Evolution of insect mushroom bodies: old clues, new insightssfarris/2005FarrisASDRev.pdfmushroom...

Evolution of insect mushroom bodies: old clues, new insights

Sarah M. Farris*

Department of Biology, West Virginia University, 3139, Life Sciences Building, 53 Campus Drive, Morgantown, WV 26506, USA

Received 20 October 2004; accepted 7 January 2005

Abstract

The mushroom bodies are a morphologically diverse sensory integration and learning and memory center in the brains of various

invertebrate species, of which those of insects are the best described. Insect mushroom bodies are composed of numerous tiny intrinsic

neurons (Kenyon cells) that form calyces with their dendrites and a pedunculus and lobes with their axons. The identities of conserved

Kenyon cell subpopulations and the correlations between morphological and functional specializations of the mushroom bodies are just

beginning to be elucidated, providing insight into mechanisms of mushroom body evolution. Comparisons of mushroom body organization

in different insect lineages reveal trends in the evolution of subcompartments correlated with the elaboration, reduction, acquisition or loss of

Kenyon cell subpopulations. Furthermore, these changes often appear correlated with variation in type and strength of afferent input and in

behavioral ecology. These and other features of mushroom body organization suggest a striking convergence with mammalian cortex, with

Kenyon cell subpopulations displaying evolutionary modularity in a manner reminiscent of cortical areas.

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Keywords: Afferents; Behavioral ecology; Brain evolution; Comparative anatomy; Convergence; Cortex

1. Introduction

Dujardin (1850) first described the insect mushroombodies and the correlation between their increased size andthe acquisition of social behavior in the Hymenoptera. Thisprovocative connection paved the way for a rich body ofinvestigations into mushroom body function that soonrevealed the importance of these neuropils in a number ofcomplex behaviors such as sensory integration (Li andStrausfeld, 1997, 1999; Schildberger, 1984), place memoryand motor control (Mizunami et al., 1993), visual navigationduring foraging (Capaldi et al., 1999 for review), and certaintypes of learning and memory, particularly olfactoryassociative and context-dependent learning and memory(reviewed in Waddell and Quinn, 2001; Heisenberg, 2003).With the establishment of the fruit fly Drosophilamelanogaster as a key molecular genetic model system,the insect mushroom bodies have become an importantsource of information on the molecular basis and cellularlocalization of learning and memory (Zars et al., 2000;

Pascual and Preat, 2001; McGuire et al., 2003). Morerecently, insect mushroom bodies have emerged as a modelof neuronal plasticity and development (Watts et al., 2003,2004; Zhu et al., 2003; Awasaki and Ito, 2004; reviewed inFarris and Sinakevitch, 2003).

The early mushroom body literature is a treasure trove ofcomparative anatomy. As suggested by Dujardin’s work,mushroom body morphology is enormously diverse acrossthe insects and in some cases robustly correlated withbehavioral ecology. Unfortunately, the technical limitationsof older studies led to difficulties with interpretation or evenerroneous conclusions about the anatomy, connectivity andevolution of insect mushroom bodies. In particular, a lack ofknowledge about the cellular makeup of the insect brainmade the identification of homologous structures and tractsacross taxa a difficult task. This began to change in the1970s and 80s with detailed analyses of mushroom bodyintrinsic neurons, now termed Kenyon cells, that allowedthe general groundplan of mushroom body organization tobe elucidated (Pearson, 1971; Schurmann, 1973; Strausfeld,1976; Mobbs, 1982). The more recent focus on cellular,developmental and gene expression analyses in modelspecies has resulted in relatively fewer forays intocomparative studies, however, and as a result currentunderstanding of mushroom body morphological and

Arthropod Structure & Development 34 (2005) 211–234

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functional evolution remains obscure. Nevertheless,detailed investigations of four distantly related insectspecies, the cricket Acheta domestica (Orthoptera), thecockroach Periplaneta americana (Dictyoptera), the fruitfly D. melanogaster (Diptera) and the honeybee Apismellifera (Hymenoptera) have built up a strong theoreticalframework from which evolutionary hypotheses may begenerated and tested via the appropriate speciescomparisons.

Detailed accounts of the history of insect mushroombody research and the evolutionary implications of modernstudies are already available (Strausfeld et al., 1995, 1998).In the years since these reviews were published, however,further significant progress has been made in the study of thecellular organization of mushroom body circuitry. Thepresent article seeks to integrate earlier studies of mushroombody anatomy with these more recent advances, with thegoal of identifying evolutionary trends and suggestingpossible cellular and functional correlates of these trends. Inlight of these comparisons, the high degree of convergenceof insect mushroom bodies with the vertebrate cortex interms of function, structure and developmental mechanismsbecomes increasingly evident (Mizunami et al., 1997;reviewed in Strausfeld et al., 1998; Mizunami et al.,2004). Further comparative studies to test and expand onhypotheses about mushroom body evolution will thusprovide insight into the interplay between structure andfunction during evolution of higher brain centers in animals.

2. What are mushroom bodies?

The term ‘mushroom bodies’ applies to complexneuropils in the brains of several invertebrate groups,including insects. Flogel (1878; reviewed in Strausfeldet al., 1998) defined mushroom bodies as containinghundreds to thousands of tiny cell bodies (globuli cells)that supply a lobed neuropil with their parallel fibers.According to this definition, mushroom bodies have beendescribed in chelicerates, diplopods, chilopods and somenon-insect hexapods (Arthropoda), as well as in three otherinvertebrate phyla, the Onychophora, the Annelida and thePlatyhelminthes (Holmgren, 1916; Strausfeld et al., 1995;Strausfeld, 1998). The putative homology of the crustaceanhemiellipsoid bodies, which are composed of globuli-likecells and are functionally similar to mushroom bodies, butlack the parallel fiber-containing lobes, is still under debate(Strausfeld, 1998; McKinzie et al., 2003). Considering thelack of mushroom bodies in the most basal hexapod lineages(Hanstrom, 1940), and assuming that crustacean hemiellip-soid bodies are not homologous to mushroom bodies, itseems likely that mushroom bodies arose independentlymore than once in the invertebrates (Strausfeld, 1998).Furthermore, evidence suggests that mushroom bodies havebeen acquired twice in the Hexapoda alone: in the non-insect Diplura and in the Insecta after the divergence of the

most basal extant insect lineage, the apterygote Archaeo-gnatha (Hanstrom, 1940). Until the taxonomic position ofthe Diplura is resolved, however, it must also be consideredthat mushroom bodies arose once within the hexapods andwere secondarily lost in the archaeognathan insects. Withinthe remaining insect species (the apterygote Zygentoma, thePalaeoptera and the Neoptera; see Fig. 1 for an overview ofinsect phylogeny), the mushroom bodies share a commongroundplan in terms of subcompartment specialization,cellular makeup, and afferent and efferent connectivity,which will be the focus of this article.

3. Methods

The following histological methods were used to revealmushroom body anatomy as seen in the figures accompany-ing this article.

3.1. Cason’s staining

Brains were dissected from the head capsule in physio-logical saline and fixed for 1 h 15 min to 2 h in Carnoy’sfixative at room temperature. After fixation brains weretransferred to 70% ethanol and refrigerated until furtherprocessing could take place. Brains were dehydrated in anascending series of ethanols, cleared in xylene andembedded in paraffin prior to sectioning at 10 mm thickness.Sections were mounted on slides and stained using Cason’sstain according to the protocol of (Kiernan, 1990), thencoverslipped in xylene-based mounting medium.

3.2. Anti-DC0 and phalloidin staining

Anti-DC0 antibody, a generous gift of Dr DanielKalderon, was used to label mushroom bodies in the brainsof a wide variety of insects. The antibody is derived againstthe catalytic subunit of protein kinase A in D. melanogaster(Skoulakis et al., 1993), and was visualized in this studywith Texas Red-conjugated goat anti-rabbit secondary anti-body (Molecular Probes, Eugene, OR, USA). Alexa 488-conjugated phalloidin, a fungal toxin that binds to filamentousactin, selectively labels ingrowing axons in the mushroombodies (Kurusu et al., 2002; Farris et al., 2004). This stainingallowed the location of older vs. younger Kenyon cells in thelobes to be determined. Double-stained preparations wereviewed on a Zeiss LSM 510 confocal microscope. A moredetailed double staining protocol is available in Farris andStrausfeld, (2003) and Farris et al., (2004).

4. Overview of insect mushroom body organization

The basic groundplan of the insect mushroom bodies andtheir spatial relationship to other brain structures is shown inFig. 2. The cell bodies of mushroom body intrinsic neurons,

S.M. Farris / Arthropod Structure & Development 34 (2005) 211–234212

called Kenyon cells, form a dense cluster in the dorsopos-terior protocerebrum. Each Kenyon cell produces a singleneurite that provides dendritic specializations to the calyxproximally and axon-like processes to the lobes distally.The calyx or calyces reside more or less anteroventral to thecell body region, and are surrounded by Kenyon cellsomata. The calyces receive sensory afferents, neuromodu-latory, and feedback elements (Fig. 2; Mobbs, 1982;Hammer, 1993; Grunewald, 1999; Li and Strausfeld,1999; Strausfeld and Li, 1999a). In this article two typesof calyces will be differentiated: primary calyces, made upof Classes I and II Kenyon cells, and accessory calyces,made up of Class III Kenyon cells (see Table 1 for Kenyoncell subpopulation definitions).

Anteroventral to the calyx, Kenyon cell processes funnelinto the pedunculus, where in some species they receiveadditional afferents from elsewhere in the protocerebrum(Schurmann, 1970, 1973; Strausfeld and Li, 1999a). Stillmore distal to the pedunculus are the lobes, made up ofKenyon cell axons and representing the primary outputregion of the mushroom bodies, although again afferentinput from the protocerebrum is targeted to this compart-ment as well (Li and Strausfeld, 1997). It is important topoint out that although the processes of Kenyon cells in thelobes are typically referred to as ‘axons’, evidence suggests

that they are both pre- and post-synaptic to extrinsic neuronsand to each other, and may thus be more properly regardedas amacrine-like (Strausfeld et al., 1998; Strausfeld, 2002).

A brief review of mushroom body development isessential for understanding spatial relationships betweenKenyon cell subpopulations and their processes in thecalyces and lobes; a more substantial review can be found inan earlier article by Farris and Sinakevitch (2003). Themushroom bodies of neopteran insects are composed ofdistinct Kenyon cell subpopulations that are producedsequentially during development by dedicated mushroombody neuroblasts (Ito et al., 1997; Lee et al., 1999). Kenyoncell bodies are passively pushed away from these proli-ferative centers as development progresses (Farris et al.,1999; Cayre et al., 2000; Farris and Strausfeld, 2001). As theneuroblasts are situated central to each calyx, the resultingarrangement of Kenyon cell bodies is age-based with thefirst-born subpopulations lying farthest away from the calyxcenter. An age gradient is also observed in the pedunculusand lobes, as newborn Kenyon cell axons enter thesesubcompartments via a centrally or posteriorly locatedingrowth core (Farris and Strausfeld, 2001; Kurusu et al.,2002; Malaterre et al., 2002; Farris et al., 2004). Theintegration of new axons into the lobes at a fixed pointprovides the basis for an age-based organization of axon

Fig. 1. Overview of the phylogenetic groupings of insect orders. Orange branches, Apterygota; green branches, Palaeoptera; blue branches, hemimetabolous

Neoptera; purple branches, holometabolous Neoptera. Figure is adapted fromWhiting et al. (1997) with permission of the author and of Taylor & Francis, Inc.

S.M. Farris / Arthropod Structure & Development 34 (2005) 211–234 213

Fig. 2. Overview of insect mushroom body organization, using the honeybee Apis mellifera as an example. A. Ethyl gallate-stained sagittal section of onehemisphere of the brain, showing the orientation of the mushroom bodies in the dorsal protocerebrum. Kenyon cell bodies surround the double calyces, which

contain the Kenyon cell dendrites. Kenyon cell axons funnel into the pedunculus (overlaid Golgi impregnation) and bifurcate (arrow) to form a medial and a

vertical lobe. Reproduced with permission from Farris et al., 2004, Journal of Comparative Neurology (copyright 2004, Wiley-Liss Inc.). B. Bodian-stained

frontal section of one hemisphere of the brain. The vertical lobe is not visible in this plane of section. Axes in the upper right corner of each panel indicate the


subpopulations via passive displacement of older fibers byprogressively younger fibers. Aside from being defined bybirthdate, Kenyon cell subpopulations are morphologicallyand molecularly unique, forming structural and functionalsubcompartments in the calyces and lobes.

Kenyon cell subpopulations display a bewilderingvariety of morphologies across and within taxa. Classifi-cation schemes have been proposed based on developmentalhistory, gene expression, neurotransmitter identity, afferentinput and process morphology. Using these characteristics,broadly analogous and possibly homologous subpopulationsmay be linked across taxa (Table 1). This article will refer toKenyon cell subpopulations in terms of a relatively simpleclassification derived from studies of the cockroachPeriplaneta, in which three broad intrinsic neuron types,termed Classes I, II and III, exist (Strausfeld and Li, 1999b;Farris and Strausfeld, 2003). This classification scheme willbe used to provide a conceptual framework at the cellular andsubcellular level for establishing relationships among mush-room body substructures in widely divergent insect taxa.

5. Mushroom body calyces

5.1. Morphology and organization of Kenyon cell dendrites

Dendritic morphology is a key characteristic defining

Kenyon cell subpopulations within species. The Classes III,II and I Kenyon cells, respectively, of Periplaneta are mosteasily differentiated by the trajectories of their proximalneuriltes, which travel along the outer primary calyxsurface, directly through the calyx neuropil or along theinner calyx surface (Fig. 3A and B; Strausfeld and Li,1999b; Farris and Strausfeld, 2003). Similar morphologiesdefine Kenyon cell subpopulations in Apis (Mobbs, 1982;Strausfeld, 2002), Acheta (Schurmann, 1973) and the scarabbeetle Pachnoda marginata (Coleoptera; Larsson et al.,2004). In the highly derived brachyceran fly D. melanoga-ster, however, all neurites pass along the outer surface of theprimary calyx (Strausfeld et al., 2003; Zhu et al., 2003),although the Drosophila mushroom bodies clearly containmore than a single Kenyon cell subpopulation includingputative Classes I and II cells. The use of proximal neuritemorphology as a primary indicator of Kenyon cellsubpopulation homology must, therefore, be applied withcare to comparisons among higher-level taxa.

Regardless of proximal neurite trajectory, the dendriticarbors of Classes I and II neurons characteristically overlapwithin the primary calyx neuropil (Mobbs, 1982; Mizunamiet al., 1998a; Strausfeld and Li, 1999b; Strausfeld, 2002;Strausfeld et al., 2003; Zhu et al., 2003; Ehmer andGronenberg, 2004; Tanaka et al., 2004). In Apis, Drosophilaand Periplaneta, Class II dendrites display a relativelyhomogeneous morphology and as a whole sample all

dorsal (D), ventral (V), anterior (A), posterior (P), medial (M) and lateral (L) directions. C. Schematic diagram of a generalized insect brain showing location ofthe mushroom bodies relative to other brain neuropils, and the trajectories of afferent (left hemisphere), efferent and recurrent (right hemisphere) extrinsic

neurons. Acc ca—accessory calyx, ant lo—antennal lobe, Cc—central complex, Kcb—Kenyon cell bodies, L Ca—lateral primary calyx, lo—lobula of the

optic lobe, lo glom—lobus glomerulatus, M—medial lobe, M Ca—medial primary calyx, me—medulla of the optic lobe, P ca—primary calyx, Pe—

pedunculus, pr—protocerebrum, sog—subesophageal ganglion, trito—tritocerebrum, V—vertical lobe. Scale bars A and BZ100 mm.

Table 1Classification of Kenyon cell subpopulations in the insect mushroom bodies

Kenyon cell

type charac-

teristics

Class I Class II Class III

General com-ments

Largest population of Kenyon cells, typi-cally consisting of many further subdivi-

sions according to neurotransmitter identity

and dendritic regionalization in the calyx

Also called ‘clawed’ or ‘g’ kenyon cells.Their distinct process morphology appears

to be particularly highly conserved across

widely divergent species. May represent an

ancestral cell type

First identified in some, but not allDictyoptera. Presence or absence is extre-

mely variable across the insects

Development Last born. Cell bodies and processes are

closest to the primary calyx center and

ingrowth core

First born, unless Class III cells are present.

Undergo metamorphic reorganization in

holometabolous insects

First born when present

Proximal

neurite

Often projects along the inner wall of the

primary calyx

Often projects directly through the synaptic

neuropil of the primary calyx

Projects along the outer surface of the

primary calyx

Dendrites Terminal morphology is extremely variable.

Dendritic arbors are segregated in theprimary calyx, according to afferent input

Distinct clawed terminals. As a whole, Class

II cells sample all regions and afferentsources of the primary calyx

Terminal morphology is variable. Usually

form a separate posteroventral ‘accessorycalyx.’ Associated with gustatory afferents

Axons Bifurcated to supply medial and vertical

lobes. Form distinct layers representing

different calycal subcompartments

Form a distinct, glial-delimited ‘g’ lobe orlayer representing the entire calyx. May be

unbranched in Holometabola

Often form a pedunculus and lobe system

that is separate from that of class I and II

cells

Morphological characteristics of Classes I, II and III Kenyon cell subpopulations. Mushroom body intrinsic neurons can be broadly classified according todevelopmental, structural and functional characteristics. Different insect species may display elaboration, reduction or absence of a given Kenyon cell

subpopulation. Each subpopulation may also consist of additional subdivisions.


regions of the primary calyx. In contrast, Class I cells areparsed into further cohorts with distinct dendritic mor-phologies that are more or less segregated into calyxcompartments defined by afferent input. These compart-ments may reflect afferents arising from different regionswithin a single sensory neuropil, afferents representing

entirely different sensory modalities, or both. Afferentsubcompartmentalization is particularly obvious in thesocial Hymenoptera, in which visual and olfactory afferentstarget specific, non-overlapping regions of the primarycalyx (Gronenberg, 2001) that correspond to the dendrites ofdistinct Class I cohorts (Strausfeld et al., 2000; Strausfeld,

Fig. 3. Frontal sectioned Golgi impregnations of Kenyon cell dendrites. A and B, Periplaneta americana, C, Acheta domestica. A. Class II (arrowheads) and

Class I neurites (arrows) in the primary calyces. Class I neurites line the inner calyx surface, while those of Class II cells pass through the synaptic neuropil ofthe calyx. B. Class I (arrowheads) and Class III neurites (arrows), the latter of which pass along the outer surface of the calyx cups and form a separate

accessory calyx ventral to the primary calyces. C. Comparison of Classes I, II and III dendritic specializations at the junction between the primary and

accessory calyces of the cricket mushroom bodies. Ca—primary calyx, Pe—pedunculus. Scale bars for A and BZ50 mm, CZ20 mm.


2002). In other insects Class I cohort dendritic fields are notas precisely defined and there is some degree of overlap(Mizunami et al., 1998a; Strausfeld and Li, 1999b; Ehmerand Gronenberg, 2004).

In contrast to the overlapping dendritic arbors of ClassesI and II cells, the dendrites of Class III cells are nearlyalways segregated into a separate accessory calyx posi-tioned posteroventral to the primary calyx (Kuhnle, 1913;Pflugfelder, 1936; Weiss, 1981; Farris and Strausfeld,2003). In the Lepidoptera, Class III neurons form adistinct accessory calyx in the butterflies (Ali, 1974), butappear to be integrated into the primary calyces of moths(Bretschneider, 1924a,b; Pearson, 1971).

The morphology of Kenyon cell dendritic terminals isgreatly variable, and the functional correlates of thesemorphologies are mostly unknown (Fig. 3C). Only the ClassII cells appear to be united by a single terminal type acrosstaxa, the unique ‘claws’ that have caused these neurons toalso be termed clawed Kenyon cells (Pearson, 1971;Schurmann, 1973; Strausfeld, 1976, 2002; Strausfeld andLi, 1999b; Strausfeld et al., 2003). In contrast, neither ClassI nor Class III cells may be defined by a single terminalmorphology. Class I cohorts vary in terminal type withinspecies, and the relationship among different Class I cohortsbetween species is not yet known. Some conserved motifsexist, however, as Class I cells studded with dendritic spinesand sparsely decorated with finely curling terminals havebeen observed in Acheta, Periplaneta and Apis (Schurmann,1973; Mizunami et al., 1998a; Strausfeld and Li, 1999b;Strausfeld, 2002; Strausfeld et al., 2003). Class III neuronshave only been described in detail for Periplaneta, and sharemany common developmental and morphological traits withthe putative Class III Kenyon cells making up the accessorycalyx of Acheta, yet there is little resemblance between themorphologies of their dendritic terminals (Schurmann,1973; Farris and Strausfeld, 2003).

Synapses within the calyx neuropils are organized intoglomeruli. In the firebrat Thermobia domestica, whichbelongs to the basal insect Order Zygentoma, theseglomeruli are few and large (10–15 mm), and are locatedon the outer surface of the calyx (Farris, 2005). In mostNeoptera, however, these structures are much smaller (andthus termed ‘microglomeruli’) and extremely numerous.The combined results of immunological and electronmicroscopical studies in Acheta, Apis andDrosophila revealthat microglomeruli are primarily composed of a cholin-ergic bouton from an afferent axon, surrounded by a numberof f-actin containing Kenyon cell dendrites and GABAergicterminals (Ganeshina and Menzel, 2001; Yusuyama et al.,2002; Frambach et al., 2004). Thus, microglomerulirepresent a region of convergence for excitatory andinhibitory inputs onto Kenyon cell dendrites.

5.2. Afferents to the calyces

Two main types of calycal afferents have been described:

afferents arising from primary sensory neuropils, andmultimodal afferents arising from higher-order centers inthe protocerebrum. In addition, GABAergic feedbackneurons may directly or indirectly connect the mushroombody lobes to the calyces, and aminergic and peptidergicneurons arising from elsewhere in the brain and subesopha-geal ganglion innervate the calyces. As previouslydescribed, many of these afferents are targeted to specificregions of the calyces corresponding to the dendritic fieldsof distinct Kenyon cell subpopulations; their variouspermutations across taxa are described in detail below.

Perhaps the largest source of afferents to the mushroombody calyces derives from primary sensory neuropils (Figs.1C and 4). As with most insect sensory neurons, theseafferents are cholinergic (Yusuyama et al., 2002), and thepost-synaptic Kenyon cells express acetylcholinesterase andnicotinic acetylcholine receptors (Bicker, 1999; Goldberget al., 1999; Shapira et al., 2001; Homberg, 2002; Thanyet al., 2003). In terrestrial insects, antennal projectionneurons carrying olfactory and mechanosensory informationfrom the glomerular antennal lobe of the deutocerebrum area dominant source of input to the primary calyces (Figs. 1Cand 4; Mobbs, 1982; Schildberger, 1984; Ito et al., 1998;Strausfeld and Li, 1999a; Frambach and Schurmann, 2004).Axons of antennal projection neurons travel ipsilaterallythrough the central protocerebrum via a distinctive innerantennocerebral tract (iACT; Homberg et al., 1989). TheiACT curves beneath the primary calyces, where it providesnumerous tributaries, and ends in the lateral protocerebrum.The iACT may also be split into medial and lateralcomponents that both supply the primary calyces (forexample in the Hymenoptera; Mobbs, 1984). Axons ofantennal projection neurons are often further parsed intocalycal subdivisions according to the groups of antennallobe glomeruli that they represent (Strausfeld and Li, 1999a;Gronenberg, 2001; Tanaka et al., 2004).

In some insects such as Drosophila, the antennal lobesare the sole source of input from primary sensory neuropilsto the mushroom bodies (Ito et al., 1998). Other insects mayreceive additional afferents from primary neuropils repre-senting different sensory modalities. The tritocerebral tract(TT) in neopteran insects contains neurons carryingmechanosensory and gustatory information from thetritocerebrum, non-olfactory deutocerebrum and possiblythe subesophageal ganglion (Pflugfelder, 1936; Jawlowski,1954; Weiss, 1981; Schroter and Menzel, 2003; Frambachand Schurmann, 2004), which in turn are innervated bysensory afferents from the mouthparts (Ernst et al., 1977;Ignell et al., 2000). The TT travels with the iACT ventrallythen takes a more lateral trajectory at the level of themushroom body lobes (Schroter and Menzel, 2003;Frambach et al., 2004). In hemimetabolous insects afferentsfrom these centers are often targeted to the Class III Kenyoncell-containing accessory calyx, if present (Pflugfelder,1936; Weiss, 1981; Frambach and Schurmann, 2004). InApis, which lacks accessory calyces in the adult (Farris


et al., 2004), TT afferents appear to be targeted to a narrowsubcompartment in the primary calyces (Schroter andMenzel, 2003; Fig. 3A). This calyx zone corresponds tothe dendritic arborizations of Class II and a distinct cohortof Class I Kenyon cells (Strausfeld, 2002).

Visual input to the mushroom bodies may be observed,but to widely varying degrees. A ‘tractus opticus globularis’(TOG) from the medulla to the primary calyces or, in thosewithout calyces, to the pedunculus, has been described inthe older literature for the Odonata, Isoptera, Orthoptera andTrichoptera (Hanstrom, 1930, 1940; Ehnbom, 1948;Schurmann, 1973). These conclusions were primarilydrawn from tissue prepared with general histological stains.To date, no modern tracing studies have been performed toverify that the TOG (referred to as the anterior superioroptic tract in more recent studies) actually suppliescollaterals to the mushroom bodies in these insects. In thecockroach Periplaneta only a single neuron from the opticlobe medulla to the mushroom body primary calyx has beenidentified (Strausfeld and Li, 1999b), suggesting that despitethe proximity of the TOG to the mushroom bodies,innervation of the calyces by TOG axons may in fact besparse in some species.

In contrast, it has been amply demonstrated in the socialHymenoptera such as bees, wasps, and many ants that theprimary calyx receives a great deal of visual input suppliedby three optic tracts (the anterior superior optic, anteriorinferior optic and lobula tracts) originating from differentareas of the optic lobes and projecting both ipsi- and contra-laterally (Mobbs, 1982; Gronenberg, 2001; Ehmer andGronenberg, 2002; Lopez-Riquelme and Gronenberg,2004). The finely divided calycal zones receiving these

terminals correspond not only to the dendritic fields ofdifferent cohorts of Class I Kenyon cells, but also toparticular levels of the dendritic tree within a single Kenyoncell cohort (Ehmer and Gronenberg, 2002; Strausfeld,2002).

Afferents to the primary calyx originating from higherprocessing centers in the protocerebrum rather than fromprimary sensory neuropils have been described in Peripla-neta and Acheta and are typically multimodal (Schildberger,1984; Strausfeld and Li, 1999a). The multimodality of theseafferents likely arises from the sampling of differentprotocerebral regions that themselves receive inputs fromprimary sensory neuropils (Li and Strausfeld, 1997).

Dense GABAergic innervation of the primary calyceshas been reported in a number of insect species, as has theexpression of GABA receptors by Kenyon cells, and likelyrepresents a highly conserved component of the mushroombody pathway (Bicker et al., 1985; Homberg et al., 1987;Leitch and Laurent, 1996; Brotz et al., 1997; Nishino andMizunami, 1998; Yamazaki et al., 1998; Grunewald, 1999;Strausfeld and Li, 1999a; Schurmann, 2000; Ganeshina andMenzel, 2001; Homberg, 2002). The protocerebral neuronsproviding these GABAergic profiles have two basicmorphologies: dendrites in the lobes and axons in theprimary calyces, or dendrites in the lateral protocerebrumand axons in the primary calyces. The former are interpretedto be feedback neurons and their morphology is particularlywell studied in the Hymenoptera (Mobbs, 1982; Gronen-berg, 1987; Grunewald, 1999; Ganeshina and Menzel,2001), where they have been shown to form a recurrentconnection between the axons and dendrites of a givensubpopulation of Kenyon cells (Grunewald, 1999;

Fig. 4. Serial reconstruction of golgi impregnated frontal sections showing the iACT (blue arrows) supplying afferents to the dorsal zone of the mushroom bodycalyx (red arrowheads) and the lateral protocerebrum (red arrow) in Periplaneta americana. Ca—calyx. Scale barZ50 mm.


Strausfeld, 2002). Electron microscopic evidence suggeststhat information flow in these neurons is mostly, but notalways, from the lobes to the primary calyces (Leitch andLaurent, 1996; Ganeshina and Menzel, 2001). The primarycalyces of Periplaneta and Drosophila mushroom bodiesalso show extensive innervation by large GABAergic cellswith dendrites in other regions of the protocerebrum, ratherthan the lobes (Ito et al., 1998; Yamazaki et al., 1998;Strausfeld and Li, 1999a; Strausfeld et al., 2003). It has beenproposed that these GABAergic cells may still function asfeedback neurons due to the overlap of their dendrites withthe terminals of mushroom body lobe efferents in theprotocerebrum (Strausfeld and Li, 1999a).

Aminergic neuromodulatory neurons to the primarycalyces appear widespread across the insects, againsuggesting a conserved aspect of mushroom body circuitry(Homberg, 2002; Strausfeld et al., 2003). The octopamin-ergic VUM neuron arising from the subesophageal ganglionand connecting the antennal lobes, calyces and lateralprotocerebrum has been suggested to play a pivotal role inolfactory conditioning in Apis (Hammer, 1993, 1997),although homologues in other insects have yet to beidentified. Similarly, peptidergic neurons such as thoseutilizing the neurotransmitter tachykinin are targeted to theprimary calyces in species of Orthoptera and Dictyoptera,but not in the dipteran Drosophila (Nassel et al., 2000; Isaacand Nassel, 2003; Winther et al., 2003).

5.3. Evolutionary trends in Kenyon cell subpopulations andcalyx morphology

Mushroom body intrinsic neurons resembling Classes Iand II Kenyon cells have been described in a wide variety ofinsects and likely represent highly conserved components ofthe mushroom bodies. A possible exception to the abovegeneralization occurs in the scarab beetle Pachnoda, whichdoes not appear to possess Kenyon cells matching the ClassI suite of traits (Larsson et al., 2004). In contrast, instancesof Class III neurons are widely scattered across the insects,and highly variable in number in the lineages in which theydo occur. For example, within the Order Dictyoptera, ClassIII Kenyon cells are present in small numbers in the basalcockroaches such as Periplaneta, enormously elaborated inthe termites, and completely lacking in the mantises andhigher cockroaches (Farris and Strausfeld, 2003).

Where are the Class III Kenyon cells in other insects?The existing literature suggests some intriguing possibili-ties. The mushroom bodies of Apis receive TT afferents,which target Class III cells in insects such as Acheta, to asmall subcompartment of the primary calyx (Schroter andMenzel, 2003). This suggests that a subpopulation of Class Icells are functioning as Class III cells do in other insectspecies, perhaps explaining how Class III cells themselvesmay be lost in some insects, but their sensory processingfunctions maintained. A related hypothesis is that Class IIIcells of some species appear early in development to

establish the necessary circuitry with TT afferents, which istransferred to another population of Kenyon cells later indevelopment after the Class IIIs themselves have died.Again in Apis there is evidence to suggest that Class III-likestructures are produced early in development but degenerateshortly afterwards, providing a potential explanation for theabsence of Class III neurons in the adult (Farris et al., 2004).An additional possibility is that all insects have ‘covert’Class III cells that are fully integrated into the mainmushroom body neuropil, perhaps supported by evidencefrom the moth Sphinx ligustri in which a subpopulation ofKenyon cells with dendrites in the primary calyx form aseparate ‘Y-tract’ and lobe system reminiscent of thatproduced by Class III cells in Periplaneta (Pearson, 1971).Finally, it is possible that early-born Kenyon cell subpopu-lations receiving TT afferents have been acquired manytimes independently, making Class III Kenyon cells asdefined a ‘paraphyletic’ grouping. In order to answer thesequestions and achieve a more definitive assignment ofKenyon cell subpopulation identities across widely diver-gent taxa will be greatly facilitated by the development ofsubpopulation-specific gene expression markers, the exist-ence of which is suggested by various GAL4 lines inDrosophila (for example see Tanaka et al., 2004; Fig. 4A).

As might be expected from the observed variability inKenyon cell subpopulations across taxa, the overall shape ofmushroom body calyces is also evolutionarily malleable, asshown in Figs. 5–7. Figs. 5 and 6 show variation in primaryand accessory calyx morphology, respectively, while Fig. 7is a schematic representation of the distribution of differentcalyx morphologies across the insects. Calyx morphology isnot always correlated with the current understanding ofinsect phylogeny, suggesting that different calyx structuresarose independently in multiple insect lineages. This is wellillustrated by the phylogenetic distribution of single anddouble primary calyx neuropils (Fig. 5A–D). Doubled,deeply cup-shaped primary calyces are observed in twowidely divergent groups, the hemimetabolous cockroaches(Fig. 5A), and the holometabolous social Hymenoptera (Fig.5B). Developmental and anatomical studies of Kenyon cellcomposition and afferent input have shown that doubleprimary calyces are identical or ‘equivalent’ (Farris et al.,1999; Gronenberg and Holldobler, 1999; Strausfeld and Li,1999a; Farris and Strausfeld, 2001). Likewise, singleprimary calyx neuropils are distributed across the insects,being found in such widely divergent groups as theZygentoma (Fig. 5C) and the Diptera (Fig. 5D). A clue tothe relationship between these different morphologies lies inthe fact that in the majority of species with single calyces,including the basal Zygentoma, these neuropils are suppliedby two Kenyon cell tracts arising from the progeny of twomushroom body neuroblasts or neuroblast clusters (Han-strom, 1940; Farris, 2005). This suggests that the initialduplication of mushroom body neuroblasts leading to theformation of two Kenyon cell groups per hemisphereoccurred very early in insect evolution.


The potential for primary calyx doubling via theformation of two distinct clusters of Kenyon cells, therefore,appears to be an ancestral characteristic of insect mushroombodies. How does calyx doubling occur and why is itobserved in some lineages but not others? The acquisition ofdoubled and cup-shaped calyces, like the cortical gyri andsulci, would represent an increase in calyx volume and totalsynaptic surface, suggesting that, as also observed in themammalian cortex (Harrison et al., 2002), this morphologymay be correlated with increased Kenyon cell number andthus relative mushroom body size in species. This issupported by the fact that the above-mentioned cockroachesand social Hymenoptera have among the largest mushroombodies, containing approximately 170,000 Kenyon cells per

hemisphere, while those of the Diptera are among thesmallest with approximately 2500 Kenyon cells per hemi-sphere in Drosophila (Neder, 1959; Witthoft, 1967; Ballinget al., 1987). Furthermore, intermediate steps in the trendtowards acquisition of double primary calyces are nicelyrepresented in the scarabaeid beetles (Gooßen, 1951; Fig.5E–G), in which certain species have acquired doubledcalyces, some of which are quite convoluted, although theydo not form the deep cups observed in the socialHymenoptera. Rather, as pointed out by Jawlowski(1960), the mushroom bodies of scarabs with doublecalyces are strikingly similar to those of more basalHymenoptera such as sawflies. The scarabaeid and hyme-nopteran lineages thus seem to capture different stages of

Fig. 6. Morphology of accessory calyces revealed by anti-DC0 immunostaining. A. Diffuse dendritic arbors (arrow) arising from the pedunculus in Periplaneta

americana. B. Dense spherical accessory calyx opposite the single primary calyx in the milkweed bug Oncopeltus fasciatus (Heteroptera). C. Diffuse dendrites

arising from a separate ‘Y-tract’ ventral to the triple primary calyces in a green lacewing (Chrysopidae; Neuroptera). A satellite neuropil that does not appear to be

directly linked to the mushroom bodies is also heavily labeled (arrowhead). D and E. Variability in accessory calyx anatomy in the Orthoptera. D. A large accessorycalyx neuropil encircles the posterior and lateral primary calyx in the cricket Acheta domestica (cross-section shown). E. A cup-shaped accessory calyx in the

grasshopper Schistocerca americana. F. A small accessory calyx encircles the pedunculus in the camel cricket Tachycines asynamorus. Arrows-accessory calyx.

PCa—primary calyx, Pe—pedunculus. All sections sagittal except B (horizontal) and C (frontal). Scale bars A and BZ20 mm, C, D and FZ50 mm, EZ100 mm.

Fig. 5. Diverse morphologies of the mushroom body primary calyx. A and B. Double, cup-shaped calyces of the cockroach Periplaneta americana (A) and the

honeybee Apis mellifera (B). C and D. Single calyces of the firebrat Thermobia domestica (C) and a flesh fly (D, Sarcophagidae; Diptera). Arrows in C,glomeruli of the calyx; in D, four Kenyon cell tracts arising from four clonally equivalent groups of Kenyon cells contributing to the calyx. E–G. Variability in

calyx morphology in a single family of beetles, the Scarabaeidae (Coleoptera). Arrows denote the two Kenyon cell tracts supplying the calyx or calyces. E.

Single knob-shaped calyx neuropil in the dung beetle Aphodius spp. F. Double knob-shaped calyx neuropils in the dynastine beetle Cyclocephala spp. G.

Double, convoluted calyx neuropil in the melolonthine beetle Maladera castanea. H and I. Calyx-less mushroom bodies. H. Thin pedunculus arising fromKenyon cells in the brain of a cicada (Tibicen spp.; Heteroptera). I. Pedunculus formed of multiple thick axon tracts decorated with microglomeruli in a

dragonfly (Perithemis tenera; Odonata). Ca—calyx, Kcb—Kenyon cell bodies, Pe—Pedunculus. A–C, E–G is Cason’s staining, D, H and I anti-DC0 and

phalloidin double labeling. All sections frontal except I (sagittal). Scale bars A and HZ50 mm, BZ100 mm, CZ10 mm, D, E–G and IZ20 mm, HZ50 mm.


Fig. 7. Distribution of calyx morphologies across the insect orders and families. Star (*) indicates that an accessory calyx and/or Class III Kenyon cells are also

present.


the evolution of calyx doubling. This opens up intriguingquestions as to what selective pressures may generaterelatively larger mushroom bodies in these lineages, andwhether trends towards increased neuropil size are associ-ated with the acquisition of new Kenyon cell subpopulationsor the expansion of one or more existing subpopulations.

Given the amazing diversity of insects, it should not besurprising that such a simple relationship between mush-room body size and calyx morphology does not represent ahard and fast rule. Of particular note are the termites, whichlike the social Hymenoptera display an enormous develop-ment of the mushroom bodies perhaps correlated with theacquisition of social behavior (Hanstrom, 1930; Farris andStrausfeld, 2003). Unlike the social Hymenoptera, however,increased mushroom body size has been accomplished inthe termites primarily by elaboration of the lobes (Farris andStrausfeld, 2003), with a concomitant trend towards calyxfusion (Hanstrom, 1930). Lobe circuitry, therefore, appearsto have been preferentially enhanced, perhaps at the expenseof calycal circuitry.

In other insect lineages, existing evidence suggests atrend towards decreased, rather than increased mushroombody size and complexity. In the Psocoptera some speciespossess mushroom bodies resembling those of the closelyrelated Hemiptera, while in others the primary calyx isreduced or even lost (Jentsch, 1940). In the Diptera, thesmall mushroom bodies possess a single primary calyxsupplied by four Kenyon cell tracts representing the progenyof four equivalent neuroblasts (Ito et al., 1997; Strausfeldet al., 2003; Zhu et al., 2003; Fig. 5D). The four tracts travelalong the outer surface of the primary calyx, essentiallyinside-out in relation to that of more basal insects likeThermobia and suggesting a secondarily derived fusion ofancestrally separate calycal neuropils. Two- and four-foldsymmetry in gene expression patterns generated byenhancer trap studies in Drosophila support the hypothesisthat the primary calyx is derived from two ancestral calyces,each additionally composed of two hemicalyces (Yanget al., 1995). In Periplaneta and Apis, hemicalyces arereadily recognizable as a glial barrier dividing the Kenyoncell bodies within each primary calyx (Strausfeld and Li,1999b; Strausfeld, 2002). On either side of the barrier,Kenyon cells provide dendrites only to the calyx neuropil onthe same side.

Finally, rare developmental events may be behind ascattering of more unusual primary calyx morphologies. Incontrast to the near-universal two or four Kenyon cellgroups per hemisphere, the Orthoptera have just a single cellgroup while the Dermaptera, Neuroptera and dytiscidColeoptera have three (Holste, 1910; Kuhnle, 1913;Jawlowski, 1936; Hanstrom, 1940; Cayre et al., 2000;Malaterre et al., 2002; Farris, 2005; Fig. 6C). A simpleexplanation for the acquisition of similar calyx mor-phologies in the latter three widely divergent insect groupsis that a third group of mushroom body progenitor cells hasbeen acquired independently in each, resulting in three

groups of Kenyon cells and thus three primary calyces.Histological evidence from the larval brain of the divingbeetle Dytiscus marginalis suggests that this is indeed thecase (Jawlowski, 1936). Furthermore, in all of the above-mentioned taxa the three calyces are fused and of a similarsize, indicating equivalence such as that observed for moretypical double primary calyces. This is an importantdistinction in light of the presence in some species of non-equivalent accessory calyces formed by a distinct Kenyoncell subpopulation, as described below. In the Orthoptera,Kenyon cells derive from a single progenitor cell group(Cayre et al., 2000), suggesting that a second proliferativecenter may have been lost in the orthopteran ancestor. Suchan event is observed during embryonic development in theclosely related Phasmatodea, in which one of two neuroblastgroups degenerates prior to hatching and the nymphsubsequently possesses a single calyx neuropil (Malzacher,1968). Interestingly, in this latter taxon a further oddity isobserved in the apparent defasciculation of Kenyon cellfibers, which enter the calyx seemingly at random viascattered small tracts (Hanstrom, 1940). A similar pheno-type is observed in fasciclin II mutants of Drosophila(Kurusu et al., 2002), suggesting a possible single-genebasis for this unusual calyx morphology.

As with single and double primary calyces, insect speciesentirely lacking mushroom body calyces encompass avariety of divergent lineages (Fig. 5H and I). Interestingly,many of these species, though not all, share an aquaticlifestyle at least in the juvenile stage (Graichen, 1936;Pflugfelder, 1936; Hanstrom, 1940; Ehnbom, 1948; Straus-feld et al., 1998). In most species the loss of mushroom bodycalyces is most likely secondary and is associated with theconcomitant reduction of the antennae and reduction or lossof glomerular antennal lobes (Hanstrom, 1940; Strausfeldet al., 1998). It is not clear whether the iACT is also lost; it ispossible that this tract and the TT may remain to carry non-olfactory deutocerebral and tritocerebral afferents to thepedunculus or to the lateral protocerebrum (Hanstrom,1940; Farris, 2005). In any case, the strong correlationbetween reduced antennae, loss of the glomerular antennallobe and loss of the calyx supports the notion that thecalyces function primarily with regard to olfactory process-ing (Strausfeld et al., 1998).

Among those insects sharing this ensemble of charactersare the Odonata and the Ephemeroptera, together classifiedas the Palaeoptera. The basal phylogenetic position of thePalaeoptera has been interpreted to suggest that calyx-lessmushroom bodies with afferents to the pedunculus representan ancestral organization of the mushroom body circuit inthese primitively anosmic insects (Strausfeld et al., 1998).Recent evidence from the firebrat Thermobia, a representa-tive of the most basal group of mushroom body-possessinginsects, may suggest otherwise (Farris, 2005). Thermobiapossesses a single calyx neuropil that is supplied by a tractof deutocerebral origination with a trajectory highlyreminiscent of that of the iACT. The deutocerebral neuropil,


although largely composed of a columnar region thought toprocess mechanosensory afferents from the antenna, alsocontains a small region of robust glomeruli very similar tothose of the neopteran antennal lobe (N. J. Strausfeld,personal communication; Farris, 2005). It is, therefore,possible that the Thermobia mushroom bodies may indeedhave served olfactory processing functions. The presence ofolfactory processing circuitry in basal Thermobia suggeststhat the absence of a mushroom body calyx and glomerularantennal lobe may always be derived, even in thePalaeoptera. It is also possible, however, that mushroombody calyces arose at least twice independently in theinsects, once in the Zygentoma and once in the Neoptera.The presence of calyx-like microglomeruli in the dragonflypedunculus, suggesting that this may represent the primarysite of afferent input in the Palaeoptera, may lend support tothe latter hypothesis (Fig. 5I).

In keeping with the theme of mushroom body diversity,accessory calyces are found in a scattered complement ofinsect taxa, and tend to be characteristic of particularlineages at the familial or ordinal level (Figs. 6 and 7). Asopposed to double primary calyces, accessory calyces arenon-equivalent to primary calyces, being composed of ClassIII Kenyon cells and receiving afferents via the TT, ratherthan olfactory inputs from the ACT (Pflugfelder, 1936;Jawlowski, 1954; Farris and Strausfeld, 2003; Frambachand Schurmann, 2004). As observed with the primary calyx,the accessory calyx may be secondarily lost in lineages thathave acquired an aquatic or semi-aquatic lifestyle, as isdocumented for various species of Hemiptera (Graichen,1936; Parsons, 1960).

Accessory calyces are nearly always located postero-ventral to the primary calyx, and are smaller than the latterstructure (Fig. 6A–C). The accessory calyx may becomposed of a diffuse network of dendrites or may be asolid mass similar to the primary calyx (compare Fig. 6Aand B). Due to the early birthdate of Class III Kenyon cells,the cell bodies supplying accessory calyces lie at the outerperimeter of the mushroom bodies, as do the neurites andaxons in the pedunculus and lobes (Schurmann, 1973;Malaterre et al., 2002; Farris and Strausfeld, 2003). Aspreviously mentioned, Class III dendrites in the mothSphinx ligustri appear to be integrated into the primarycalyx rather than forming a separate structure (Pearson,1971). The presence of this Kenyon cell subpopulation isnevertheless betrayed by a separate ‘Y-tract’ running fromthe calyx to the vertical lobe. In other Lepidoptera such asthe butterfly Pieris brassicae there is no Y-tract, but adistinct accessory calyx is present (Ali, 1974). Interestingly,a combination of the two lepidopteran configurations isobserved in the green lacewing (Neuroptera; Fig. 6C),which possesses both a separate accessory calyx and aY-tract feeding into the vertical lobe.

Calycal subcompartments display evolutionary variabil-ity in size and number that is strongly correlated with thebehavioral ecology of a particular lineage. For example,

mole crickets (Gryllotalpidae) are burrowing insects with alifestyle similar to that of the mammal of the same name.The gryllotalpid accessory calyx is enormously expandedrelative to the primary calyx (Jawlowski, 1954; Panov,1966; Weiss, 1981), suggesting that the undergroundlifestyle of these creatures has relegated airborne olfactionto a relatively minor role but elevated the importance ofgustation and mechanosensation in terms of the need forhigher sensory processing via the mushroom bodies. Inmammalian moles, the relative sizes of areas in the sensorycortex display similar adaptations for the burrowinglifestyle, most notably the extensive elaboration of soma-tosensory cortex representing afferents from tactile organson the nose and the concomitant diminishment of visualcortex (Catania, 2000).

The dependence on visual cues for spatial navigationwhen foraging from a central nest source has likely driventhe acquisition of enlarged and highly subcompartmenta-lized visual input zones in the calyces of the bees and wasps(Gronenberg and Holldobler, 1999). In contrast to thevisually-dependent bees and wasps, however, many antspecies rely more on olfactory cues for spatial navigation.These species display a reduction of visual inputs to themushroom bodies and a corresponding elaboration andsubcompartmentalization of olfactory inputs (Gronenbergand Holldobler, 1999; Ehmer and Gronenberg, 2004). TheHymenoptera thus provide a robust example of two motifsthat have been repeatedly demonstrated in the evolution ofthe vertebrate cortex: the elaboration of specific sensoryinputs and a concomitant expansion of their target regions inhigher processing centers (as also described above for themole cricket), and the further subcompartmentalization ofthese target regions (Kaas, 1995; Catania, 2000; Finlayet al., 2001). In addition, the overall size and complexity ofthe Hymenopteran mushroom bodies may reflect theincreased processing capacity necessary for complex socialinteraction in these insects, as was originally proposed byDujardin (1850). Large and complex cortices are alsostrongly associated with the acquisition of social behavior invertebrate groups such as the primates (reviewed by Kaas,1995).

6. Mushroom body pedunculus and lobes

6.1. Morphology of Kenyon cell axons

As with Kenyon cell dendrites, the trajectories of Kenyoncell axons are subcompartmentalized within the lobes andare variable in their morphologies both within and betweenspecies. In most instances, Kenyon cell axons bifurcate inthe distal pedunculus, with one branch entering the mediallobe and one the vertical lobe (Figs. 1 and 8). The highdegree of conservation of this axon morphology isillustrated by the presence of distinct medial and verticallobes in nearly every insect lineage (Fig. 8A–C). Most of the


observed lobe structures generally represent more or lesselaborate variations on this common theme (Fig. 8D–F). Incontrast, when Class III Kenyon cells are present, theiraxons form physically separate tracts in the pedunculus and/or lobes. In crown termite species, for example, Class IIIaxons are particularly enormously elaborated and effec-tively duplicate the volume of the medial and vertical lobes,which form convoluted folds dominating the protocerebrum(Farris and Strausfeld, 2003; Fig. 8D).

Perhaps one of the most unusual lobe morphologies isfound in some species of polistine wasp (Family Vespidae,Order Hymenoptera; Jawlowski, 1959; Ehmer and Hoy,2000). In these insects the medial axon branches fromKenyon cells in each calyx are segregated and directedposteriorly through the pedunculus to form two lobes, whileaxons of the vertical lobe are oriented ventrally so that theyresemble (and were previously mistaken for) a medial lobe.Note that although atypical, this lobe morphology still hasthe typically bifurcated Kenyon cell axon as its basis.Interestingly, it has been shown in the polistine waspPolistes apachiensis that this adult lobe morphology resultsfrom a massive reorganization of Apis-like medial andvertical lobes during the mid-pupal stage (B. Ehmerunpublished observations; Farris et al., 2004), clearlyshowing how polistine lobe structure derives from themedial and vertical lobe orientation typifying the remainderof the Hymenoptera.

Metamorphic reorganization that gives rise to diversity inKenyon cell axon trajectory is well-documented in anotherholometabolous insect, the fruit fly Drosophila, in whichmost Class II Kenyon cells provide a single unbranchedaxon to the medially directed g lobe in the adult (Yang et al.,1995; Strausfeld et al., 2003). In the Drosophila larva, ClassII axons are bifurcated as usual, but degenerate and regrowsolely in the medial direction during metamorphosis (Leeet al., 1999). Golgi impregnations also suggest thatunbranched subpopulations of Class II Kenyon cells existin the lepidopteran Sphinx ligustri and in Apis, indicatingthat they may be widespread in holometabolous insects(Pearson, 1971; Strausfeld, 2002). In Apis there is alsoevidence for degeneration of Class II axons duringmetamorphosis, but reshaping of lobe structure is muchless dramatic in this insect than in Drosophila (Farris et al.,2004).

6.2. Neurotransmitter identities of Kenyon cells

The amino acid transmitters glutamate, aspartate andtaurine have been localized to Kenyon cells (Schurmannet al., 2000; Sinakevitch et al., 2001; Strausfeld et al., 2003),as have peptide transmitters such as Phe-Met-Arg-Phe-NH2

(FMRFamide) and gastrin cholecystokinin (GCCK; Schur-mann and Erber, 1990; Strausfeld et al., 2000). In addition,components of the pathway for production of the retrogradetransmitter nitric oxide may be present (O’Shea et al., 1998;Ott and Elphick, 2002). Complex and combinatorial

patterns of neurotransmitter usage create fine partitions ofKenyon cell axons in the lobes, often revealing furtherfunctional subcompartments beyond the three broad Ken-yon cell classes (see below).

Each Kenyon cell subpopulation displays an affinity forantibodies against multiple putative neurotransmitters, somecharacteristics of which are conserved across taxa. Forexample, glutamate immunoreactivity is particularly strongin the latest-born Class I Kenyon cells, whose axons occupythe ingrowth core (Sinakevitch et al., 2001; Strausfeld et al.,2003). During development the core contains an ever-changing population of axons, with older axons beingconstantly pushed to more peripheral layers by newerprocesses (Farris and Strausfeld, 2001). Accordingly,glutamatergic neurotransmitter identity is a transient aspectof Kenyon cell development that is lost or significantlyreduced in mature axons. Class I Kenyon cells outside of thecore instead show affinities for antibodies against the aminoacids taurine, aspartate, or both; the strength of which variesaccording to further cohort divisions among this generalclass of intrinsic neurons (Sinakevitch et al., 2001;Strausfeld et al., 2003). Class II cells, in contrast, displaylow or absent immunoreactivity for all three putativetransmitters. Class III neurons in the cockroach may containhigh levels of aspartate, particularly in their medial lobebranches (Sinakevitch et al., 2001), while in the cricket,evidence suggests that Class III neurons contain glutamate(Schurmann et al., 2000).

The distribution of FMRFamide and GCCK immuno-reactive Kenyon cells has been best described in thehoneybee (Schurmann and Erber, 1990; Strausfeld et al.,2000) and the cockroach (Strausfeld and Li, 1999b). As withthe amino acid transmitters, a complex pattern of high, lowor absent levels of immunoreactivity appears to differentiatethe Classes II and I populations, as well as defining furthercohorts of Class I cells. There is no evidence thus far ofpeptide transmitters in Class III Kenyon cells.

NADPH-diaphorase histochemistry has been used todemonstrate the use of nitric oxide by Kenyon cells,although the subpopulation affiliations vary. In the roachPeriplaneta americana, enzyme activity is localized toClass III neurons (Ott and Elphick, 2002), while in thelocust Schistocerca gregaria a cohort of Class I cells whoseaxons lie just outside the ingrowth core are labeled (O’Sheaet al., 1998).

6.3. Layers and lobes

Kenyon cell dendrites are segregated according tosubpopulation identity, source of afferent input and birth-date. The same is true for the lobes, in which the axons ofeach Kenyon cell subpopulation as represented in thecalyces define a morphological and functional layer (Leeet al., 1999; Strausfeld and Li, 1999b; Strausfeld et al.,2000; Farris and Strausfeld, 2001; Fig. 9A). Stratification ofaxons in the pedunculus and lobes is respected by afferents


to and efferents from these regions. Whereas calycal zonesdefined by Kenyon cell dendrites may be overlapping(Mizunami et al., 1998a; Strausfeld and Li, 1999b), thepedunculus and lobes display a stricter segregation in whichindividual Kenyon cell axons do not cross from one layer toanother (Mizunami et al., 1998b; Strausfeld and Li, 1999b;Strausfeld, 2002). Immature Class I axons are the only

known exception to this rule, producing in Periplaneta andApis thin collaterals extending from the ingrowth coreacross adjacent layers (Farris and Strausfeld, 2001; Farriset al., 2004). As with glutamate immunoreactivity, thesecollaterals are transient features of immature axons andare not observed on axons residing outside of the ingrowthcore.

Fig. 8. Morphologies of mushroom body lobes revealed by anti-DC0 immunostaining. A–C. The formation of a medial and vertical lobe by the bifurcation ofKenyon cell axons is highly conserved across the insects. A. A damselfly (Enallagma spp.; Odonata). B. A cicada (Tibicen spp.; Heteroptera). C. A flesh fly

(Sarcophagidae; Diptera). D–F. Regions of the lobes formed by the processes of Class III Kenyon cells (arrows). D. The enormously elaborated lobes of the

termite Gnathamitermes perplexus (Dictyoptera) appear doubled due to the expansion of Class III Kenyon cells. E. Class III axons in the ‘Y-tract’ of a green

lacewing (Chrysopidae; Neuroptera) encircle the vertical lobe, as do those forming the lobelet of Periplaneta americana (F). Ca—primary calyx, M—mediallobe, V—vertical lobe, Pe—pedunculus, I, II-Class I and II Kenyon cells. All sections frontal except F (sagittal). Scale bars A–C and FZ50 mm, DZ20 mm,

EZ25 mm.


In addition to neurotransmitter identity, layers in theKenyon cell subpopulations may also be differentiated bythe presence or absence of proteins like protein kinase A(Crittenden et al., 1998) (Figs. 5D, H and I, 6, 8 and 10B–D),as well as by non-specific histological methods such as ethylgallate and reduced silver staining (Weiss and Edwards,1974; Mizunami et al., 1997; Strausfeld and Li, 1999b). Inthe latter case, light and dark banding of the lobes inPeriplaneta has been correlated with ultrastructural reaturessuch as amount and kind of synaptic vesicle and width of theconstituent axons (Iwasaki et al., 1999; Strausfeld and Li,1999b).

The mushroom bodies of the Diptera, as represented bythe fruit fly D. melanogaster, are made up of three or morelobe systems rather than a single medial–vertical lobe pair.In these insects, each lobe or lobe pair (termed g, a 00/b 00,a 0/b 0, a/b and a/b core) displays differential patterns ofefferent output, developmental organization, geneexpression and process morphology like those that defineKenyon cell layers in the lobes of insects such as Apis andPeriplaneta. It is, therefore, highly likely that the lobesystems of Drosophila are equivalent to the layers makingup the lobes of other insects (Strausfeld et al., 2003).

6.4. Extrinsic connections to the pedunculus and lobes

Extrinsic neurons to the lobes have been systematicallycharacterized in a few species, most notably Apis andPeriplaneta (Mobbs, 1982; Rybak andMenzel, 1993; Li andStrausfeld, 1997, 1999; Strausfeld, 2002). Unlike afferentsto the calyx, lobe extrinsic neurons do not form largedistinct tracts. Instead, they arise from multiple protocer-ebral areas and individual cells may have exceedinglycomplex patterns of connectivity encompassing severalother protocerebral regions in addition to the mushroombodies. These features have made it somewhat more difficultto definitively assess relationships among lobe extrinsicneurons, both within and between species. Nevertheless,many aspects of overall extrinsic neuron organization maybe recognized across taxa.

As mentioned earlier, Kenyon cell processes in thepedunculus and lobes are typically termed ‘axons’ forsimplicity, although there is ample evidence that they arenot axons in the strictest sense, but rather are pre- and post-synaptic to extrinsic neurons as well as to one another asdemonstrated by electron microscopy (Schurmann, 1970;Strausfeld and Li, 1999b). Afferents and efferents to thelobes are characterized by their elaborate and often beautifulpatterns of innervation, varying tremendously in thecombinations of Kenyon cell subpopulations sampled, andat which position along the length of the axon theseconnections are formed (Mobbs, 1982; Rybak and Menzel,1993; Li and Strausfeld, 1997, 1999; Ito et al., 1998;Strausfeld, 2002;Fig. 9B). Lobe afferents arise from variousprotocerebral areas, and like protocerebral afferents to theprimary calyx are multimodal (Li and Strausfeld, 1997; Ito

et al., 1998; Strausfeld, 2002; Larsson et al., 2004).Described thus far in Periplaneta, Drosophila, Pachnodaand Apis, lobe afferents are widespread and must essentiallyconstitute the sole source of input to calyxless mushroombodies (Strausfeld et al., 1998). Among mushroom bodyefferents, some examples exist of uniquely identifiedneurons such as the Pe1 neuron of the honeybee (Mauels-hagen, 1993; Rybak andMenzel, 1998), which is recognizedfor its response plasticity during mushroom body-mediatedlearning and memory. Other lobe efferents are character-istically multimodal, sometimes possessing dendritic arborsoutside of the mushroom bodies as well as within (Erber,1978; Schildberger, 1984; Li and Strausfeld, 1997; Li andStrausfeld, 1999). Efferent axons project to protocerebralregions surrounding the mushroom bodies, with a fewproviding descending collaterals (Li and Strausfeld, 1997).The lobes may also be invaded by the processes ofaminergic neuromodulatory cells (Schurmann and Klemm,1984; Homberg, 2002) and the dendrites of GABAergicfeedback neurons as previously discussed.

In some insects such as Apis (Fig. 9C and D), Periplaneta(Li and Strausfeld, 1999) and Acheta (Schurmann, 1970,1973), protocerebral afferents may also invade the pedun-culus. The post-synaptic nature of Kenyon cell processes inthe pedunculus (often called the ‘neck’ in the honeybee) isclearly indicated by the spines decorating their length (Fig.9D). In Periplaneta, a group of eight GABAergic efferents(four per hemisphere) provide extensive collaterals to thepedunculus and respond solely to mechanosensory stimu-lation, thus representing the only instance of unimodal inputto non-dendritic Kenyon cell processes (Strausfeld and Li,1999a,b). In at least one insect taxon in which calyces aremissing, the Odonata, extensive afferents to the pedunculus,some possibly arising from primary sensory neuropils, arereported (Hanstrom, 1940). Pedunculus afferents are not aubiquitous features of insect mushroom bodies, however, asthey are entirely lacking in Drosophila (Ito et al., 1998).

One additional type of neuron has been described in thepedunculi of Apis and Pachnoda (Mobbs, 1982; Strausfeld,2002; Larsson et al., 2004). The ‘exotic’ or ‘elaborate’intrinsic neurons are clearly not Kenyon cells, as their cellbodies are located distant from the mushroom bodies, butlike Kenyon cells they provide all or most of their processesto the mushroom bodies. Two distinct types exist in Apis,both of which invade the pedunculus and a calycalsubcompartment called the basal ring, and one of whichprovides thick axons to the g layer of the vertical lobe aswell (Strausfeld, 2002). In Pachnoda, Golgi impregnationsreveal a large neuron with processes suffusing nearly theentire axon field of one Kenyon cell subpopulation (Larssonet al., 2004). The presence of similarly unusual neurons in twodisparate insect orders hints at a conserved function, atantalizing hint of which may be suggested by studies inDrosophila. Like the exotic intrinsic ofApis andPachnoda thetwo DPM neurons of Drosophila have distantly located cellbodies and densely innervate a large proportion of the


mushroom body lobe system. They are presumed to serve aneuromodulatory function via a peptide transmitter expressedby the amnesiac gene (Waddell et al., 2000); as the namesuggests, theamnesiacgenewas isolated in screens of learningand memory mutants (Quinn et al., 1979). This evidencesuggests that the DPM neurons play an important role inlearning and memory, and their similarity to the exoticintrinsic may indicate a homologous functional relationship.

6.5. Evolutionary trends in pedunculus and lobemorphology

In the Neopteran insects described above, layeredsubdivisions in the lobes each represent the axonalramifications of a single Kenyon cell subpopulations (Fig.10A and B). The lobes of the basal insect Thermobia aremade up of many bulb-like subdivisions termed ‘trauben’(meaning ‘grapes;’ a term coined by Bottger (1910) in hisdescription of the mushroom bodies of a related species,

Lepisma saccharina). Close inspection at the cellular level,however, reveals a very different organization of Kenyoncell axons, in which the trauben are formed by the extensivebranching pattern of a single Kenyon cell type (Fig. 10C;Farris, 2005). Individual Thermobia trauben, therefore,cannot represent calyx-level differences in afferent input asdo the layered subdivisions of the Neopteran lobes.

Despite the current absence of knowledge about lobeextrinsic neurons in Thermobia, some speculation on thefunctional significance of the trauben architecture ispossible. For example, each of the trauben could representa local circuit like those identified in neopteran insects andconsisting of lobe afferents, Kenyon cell to Kenyon cellinteractions and lobe efferents (Li and Strausfeld, 1997;Strausfeld and Li, 1999b; Strausfeld, 2002). Diversity inafferent inputs and processing outputs would thus occur atthe level of the trauben rather than the calyx in Thermobia,with sensory inputs to the calyx pathway perhaps addingcontext-dependent information to these outputs. Such a

Fig. 9. Organization of Kenyon cell axons and extrinsic neurons in the pedunculus and lobes. A. Axons of Classes I and III Kenyon cells (arrows) in Acheta

domestica forming distinct layers in the medial and vertical lobes. Note the short side branches of Class III axons in the medial lobe, lacking in the Class Iprocesses. B. Two lobe efferents in Periplaneta americana. The left arbor shows selective innervation of alternating layers (arrowheads). The right arbor

appears to selectively innervate dorsal layers (white arrows), with a more even arborization pattern across more ventral layers (black/white arrow). C and D.

Pedunculus afferents in Apis mellifera (Hymenoptera). C. Thin afferent processes (arrows) invade the pedunculus, made up of the parallel-projecting axons of

Kenyon cells. Terminals of primary sensory afferents in the calyx are also visible (arrowheads). Box indicates area of closeup in (D). D. Post-synapticspecializations (spines) on Kenyon cell ‘axons’ in the pedunculus (small arrows). Ca—calyx, Pe—pedunculus. Large arrow-afferent processes. All frontal

sections of Golgi impregnated brains. Scale bars A–CZ20 mm, DZ10 mm.


‘tuning’ role for the mushroom body calyx has already beenproposed in neopteran insects (Strausfeld and Li, 1999b;Strausfeld, 2001). Thermobia Kenyon cells are alsoorganized in a manner reminiscent of the Class II Kenyoncells of neopterans, neither of which show afferent-specific

partitioning in the calyces and which output to homo-geneous subdivisions in the lobes (the trauben in Thermobiaand the single g layer in the Neoptera). Both local circuits inthe lobes and the simpler processing circuits represented byClass II Kenyon cells have been proposed to represent

Fig. 10. Subdivisions in the mushroom body lobes and their relationship to Kenyon cell subpopulations. A and B. Layers representing the axons of separateKenyon cell subpopulations. A. Anti-taurine immunostaining reveals high and low affinity cohorts (arrows) of Class I Kenyon cells in the vertical lobe of the

cockroach Periplaneta americana. Class II cells show a uniform low affinity for the antiserum. The ingrowth core is located at the posteriormost margin of the

vertical lobe (arrowhead). B. Anti-DC0 immunostaining reveals layers of differing affinity (arrows) in the vertical lobe of the honeybee Apis mellifera

(Hymenoptera). C and D. Anti-DC0 staining of bulbous subdivisions (‘trauben,’ indicated by arrows) formed by multiple branches of a single Kenyon cellsubpopulation. C. The trauben of the medial, vertical and horizontal lobe in Thermobia domestica (Zygentoma) are simultaneously supplied by branches from

the axons of a single population of Kenyon cells. D. A similar morphology is observed in Oncopeltus fasciatus (Heteroptera), although calyx morphology

suggests that the lobe subdivisions are comprised of at least two distinct Kenyon cell subpopulations. M—medial lobe, V—vertical lobe, Pe—pedunculus. A

and D sagittal sections and B and C frontal sections. Scale bars A and DZ50 mm, BZ100 mm, CZ25 mm.


ancestral modes of mushroom body organization, hypo-theses that are supported by their prevalence in thephylogenetically basal Thermobia.

Based on the findings in Thermobia, the acquisition ofmorphologically and functionally distinct Kenyon cellsubpopulations appear to represent a neopteran innovation(Farris, 2005). The acquisition of Kenyon cell subpopu-lations would allow the segregation of afferent input to thecalyces and thus their outputs in the lobes, the generation ofparallel processing pathways for rapid processing ofparticular modalities, and increase the potential poolof integrative functions by increasing the types and numbersof combinatorial outputs generated in the lobes. In insectlineages in which a trend towards increased mushroom bodysize is observed, as in the cockroaches and socialHymenoptera, functional and morphological modularity iseven more pronounced (Fig. 10A and B; Strausfeld and Li,1999a,b; Strausfeld, 2002). Once again, structure–functionrelationships in mushroom body evolution appear to mirrorthose in cortical evolution, as the evolution of larger corticesis accompanied by a trend towards increased specializationof processing circuits, as demonstrated by the increasinglyfine subdivision of regions such as the visual cortex ofprimates into cortical areas (Kaas, 1995).

Interestingly, there is some evidence to suggest that bothtrauben and distinct Kenyon cell subpopulations coexist inat least one neopteran insect lineage, the Hemiptera (Fig.10D; Pflugfelder, 1936; Johansson, 1957). Further studies ofinsects such as these will be useful in determining thedifferences in extrinsic circuitry between these two verydifferent modes of mushroom body organization.

How commonly is the pedunculus a target of afferentinput? Afferents to the pedunculus and lobes are documen-ted in Periplaneta, Acheta and Apis, none of which arisefrom primary sensory neuropils (Schurmann, 1970; Straus-feld and Li, 1999b; Strausfeld, 2002). This is of interest inlight of the reports of Hanstrom (1940) that inputs from theACT and TOG are directed to the pedunculus in calyxlessinsects. The close proximity of tracts to the mushroombodies as revealed by general histology is not alwaysindicative of innervation, however, and the extreme thinnessof the pedunculus in many calyxless insects is in contrast tothe robust architecture of the pedunculus in species in whichafferents have been demonstrated (compare the pedunculusof the cockroach in Fig. 5A with that of the calyxless cicadain 5H). Following the same line of reasoning, afferentinnervation may be poorly developed in many insects inwhich the pedunculus is spindly in appearance. In support ofthis correlation, a comprehensive survey of extrinsicneurons to the Drosophila mushroom bodies failed toreveal any connections to the thin pedunculus (Ito et al.,1998). It, therefore, seems likely that afferents to thepedunculus represent a derived state that has arisenindependently in a number of insect lineages. As an aside,afferent innervation of the mushroom bodies in calyxlessinsects has yet to be determined, and it would be interesting

to know whether primary sensory afferents are lost, leavingonly multimodal protocerebral afferent to the lobes, or ifprimary sensory afferents are entirely redirected to lobeneuropil.

Despite their different organizations, the mushroom bodylobes are crucial to the functioning of these structures asdemonstrated by their ubiquity across the insects. Althoughnever entirely absent, evolutionary trends towards theirsimplification are documented in the Psocodea, whichcontain the parasitic lice (Phthiraptera) and the bark andbook lice (Psocoptera). The mushroom bodies of somepsocopteran lineages show a gross simplification of lobemorphology (Jentsch, 1940). Certain species of thePsocoptera possess mushroom bodies much like those ofhemipterans, with a vertical lobe and numerous trauben-likesubdivisions of the medial lobe. Other species showsimplification of the medial lobe due to the loss of thesetrauben, and in some cases the vertical lobe appears to beabsent. The Phthiraptera also possess minute mushroombodies, containing both a medial and a vertical lobe(Hanstrom, 1940). In all cases of lobe reduction, the calyxis additionally greatly reduced or absent, so that theobserved trends may be more accurately described as anoverall diminishment of the mushroom bodies. ThePsocodea are additionally characterized by an exceedinglysmall body size, in addition to having acquired a parasiticlifestyle in the case of the Phthiraptera. It is likely thatacross the insects miniaturization and parasitism aregenerally correlated with the reduction and perhaps loss ofhigher brain regions like the mushroom bodies, as has beendocumented for other morphological elements across avariety of taxa (Schmidt and Roberts, 1989; Yeh, 2002).

7. Convergence with vertebrate brain centers and futuredirections

The structure–function relationships and trends in mush-room body evolution described above illustrate manystriking similarities between the insect mushroom bodiesand the mammalian cortex, suggesting convergencebetween these higher sensory processing centers at multiplelevels. The primary difference when considering theseconvergences lies in what is considered a functional unit ineach system. Cortical layers represent a delegation ofdifferent processing functions, with neurons in layers V andVI generating efferent output, those in layer IV receivingafferent input, and those in layers II and III specializing incortico–cortical integration (reviewed in Kornack, 2000). Incontrast, each individual Kenyon cell, in concert with anarray of extrinsic neurons, accomplishes all of thesefunctions. In the mushroom bodies, afferent, efferent andKenyon cell to Kenyon cell connectivity occurs at the levelof cellular subcompartments representing the calyces,pedunculus and lobes, rather than at the level of specializedcell types arranged in cortical layers. Nevertheless, the basic


functions are identical: in both structures sensory input isreceived and selectively integrated, with outputs comprisinga variety of complex and coordinated motor and cognitivebehaviors.

Kenyon cell subpopulations, particularly cohorts of ClassI cells, form segregated processing circuits in the lobes andcalyces representing different afferent modalities such asgustation, olfaction and vision. These zones may be furthersubdivided according to different aspects of a givenmodality, for example, inputs from the different parts ofthe medulla and lobula of the optic lobe in Apis (Ehmer andGronenberg, 2002). In the mammals, broad cortical areasare defined by the sensory modality of their afferents, andmay be further subdivided in a lineage-specific manner intosmaller parallel processing units (Kaas, 1995; Finlay et al.,2001).

Kenyon cell subpopulations may, therefore, be regardedas the functional equivalents of cortical areas, a hypothesisthat is supported by the parallel evolution of these functionalmodules in the insect mushroom bodies and the mammaliancortex. Shared evolutionary trends include the correlationbetween behavioral ecology, sensory afferents and theconcomitant expansion or diminishment of Kenyon cellsubpopulations/cortical areas; the acquisition of increasednumbers of Kenyon cell subpopulations/cortical areas withincreased brain size; and the general association betweenincreased brain size and the folding of the synaptic neuropil.Further examples of convergence at the molecular anddevelopmental level that give rise to these structural andfunctional similarities are likely to be uncovered by futurestudies. The innumerable comparative opportunitiesafforded by the behaviorally and taxonomically diverseinsects are poised to provide novel insight into mechanismsof brain and behavioral evolution that are conserved acrosssuch disparate phyla as the arthropods and the vertebrates.

Acknowledgements

The author thanks Mr Jonathan Benincosa, Ms AijunQiu, Mr Nathan Roberts and Ms Kelly Todd for assistancewith histology. Dr Ronald Bayline and two anonymousreviewers provided helpful comments and suggestions onthe manuscript.

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