Structural, Functional and Developmental Convergence of ...sfarris/2008FarrisScarabConverg.pdf · with backbones. In vertebrates, these functions are per-formed by higher brain centers

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Original Paper

Brain Behav Evol 2008;72:1–15 DOI: 10.1159/000139457

Structural, Functional and Developmental Convergence of the Insect Mushroom Bodies with Higher Brain Centers of Vertebrates

Sarah M. Farris

Department of Biology, West Virginia University, Morgantown, W. Va. , USA

time for development of these brain centers is protracted. Taken together, these findings extend our understanding of how evolutionarily constrained neural substrates might con-verge under shared adaptive landscapes, even after 600 mil-lion years of divergence, and even at the level of higher brain centers that generate complex behaviors.

Copyright © 2008 S. Karger AG, Basel

Introduction

There is little doubt today that the basic elements of the bilaterian nervous system were laid down prior to the protostome-deuterostome split. Deep organizational and functional homology is illustrated by the roles of tran-scription factors in patterning the embryonic nervous system and the ubiquity of ion channels and synaptic ma-chinery that sustain neural activity [Salkoff et al., 1992; Ortellis and Lunt, 1995; Lichtneckert and Reichert 2005; Caveney et al., 2006; Mizutani et al., 2006; Denes et al., 2007; Sakarya et al., 2007]. Upon this shared groundplan, functionally and structurally convergent circuits have arisen in distantly related species in response to similar selective pressures upon processing capabilities. For ex-ample, lateral inhibitory circuits and topographic repre-sentations are common elements of sensory processing pathways in both protostome and deuterostome nervous systems [Hartline et al., 1956; Murphey et al., 1980; Squire et al., 2003; Catania and Henry, 2006]. Given that the pe-ripheral sensory structures served by these circuits are

Key Words

Beetle � Convergence � Generalist � Learning � Mushroom bodies � Neuroblasts � Precocial � Scarabaeidae � Vision

Abstract

Convergence of higher processing centers has been pro-posed for insects and vertebrates, but the extent of these similarities remains controversial. The present study demon-strates that one higher brain center of insects, the mush-room bodies, displays a number of similarities with mamma-lian higher brain centers that are arguably the products of adaptation to common behavioral ecologies, despite their deeply divergent origins. Quantitative neuroanatomy, im-munohistochemistry, fluorescent tract tracing and BrdU la-beling are employed to investigate the relationships among behavioral ecology and mushroom body size, sensory input and mode of development in one taxon, the scarab beetles (Coleoptera: Scarabaeidae). Comparisons are extended to a taxon in which similar mushroom body architectures have arisen independently, the cockroaches (Dictyoptera), and to published accounts of vertebrate brain evolution. This study demonstrates that evolutionary increases in higher brain center size and intrinsic neuron number are associated with flexibility in food acquisition behaviors in both vertebrates and insects. These evolutionarily expanded higher brain centers are divided into novel structural subcompartments that acquire novel processing functions. Increased numbers of neurons comprising enlarged higher brain centers are generated by expanded neural precursor pools, and the

Received: December 29, 2007 Returned for revision: January 6, 2008 Accepted after revision: February 16, 2008 Published online: June 17, 2008

Sarah M. Farris Department of Biology, West Virginia University 3139 Life Sciences Building, 53 Campus Drive Morgantown, WV 26506 (USA) Tel. +1 304 293 5201, Fax +1 304 293 6363, E-Mail [email protected]

© 2008 S. Karger AG, Basel0006–8977/08/0721–0001$24.50/0

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Brain Behav Evol 2008;72:1–152

highly divergent and scattered across the bilaterian fam-ily tree, it is clear that they are not shared through com-mon descent. Rather, lateral inhibitory circuits are adap-tations for increasing sensory resolution [Kandel et al., 1991], and topographic maps allow sensory modalities that are perceived as vector quantities to be represented intact within the brain [Kaas, 1997]. Both circuits make use of the developmental, cellular and functional build-ing blocks shared by all bilaterians, which under similar selective pressures for function have produced conver-gence in structure.

A particularly spectacular example of structural and functional convergence is observed in the insect and ver-tebrate olfactory systems [Hildebrand and Shepherd, 1997; Strausfeld and Hildebrand, 1999; Eisthen, 2002; Ache and Young, 2005]. Both are adapted for detecting airborne odors, and consist of sensory receptor cells that project to an olfactory bulb or antennal lobe composed of dense synaptic associations termed glomeruli. These glomeruli consist of synapses among receptor neurons, ascending projection neurons, and inhibitory local inter-neurons. Independent origins of olfactory receptor pro-teins and perhaps even their downstream effectors in in-sects and vertebrates suggest that the striking similarity of olfactory systems is truly a product of convergence, rather than homology [Benton, 2006; Vosshall and Stock-er, 2007]. As with simpler circuits such as those respon-sible for lateral inhibition, convergence of olfactory sys-tems composed of hundreds to millions of neurons has resulted from adaptation to a common functional role.

The higher brain centers of vertebrates such as the hip-pocampus, cerebellum and cerebral cortex are stunning in their cellular and functional complexity. Comparative studies are only beginning to uncover how these struc-tures, composed of millions to trillions of cells, have been adapted into species-specific circuitry. The cerebral cor-tex has been the subject of much of this research, given its enormous expansion in our own taxon, the Primates, and in other groups with complex social architectures such as the cetaceans. Comparisons among mammals demon-strate that sensory modalities that are of particular im-portance to a species, such as vision in primates or the sense of touch from the specialized ‘star’ of the star-nosed mole, are more abundantly represented in the cortex than are less-used modalities [Kaas, 1995; Catania, 2005; Cat-ania and Henry, 2006]. Relative size of the telencephalon or of the entire brain is also larger in vertebrate species that navigate complex environments or that demonstrate the capacity for flexible, ‘innovative’ behavior, often re-lated to food acquisition or social interactions [Burish et

al., 2004; Lefebvre et al., 2004; Iwaniuk and Hurd, 2005; Sol et al., 2005a, b; Marino et al., 2007; Pollen et al., 2007; Yopak et al; 2007]. These adaptive modifications have oc-curred predictably and independently in many vertebrate lineages, having been documented by the above studies in primates, cetaceans, several bird taxa, and both bony and cartilaginous fish.

The need to perceive the environment in the context of multiple external cues and internal motivational states, and to flexibly synthesize this information so as to direct the appropriate behaviors, is not restricted to animals with backbones. In vertebrates, these functions are per-formed by higher brain centers such as the cerebral cor-tex, hippocampus and cerebellum. The notion persists, however, that invertebrates such as insects possess little higher-level processing capability. It has been known since the work of Dujardin [1850], however, that insects do possess higher brain centers, and that variation in size and shape of one such brain region, the mushroom bod-ies, might be associated with complex behaviors such as sociality. The mushroom bodies consist of parallel-pro-jecting arrays of hundreds to many thousands of cells, whose outputs integrate multimodal sensory informa-tion and coordinate plasticity in responses, for example by mediating learning and memory formation [Straus-feld et al., 1998; Heisenberg, 2003; Davis, 2005].

The present account documents, experimentally and via comparisons with the literature, instances of devel-opmental, structural, and functional convergence be-tween vertebrate higher brain centers and the insect mushroom bodies. Although previous investigations have focused on comparisons across families, classes or perhaps orders of animals, this is the first effort to ex-pand the scope of brain evolutionary studies to include comparisons across phyla. Insect mushroom bodies are ideally suited for studies of evolutionary variation in brain and behavior, as the exceedingly diverse life histo-ries of insects provide the opportunity for investigation of almost any behavioral specialization. The functions of mushroom bodies, particularly in olfactory processing and learning and memory, have been extensively ex-plored, and a wealth of model systems studies has pro-vided a detailed understanding of their structure and connections in the brain [Strausfeld and Li, 1999a, b; Strausfeld, 2002; Zhu et al., 2003; Strausfeld et al., 2003]. Developmental studies have also become increasing-ly more detailed, particularly with the application of MARCM [mosaic analysis with a repressible cell marker; Lee and Luo, 1999; Lee et al., 1999], technology in Dro-sophila for labeling the processes of single neurons in a

Evolution and Development of Higher Brain Centers

Brain Behav Evol 2008;72:1–15 3

birthdate-dependent manner. Comparative studies have drawn together the findings from these diverse model systems, ranging from the cockroach Periplaneta ameri-cana to the fruit fly Drosophila melanogaster to the hon-ey bee Apis mellifera , into a coherent picture of the con-served groundplan for mushroom body organization, development and function [Strausfeld et al., 1998; Farris and Sinakevitch, 2003; Farris, 2005a].

Just as in vertebrate higher brain centers, the mush-room bodies are highly morphologically variable across the class Insecta, although they always retain features of the ancestral groundplan [Strausfeld et al., 1998; Farris, 2005a]. Variation in mushroom body structure often can not be explained by known phylogenetic relationships among species, but instead appear to be associated with taxon-specific ecologies such as aquatic life stages, social-ity or generalist feeding preferences [Strausfeld et al., 1998; Farris and Roberts, 2005].

This study focuses on the adaptations of life history, development and sensory inputs that have accompanied the evolution of a particularly striking feature of some insect mushroom bodies: the enlargement and doubling of dedicated sensory input neuropils called the calyces. Comparisons between species with doubled calyces and species with ancestral single calyx neuropils will be pri-marily focused on the family Scarabaeidae. A previous study has demonstrated that generalist plant-feeding scarab subfamilies, alone among the Superfamily Scara-baeioidea, have acquired greatly enlarged mushroom bodies with double calyces, whereas specialist dung-feeding scarab subfamilies retain the ancestral small mushroom bodies with single calyces [Farris and Rob-erts, 2005]. In this study BrdU immunolabeling experi-ments are used to compare patterns of neurogenesis dur-ing larval and pupal development in two species, the gen-eralist plant-feeding scarab Popillia japonica , and the specialist dung-feeding scarab Onthophagus hecate . Flu-orescent tracer experiments are used to assess the acqui-sition of novel inputs by the enlarged calyces of several species of generalist scarabs, in comparison with the spe-cialist Onthophagus . All of these results are matched with the life histories of each species, and compared with divergent insect lineages that have independently ac-quired similar modifications of the mushroom bodies, such as the cockroaches. Finally, the relationships among mushroom body development, sensory input and behav-ioral ecology in scarabs and other insects will be com-pared with other published studies to support the case for convergent evolution of insect and vertebrate higher brain centers.

Materials and Methods

Insects For developmental studies, Popillia japonica (Rutelinae) and

Onthophagus hecate (Scarabaeinae) were used due to the ease with which they could be acquired and induced to breed in a labora-tory setting. To collect Popillia japonica eggs, adult beetles were housed in cages filled with 2–3˝ of damp soil and fed grape leaves ( Vitis sp.) and raspberries (Rubus idaeus) ad libitum. Eggs were collected from the soil approximately 1 week after the colony was established. Onthophagus hecate adults were collected from fresh dog dung (Canis familiaris) that was gathered in the Morgantown area. After it was determined that the adults would not breed on the dog dung, eggs were collected by placing adults on the dung of white-tailed deer (Odocoileus virginianus) . The dung was en-closed in a 5-gallon plastic bucket containing approximately 8˝ of soil. After 3–4 days, dung balls containing eggs were removed from tunnels beneath the dung. Both Onthophagus and Popillia eggs, once collected, were incubated on a damp paper towel in a plastic Petri dish at 21 ° C until hatching.

For comparisons of mushroom body volume and sensory in-put in generalist scarab species, the goal was to represent as wide a range of species as possible. Adult Popillia japonica (Rutelinae), Maladera castanea (Melolonthinae) and Cyclocephala borealis (Dynastinae) were selected due to their easy availability in the Morgantown, WV area. Cotinus mutabilis (representing the Ce-toniinae) and Chrysina (Plusiotis) gloriosa (a secondarily special-ist ruteline) were selected to further broaden the species sample. These species were obtained as adults from Hatari Invertebrates (Portal, Ariz., USA).

Enlarged mushroom bodies with doubled calyces have inde-pendently evolved in the cockroaches (Dictyoptera). Two species were used to demonstrate the anatomy of these enlarged mush-room bodies and their development. Blaberus giganteus (Dicty-optera: Blaberidae) and Periplaneta americana (Dictyoptera: Blattidae) were maintained in colonies (the former started from adults generously provided by Dr. Roy Ritzmann, the latter start-ed from adults obtained from Carolina Biological Supplies, Burl-ington, N.C., USA) reared on Meow Mix � brand cat food (Meow Mix Co., Seacaucus, N.J., USA) and provided with water ad libi-tum via a damp paper towel. The colonies were housed in venti-lated plastic cages in an incubator set at 28 ° C with a 12: 12 light:dark cycle.

Histology and Stereology Brains of adult beetles were collected and processed for paraf-

fin histology and Cason’s staining exactly as described by Farris and Roberts [2005]. Histological preparations were viewed on a Zeiss AxioSkop 2 brightfield microscope, and images captured with a Zeiss AxioCam MRC5 camera and Axiovision 4 software (Carl Zeiss AG, Oberkochen, Germany). Volume measurements and Kenyon cell counts for Onthophagus and Popillia brains were also performed as described by Farris and Roberts [2005]. Statis-tical analyses and graphs were prepared using JMP TM software (SAS Institute Inc., Cary, N.C., USA). Tests used were a 1-way ANOVA followed by a Student t test ( Onthophagus vs. Popillia comparisons, fig. 2 ) or linear regression and analysis of residuals (generalist scarab comparisons, fig. 4 ).

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Fluorescent Dextran Fills Beetles were cold anesthetized, and brains removed rapidly in

cold physiological saline [O’Shea and Adams, 1981]. The tip of a pulled glass electrode was broken against a glass slide and then coated in a thick paste consisting of 6 5% Texas Red- or fluores-cein-conjugated 3000 MW dextran (Molecular Probes, Inc. (In-vitrogen), Eugene, Oreg., USA). Dextrans were applied to the op-tic lobes or antennal lobes by poking the electrode into the respec-tive neuropils by hand. After dye application, the brains were incubated in the dark in room temperature physiological saline for 4 h on a rapidly rotating orbital shaker. Brains were then fixed at least overnight in 4% paraformaldehyde in phosphate buffered saline (PBS; pH 7.2) at 4 ° C in the dark.

After fixation, tissue was washed 3 ! 10 min in PBS, embed-ded in 8% agarose and cut into 70 � m sections on a Leica VT1000 vibratome. Sections were washed overnight in PBST (PBS + 0.5% Triton X-100), then cleared in 60% glycerol in PBS for 30 min and 80% glycerol in PBS for 1 h prior to coverslipping. Tissue was viewed and scanned on an Olympus Fluoview 1000 laser scanning confocal microscope. Staining images in 2D were made from compressed Z-stacks using the Fluoview software, and adjusted for brightness and contrast using Adobe � Photoshop � 7.0 for Macintosh (Adobe Systems Incorporated, USA).

DiI Fills Brains of the generalist scarab Cotinus mutabilis were dissect-

ed out in cold physiological saline and stored in 4% paraformal-dehyde in PBS for several weeks. Tissue was washed in PBS three times 10 min prior to application of DiI (1, 1 � -dioctadecyl-3,3,3 � ,3 � -tetramethylindocarbocyanine perchlorate; Molecular Probes). Crystals of DiI were pushed into the optic lobes using the broken tip of a pulled glass electrode. After DiI application, brains were stored in PBS in a foil-wrapped scintillation vial for 1 week at room temperature to allow diffusion of the dye. DiI labeled tissue was prepared for confocal microscopy as described above for dex-tran fills, with the exception that the overnight PBST wash was omitted to prevent leaching of the lipophilic dye.

BrdU Immunolabeling Newly hatched Onthophagus and Popillia larvae were anesthe-

tized with cold, and a drop of 25 mg/ml BrdU (5 � -bromo-2-deoxy-uridine) in physiological saline was placed on the mouthparts. Larvae were allowed to ingest the solution for 30 min as they re-covered from the cold, and then placed in a clean dish for 4 h. After treatment, heads were removed and fixed whole in Carnoy’s fixative for 2 h. Processing for paraffin embedding and sectioning and BrdU immunodetection was performed as described by Far-ris and Strausfeld [2001], with the exception that fluorescent sec-ondary detection using a Texas Red-labeled antibody was used rather than enzymatic detection. Autofluorescence generated by the Argon laser of the confocal microscope was used to visualize nonstained tissue. Scans of sequential tissue sections were col-lected on the confocal and merged and adjusted for brightness and contrast in Adobe Photoshop.

Popillia pupae were injected with 25 mg/ml BrdU and allowed to incorporate the label for 4 h at room temperature. Brains were then dissected and fixed overnight in 4% paraformaldehyde in PBS, after which they were washed and processed for vibratome sectioning as described above. Agarose sections at 70 � m were washed 3 ! 10 min in PBST, incubated for 45 min in 2N HCl to

denature DNA and blocked for 1 h in 10% normal goat serum, prior to overnight incubation at room temperature on an orbital shaker in a 1: 1000 concentration of mouse anti-BrdU primary antibody (Becton-Dickinson, Franklin Lakes, N.J., USA) in 1% NGS in PBST. At the same time, sections were incubated in a1: 1000 concentration of rabbit anti-DC0 (a generous gift of Dr. Daniel Kalderon). The latter antibody was raised against the cat-alytic subunit of Drosophila melanogaster protein kinase A, and is an excellent marker of developing and adult Kenyon cells [Skou-lakis et al., 1993; Farris et al., 2004; Farris, 2005a, b]. The next day, sections were washed 6 ! 20 min in PBST and then incubated in Alexa 488 conjugated goat anti-mouse and Texas Red conjugated goat anti-rabbit secondary antibodies at a 1: 250 concentration in 1% NGS in PBST overnight (both secondaries from Molecular Probes). The following day, tissue was washed 6 ! 20 min in PBST, cleared in glycerol and imaged on the confocal as described for dextran fills.

In order to compare neurogenesis patterns with those of scar-ab beetle larvae, brains of first instar Blaberus giganteus nymphs were dissected in physiological saline and placed in 25 mg/ml BrdU in physiological saline for 4 h at room temperature on an orbital shaker. Brains were then washed rapidly in physiological saline and fixed in 4% paraformaldehyde overnight, after which they were processed for BrdU immunostaining as described for Popillia pupae. Texas Red conjugated goat anti-mouse secondary antibody was used to visualize BrdU labeling, and Argon laser-generated autofluorescence illuminated the surrounding tissue.

Anti-DC0 Immunostaining of Paraffin Embedded Tissue For comparison with the brains of adult scarab beetles, the

brains of adult cockroaches (Periplaneta americana) were re-moved in physiological saline and fixed for 2 h in Carnoy’s fixa-tive, then processed for paraffin embedding and sectioning. The 10 � m sections were mounted on slides, deparaffinized, rehydrat-ed, and washed in PBS. Slides were incubated with blocking solu-tion and rabbit anti-DC0 primary as described above. Signal was detected using a horseradish peroxidase conjugated goat anti-rab-bit secondary (Jackson Immunochemicals, West Grove, Pa., USA) and the chromogenic substrate 3,3 � -diaminobenzidine (Sigma-Aldrich, St. Louis, Mo., USA).

Results

Distribution of Large, Double-Calyx Mushroom Bodies across the Insecta The mushroom bodies of all insects might be recog-

nized according to their conserved cellular organization, consisting of hundreds to thousand of minute, granule cell-like intrinsic neurons with parallel projecting pro-cesses [Strausfeld et al., 1998]. A survey of the insects re-veals substantial variation upon this groundplan, how-ever, some of which is not clearly associated with phylo-genetic relationships, suggesting lineage-specific selective pressures on mushroom body structure ( fig. 1 ). For ex-ample, the mushroom bodies of cockroaches (Dictyop-



Archaeognatha Zygentoma Paleoptera Orthoptera Dictyoptera Paraneoptera Hymenoptera Coleoptera Neuropterida Diptera Lepidoptera

C

Fig. 1. Large insect mushroom bodies and their distribution across taxa. A , B Anti-DC0 staining of the mushroom bodies in the brain of the cockroach, Periplaneta americana . The lobes (V, M; made up of Kenyon cell axons), take up a large propor-tion of the brain ( A ); the calyces (Ca; com-posed of Kenyon cell dendrites) are dou-bled and cup-shaped ( B ). C Large mush-room bodies (circles), as characterized by doubled, convoluted calyces, are found only in three distantly related taxa: the cockroaches (Dictyoptera), higher Hyme-noptera (ants, bees and wasps) and the scarab beetles (Coleoptera). Insect phylo-genetic tree is adapted from Grimaldi and Engel [2005]. Kcb = Kenyon cell bodies;Pe = pedunculus of the mushroom bodies; V = vertical lobe; M = medial lobe. Scale bars = 100 � m.

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tera) are strikingly enlarged, with almost 175,000 intrin-sic neurons (Kenyon cells) per hemisphere [Neder, 1959] and doubled and convoluted sensory input neuropils called calyces [Strausfeld and Li, 1999a] ( fig. 1 A, B). Large mushroom bodies with double calyces are also found in the apocritan Hymenoptera [ants, bees and wasps; Witt-höft, 1967; Gronenberg, 2001] and in scarab beetles [Co-leoptera; Farris and Roberts, 2005]. Mapping the occur-rences of large mushroom bodies onto a simplified phy-logenetic tree of the Insecta [Grimaldi and Engel, 2005] reveals that these taxa are not closely related, and are un-likely to have acquired this particular trait through a common ancestor. The enlarged mushroom bodies of these divergent taxa are thus examples of parallel evolu-tion, having arisen independently from common ances-tors with smaller mushroom bodies and single calyx neu-ropils.

Relationships between Mushroom Body Size, Subcompartmentalization and Acquisition of Visual Inputs in Generalist and Specialist Scarabs: Stereological and Fluorescent Tracer Experiments A previous study demonstrated profound differences

in brain structure between scarab beetles with a general-ist, plant-feeding ecology (families Dynastinae, Ceto-niinae, Rutelinae and Melolonthinae) and those with a specialist, dung-feeding ecology (families Aphodiinae and Scarabaeinae) [Farris and Roberts, 2005]. Qualitative and quantitative comparisons were made between Ca-son’s stained preparations of just two species of scarab beetle, the specialist Onthophagus hecate (Scarabaeinae) and the generalist Popillia japonica (Rutelinae), that em-phasized the dramatic differences in mushroom body morphology related to the two feeding ecologies ( fig. 2 ). Onthophagus , a dung-feeding scarab, possessed a small, ovoid calyx and thin, knobbed medial and vertical lobes ( fig. 2 A, C). Popillia , a plant-feeding scarab that has been documented to feed on the leaves, flowers and fruits of over 300 plant species [Potter and Held, 2002], displayed a greatly elaborated lobe system and two distinct calyces characterized by dorsal and ventral subdivisions ( fig. 2 B, D). Relative mushroom body volume was approximately three times greater in Popillia than in Onthophagus , and Kenyon cell number was an order of magnitude greater in Popillia ( fig. 2 E, F; 1-way ANOVA F = 371.67, p ! 0.0001 and F = 99.70, p ! 0.0001 respectively).

Further examination of histological preparations of the mushroom bodies revealed clear subcompartments in the large calyces of the generalist Popillia that were lacking in the smaller calyx of the dung beetle Onthopha-

gus ( fig. 2 A, B, arrows). The presence of significant optic lobe input to the mushroom bodies of aculeate Hyme-noptera [Gronenberg, 2001], which forage diurnally as do most generalist scarabs, suggested that the novel subcom-partments in Popillia might also be receiving optic lobe input. As predicted, applications of fluorescent dextrans to the vision-processing optic lobes of both Popillia and Onthophagus adults revealed that the Popillia mushroom bodies alone received input from these neuropils ( fig. 3 A, B). Dextran-labeled optic lobe outputs in both species targeted several areas of the protocerebrum, but only in Popillia did one tract target the ventral subcompartment of the mushroom body calyces. Additional subcompart-ments in Popillia, as well as other generalist species test-ed, were observed to receive olfactory input from the an-tennal lobes. This was demonstrated by dextran fills ap-plied to the antennal lobes of the melolonthine Maladera castanea ( fig. 3 E, olfactory; fig. 3 F, visual). Dextran fills similarly placed in the optic lobes of Onthophagus adults revealed no visual innervation of the calyx (n = 3 indi-viduals, with dextrans applied to the optic lobes of both brain hemispheres; fig. 3 C, D).

Evolutionary Variability of Mushroom Body Size and Visual Inputs in Generalist Scarab Species: Stereological and Fluorescent Tracer Experiments The impact of the generalist feeding ecology on mush-

room body size and sensory input was further illustrated by comparisons among generalist scarab species. Linear regression and residual analysis of volume estimates cal-culated from Cason’s stained preparations revealed the relationship between body length and total central brain volume ( fig. 4 A) and body length and mushroom body volume ( fig. 4 B) in twenty-three individuals representing five species and four generalist subfamilies of the Scara-baeidae [Farris and Roberts, 2005]. Both brain measures correlated highly with body length (R 2 = 0.9521, p ! 0.0001 and R 2 = 0.8867, p ! 0.0001 for comparisons of central brain volume and mushroom body volume, re-spectively). Interestingly, measurements of brain and mushroom body size fell below the regression line for the species Cyclocephala borealis (Dynastinae), although the difference was not significant according to residual anal-ysis (confidence curves set at � = 0.05). Histological prep-arations of brains from scarabs belonging to generalist subfamilies revealed that Maladera castanea (Melolon-thinae; fig. 3 E inset) and Cotinus mutabilis (Cetoniinae; fig. 4 C) possessed mushroom bodies like those of Popil-lia , with large, convoluted calyces divided into clear sub-compartments. In contrast, the calyces of Cyclocephala



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Fig. 2. Qualitative and quantitative differences in scarab beetle mushroom bodies. A Cason’s staining of a single, ovoid calyx (Ca) topped by a small number of Kenyon cell bodies (Kcb) in the brain of the specialist dung feeding scarab Onthophagus hecate . B Dou-bled, shallow cup-shaped calyces of the generalist herbivore Popil-lia japonica , surrounded by a cloud of tightly-packed Kenyon cell bodies. Arrowheads indicate histologically distinct subcompart-ments in the ventral calyces. C , D Mushroom body lobes of On-thophagus ( C ) and Popillia ( D ) illustrating the difference in size and thickness between the two species. E Total mushroom body

neuropil volume relative to total central brain volume in the spe-cialist Onthophagus vs. the generalist Popillia (n = 8 for each spe-cies) demonstrates the dramatic increase in mushroom body size in the generalist. F Kenyon cell number was approximately 7-fold less in Onthophagus than in Popillia . Significant differences indi-cated by * as determined by 1-way ANOVA followed by a Student t test. Pe = Pedunculus; V = vertical lobe; M = medial lobe. Verti-cal black lines in A , C and D = midline of the brain. Scale bars = 20 � m.

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Fig. 3. Patterns of calyx innervation in generalist and specialist scarabs. A , B Low-power ( A ) and high-power ( B ) views of axons filled with dextrans applied to the ipsilateral optic lobe (OL) in Popillia . Innervation of the ventral portion on the calyces (Ca), particularly in the lateral calyx, is indicated by arrowheads. C , D Bilateral optic lobe fills in two Onthophagus individuals revealed no innervation of the calyx; rather, labeled tracts passed by the calyx without producing collaterals (arrowheads in D ). E Dextran applied to the ipsilateral optic lobes of another generalist species,

Maladera castanea , also filled inputs to two regions of the ventral calyces (arrowheads). Cason’s staining (E inset) showed doubled calyces (arrows) with subcompartmentalization in this species. F Dextran applied to the antennal lobe of Maladera castanea filled inputs to four dorsal and one ventral subcompartment of the ca-lyx (arrowheads), and appeared to be nonoverlapping with the visual inputs from the optic lobes. Scale bars = 50 � m ( A , C , D ), 20 � m ( B , E , E inset, F ).



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Fig. 4. Specialization or loss of adult feeding is associated with a decrease in mushroom body size and complexity. A , B Linear re-gression fit of the relationship between body length and central brain volume ( A ) and mushroom body volume ( B ) in generalist scarabs. Circles at lower left of graphs indicate Cyclocephala bo-realis individuals; the circles at upper right highlight a single spec-imen of Chrysina gloriosa . Measurements for both species fell be-low the predicted regression line, but significantly so only for Chrysina (residuals analysis, � = 0.05). C Cason’s staining of Co-tinus mutabilis demonstrated distinct subcompartmentalization of the double calyces (arrows). D Cason’s staining of the calyces of

Cyclocephala , which although doubled, appeared flattened and without the subcompartments characteristic of other generalist species. E DiI filling of the ipsilateral optic lobe in Cotinus re-vealed massive output to the mushroom bodies, particularly to the lateral (left) calyx (arrows). F Application of fluorescent dex-trans to the ipsilateral optic lobe (large arrows) and antennal lobe (small arrows) labeled diffuse olfactory inputs and no visual in-puts to the calyces in Cyclocephala (indicated by dotted ovals). Although Cyclocephala belongs to a generalist subfamily (Dynas-tinae), this species does not feed as an adult. Scale bars = 50 � m ( C ), 20 � m ( D–F ).

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mushroom bodies were flattened and lacked any discern-able subcompartments ( fig. 4 D). Dextran fills applied to the optic lobes of these species confirmed the presence of visual input to calycal subcompartments in Maladera ( fig. 3 F) and Cotinus , the latter revealing a novel dorsal compartment in the lateral calyx of this species ( fig. 4 E). Optic lobe fills in Cyclocephala brains, however, revealed no input from these neuropils to any part of the calyces

( fig. 4 F, dotted ovals), although fibers leaving the optic lobes and bypassing the calyces were visible (large ar-rows). In contrast, fluorescent dextrans applied to the ol-faction-processing antennal lobes revealed ascending fi-bers and their collaterals projecting diffusely throughout the calyces (small arrows). One potential explanation for the total loss of visual input to the calyces in Cyclocepha-la is that this species has an abbreviated, non-feeding

Fig. 5. Mushroom body development in generalist and specialist scarabs. A , B Four hour BrdU incorporation in newly hatched lar-vae of Onthophagus ( A ) and Popillia ( B ). A Two large proliferating cells (arrows) associated with a small number of smaller ganglion mother cells were present in each hemisphere of the Onthophagus brain. Proliferation as indicated by BrdU incorporation was evi-dent in neuroblasts outside of the mushroom bodies, but no neu-ropil could be detected at this stage. B Two dense clusters of pro-liferating cells (likely neuroblasts and ganglion mother cells) were present in each hemisphere of the Popillia brain (arrows). The ca-lyces were also visible (Ca, outlined), indicating that Kenyon cell

production and process outgrowth had begun prior to hatching. C Mushroom body calyces (Ca) and neuroblasts (arrows) of a Bla-berus giganteus first instar nymph, illustrating the similarities in neuroblast proliferation in this distantly related species. D BrdU (nuclei, arrows) and anti-DC0 labeling (calyces, Ca) of an early Popillia pupa revealed a large number of mitotically active cells forming two clusters, one associated with each calyx. These clus-ters appeared to consist both of larger neuroblasts (arrowheads) and smaller ganglion mother cells or newborn Kenyon cells (ar-rows). Scale bars = 20 � m ( A–C ), 50 � m ( D ).



adult stage [Ritcher, 1957; Krischik and Davidson, 2004]. Cyclocephala adults therefore are unlikely to rely on vi-sual cues to learn the location of food or other resources in their environment. Interestingly, mushroom body vol-ume and central brain volume measurements that did fall significantly below the regression line according to re-sidual analysis were observed for a single specimen of Chrysina (Plusiotis) gloriosa ( fig. 4 A, B; confidence curves set at � = 0.05). Chrysina is a highly specialist feeder on the leaves of juniper trees [Arnett et al., 2002], a much narrower host range than that of the other ruteline ana-lyzed here, Popillia japonica .

Comparisons of Early Neurogenesis Patterns in the Mushroom Bodies of Generalist and Specialist Scarabs: BrdU Incorporation Experiments and Comparisons with Cockroaches Treatment of newly hatched Onthophagus and Popillia

larvae with the mitotic cell marker 5 � -bromo-2-deoxyuri-dine (BrdU) revealed distinct differences in mushroom body precursor pools ( fig. 5 A, B). Two large neuroblasts ( fig. 5 A, arrows) associated with four to six smaller gan-glion mother cells (GMCs) were labeled in each hemi-sphere of the Onthophagus brain, as were a number of other protocerebral neuroblasts. In contrast, BrdU treat-ment of newly hatched Popillia larvae labeled two com-pact clusters containing several neuroblasts and smaller GMCs (arrows, fig. 5 B). Small numbers of single neuro-blasts as observed in Onthophagus are typically observed in insects in which the mushroom bodies are relatively small and simple [Farris and Sinakevitch, 2003; Urbach and Technau, 2003]. Neuroblast clusters, in contrast, are characteristic of insects with large mushroom bodies, such as cockroaches, crickets and honey bees ( fig. 5 C).

Comparisons of the Timeline of Mushroom Body Development in Generalist and Specialist Scarabs: BrdU Incorporation Experiments and Life Cycle Comparisons Four-hour BrdU treatments of Popillia at the early pu-

pal stage revealed that the number of mitotically active cells was enormously increased ( fig. 5 D) compared with the newly hatched larva ( fig. 5 B). It appeared that many of the labeled cells in the pupal mushroom bodies had smaller cell bodies, reminiscent of GMCs or Kenyon cells ( fig. 5 D, arrows), suggesting that neuron production was occurring at a rapid rate in this developmental period. Finally, a review of the literature showed that generalist scarab beetles such as Popillia develop at a much slower rate than do specialists such as Onthophagus . The total

time for development from egg to adult was greatly in-creased in Popillia , taking place over the course of a year, whereas the life cycle of the dung beetle Onthophagus taurus (a species closely related to Onthophagus hecate ) lasted just over a month (table 1) [Emlen and Nijhout, 1999; Potter and Held, 2002].

Discussion

The Relationship between Behavioral Ecology and Large Brains and Higher Brain Centers Behavioral ecologies that require complex sensory

processing, decision-making, place memory and learn-ing are associated with the evolution of larger brains and/or higher brain regions in birds and mammals [Krebs et al., 1989; Reader and Laland, 2002; Lefebvre et al., 2004; Sol et al., 2005a]. As in vertebrate higher brain centers, the insect mushroom bodies function in associative, con-text dependent and spatial learning and memory [Mizu-nami et al., 1998; Liu et al., 1999; Roman and Davis, 2001], and in sensory integration [Schildberger, 1984; Li and Strausfeld, 1997, 1999]. In both vertebrates and insects, reliance upon these behaviors and associated enlarge-ment of the relevant brain centers might be encountered in the context of food acquisition. Examples in verte-brates include large hippocampi in food-caching parid birds that must remember the locations of their hidden food stores, [Krebs, 1990] and larger total brain sizes in

Table 1. Development times from egg to adult for two scarab bee-tle species

Onthophagus taurus Popillia japonica

Egg – L2 7.881.3 days Egg 10–14 daysL1 14–21 daysL2 21–28 days

L3 stage I 7.082.5 days L3 pre-diapause �28 daysL3 stage II 1.881.3 days L3 post-diapause 28–56 daysL3 stage III 1.080.6 daysL3 stage IV 1.680.6 daysL3 prepupa 1.680.5 days L3 prepupa 10 daysPupa 8.781.0 days Pupa 7–17 days

Total 21.7437.3 days 118–174 days

The specialist dung feeding scarab, Onthophagus taurus, pro-ceeds more rapidly through each developmental stage than does the generalist herbivore Popillia japonica [Emlen and Nijhout, 1999; Potter and Held, 2002].

Farris


birds that display flexible and innovative food acquisition behaviors [Sol et al., 2005b]. In insects, large mushroom bodies are found in generalist plant-feeding scarab bee-tles that must assess and select among many potential host plants, perhaps learning the locations of particular-ly suitable hosts for repeated visits [Farris and Roberts, 2005].

The Evolutionary Acquisition of Novel Subcompartments Subserving Novel Functions in Large Higher Brain Centers It has been noted in mammals that as brains and brain

regions increase in size, they become increasingly sub-compartmentalized [Kaas, 1995; Northcutt and Kaas, 1995; Catania, 2005; Changizi and Shimojo, 2005; Strei-dter, 2005]. One potential explanation for this evolution-ary trend is that as neuron number increases in large brains, it becomes prohibitively expensive energetically to build and maintain connections among them all. Net-works therefore evolve to minimize long-distance con-nections while maintaining local circuits, the latter of which may diverge into distinct functional subcompart-ments [Ringo, 1991; Kaas, 2000]. These subcompart-ments might act as parallel processing units for different aspects of a single computational task, as observed in the 30+ functional subdivisions of the macaque visual cortex [Kaas, 1995; Northcutt and Kaas, 1995], or acquire en-tirely new functions, such as the specialization of Broca’s area of the cortex for language processing in humans [Cooper, 2006].

Energetic costs of maintaining neuronal connectivity are likely to represent a universal constraint on brain evo-lution, so it is perhaps unsurprising to find that the larg-est insect mushroom bodies are highly subcompartmen-talized as well. For example, FMRFamide-like immuno-histochemistry reveals twelve distinct modules of Kenyon cells in the mushroom body lobes of the honey bee Apis mellifera , but just six in the much smaller mushroom bodies of the moth Spodoptera littoralis [Strausfeld et al., 2000; Sjöholm et al., 2006]. The calyces of social Hyme-noptera such as the honey bee are also subdivided into subcompartments, each comprising characteristic arrays of Kenyon cell dendrites and their sensory inputs, includ-ing visual inputs [Gronenberg and Hölldobler, 1999; Ehmer and Gronenberg, 2002; Strausfeld, 2002; Lòpez-Riquelme and Gronenberg, 2004].

Visual inputs to the mushroom bodies of most other insects appear to be rare and, if present, minimal [Straus-feld and Li, 1999b]. The presence of significant visual in-put to social hymenopteran mushroom bodies has thus

been attributed to the particular reliance of these species on visual cues for learning landmarks used in spatial ori-entation and navigation relative to a central nest [Gronen-berg and Hölldobler, 1999]. In generalist scarabs, the di-urnally foraging Popillia shows color preferences when choosing floral hosts [Held and Potter, 2004], indicating that visual cues are of at least some importance to this insect. It is therefore tempting to speculate that over the course of their four-week lifespan (which is relatively long for an insect), Popillia adults might use visual informa-tion to identify and remember palatable food sources. In contrast, dung beetles such as Onthophagus rely primar-ily on olfactory cues to locate their frequently cryptic and ephemeral food source [Martin-Piera and Lobo, 1996; Dormont et al., 2004]. Ball-rolling species do use polar-ized moonlight for orientation when rolling a ball of dung away from a central source [Dacke et al., 2004], but in lo-custs neurons conveying polarization information from the optic lobes have been shown to project to another in-tegrative region called the central complex [Heinze and Homberg, 2007]. Given their lifestyle it is therefore un-likely that dung beetles such as Onthophagus rely heavily on visual associative learning or spatial learning. These differences in behavioral ecology appear to be reflected in the presence (Popillia) or absence (Onthophagus) of di-rect input from the vision-processing optic lobes to the mushroom body calyces. This suggests that the capacity for mushroom body-mediated visual learning has been selected for in generalist scarabs such as Popillia , but not in specialists such as Onthophagus .

What could be the significance of the apparent de-crease in mushroom body size and concomitant loss of visual input to the calyces in Cyclocephala ? One possibil-ity is suggested by the observation that Cyclocephala is nocturnal [Krischik and Davidson, 2004]. This lifestyle might be expected to select for a greater reliance on olfac-tory rather than visual cues for host plant location, and thus a loss of visual input to the calyces. This is unlikely to be the correct explanation, however, as the generalist Maladera castanea also flies at night and yet receives sig-nificant optic lobe input to the ventral calyx ( fig. 4 E). In-stead, the primary manner in which Cyclocephala borea-lis differs from all other generalist species analyzed is that they are non-feeding and live only a few days as adults [Ritcher, 1957; Krischik and Davidson, 2004]. According to the hypothesis that large mushroom bodies with vi-sual input are adaptations to a generalist feeding ecology, species in which adults are short-lived and non-feeding would undergo a release upon the selective pressure for maintaining a neural substrate for complex sensory inte-



gration and learning and memory functions. Similarly, specialist feeders, whether diurnal or nocturnal, are like-ly to rely on a small number of definitive odor cues that allow robust, reliable and rapid detection of the host plant [Bernays, 2001]. The extreme specialist lifestyle of the ruteline Chrysina gloriosa could thus be sustained with a lesser reliance on complex cognition and the underlying neural circuitry, leading to the observed evolutionary de-crease in mushroom body size.

Developmental Modifications Associated with the Evolution of Large Higher Brain Centers In mammals, evolutionary changes in brain size have

targeted developmental pathways regulating precursor proliferation dynamics and neuronal survival [Kornack and Rakic, 1998; Kouprina et al., 2004]. Accelerated adap-tive evolution of genes regulating neural precursor num-bers [Kouprina et al., 2004; Evans et al., 2004a, b; Fish et al., 2006] and increased numbers of non-differentiating progenitor cell divisions leading to a larger neural pre-cursor pool are observed in primates [Kriegstein et al., 2006] that have undergone a dramatic evolutionary ex-pansion of the cerebral cortex. In addition, a longer pe-riod of cortical development in primates as opposed to mammals such as rodents allows time for additional pre-cursor divisions, and thus an increase in neuron produc-tion and total cortical volume [Finlay and Darlington, 1995; Kornack and Rakic, 1998]. The present account demonstrates that both an increase in neural precursor number and in brain region developmental time is ob-served in the large mushroom bodies of Popillia , in com-parison with the smaller mushroom bodies of Onthopha-gus .

In mammals, cortical precursors undergo a period of symmetric divisions in which they increase their num-bers, and then switch to asymmetric, neurogenic divi-sions [Rakic, 1995]. Changes in precursor proliferation dynamics, such as number of symmetric vs. asymmetric divisions and total number of cell divisions, have been proposed as evolutionary mechanisms for changes in ver-tebrate cortical size [Kornack and Rakic, 1998; Kriegstein et al., 2006]. In the honey bee mushroom bodies, four proliferating clusters consisting of approximately 50 neu-roblasts each at hatching increase in number via symmet-ric divisions throughout the larval stage [Farris et al., 1999]. By the onset of metamorphosis, each cluster con-tains nearly 500 neuroblasts for a total of 1,000 neuro-blasts per mushroom body. This enormous number of neuroblasts generates approximately 170,000 Kenyon cells in a period of active neurogenesis that lasts only ten

days [Witthöft, 1967; Farris et al., 1999]. In the scarab Popillia an increase in neuroblast number, probably due to the novel acquisition of a period of symmetric divi-sions in the early larva, further combined with an in-creased duration of mushroom body neurogenesis, pro-vides a sufficient developmental basis for increased Ken-yon cell number and mushroom body size. That these developmental mechanisms are associated with increases in higher brain region size in mammalian lineages sug-gests that convergent developmental mechanisms at the cellular (and perhaps molecular) level underlie conver-gent increases in the size and complexity of higher brain centers.

Conclusions

In both insects and vertebrates, the capacity for flexi-ble behavior is associated with the acquisition of large higher brain centers. Enlargement of these brain regions is accompanied by their increasing subcompartmental-ization and acquisition of new processing capabilities, and an enlargement of the neural precursor pool and ex-tension of the developmental period during which the brain develops. These commonalities exist despite 600 million of years of divergence between protostomes (in-sects) and deuterostomes (vertebrates). Together, the ob-servations of this account suggest that developmental, structural and functional constraints that have acted during vertebrate brain evolution also operate on inver-tebrate nervous systems. These conserved constraints, hallmarks of the shared ancestry of the bilaterian ner-vous system, predictably shape adaptations of higher brain centers that emerge under similar selective pres-sures for behavior.

Acknowledgements

The author thanks Mr. Todd Stueckle for assistance with sta-tistical methods and Mr. Glenn Farris and Mr. Justin Miller for assistance with insect collecting; Dr. Daniel Kalderon for provid-ing anti-DC0 antibody and Dr. Roy Ritzmann for providing Bla-berus giganteus ; and Dr. Ronald Bayline for helpful comments on this manuscript. This research was funded in part by a West Vir-ginia University Faculty Senate Grant.

Farris


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Structural, Functional and Developmental Convergence of ...sfarris/2008FarrisScarabConverg.pdf · with backbones. In vertebrates, these functions are per-formed by higher brain centers