabaeids. Sampling frequency for perimeter measurements was deter- We discovered that calyx morphology was tightly linked to mined in the same manner as for volume estimations. feeding ecology; lissencephalic calyces were always observed in To calculate Kenyon cell number, 50–100 individual Kenyon scarabaeid species belonging to dung-feeding (coprophagous) sub- cell soma were selected from throughout the Kenyon cell body families, whereas gyrencephalic calyces were always observed in plant-feeding (phytophagous) subfamilies (Fig. 2; also see support- region and their diameters measured by using the AXIOVISION 4 ing information) (30). Phytophagous scarabs are highly generalist, software. An average Kenyon cell body diameter was calculated feeding on the foliage, flowers, and fruits of a wide range of host from these data and subsequently used to calculate an average plants. By contrast, coprophagous scarabs and species belonging to Kenyon cell body volume for that brain. The total volume of the related nonphytophagous taxa are relatively more specialized in Kenyon cell body region was then divided by the average their feeding habits and exhibited lissencephalic calyces. The wood- individual Kenyon cell body volume to provide an estimate of feeding Passalidae, however, were exceptional in possessing some- Kenyon cell number for that brain. what enlarged and flattened calyces, perhaps reminiscent of an Finally, the total number of antennal lobe glomeruli was early stage in calyx splitting. Nevertheless, the overall distribution of counted from serial sections of male brains only, to control for calyx morphologies across the taxa sampled strongly supports the the likely presence of sex-specific glomeruli. inference that robustly gyrencephalic calyces evolved in accompa- Measurement calculations and graphs were generated by using niment with a derived and highly generalist feeding ecology in the Microsoft EXCEL X for Mac (Microsoft, Redmond, WA), then Scarabaeidae. exported to JMP software (SAS Institute, Cary, NC) for statistical Additional comparisons of scarab calyces and their major analyses. Measured calyx surface areas were compared with sensory input source, the antennal lobes, provided further insight calculated calyx surface areas, the latter derived from the into the potential behavioral significance of large mushroom measured calyx volume and the equation for the surface area of bodies in generalist scarabs. We found that antennal lobe volume a sphere (ϭ 4␲r2). Brain measurement ratios for species with relative to total central brain volume was actually larger in single vs. double calyces were tested for significant differences by beetles with lissencephalic calyces than in those with gyrence- using one-way ANOVA. All statistical comparisons were con- phalic calyces (Fig. 3A; one-way ANOVA F ϭ 12.306, P ϭ 0.001), sidered significant at P Յ 0.05. and there was no difference in the number of antennal lobe

17396 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508430102 Farris and Roberts Downloaded by guest on September 30, 2021 Fig. 3. Differences in calycal circuitry in single and double calyces. (A–D) Average brain measurement ratios for beetles with single calyces (black bars) versus double calyces (white bars). (A) Antennal lobe volume relative to central brain volume. (B) Number of antennal lobe glomeruli (male beetles only). (C) Calyx volume relative to antennal lobe volume. (D) Calyx volume relative to Kenyon cell number. Error bars in A–D represent standard errors of the mean. *, Significant

difference (P Յ 0.05) as determined by one-way ANOVA. ns, no significant difference. (E) Microglomeruli in the double calyx of Popillia japonica (Rutelinae). EVOLUTION Two sizes of microglomeruli (small and large circles) occupy distinct calycal subcompartments (separated by black line). (F) Microglomeruli (circles) in the single calyx of Canthon sp. (Scarabaeinae). (Scale bars: 10 ␮m.)

glomeruli in males (Fig. 3B; one-way ANOVA F ϭ 0.048, P ϭ convergence of Kenyon cell dendrites, and extrinsic inhibitory 0.829). This suggests that calyx gyrencephaly is unlikely to be processes about a single antennal projection neuron terminal associated with greatly increased numbers of antennal lobe (31). Microglomeruli were smaller and more densely packed in neurons projecting to the calyx. the calyces of generalist scarabs (Fig. 3 E and F). Calycal Instead, our data suggested that individual afferent neurons subcompartments were also observed in gyrencephalic mush- might form with more Kenyon cells in the mushroom room bodies (Fig. 3E), perhaps indicating the segregation of bodies of generalist species. Generalist scarabs had nearly four olfactory processing inputs or acquisition of other afferent times as much calyx volume per unit of antennal lobe volume modalities such as vision or gustation. compared with coprophagous scarabs (Fig. 3C; one-way ANOVA F ϭ 133.017, P Ͻ 0.0001) and significantly more Discussion Kenyon cells per unit of calyx volume (Fig. 3D; one-way ANOVA Our findings suggest that gyrencephalic calyces in scarabaeid F ϭ 4.883, P ϭ 0.032). This latter measurement was supported beetles, and perhaps other lineages such as the cockroaches and by high-magnification images of calycal microglomeruli, sites of the social Hymenoptera, have arisen in conjunction with an

Farris and Roberts PNAS ͉ November 29, 2005 ͉ vol. 102 ͉ no. 48 ͉ 17397 Downloaded by guest on September 30, 2021 Fig. 4. Differences in calyx structure in generalist and specialist predators. (A) Tripartite gyrencephalic calyx (brackets) of the generalist predator Scarites subterraneus (Carabidae). (B) Lissencephalic calyx of the carabid Scaphinotus elevatus, a specialist predator of snails and slugs. (C) Minute lissencephalic calyx of the ladybird beetle Harmonia axyridis (Coccinellidae), a specialist predator of aphids and scale insects. Arrowheads, Kenyon cell body regions. (Scale bars: A, 50 ␮m; B and C,20␮m.)

evolutionary increase in Kenyon cell number, neuropil volume, cockroaches (38, 39). Similarly, gyrencephalic calyces are ob- and neuropil surface area. Similar trends are observed in served in basal nonsocial Hymenoptera such as sawflies (40). mammals and are particularly pronounced in lineages such as Sociality, therefore, does not appear to be a robust predictor of primates, in which increased size of the cerebral cortex relative calyx morphology. to total brain size is strongly correlated with an increase in gyral In scarabaeids, calyx morphology was uniformly associated complexity (20, 21). Nonhomologous brain centers like the with feeding ecology. We propose that large gyrencephalic mushroom bodies and the cerebral cortex can thus adopt strik- calyces have evolved in response to selective pressure for be- ingly similar architectures when shared principles of cellular havioral flexibility that would be imparted by the above neural organization are acted on by similar selective pressures. properties and that would be beneficial to a generalist feeding Gyrencephalic calyces are not associated with a concomitant ecology in a long-lived mobile insect. For example, the adult increase in the volume of the antennal lobes, suggesting that the Japanese beetle Popillia japonica is long-lived (4–6 weeks) and evolution of large mushroom bodies in highly generalist phyto- feeds on the tissues of Ͼ300 different host plant species (41), phagous beetles was not driven by the acquisition of inputs from which it locates by using olfactory cues such as host plant volatiles more olfactory afferent neurons. Instead, gyrencephalic calyces induced by feeding damage (42) and visual cues (43). Despite this may represent an adaptation for enhancing the computational wide host range, Popillia clearly prefers certain plant species capacity of the mushroom bodies in three ways: by increasing the while rarely feeding on others (44), suggesting that these insects number of inputs from individual afferent neurons, by subcom- are actively discriminating and choosing among many potential partmentalization for segregation of specialized functions, and food sources by using additional sensory cues. A long-lived insect by providing additional substrate for complex learning compu- may also need to integrate this information with that regarding tations and the storage of memories. Regarding the first possi- food source handling, location, and availability, storing this bility, modeling studies have proposed that small groups of knowledge for future encounters. Cockroaches and social Hy- antennal projection neurons converge combinatorially on pop- menoptera are similarly long-lived and flexible in their feeding ulations of Kenyon cells to form odor-discriminating ‘‘functional behaviors and have arrived at a similar neural solution. subunits’’ (32). Increasing the number of Kenyon cells would In contrast, dietary specialists using a few highly specific thus generate a greater capacity for combinatorial synaptic cues to rapidly and unambiguously detect one or a closely inputs from the antennal lobe and the potential to discriminate related group of host species would be expected to have fewer among larger numbers of odors. Regarding the second possibil- requirements for sensory discrimination and integration and to ity, calycal subcompartments like those observed in generalist have a more hard-wired capacity for handling the host plant. scarabs are found in the social Hymenoptera (6, 11), where they These insects would not be predicted to undergo such strong are characterized by distinct afferent modalities and Kenyon cell selection for large mushroom bodies. Our results support this populations. This organization suggests specialization of func- prediction, and we suggest that the mushroom bodies are an tions that may than be selectively integrated via combinatorial important component of the ‘‘larger and more sophisticated outputs from the mushroom body lobes (11). Subcompartmen- nervous system’’ (45) that is a hypothesized requirement for talization coincident with evolutionary expansion of higher brain decision-making efficiency in insects with generalist feeding regions is also evident in mammals, as exemplified by the ecologies (46). elaborate functional subpartitions in the enlarged visual cortex The hypothesis that cognitive adaptations for generalist phy- of primates (33). Regarding the third possibility, unique cogni- tophagy played a key role in the evolution of gyrencephalic tive capabilities of honey bees, such as perception of generalized calyces would equally predict that long-lived mobile generalist and abstract rules about multiple stimuli in associative learning feeders of any kind might display a similar evolutionary trend. paradigms (34, 35), suggest a role for the gyrencephalic mush- Generalist and specialist carnivorous beetles illustrate that this room bodies of these and perhaps other insects in enabling is indeed the case (Fig. 4). The generalist predator Scarites complex learning and memory functions. subterraneus possesses a gyrencephalic tripartite calyx capped by Sociality has been proposed to drive the evolution of large a dorsally protruding Kenyon cell body region (Fig. 4A). In higher brain centers in both insects and vertebrates (36, 37). In contrast, two specialist predators, Scaphinotus elevatus (Fig. 4B) insects, gyrencephalic calyces and large mushroom bodies are and Harmonia axyridis (Fig. 4C), possess lissencephalic calyces observed in cockroaches, scarab beetles, and social hymenopter- topped by smaller aggregates of Kenyon cell bodies. ans. Cockroaches, although gregarious, are not considered social Despite these robust results, feeding ecology is unlikely to be at the level of ants, bees, and wasps. Elaborated mushroom body equally predictive of mushroom body morphology across the lobes are found in the eusocial termites, but the calyces appear highly diverse insects. For example, it is unknown how quickly to be reduced and fused relative to those of their sister taxon, the changes in the brain can track changes in behavior. At the gross

17398 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0508430102 Farris and Roberts Downloaded by guest on September 30, 2021 anatomical level, it is possible that an evolutionarily recent and maintain populations when colonizing new environments switch in feeding ecology may not produce measurable differ- (48). The worldwide ubiquity of cockroaches, social Hymenop- ences in mushroom body morphology when compared with tera, and phytophagous scarabs, many of which are economically related species retaining the ancestral feeding ecology. Devel- important invasive pests due to their colonization success, speaks opmental constraints may need to be overcome before signifi- to a similar role for large gyrencephalic mushroom bodies. cant enlargement or diminishment of the mushroom bodies Behavioral innovation is therefore likely to be a driving factor in could occur. Additionally, other complex behaviors such as the evolution of large higher processing centers in the brains of social interaction and spatial navigation, also require advanced both invertebrates and vertebrates. capabilities for sensory integration, discrimination, learning, and memory. Insects displaying these behaviors might be expected to We thank Ms. Marissa Smith for assisting with data analysis, exhibit calyx gyrencephaly regardless of feeding ecology. Drs. Ronald Bayline and Kevin Daly for fruitful discussions, and In vertebrates such as primates and birds, large and typically Drs. Susan Fahrbach and Gene Robinson for comments that greatly gyri-rich cerebral cortices are correlated with the capacity for improved this manuscript. N.S.R. was supported by Howard Hughes behavioral innovation (particularly in terms of novel food source Medical Institute Undergraduate Science Education Program Grant usage) and success in invading novel environments (36, 47). 52002683, awarded to Washington and Jefferson College. S.M.F. was Polyphagous insects are also more likely to successfully establish supported by West Virginia University.

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