The Journal of Comparative Neurology 513:265–291 (2009)

Ground Plan of the Insect Mushroom Body: Functional and Evolutionary Implications

1 2 1 3 NICHOLAS J. STRAUSFELD, * IRINA SINAKEVITCH, SHEENA M. BROWN, AND SARAH M. FARRIS 1Arizona Research Laboratories, Division of Neurobiology, University of Arizona, Tucson, Arizona 85721 2IBDML-UMR 6216, Case 907 Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France 3Department of Biology, West Virginia University, Morgantown, West Virginia 26506

ABSTRACT which olfaction is redundant. Examples are cicadas and In most insects with olfactory glomeruli, each side of the mayflies, the latter representing the most basal lineage of brain possesses a mushroom body equipped with calyces winged insects. Mushroom bodies of another basal taxon, supplied by olfactory projection neurons. Kenyon cells pro- the Odonata, possess a remnant calyx that may reflect the viding dendrites to the calyces supply a pedunculus and visual ecology of this group. That mushroom bodies persist lobes divided into subdivisions supplying outputs to other in brains of secondarily anosmic insects suggests that they brain areas. It is with reference to these components that play roles in higher functions other than olfaction. Mush- most functional studies are interpreted. However, mush- room bodies are not ubiquitous: the most basal living in- room body structures are diverse, adapted to different ecol- sects, the wingless Archaeognatha, possess glomerular an- ogies, and likely to serve various functions. In insects whose tennal lobes but lack mushroom bodies, suggesting that the derived life styles preclude the detection of airborne odor- ability to process airborne odorants preceded the acquisi- ants, there is a loss of the antennal lobes and attenuation or tion of mushroom bodies. Archaeognathan brains are like loss of the calyces. Such taxa retain mushroom body lobes those of higher malacostracans, which lack mushroom bod- that are as elaborate as those of mushroom bodies ies but have elaborate olfactory centers laterally in the brain. equipped with calyces. Antennal lobe loss and calycal re- J. Comp. Neurol. 513:265–291, 2009. gression also typify taxa with short nonfeeding adults, in © 2009 Wiley-Liss, Inc.

Indexing terms: memory; learning; insect; mushroom bodies; sensory integration; evolution

The present account addresses the following question: by neurons from the olfactory lobes. The calyx is a distal what constitutes an insect mushroom body such that its or- neuropil of the mushroom body composed of the dendrites of ganizational ground plan can be reconciled with current mod- this center’s intrinsic neurons (see Fig. 1). In many species of els of its supposed functional properties? The reason why this insects, the calyx is supplied with olfactory input from the brain center must be subject to special scrutiny is in large part antennal lobes, but, in some, it also receives relays from the a result of its acquired reputation as an odorant processor and visual system, and, in one species of gyrinid beetle, visual as a higher brain center that plays a cardinal role in cognition. afferents are the exclusive inputs (Strausfeld, unpublished Common opinion is that mushroom bodies play an indispens- data). The calyx leads to a stalk and a system of lobes that are able role in olfactory discrimination as well as in olfactory formed by the axon-like extensions of intrinsic neurons (see learning and memory (Heisenberg, 1998, 2003; Perez-Orive et Fig. 1). Because for most investigators this organization de- al., 2002; Akalal et al., 2006). The latter is suggested from fines the mushroom body (Flo¨ gel, 1878; Kenyon, 1896; memory deficits caused by targeted disruption of mushroom body development. by suppression of the cyclic AMP cas- cade within its neuropils, or by selective inhibition of protein Grant sponsor: Human Frontiers Science Program; Grant number: signaling or synaptic transmission in its intrinsic neurons, the RG0143/2000-B; Grant sponsor: National Science Foundation; Grant num- ber: IBN 936729; Grant number: IBN9726957; Grant sponsor: National Kenyon cells (deBelle and Heisenberg, 1994; Zars et al., 2000; Institutes of Health NCRR; Grant number: 2-PO1 NS28495-11. McGuire et al., 2001; Ferris et al., 2006). Studies of one N.J.S. and S.M.F. contributed equally to this work. neopteran insect in particular, the locust Schistocerca ameri- *Correspondence to: Dr. Nicholas. J. Strausfeld, ARL Division of Neurobiol- cana, have led to the idea that the mushroom bodies play ogy, 611 Gould-Simpson Bldg., University of Arizona, Tucson, AZ 85721. E-mail: fl[email protected] crucial roles in the encoding and representation of specific Received 27 July 2008; Revised 19 September 2008; Accepted 19 No- odors (Laurent et al., 2001; Cassenaer and Laurent, 2007). vember 2008 However, one problem with these and related studies is that DOI 10.1002/cne.21948 they focus on mushroom bodies that possess a calyx supplied Published online in Wiley InterScience (www.interscience.wiley.com).

© 2009 Wiley-Liss, Inc. The Journal of Comparative Neurology 266 N.J. STRAUSFELD ET AL.

Figure 1. Summary diagram of the principal elements of mushroom bodies in dicondylic insects. Top: Mushroom bodies with paired calyces (Ca) equipped with Kenyon cells from cell bodies known as globuli cells (gl). Kenyon cells (magenta) have dendritic branches in the calyces, which also receive inputs from sensory neuropils (green): diagrammed here are afferents from the antennal lobes that terminate laterally outside the calyx in the lateral horn neuropil of the protocerebrum (L ho). Afferents and efferents (aff, eff; brown) also supply and extend from various locations in the lobes (see Strausfeld, 2002). Bottom: Calyxless mushroom body with globuli cells (gl) providing Kenyon cells (Kc) with axon-like branches to the vertical and median lobes (V, M). Inputs (afferent; aff) and outputs (efferents; eff) extend to and from various locations along the lobes.

Sa´ nchez y Sa´ nchez, 1933), practically all studies over the last The widely held notion that the calyces receive the mush- 3 decades have been interpreted under the assumption that room body’s inputs and its lobes provide its outputs is, how- the calyces alone receive the mushroom body’s inputs and ever, conceptually flawed. Frederick Kenyon in 1896 already the lobes provide its outputs (Menzel, 2001). The general view proposed that other neurons supply terminals to the lobes then is that because Kenyon cell dendrites in the calyces are (Fig. 1). Four recent studies (Ito et al., 1998; Li and Strausfeld, clearly postsynaptic and are the recipients of information 1999; Strausfeld, 2002; Tanaka et al., 2008) unequivocally (Steiger, 1967) their axon-like extensions in the pedunculus demonstrate that, in addition to providing outputs (efferents), and lobes must be overwhelmingly presynaptic, imparting the mushroom body’s lobes receive abundant inputs (affer- information to efferent neurons (Menzel and Manz, 2005; Cas- ents) as well as the aforementioned modulatory neurons. In- senaer and Laurent, 2007). For example, a major role of the deed, if there were no such inputs, the observer would be hard mushroom bodies in mediating learned behaviors in the honey pressed to explain why, in a mushroom body whose calyces bee has been attributed to one type of efferent neuron, called receive almost exclusively olfactory relays, the efferent neu- “Pe1,” recordings from which demonstrate a decrease in re- rons from its lobes can respond to modalities other than sponsiveness as a result of an acquired association of an olfaction, such as acoustic, visual motion, and tactile (Li and olfactory and gustatory stimulus, presumably at the level of Strausfeld, 1997, 1999). Such recordings have been done the calyx (Mauelshagen, 1993; Menzel and Manz, 2005; Okada extensively in cockroaches, where some 14 unique types of et al., 2007). Similarly, it has been proposed that the response efferent neurons responding to a variety of sensory modalities properties of a population of efferents in the locust mushroom have been identified morphologically and functionally. bodies, called “␤-L neurons,” specifically maintain the syn- If it is accepted that mushroom bodies in general give rise to chrony of oscillations that are characteristic of odor represen- efferent neurons that respond to multimodal stimuli, this tations relayed to the calyx (Cassenaer and Laurent, 2007). raises pivotal questions about the basic structure of these Models of how mushroom bodies might function as memory centers and thus their functional organization and evolution. If centers also stress the afferent supply to the calyces as being mushroom body calyces generally receive most of their inputs of crucial importance in associating olfactory stimuli with a from the antennal lobes, what is the role of this specialized conditioned stimulus, either a reward or punishment, that is stream of olfactory input with regard to the mushroom body’s delivered by modulatory neurons that globally innervate the ability to integrate other types of multimodal information re- mushroom body lobes (Schwaerzel et al., 2002, 2007). ceived by its lobes? Furthermore, does the massive innerva- The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 267 tion of the mushroom body calyx by olfactory afferents in feld et al., unpublished data). We argue that this organization, phylogenetically divergent species, such as the locust, cock- which represents the Zygentoma ؉ Pterygote ground plan of roach, fruit fly, and honey bee, support the contention that this the mushroom bodies, is independent of the presence of a brain region evolved and continues to function primarily as an calyx. This is discussed with respect to orthodox views of olfactory processing center? If so, it would be expected that, mushroom body function and with respect to a plausible first, mushroom bodies initially arose in conjunction with the scenario of mushroom body evolution. origin of antennal lobes and their projection neurons. Second, species lacking antennal lobes should also lack mushroom bodies. In support of the first scenario, recent studies of the A NOTE ON TERMINOLOGY basal apterygotes Thermobia domestica and Lepisma sac- The uniramous deutocerebral appendages of insects are charina reveal mushroom bodies with calyces that receive known as the “antennae,” which are homologous to the “first input from small glomerular antennal lobes (Farris, 2005; antennae” of crustaceans, which are called “antennules” Schactner et al., 2005; Farris, unpublished). However, classi- (Boxshall, 2004), which are also homologous to the chelicerae cal studies by Hanstro¨ m et al. (1940) on representatives of the of the chelicerates (Boxshall, 2004). The paired appendages most basal group of insects, the Archaeognatha, show no that supply the crustacean tritocerebrum are biramous, and evidence of mushroom bodies, although descriptions of this the accepted term for these is “antennae”, or sometimes group identify antennal lobes. As will be described here, the “second antennae” (Boxshall, 2004). These appendages have absence of mushroom bodies but presence of antennal glo- been lost in the insects, although rudiments can appear tran- meruli has been confirmed in the Archaeognatha by using siently during embryogenesis (Tamarelle, 1984). Thus, the sin- modern immunofluorescence techniques that can better re- gle pair of deutocerebral appendages in insects ought to be veal the detailed structure of the brain. Regarding the notion termed the “antennules.” However, the unfortunate use of the that insects without antennal lobes should lack mushroom term “antennae” by insect morphologists has a long tradition bodies, it was demonstrated over 130 years ago that insects and because of this has been retained for this account, bear- that lack antennal lobes nevertheless possess mushroom ing in mind that insect antennae and crustacean “antennae” bodies (Flo¨ gel, 1878). Curiously, although the antennal lobes are not homologous. and calyces are reduced or absent in these mostly aquatic Intrinsic neurons of the mushroom bodies are usually re- species, the mushroom body lobes remain (Fig. 1) and pre- ferred to as “Kenyon cells,” following Howse’s (1974) first use sumably serve processing functions other than the perception of the eponymous term. Here, both terms are used inter- of odors (Strausfeld et al., 1998). That such insects are wholly changeably. “Extrinsic neuron” or “element” is a generic term “anosmic” is not, however, proven; they may detect water referring to afferent and efferent profiles that extend to and soluble molecules via gustatory pathways, and a recent ac- from the lobes at different levels. count of the antennae of the dragonfly identifies pore sensilla that may represent hygro- and chemoreceptors (Rebora et al., 2008). Finally, in insects that do possess calyces, these struc- MATERIALS AND METHODS tures are remarkably diverse in morphology and across spe- Insects cies. Particularly, Hymenoptera and Coleoptera can receive Adult Machilis germanica (Machilidae, Archaeognatha) substantial gustatory and/or visual input to their calyces in were collected from colonies located in damp limestone walls addition to, or instead of, an olfactory input (Schro¨ ter and in the gardens of the Botanical Gardens, University of Wu¨ rz- Menzel, 2003; Strausfeld et al., 1998; Farris, 2005; Strausfeld burg. Damselflies and dragonflies (Calopteryx splendens, Li- et al., unpublished data). Clearly, then, calyces are not dedi- bellula depressa, Odonata) and mayflies (Potamanthus luteus, cated solely to olfactory processing. Ephemeroptera) were captured near streams and rivers in the This account compares four types of insect brains, repre- vicinity of Wu¨ rzburg. Dragonflies (Perithemis tenera, Libelluli- senting key phylogenetic positions and behavioral ecologies, dae, Odonata) and Japanese beetles (Popillia japonica, Scar- to demonstrate that irrespective of whether or not mushroom abaeidae, Coleoptera) were collected in the Morgantown, bodies are equipped with calyces their lobes reveal a common West Virginia, region. Individuals of Lepisma saccharina (Zy- ground plan of organization. The species selected include gentoma) were caught locally in Arizona. Small numbers of “typical” terrestrial neopterans with glomerular antennal adult tiger beetles (Cicindela ocellata ocellata, Cicindelidae), lobes, aquatic neopteran insects that have secondarily lost soldier beetles (Chauliognathus lecontei), fig beetles (Cotinus their antennal lobes, basal palaeopteran insects that also lack mutabilis), and diving beetles (Thermonectus marmoratus antennal lobes, and the most basal of insects: the primitively [also referred to as Thermonectes marmoratus]) were col- wingless apterygotes. The present observations are also com- lected locally in the vicinity of Tucson or at the Willcox Playa pared with published accounts of mushroom bodies in addi- and from cattle tanks near Nogales. Terrestrial and aquatic tional species of terrestrial neopterans that span the insect hemipterans (the cicada Diceroprocta semicincta and back- phylogenetic tree. swimmer Notonecta undulata) were obtained in riparian re- The present results demonstrate that what unites the mush- gions near Arivaca, Arizona. Specimens of Notonecta glauca room bodies of all insects from the apterygote Zygentoma were obtained from pools of the University of Wu¨ rzburg bo- forward is that the mushroom body lobes comprise systems tanical gardens. Blowflies (Phaenicia sericata) were obtained of parallel axon-like processes that are supplied by the termi- from breeding colonies in the ARL Division of Neurobiology. nals of afferent neurons from protocerebral centers. These Adult Thermobia domestica (Zygentoma) were collected from synapse onto Kenyon cells, and the Kenyon cells synapse breeding colonies maintained at 35°C on a 12:12-hour light: onto the dendritic trees of numerous efferent neurons (Straus- dark cycle. The Journal of Comparative Neurology 268 N.J. STRAUSFELD ET AL.

Histology 4% paraformaldehyde in phosphate-buffered saline (PBS; from tablets; pH 7.4; Sigma, St. Louis, MO). Dissected out Golgi impregnations. We employed the combined Golgi brains were stored in fixative until processing for immuno- Colonnier and Golgi Rapid procedures described by Li and staining. They were washed in PBS and then embedded in 7% Strausfeld (1997) and Farris and Strausfeld (2001). This agarose. Agarose blocks were sectioned on a vibratome at method provides crisp impregnation of neurons in all the 50–70 ␮m and the sections washed in PBS containing 0.1% brains so treated and is a “ubiquitous” Golgi procedure that Triton X-100 (PBST). Sections were blocked in PBST contain- requires no further modification. The numbers of neurons impregnated can, however, be increased by a second cycle of ing 10% normal goat serum (NGS). The primary antiserum was osmium-dichromate treatment, followed by a second silver used at a concentration of 1:1,000 in PBS. After overnight impregnation. The method used is as follows. After opening of incubation at 20°C, sections were washed in PBST and incu- the heads of cooled animals submerged in 2.5% potassium bated in Texas red-conjugated goat antirabbit secondary an- dichromate containing 1.3 g sucrose/100 ml solution, fat bod- tibody (Molecular Probes, Eugene, OR) at a concentration of ies and trachea are cleaned off the brain’s surfaces, and then 1:500. In the confocal images, anti-DCO labeled neurons are the brain was removed and placed in a 1:5 admixture of 25% shown red, magenta, or pink-brown. For DCO/phalloidin dou- glutaraldehyde and 2.5% potassium dichromate containing ble staining, Oregon green-conjugated phalloidin (Molecular 1.3 g sucrose/100 ml (4 days at 4°C), followed by a second Probes) was also added at 1:500 concentration. After 12 chromation (3 days at 4°C) in 2.5% potassium dichromate and hours, sections were washed in PBST and cleared in 60% 1% osmium tetroxide (99:1). Tissue was washed for 5 seconds glycerol in PBS for 1 hour, followed by 80% glycerol in PBS for in distilled water and then immersed in 0.75% silver nitrate in 1 hour. Sections were mounted on slides in 80% glycerol and distilled water or in borax-borate buffer adjusted to pH. 7.2. coverslipped, using clear nail polish to seal the edges of the After 24 hours of impregnation, tissue was dehydrated, em- slide (Rodriguez and Deinhardt, 1960). In confocal images, bedded in plastic, and serial sectioned. Some brains of T. phalloidin labeling is shown green. marmoratus were processed with double impregnation to in- Taurine and aspartate. Antisera against taurine and as- crease the number of elements impregnated in the mushroom partate (GEMAC, Talence, France) were separately generated bodies. After the first silver nitrate treatment, tissue was in rabbits immunized with L-aspartate or taurine conjugated to washed in distilled water and placed again in dichromate- poly-L-lysine (MW 60,000) by means of glutaraldehyde and osmium for 3 days and then placed in 0.75% silver nitrate. purified by adsorption on the glutaraldehyde-treated protein Reduced silver staining. Bodian’s (1936) original method carriers (Campistron et al., 1986a,b). Affinity and specificity was used on brains fixed in AAF (85 ml 98% ethanol, 5 ml tests were performed on thyroglobulin-linked immunoabsor- glacial acetic acid, 10 ml 35% formaldehyde), dehydrated, bent assay (ELISA) and on rat cerebellum (Campistron et al., cleared in terpineol, and embedded in Paraplast Plus (Sher- 1986a,b) to demonstrate negligible cross-reactivity to gluta- wood Medical, St. Louis, MO). No species-specific modifica- mate, GABA, taurine (for tests of antiaspartate), aspartate (for tions were necessary, and all brains on which the method was tests of antitaurine), glycine, and ␤-alanine. For the present used stained with equal clarity. study, brains were fixed overnight in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) and then embedded in 7–8% Immunolabeling agarose or gelatin/albumin and vibratome sectioned at 60– It is not the aim of this account to ascribe epitopes to 100 ␮m. After being washed in PBST, sections were blocked neuropils. Rather, immunolabeling is used here exclusively as in PBST containing 10% normal swine serum (Dako Corp., a histological label to resolve brain regions, such as the mush- Carpinteria, CA). Sections were incubated overnight with as- room bodies, by DCO. Brains were labeled with antisera that partate antiserum (1:500) or taurine antiserum (1:500) at 20°C. in Drosophila and the cockroach Periplaneta americana have After washing in PBST, secondary goat anti-rabbit immuno- been demonstrated to reveal specific subdivisions of the globulins conjugated to Texas red (1: 250; Jackson Immunore- mushroom body lobes (Sinakevitch et al., 2001; Strausfeld et search Laboratories, West Grove, PA) or anti-rabbit immuno- al., 2003; Farris et al., 2004; Sjo¨ holm et al., 2005; Farris, 2005). globulins conjugated to Alexa 568 (Molecular Probes) were After labeling with secondary antibody conjugated to a fluo- applied overnight. After washing in PBST, sections were rescent dye, immunolabeled material was observed with a mounted on slides and coverslipped under 80% glycerol. Zeiss Pascal three-line confocal microscope. Preparations incubated in the absence of primary antiserum Anti-DCO and phalloidin. Polyclonal antibody (gener- showed no staining. Specificity of immunolabeling was con- ously provided by Dr. Daniel Kalderon; see Lane and Kal- trolled as described by Campistron et al. (1986a,b): taurine deron, 1993) raised against DCO (the catalytic subunit of (Sigma; T9931), L-aspartic acid (Sigma; A-8949), and Drosophila cAMP-dependent protein kinase) reliably labels all L-glutamic acid (Sigma; G-6904) were each conjugated to Kenyon cell subpopulations in the mushroom bodies of bovine serum albumin (BSA) by glutaraldehyde (Tau-G-BSA, neopteran insects (Farris and Sinakevitch, 2003; Farris and Asp-G-BSA, Glu-G-BSA). After adsorption of taurine anti- Strausfeld, 2003; Farris et al., 2004). The anti-DCO polyclonal serum with Tau-G-BSA (10–4 M, concentration with respect to antibody was generated by cloning the DCO cDNA sequence the amino acid), there was no labeling after secondary anti- into a pAR3040 T7 expression vector (Studier and Moffatt, body treatment. Adsorption of taurine antiserum with Asp-G- 1986); then, after purification of the protein from sedimenta- BSA or Glu-G-BSA did not block staining with the secondary tion of inclusion bodies, this was used to immunize rabbits antibody. Aspartate immunostaining was undiminished after (Lane and Kalderon, 1993) and the final antibody was purified incubation of the antiserum with Tau-G-BSA (10–4 M) and using DCO protein immobilized on nitrocellulose (Harlow and Glu-G-BSA (10–4 M), whereas adsorption of aspartate anti- Lane, 1988). For the present study, brains were fixed in situ in serum with 10–4 M Asp-G-BSA abolished immunostaining. The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 269

Imaging and reconstructions and Engel, 2005). Not all Coleoptera are terrestrial, however; some hunt and forage either on or beneath the water surface Histological preparations were viewed with a Leitz Orthop- as adults. Whirligig beetles and diving beetles are two exam- lan microscope equipped with a Sony DKC 5000 CCD digital ples; the second here represented by the dytiscid Thermon- camera linked to an Apple G4 computer equipped with ectus marmoratus. graphic software. Images of Golgi-impregnated neurons were Regardless of adult feeding preference, terrestrial beetles reconstructed from stacked optical sections captured at an are endowed with antennae equipped with odorant receptors, initial magnification of ؋600, employing a planapochromat oil-immersion objective. About 25–50 optical images were the axons of which supply prominent antennal lobes divided captured and layered in register, then made transparent by into glomerular domains (Hansson et al., 1999; Larsson et al., using the Photoshop darkening function, and shadows were 2001, 2003). Projection neurons from each glomerulus extend removed section by section before flattening the image. For axons into the antennocerebral tract and provide systems of larger areas, several such reconstructions were seamlessly collaterals that supply the mushroom body calyces (green montaged. This results in images showing each process ex- outlines, Fig. 2B–D,F–H; see also Larsson et al., 2004; Farris, actly in focus throughout a depth of 25–50 ␮m. Immunola- 2008a,b). These projection neurons terminate in the lateral beled images were acquired with a three-line Zeiss Pascal or protocerebrum in a region known as the “lateral horn” (Straus- an Olympus Fluoview 1000 confocal microscope. Line recon- feld, 1976). In insect orders such as the Hymenoptera, Lepi- structions of brain regions were made by tracing outlines from doptera, and Diptera, the mushroom bodies are also supplied serial sections captured with the Sony DKC 5000 CCD digital by a second (outer) antennocerebral tract, which essentially camera. Three or four images were superimposed as stacks reverses the course of the inner tract by providing collaterals by using fiducial markers such as tracheoles or prominent to the lateral horn first and terminating in the mushroom fiber tracts to maintain orientation. Each digitized stack was bodies (Tanaka et al., 2004). There is no evidence for an outer printed onto paper, and outlines were drawn in Smartsketch antennocerebral tract in the beetles surveyed so far (Larsson (Futureware Software, Inc., San Diego, CA). Images were im- et al., 2004; Farris, 2008b). ported into Photoshop as TIF files. Shading and hidden line As described from observations of scarabid beetles (Lars- elimination provided plastic images of brains viewed frontally, son et al., 2004; Farris and Roberts, 2005), the coleopteran with the neuraxial ventral surface facing to the front. Optic mushroom body, like that of other neopterans, has two caly- lobes were omitted from these reconstructions. ces ranging across various species-specific degrees of fu- sion; from a single neuropil to two completely separate neu- ropils (Fig. 2I). Regardless of calyx morphology, distinct RESULTS populations of Kenyon cells provide an equal contribution to “Olfactory” mushroom bodies of terrestrial the pedunculus and its division into the medial and vertical insects equipped with calyces, as exemplified by lobes (Larsson et al., 2004). Reduced silver staining and Golgi coleopterans impregnation reveal that, in insects such as cockroaches, flies, honey bees, and beetles, afferents enter the lobes from Olfaction is a key sensory modality for insects that possess regions of the protocerebrum, whereas the dendrites of effer- antennae equipped with olfactory sensilla for detecting odors ent (output) neurons project from the lobes to defined proto- associated with food and conspecifics. Specific odorant re- cerebral regions (Li and Strausfeld., 1997, 1999; Ito et al., ceptor neurons that recognize a characteristic set of ligands 1998; Strausfeld, 2002). The same principal organization of target a specific antennal lobe glomerulus having a unique afferents to and efferents from the lobes is observed in the identity and position (Chambille and Rospars, 1985). This or- mushroom bodies of terrestrial beetles (Larsson et al., 2004). ganization, best known from the fruit fly Drosophila melano- In summary, mushroom bodies of terrestrial beetles reflect gaster (Fishilevitch and Vosshall, 2005; Couto et al., 2005), is the ability of these species to detect odors. These beetles probably ubiquitous across the Neoptera, although genes en- have pronounced calycal neuropils that are supplied by col- coding odorant receptor channels in any one taxon will be distinct from those of another (Robertson and Wanner, 2006; laterals of olfactory relay (projection) neurons. Variations in Wanner et al., 2007). Typically, each glomerulus provides pro- this basic ground plan occur, however, and are associated jection neurons to the mushroom body calyces. This organi- with species-specific behavioral ecologies. In feeding gener- zation typifies terrestrial dicondylic insects. alist scarabaeids, for example (Fig. 2J), separation of the two “Terrestrial” is used here to denote an animal that spends calyces is pronounced and their neuropils are further elabo- most if not all of its adult life above water and that forages or rated into two discrete zones, each supplied by a distinct type hunts on land. Here, paradigmatic examples of terrestrial spe- of projection neuron terminal. One zone is supplied from the cies are coleopterans: the predatory tiger beetle Cicindela antennal lobes; the other is supplied by projection neurons ocellata (see Fig. 3A), the herbivorous scarab beetle Cotinus that originate in the optic lobes (Farris and Roberts, 2005; mutabilis (Fig. 2I,J), and the pollen-feeding soldier beetle Farris, 2008b). Chauliognathus leconteis (Fig. 2A–H,K), all of which have mushroom bodies with paired calyces that are supplied by Mushroom bodies of aquatic coleopterans one antennocerebral tract comprising the axons of relay neu- It is commonly held that the olfactory supply from the an- rons from glomerular antennal lobes (see schematic in Fig. 2A tennal lobes determines the primary functional role of the and micrographs in Fig. 2B–D,F–H). The order Coleoptera mushroom body in olfactory processing. However, if the func- originated in the Early Permian as the first holometabolous tion of the mushroom body were solely that of an olfactory insects and today comprises the largest number of species of center, it might be expected that an evolutionary absence of any insect order or, for that matter, any animal order (Grimaldi olfactory inputs, or the transformation of these to another The Journal of Comparative Neurology 270 N.J. STRAUSFELD ET AL.

Figure 2 The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 271 modality, should result in the diminution or even the absence lobe. Indeed, this is so, but only with regard to the appearance of the mushroom bodies. of the calyces. Whereas the calyces of aquatic species are In terrestrial species, the antennae supply two major very much reduced or in some species even absent (Berger, streams of sensory axons to the brain (Boyan et al., 2002). One 1878; Flo¨ gel, 1878), the pedunculus and lobes of aquatic carries information about olfactory stimuli to the glomerular beetles appear to differ little from those of their terrestrial antennal lobes of the deutocerebrum (Fishilevich and counterparts. They can be as large as in terrestrial ones of Vosshall, 2005); the other carries mechanosensory afferents comparable brain size (Fig. 3A,D,E) and the lobes receive from the scapus and pedicellus to striate neuropil of the inputs from the protocerebrum (Fig. 3B,C,F,G), as they do in dorsal lobes (Fig. 2K), regions that have been ascribed by terrestrial species (Larsson et al., 2004). various authors to the tritocerebrum because of their segmen- Instead of possessing Kenyon cells that are endowed with tal affinities and the passage of their heterolateral axons via densely branched dendritic trees that form a discrete calyx, in suboesophageal commissures (Viallanes, 1887; Nishino et al., most aquatic coleopterans Kenyon cells are either devoid of 2005). Only relays from the olfactory pathway, that is, via the dendrites or give rise to occasional wispy dendrites that ex- antennal lobes, normally supply the calyces. However, mu- tend far out into neuropils of the superior protocerebrum (Fig. tants of Drosophila melanogaster, such as Antennopedia, 4A). Phalloidin labeling fails to reveal microglomeruli at a distal transform the antennae into legs, resulting in a mechanosen- level that is equivalent to the calyces of a terrestrial species sory supply to the antennal lobes (Stocker and Lawrence, (Fig. 4a1). In terrestrial species, microglomeruli in the calyx 1981). In these mutant flies, transformation of the antennae (see, e.g., Fig. 2I) represent the sites of synaptic input onto into legs does not result in a loss of the mushroom bodies, Kenyon cells (Steiger, 1967; Yasuyama et al., 2002; Farris et even though the cellular organization of the antennal lobes al., 2004; Frambach and Schurmann, 2004; Groh et al., 2006; more resembles neuropil serving a thoracic appendage rather Lent et al., 2007). However, the absence of microglomeruli in than one serving an olfactory one (Stocker and Lawrence, T. marmoratus does not mean that Kenyon cells are devoid of 1981; Strausfeld, unpublished). This suggests that the deter- all manner of afferent input distally, because, as shown by mination of mushroom body neuroblasts and their production Golgi impregnations, dendrites extending out into the sur- of the proper Kenyon cell progeny assume the correct mor- rounding protocerebrum are equipped with spiny or claw-like phological fates independent of the afferent supply to the specializations indicative of postsynaptic sites (Fig. 4A). putative calyces, and although no laboratory experiments An exception to the reduced or absent calyx of aquatic have succeeded in selectively deleting the antennal lobes to beetles is found in the Gyrinidae, such as the whirligig beetle, determine whether this affects mushroom body development Dineutus sublineatus. Although its antennae supply only stri- natural selection had accomplished such experiments by the ate mechanosensory neuropils, its mushroom body neverthe- late Jurassic with the appearance of coleopteran species less has robust calyces. However, instead of being supplied adapted for life underwater. Species of aquatic beetles thus by olfactory projection neurons, the calyces are supplied by far sampled appear to have lost their antennal lobes. Slender relays from the optic lobe medullas subtending the upper annulated antennae, which the Gyrinidae (whirligig beetles) (aerial) compound eyes. In this taxon, the mushroom body use to detect surface vibrations (Bendele, 1986), supply calyces have become dominated by a sensory modality that is mechanoreceptor axons to a striate dorsal lobe neuropil cor- neither olfactory nor gustatory (Strausfeld, unpublished ob- responding to the striate dorsal lobe of terrestrial coleopter- servations). ans, which is likewise supplied by mechanosensory afferents In aquatic coleopterans, the long, parallel axon-like pro- (Fig. 2K). This region is homologous to mechanosensory neu- cesses of Kenyon cells do not differ obviously from those of ropil of the Drosophila tritocerebrum supplied by mech- terrestrial species. Kenyon cells of aquatic beetles, like those anosensory neurons from the scapus and pedicellus (Kamik- of their terrestrial cousins, appear to derive from two adjacent ouchi et al., 2006). clusters of perikarya even when the calyx is entirely absent If the mushroom bodies are olfactory processing centers, (Fig. 4A, inset a1). The two bundles of neurites converge to their organization should reflect the absence of an antennal supply a stout pedunculus, which then divides into a medial and vertical lobe. Along their lengths, the long processes of Kenyon cells are supplied by afferent endings just as they are in the lobes of terrestrial species possessing a defined calyx. Figure 2. Mushroom bodies of terrestrial Coleoptera. A–H: Reconstruction (A) Thus, as evidenced by sections cut across equivalent levels from successive reduced-silver-stained sections (B–D,F–H) of the and stained by reduced silver (Fig. 3A,D,E), the fibroarchitec- mushroom body of the soldier beetle Chauliognathus lecontei (E). ture of the lobes of aquatic coleopterans seems as elaborate I: Mushroom body of the fig beetle Cotinus mutabilis (J) labeled with as that of a terrestrial species. Golgi impregnations reveal anti-DCO to resolve Kenyon cells (magenta) and phalloidin (green), which reveals microglomeruli of the calyces (Ca). In beetles possess- inputs to the lobes (Fig. 3B,C,F,G) as they do in terrestrial ing antennal lobes (Ant Lo), projection neurons supply ascending species. In summary, the lack of an antennal lobe is usually tracts to the calyces (Ca) through the antennocerebral tract (ACT; matched by a pronounced reduction or even loss of the calyx green in A–D,F–H). In Coleoptera, each calyx is supplied by two but not by decreased elaboration of the pedunculus and identical populations of globuli cells (gl). The two calyces supply axons to the pedunculus (Ped) and the medial (M) and vertical (V) lobes. lobes. In some species, such as Chauliognathus lecontei, the concen- tric organization of each calyx is represented through the length of the Mushroom bodies of the Heteroptera: aquatic, pedunculus and its lobes. K: One of the paired glomerular antennal terrestrial, and nonfeeding terrestrial species Cotinis mutabilis, lobes (Ant Lo) of with the striate mechanosensory The Heteroptera are a diverse group of carnivorous and neuropil dorsocaudal to it (Mech S). FB, fan-shaped body of the ␮m in B (applies to B–D,F–H); 50 ␮m phytophagous species considered to be the sister group of 50 ؍ central complex. Scale bars in I,K. the holometabolous insects (Grimaldi and Engel, 2005). As The Journal of Comparative Neurology 272 N.J. STRAUSFELD ET AL.

Figure 3. A–G: Vertical and medial lobes of comparable size in a terrestrial tiger beetle (A: Cicindela ocellata) and aquatic sunburst and whirligig beetles (D: Thermonectus marmoratus; F: Dineutus sublineatus); beetles show no clear differences in terms of size relative to other brain areas. Large argyrophilic processes indicate “extrinsic” (afferent and efferent) neurons to and from the lobes. B,C,F,G: Golgi impregnations of afferent supply to the lobes of T. marmoratus and D. sublineatus. B: In T. marmoratus both the vertical lobe (cross-section in square brackets) and the medial lobe (C; brackets) receive terminals (highlighted solid arrows) from the protocerebrum. F,G. In D. sublineatus, the vertical lobe (F) and medial ؍ lobe (G) receive inputs (highlighted solid arrow) and provide outputs (open arrow). Arrowheads in F indicate Kenyon cell processes. Scale bars 50 ␮m. The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 273 previously described by Pflugfelder (1936), the mushroom calyx in either the Odonata (Fig. 4B, insets b1,b2; see also Fig. body of the terrestrial milkweed bug Oncopeltus fasciatus 7A) or the Ephemeroptera (Fig. 6A), the most basal group of (Fig. 5A) possesses a large fused calyx formed by two groups winged insects. Nevertheless, there are some interesting dif- of Kenyon cells (and a small accessory calyx supplied by a ferences between these two groups. In the Ephemeroptera, third). The larger primary calyx is supplied by afferents from each mushroom body consists of a slender pedunculus that the antennal lobe. The parallel processes of the Kenyon cells provides several bulbous structures at its base (Fig. 6A, inset continue into a typical pedunculus and lobes, the latter dec- a3). In apterygote zygentomans (see, e.g., Fig. 6, inset b1), the orated with an array of orbicular swellings (Fig. 5A). As in the mushroom bodies lobes give rise to similar bulbs or “Trauben” Coleoptera, several taxa of the heteropteran suborder (Farris, 2005), exemplified in Figure 6C,D from Ctenolepisma Hemiptera have independently adopted an aquatic life style. longicaudata. In the ephemeropteran P. luteus (Fig. 6A), an These species are here exemplified by the water strider extension of the pedunculus toward the midline gives rise to Aquarius remigis and the water boatmen Notonecta glauca tuber-like components of the medial lobe that are situated and N. undulata. These insects have also retained the mush- beneath a simple two-layered central complex (Fig. 6A, see room body lobes, but their calyces have undergone a pro- also inset a3). These swollen elements of the medial lobe (Fig. found diminution or even loss, as indicated by the complete 6A) are penetrated by axons that branch within them, sug- absence of microglomeruli (Fig. 5B), although in some species gesting both inputs and outputs at these locations. Anti-DCO the lobes are prominent (Fig. 5C,D,H). Despite the absence of and phalloidin staining demonstrates that the mayfly mush- even a vestigial calyx, there is still a trace of an antennocere- room body possesses a cluster of microglomeruli (Fig. 6A, bral tract that reaches a position at the pedunculus where a inset a2) localized within the most distal level of the pedun- calyx would be in a species equipped with antennal lobes (Fig. culus at a level where, in terrestrial neopterans, the calyx is 5B,E). normally located. The presence of microglomeruli in P. luteus Within the heteropteran suborder Homoptera, adult cicadas indicates that afferent input of some kind is targeting Kenyon are a famously short-lived terrestrial species, despite spend- cells at this level. ing up to 17 years underground as feeding nymphs (Marshall, In contrast to those of the Ephemeroptera, mushroom bod- 2001). As seen in the aquatic Hemiptera, the brain of the adult ies in the Odonata are massive structures (Figs. 4B, 7A–F). cicada Tibicen spp. reveals a robust pair of mushroom body Their intrinsic neurons derive from twinned clusters of globuli lobes (Fig. 5F) provided by a small cloud of Kenyon cell cells that provide two necks that converge to form a single somata. Despite the complete lack of microglomeruli and thus pedunculus (Fig. 7A,B). As observed in neopteran insects, but the absence of a calyx (Fig. 5G), the most distal portion of the apparently lacking both in the Ephemeroptera and possibly in pedunculus is intersected by a vestigial but recognizable an- the primitive apterygote Thermobia domestica (Farris, 2005), tennocerebral tract, as described above for the aquatic No- the odonate pedunculus and its vertical and medial lobes are tonecta (Fig. 5B,E). The tract comprises a small bundle of divided into parallel divisions. These can be separately re- neurites originating from perikarya that would, in a species solved using appropriate immunocytological markers that equipped with an antennal lobe, normally provide systems of demonstrate their similarity to divisions identified in neopteran dendrites to the antennal lobe’s glomeruli. insects, such as the cyclorrhaphan flies Drosophila melano- Mushroom bodies in Ephemeroptera and gaster (Strausfeld et al., 2003) and Phaenicia sericata (Fig. 8). Odonata This degree of similarity suggests that parallel divisions of the Until very recently, it was thought that extant bristletails mushroom bodies, composed of distinct Kenyon cell sub- (Archaeognatha), which are reminiscent of certain late Devo- populations that are generated sequentially by mushroom nian fossils, represent the earliest terrestrial insects (Laban- body neuroblasts (Lee et al., 1999), originated in the Palae- deira et al., 1988). However, the age of the first insect might optera and have been conserved in the Neoptera as part of have to be placed significantly earlier as a result of a contem- the basal ground plan of this neuropil. Because the divisions porary reinterpretation by Engel and Grimaldi (2004) of a fossil in Aeschna correspond to similar parallel divisions first iden- insect called Rhyniognatha hirsti, originally discovered in the tified in the lobes of flies, it is convenient to use the same .(1920s in Rhynie Chert, which is dated to the early Devonian. terms (␣, ␣؅, ␤, ␤؅, ␥) to indicate comparable elements (Fig. 8 The mouth parts and associated apodemes of this specimen The parallel organization of divisions in the odonate lobe is are more similar to those of primitive winged insects than to further confirmed by reduced silver preparations, which reveal those of any archaeognathan. Apart from the exciting impli- three domains throughout the length of the pedunculus and cation that winged insects had already evolved by 410 million lobes. Each domain is characterized by its distinctive arrange- years ago, the structure of this animal’s jaws suggests affin- ment of fibers and extrinsic elements (Figs. 9A–C, 10A), the ities with the Odonatoptera, a group of insects that have latter again revealing the substantial supply to the lobes by aquatic naiads (larvae) and winged imagoes. This group in- afferent neurons (Fig. 10B). cludes today’s damselflies and dragonflies (Odonata). These Studies on preemergent odonate brains, with the vital stain Palaeoptera are basal taxa that are additionally characterized methylene blue, have proposed that the most distal part of the by possessing reduced “setaceous” antennae that supply aeschnid mushroom body receives afferents from the optic axons to mechanosensory neuropils. Both damselflies and lobe (Svidersky and Plotnikova, 2004). Evidence supporting a dragonflies lack multiglomerular antennal lobes, as do their distal area that is morphologically distinct as a zone receiving naiads, yet their brains have prominent mushroom bodies an afferent supply is suggested by DCO/phalloidin staining. (Svidersky and Plotnikova 2004; Farris, 2005). DCO-positive bundles of processes originate from clusters of Golgi impregnations, reduced silver stains, and immunola- globuli cells and extend through the pedunculus (Fig. 4B, inset beling show no evidence for the presence of a neopteran-like b1). At a position just beneath where the bundles of Kenyon The Journal of Comparative Neurology 274 N.J. STRAUSFELD ET AL.

Figure 4 The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 275

-cell neurites merge to form the pedunculus proper, phalloidin reminiscent of the ␣ and ␣؅ lobes of the cyclorrhaphan mush labeling reveals punctate microglomeruli (Fig. 4B, inset b2) room body (see Fig. 8E, or their odonate equivalent, Fig. 8D). that correspond to those typifying the calyces of terrestrial The Zygentoma possess antennal lobes. These glomerular neopterans (Steiger, 1967; Yasuyama et al., 2002; Farris et al., neuropils are small relative to the layered mechanosensory 2004; Frambach et al., 2004; Groh et al., 2006; Lent et al., neuropils that are also supplied by the antennae (Schactner et 2007; Mashaly et al., 2008). As demonstrated via Golgi im- al., 2005). Nonetheless, afferents from the antennal lobes of pregnations (Figs. 4B, 7A), Kenyon cells in odonates also Thermobia project to the mushroom body calyx and from provide a very loose arrangement of dendrites that extend into there to the lateral protocerebrum via a tract that is equivalent the surrounding protocerebral neuropil, where they may re- to the inner antennocerebral tract of neopterans (Fig. 6B,D; ceive afferent relays from the visual and other sensory neuro- see also Farris, 2005, 2008a). A parallel tract originating in the pils. Dendritic branches also originate along the length of lobus glomerulatus (see Fig. 13) also carries afferents to an Kenyon cell processes in the pedunculus and extend some- accessory calyx (curved brackets, Fig. 6B), where it supplies times for scores of micrometers out from the pedunculus into just two prominent microglomeruli of the ventral calyx. Inputs other protocerebral regions (Fig. 7D–F). This arrangement is from the lobus glomerulatus to the calyces via an equivalent similar to that observed in aquatic coleopterans, where den- tract are also observed in the Orthoptera (Weiss, 1981; Fram- dritic extensions from Kenyon cells likewise reach into the bach and Schurmann, 2004). Studies of maxillary palp inputs surrounding protocerebrum (Fig. 4A). to this neuropil in the Orthoptera (Blaney and Chapman, 1969; Ignell et al., 2000) suggest that its ascending tract carries Antennal lobes and mushroom bodies in gustatory information to the calyx. It is likely that in zygento- apterygotes mans the calyces similarly receive a combination of gustatory There are two extant lineages of primitively wingless, or and olfactory information. In conclusion, the mushroom bod- apterygote, insects. These are the Zygentoma, which com- ies and their inputs in the Zygentoma are reminiscent of those prise the firebrats and silverfish, and the Archaeognatha or of orthopterans. bristletails. Recent phylogenies have placed the Zygentoma Kenyon cells have a peculiarly high affinity for anti-DCO as a sister group of the pterygote insects (Paleoptera and antibody (Farris, 2005). However, monocondylic insects, here Neoptera; Mendes, 2002; Giribet et al., 2004; Grimaldi and represented by the archaeognathan Machilis germanica (Fig. Engel, 2005), with the Archaeognatha basal to this grouping 11A), are unlike any other insect in that they uniquely lack any (Hovmo¨ ller et al., 2002; Carapelli et al., 2007). Thermobia do- evidence of mushroom bodies, including the lobes. Anti-DCO mestica, a zygentoman of the family Lepismatidae, has re- immunostaining on three individuals failed to reveal any neu- cently been demonstrated to possess mushroom bodies with rons in the brain that were labeled by this procedure, con- all of the elements of those of the terrestrial Neoptera (Farris, trasting with the anti-DCO affinity of Kenyon cells in all other 2005; see also Fig. 6B–D). The mushroom body of T. domes- insects surveyed. Phalloidin immunolabeling reveals that the tica consists of two groups of Kenyon cells, the dendrites of deutocerebrum of M. germanica looks very much like that of which supply a single calyx. Their axon-like processes extend Thermobia. It consists of a glomerular neuropil, supplied by through the pedunculus into medial and vertical lobes, where the antennal nerve, lying immediately rostral to a larger striate they provide numerous branches that constitute bulbous neuropil that is situated at the level of the tritocerebrum and Trauben as occurs in other species in this order, such as the supplied by the mechanosensory axon bundle of the antenna Australian Ctenolepisma longicaudata (Fig. 6C,D). However, it (Fig. 11C). A medial glomerular neuropil is innervated by a is not possible to discern parallel divisions in the medial lobes nerve arising from more posterior segments and thus likely of Zygentoma that in Neoptera reflect the sequential neuro- represents the tritocerebral lobus glomerulatus. These results genesis of type I–III Kenyon cell populations (Fig. 8; see also suggest that the acquisition of glomerular olfactory lobes, and Farris and Sinakevitch, 2003). Nevertheless, the vertical lobe thus the ability to process airborne odorants, occurred prior to of Ctenolepisma longicaudata terminates as two swellings the acquisition of mushroom bodies. It is also unlikely that the mushroom bodies were secondarily lost in Machilis. As we demonstrate here, in other species that have lost their caly- ces, the mushroom body lobes always persist even in species that lack antennal lobes and that have very short adult lives. Figure 4. Mushroom bodies of insects with secondary anosmia or reduced Neither condition applies to archaeognathans. However, al- olfaction. A: Golgi impregnation showing that Kenyon cells of the though there is no evidence for mushroom bodies in Machilis, diving beetle Thermonectus marmoratus (C) provide a loose arrange- the lateral horn of the protocerebrum is pronounced (S L Pr in ment of dendrites (arrowed) that extend into the surrounding proto- Fig. 11B). This suggests that in Machilis, as in other insects cerebral neuropil. DCO labeling (magenta, inset a1) shows two bun- dles of Kenyon cell neurites (arrowheads) from globuli cells projecting (Homberg et al., 1988), this part of the protocerebrum is sup- into the protocerebrum without providing a definable calyx or even a plied by relays from the antennal lobes and is likely to be remnant that, as in Odonata, would be represented by microglomeruli homologous to regions of the lateral protocerebrum that in (green, inset b1). B: Kenyon cells of the mushroom body of the banded malacostracan crustaceans receive the terminals of projec- demoiselle damselfly Calopteryx splendens (D) provide long (solid arrows) and short (s, open arrows) dendrites; the long ones extend tion neurons from the antennular lobes (Sullivan and Beltz, into the surrounding protocerebrum, whereas the short dendrites pro- 2001). vide their claw-like specializations at the pedunculus. In the dragonfly Perithemis tenera, four bundles of neurites (arrowheads in inset b1) originate from four sets of globuli cells (labeled pale magenta with DISCUSSION DCO in b1). These provide microglomeruli (labeled green with phal- loidin, inset b2) within and surrounding the head of the pedunculus, Resolving the role of the insect mushroom bodies requires ␮m in A,a1,B,b1; 20 ␮m in b2. more than a study of three “model” species, in which the 50 ؍ shown in cross-section. Scale bars The Journal of Comparative Neurology 276 N.J. STRAUSFELD ET AL.

Figure 5 The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 277 dominant position of the olfactory pathway detracts from con- tional olfactory glomeruli of the latter type. These studies siderations about other aspects of organization that may have therefore support the view that mushroom bodies indeed en- nothing at all to do with odor perception. A case in point is the code precise information about the identity of odors (Wang et gyrinid mushroom body, where the calyces have been entirely al., 2004; Szyszka et al., 2005). commandeered by the visual system (Strausfeld, unpub- Are, then, the mushroom bodies primarily olfactory centers, lished). Clues to the overall function of the mushroom bodies or might they play a broader role in multisensory integration? are likely to emerge from comparative studies that consider This question is of central concern because it is unlikely that Kenyon cell circuits in the most basal insect lineages (barring mushroom bodies evolved concurrently with the acquisition of those species that have since acquired highly derived behav- airborne odor perception. This is implied by the absence of ioral ecologies). To what degree are circuits in the lobes mushroom bodies in the Machilidae despite the presence of ubiquitous? Although this question will be considered in a olfactory glomeruli. In the Machilidae, relays from the antennal forthcoming study (Brown et al., unpublished), the present lobes supply an extensive neuropil that makes up a large discussion will consider the ground plan of the mushroom volume of the lateral protocerebrum. This area is relatively bodies, their evolution, and their likely function. much larger than that of the lateral horn, a delineated dorso- lateral region of the lateral protocerebrum that receives end- What is the functional identity of the mushroom ings from the antennal lobes of dicondylic insects (Tanaka et bodies? al., 2004) and where the organization of terminals of antennal That the mushroom bodies are olfactory processing centers lobe projection neurons best manifest odortypic representa- has been advocated primarily in studies on the orthopteran, tion (Tanaka et al., 2004; Lin et al., 2006; Jefferis et al., 2007). Schistocerca americana, in which subsets of Kenyon cells are Taken together, the protocerebral termination area for anten- thought to serve as coincidence detectors that encode odor nal lobe relays in monocondylic insects and the smaller lateral identity (Perez-Orive et al., 2002; Cassenaer and Laurent, horn of the Dicondylia are most reminiscent of neuropils in the 2007). An objection to this view is that such studies cannot malacostracan lateral protocerebrum (the medulla terminalis legitimately generalize across insects, because it is unclear and hemiellipsoid body), in which olfactory relay neurons from whether this and closely related taxa are representative even the antennular lobes terminate (Sullivan and Beltz, 2001, of the Orthoptera, because the locust’s antennal lobes are 2005). unusual in that they are aglomerular. Each olfactory receptor axon diverges to many small glomeruli and thus several pro- The mushroom body ground plan and its jection neurons (Ignell et al., 2001; Anton et al., 2002). In most elaboration in the Neoptera insects, all olfactory receptor neurons carrying a given recep- The present results demonstrate that many species of tor protein converge on a single specific glomerulus, from neopteran insects possess mushroom bodies that either lack which information is then transmitted to the mushroom bodies calyces or have drastically reduced calyces, a deficit that via a small number of mostly uniglomerular projection neurons correlates with the absence of antennal lobe glomeruli (Fig. (Bhalerao et al., 2003; Tanaka et al., 2004). However, there is 12). Such calyxless mushroom bodies occur mostly in aquatic evidence for coincidence detection and signal “sparsening” in species secondarily lacking antennal lobes. These species the mushroom bodies of the honey bee Apis mellifera and the provide the results of an experiment that cannot as yet be fruit fly Drosophila melanogaster, both of which have conven- performed in the laboratory: the selective ablation of primary olfactory neuropil. Calyxless species include diving coleopter- ans and aquatic, as well as surface, hemipterans: two exam- ples are the water boatmen (e.g., Notonecta glauca) and water Figure 5. striders (e.g., Gerris remigis), the latter relying on mech- Mushroom bodies of the milkweed bug Oncopeltus fasciatus (A) com- anosensory signals for prey and conspecific detection, orien- pared with aquatic and water-adapted hemipterans (B–H) lacking tation, and courtship (Murphey, 1971; Lang, 1980), all behav- antennal lobes and calyces. A–C,F,G show DCO/phalloidin labeling; D,E,H are Bodian stained. A: O. fasciatus mushroom body showing iors that in many terrestrial species are driven by odor globuli cells (gl), calyx (Ca), and pedunculus (Ped) extending through perception. Notwithstanding that sensory axons from the an- the lateral protocerebrum (L Pr) and giving rise to a medial lobe (M) tennae of these hemipterans supply exclusively striate mech- equipped with numerous ovoid swellings. B: Notonecta undulata. anosensory neuropils, these taxa possess large and elaborate Distal part of the pedunculus of this back-swimmer supplied by globuli cells. The absence of microglomeruli suggests complete loss mushroom bodies that lack calyces but have elaborate lobes. of calyx. The diffuse fascicle of axons (brackets) comprises the ves- At least one group of exclusively terrestrial neopterans tigial antennocerebral tract. C: N. undulata. Medial lobe of mushroom lacks antennal lobes and calyces. These are the cicadas, the body showing many swollen outgrowths. This contrasts with the me- antennae of which are minute and predominantly mech- dial lobe of the water strider Aquarius remigis, shown in H, which terminates as a single bulb. D: Aquarius remigis. The vertical lobes of anosensory, and in whose brains antennal lobes are conspic- the wider strider have two divisions (1,2) flanked by a smaller lobe uously absent. However, cicadas possess mushroom bodies (asterisk). E: N. glauca. The vestigial antennocerebral tract (brackets) with substantial medial lobes, albeit foreshortened vertical stained by reduced silver. In this species, the mushroom body is lobes (Diceroprocta; Fig. 12). The cicadas are short lived, do simple, arising from one population of globuli cells with a pedunculus ending in a system of ovoid swellings, as shown in N. undulata in C. not feed as adults, and are notable for their use of auditory The mushroom body of N. glauca is reminiscent of the ephemerop- rather than olfactory cues for mate location. This truncated teran mushroom body shown in Fig. 6A. F: Diceroprocta semicincta. and specialized adult life history may explain the loss of the The pronounced vertical (V) and medial (M) lobes of the cicada mush- antennal lobe and concomitant loss of the calyx. Diceroprocta semicincta room body that lacks a calyx. G: . Mushroom The second group of insects with calyxless mushroom bod- body globuli cells (gl), pedunculus (ped), and residual antennocerebral tract (brackets). H: A. remigis. The large bulbous medial lobe of the ies are the two ancient orders of Palaeoptera: the Ephemer- ␮m. optera, comprising the mayflies, and the Odonata, comprising 50 ؍ water strider. Scale bars The Journal of Comparative Neurology 278 N.J. STRAUSFELD ET AL.

Figure 6 The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 279

Figure 7. Mushroom body of the dragonfly Libellula depressa. A: Reduced-silver section showing two of the four bundles of neurites (arrows) arising from several thousand globuli cells and supplying the two necks (ne) of the pedunculus (ped). B: Top-down view showing the two populations of globuli cells, each supplying half of the mushroom body. The two necks of the pedunculus (ne) are surrounded by a swathe of protocerebral neuropil (Pro). There is no evidence in silver stains of a discrete calyx, but antisera against phalloidin reveal microglomeruli (Fig. 3B, inset b2). C: Golgi impregnation of a few dozen Kenyon cells supplying one pedunculus neck and then extending through the pedunculus and medial lobe. D–F: Enlargements of boxed areas in C show details of Kenyon cell dendrites that extend into the protocerebrum from the distal pedunculus .␮m 50 ؍ D) or that arise at several levels along the length of the pedunculus and medial lobe (E,F). Scale bars)

Figure 6. Comparisons of ephemeropteran (A, a2, a3) and zygentoman (B–D) the damselflies and dragonflies. The latter possess substan- mushroom bodies. A: In the mayfly Potamanthus luteus (a1), mushroom bodies are simple. A single cluster of globuli cells (gl) provides a pedun- tial protocerebra and are equipped with enormous optic lobes culus (Ped), the head of which contains an assembly of microglomeruli but lack glomerular antennal lobes and calyces. Phalloidin (green in inset a2, which is a DCO/phalloidin preparation corresponding labeling reveals copious microglomerular-like structures in to the white-outlined box in A), indicative of an attenuated calyx. The the distal pedunculus, indicating substantial afferent input to pedunculus extends medially to terminate as a few discrete swellings (arrows, also inset a3, a DCO/phalloidin preparation that corresponds to this region that is likely to originate from the optic lobes the black-outlined box in A). There is no evidence of a discrete vertical (Svidersky and Plotnikova, 2004). These microglomeruli are lobe in this taxon. B: Thermobia domestica. Mushroom bodies of the clustered within and around the passage of Kenyon cell pro- Zygentoma (inset b1, Lepisma saccharina) have a small calyx (Ca) cesses at a level in the mushroom body that, in other species, composed of two components: a dense central aggregate of micro- glomeruli (here revealed by DCO) flanked by larger ones (curved would provide the calyces. As demonstrated here, odonate brackets). These are supplied by two streams of axons from the mushroom bodies can be huge (Agrion; Fig. 12), comprising antennocerebral tract. The smaller of the two (straight brackets, left) many thousands of intrinsic neurons and subdivided into di- carries axons from the lobus glomerulatus. The larger tract carries axons of antennal lobe projection neurons. Globuli cells (gl) are orga- visions that are reminiscent of neopteran mushroom bodies. nized in two clusters. C,D: Ctenolepisma longicaudata (silverfish) pos- Among the Neoptera, the most substantial mushroom bod- sesses mushroom bodies equipped with a medial lobe from which ies with respect to the number of constituent neurons are protrude grape-like swellings called “Trauben” (T) and a prominent seen in both social and solitary Hymenoptera. Nevertheless, vertical lobe (V). Bodian stains also show the calyx (Ca) supplied by a substantial antennocerebral tract (bracket in D). EB, ellipsoid body. the mushroom bodies of the honey bee Apis mellifera are thus ␮m in A–D; 25 ␮m in a2. far the best studied (Strausfeld, 2002), and their elaboration 50 ؍ Scale bars The Journal of Comparative Neurology 280 N.J. STRAUSFELD ET AL.

Figure 8. Parallel divisions of the mushroom body lobes. In neopteran insects, here exemplified by Phaenicia serricata (B,C,E), mushroom bodies consist of several discrete parallel divisions. Antisera against aspartate, taurine, and DCO distinguish divisions of the vertical and medial lobes (B,C,E). In odonate insects, here exemplified by the dragonfly Aeschna, the same terms (␣, ␣؅, ␤, ␤؅, ␥) can be used to indicate comparable subdivisions in the lobes (A,D,F inset, H). In the fly, intense DCO immunoreactivity identifies the lobelet (E), whereas, in Aeschna, an intense DCO-positive structure at the bulb of the vertical lobe (D) indicates a vertical branch of the ␥-lobe. As in the fly (B), aspartate reveals two parallel divisions in the odonate mushroom body, best seen in cross-sections of the pedunculus (inset to F). Aspartate labels the head of the odonate pedunculus brackets, F) and the fly calyx (G). Taurine identifies the ␣؅ division in the fly (C) and a comparable swelling at the tip of the odonate vertical lobe) .␮m 50 ؍ asterisk in H). Scale bars)

represents one extreme of mushroom body evolution, in discrete concentric zones, each of which is supplied by a which the calyces are polymodal, supplied by afferents from characteristic arrangement of terminals that carry olfactory, several sensory modalities. At the other extreme are the gustatory, or visual information and invested with a charac- mushroom bodies of Ephemeroptera. These are exemplified teristic ensemble of Kenyon cell dendrites (Strausfeld, 2002; by the mushroom body of the mayfly Potamanthus luteus, Ehmer and Gronenberg, 2002; Schro¨ ter and Menzel, 2003). which lacks a calyx and originates from a small cluster of Kenyon cells associated with a particular zone send their globuli cells. In contrast, the honey bee calyx is divided into 13 parallel extensions into a corresponding stratum that extends The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 281

Figure 9. Parallel divisions of the mushroom body of the odonate Libellula depressa. A: Cross-sections of the pedunculus distinguish three parallel divisions, each of which will provides the corresponding components of the vertical (␣, ␣؅) and medial (␤, ␤؅) lobes, with the ␥ division contributing to both. Note that each division has its own characteristic fibroarchitecture and extrinsic (input and output) elements. B: Afferent neurons invading the base of the pedunculus. C: The junction of the vertical and medial lobes. Afferent neurons extending to the ␣؅/␤؅ (double .␮m 50 ؍ arrows) shown with the initial branches of two large efferent neurons arising from the ␣/␤ and ␥ divisions (single arrows). Scale bars

for the length of the pedunculus and lobes. As shown by Golgi is not divided into parallel divisions. Its lobe merely provides studies, each stratum receives afferents (inputs) that are re- several bulbous outgrowths, each of which additionally re- stricted to it, and each stratum contains layered dendrites of ceives afferents from the protocerebrum, and each presum- efferent (output) neurons that extend from it to terminate in the ably supplies outputs back to the protocerebrum. This mor- protocerebrum (Strausfeld, 2002). In essence, the honey bee phology is reminiscent of the “Trauben” formed by Kenyon mushroom body is not a single isomorphic ensemble of Ke- cell branches in the lobes of the apterygote Thermobia do- nyon cells but is a massively parallel structure that is divided mestica, although the latter does possess both a main and an into numerous subunits, each served by its own arrangement accessory calyx (Farris, 2005), the latter supplied by gustatory of inputs and providing its own outputs. Accordingly, the afferents from the lobus glomerulatus. The Ephemeroptera collar zone of the mushroom body calyx, which receives visual are acknowledged as the most “primitive” of extant winged inputs from the optic lobes (Ehmer and Gronenberg, 2002), insects, the fossil record for which extends back to the early supplies Kenyon cell processes to five parallel strata of the Permian (Grimaldi and Engle, 2005). However, although it is mushroom body’s lobes, whereas the calyx lip, which receives tempting to equate the simplicity of the ephemeropteran inputs from three clusters of antennal lobe glomeruli (Mu¨ ller et mushroom body with the basal position of this taxon, it is once al., 2002; Lo´ pez-Riquelme and Gronenberg, 2004), supplies again necessary to consider the highly derived life style of three other parallel strata of the lobes (Strausfeld, 2002). adult mayflies. As with the above-mentioned cicadas, mayflies This organization at first sight seems to be very different perish just a few hours after adult emergence, which occurs from the simple mushroom bodies of the Ephemeroptera. In en masse at streams and rivers. There are no specialized Potamanthus, there is no discrete calyx, and the pedunculus mate- or oviposition-locating mechanisms; adults also do not The Journal of Comparative Neurology 282 N.J. STRAUSFELD ET AL.

Figure 10. Parallel and local organization of the lobes of Libellula depressa. A: Golgi impregnations demonstrate that, at specific locations along the lobes, Kenyon cells provide collaterals (brackets) indicative of regions in which they participate in local circuits. B,C: Reduced silver impregnations reveal the participation of efferent (single black arrow in B, white arrows in C) and afferent (double (white and black arrows in B) neurons at the bulbous ending of the vertical (␣) lobe. The spur (sp) indicates the peduncular divide at which Kenyon cells divide to send collaterals into the .␮m 50 ؍ medial and vertical lobes. Scale bars feed, so like cicadas they have little use for olfactory cues or many divisions to provide many smaller neuroblasts that sub- higher brain centers for more complex sensory processing. sequently provide hundreds of ganglion mother cells and The extremely abbreviated adult life of mayflies (Carey, 2002) hence an enormous number of Kenyon cells (see also Farris et has likely favored the reduction of the mushroom bodies in al., 1999). In honey bees, about 175,000 Kenyon cells are this taxon, so the simplicity of these structures may not be generated for each mushroom body in which the two calyces entirely attributed to a primitive character state. are clearly separate (Wittho¨ ft, 1967). Drosophila and Apis thus The question remains, however, of how a simple mushroom represent two extremes, but Kenyon cell numbers appear to body, such as exemplified by Potamanthus, might have given have been modified many times during insect evolution (Farris rise to the kind of elaborate organization seen in many hym- and Sinakevitch, 2003; Urbach and Technau, 2003). The ease enopterans. One obvious hint is provided by the developmen- with which neuroblast number, and thus Kenyon cell number, tal history of these centers and the unusual properties of appears to respond to adaptive pressure provides a solid mushroom body neuroblasts. Usually, a neuroblast provides a mechanism for the observed variation in mushroom body size small number of neurons after producing several ganglion across the dicondylic insects. mother cell progenitors. In Drosophila, each member of a It is not currently known how differences in gene expression quartet of neuroblasts (two to each presumptive calyx) pro- produce evolutionary differences in neuroblast number, but it duces ganglion mother cells that generate about 500 daugh- is likely that proteins regulating symmetric vs. asymmetric cell ters, the Kenyon cells (Ito et al., 1997). The two fused calyces divisions, and thus neuroblast vs. neuron-producing cell divi- are each composed of the dendrites of about 1,200 Kenyon sions, are important. The product of one such gene in Dro- cells. This is numerically trivial compared with the honey bee sophila, called mushroom body defect, functions in the align- or species of ants, in which neuroblasts associated with ment of the mitotic spindle. Mutations of this gene result in a mushroom bodies behave differently. In the latter, each of the tenfold increase in mushroom body neuroblast number and an original quartet of neuroblasts (Ishii et al., 2005) undergoes enormous increase in mushroom body size (Guan et al., 2000; The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 283

Figure 11. An insect without a mushroom body, the archaeognathan Machilis germanicus (A). B: DCO/phalloidin shows no evidence of a mushroom body in the archaeognathan brain. However, there is a clearly delineated superior lateral protocerebrum (S L Pr), the organization of which is reminiscent of the medulla terminalis of a malacostracan crustacean or the lateral horn olfactory neuropil of other insects. C: The machilid antenna provides discrete olfactory glomeruli (olf glom) and a more caudal antennal mechanosensory neuropil (ant mech). Gustatory neuropil (gust) is also glomerular and is situated in the tritocerebrum. Note the simple layered arrangement of the central complex (CC), again reminiscent .␮m 50 ؍ of that of malacostracan crustaceans. Me, medulla; Lo, lobula; S M Pr, superior medial protocerebrum. Scale bars The Journal of Comparative Neurology 284 N.J. STRAUSFELD ET AL.

determinants of each subpopulation during the ontogeny of an individual fly are poorly understood at present. There is new evidence, however, that progressively decreasing con- centrations of one transcription factor, chinmo, may regulate neuroblast production of larval-born subpopulations (Zhu et al., 2003; Yu and Lee, 2007). Evolution of the mushroom body The concomitance of calyces and antennal lobes at first sight suggests that these two neuropils might have evolved in tandem with the acquisition of odorant-detecting sensory neurons. One counterargument is that the antennal lobe was already present when the first insects colonized land, but the mushroom body was not. Alternatively, the first insects might, from the outset, have been endowed with the full panoply of odorant receptors, with the antennal lobes and calyces being elements of the basal ground plan of the insect brain. How- ever, the brains of Machilidae, the most basal living lineage of insects, speak against this. Immunohistochemical labeling shows that the Machilis brain possesses glomerular antennal lobes (that is, strictly the antennular lobes; see under Termi- nology, above), but no mushroom body. Hence olfactory lobes were present in insects before the acquisition of mushroom bodies. True insects (Archaeognatha ؉ Dicondylia) are likely to have arisen from a crustaceomorph ancestor. Crucially, no extant crustacean (Entomostraca ؉ Malacostraca) possesses paired mushroom bodies, and the crustacean brain thus shares greater affinity with that of the monocondylic Machilidae than with the brain of dicondylic insects. It is the dicondylic aptery- gote Zygentoma, species such as firebrats and silverfish, whose simple detritivorous life styles are likely to be similar to those of the earliest insects, that may serve as the best rep- resentatives for what the earliest insect mushroom bodies looked like. Recent phylogenetic studies resolve the Zygentoma as a sister group of the Pterygota (Giribet et al., 2004; Grimaldi and Engel, 2005), and both groups have dicondylic mouth parts. They are thus distant from the monocondylic Archaeognatha, whose lack of a mushroom body suggests a very ancient status close to the common ancestor of the Malacostraca. The calyces of the zygentoman apterygotes Lepisma and Thermobia receive inputs from an ascending tract that origi- nates from a small cluster of glomeruli supplied by the ipsi- Figure 12. lateral antenna and that lies just rostral to striate mech- Comparisons of mushroom bodies in insects lacking a calyx and anosensory neuropil (Farris, 2005). The mushroom body of antennal lobes with an insect (Cicindela ocellata) equipped with both. Thermobia also receives gustatory input from the ipsilateral Mushroom body neuropils are shown in green, and globuli cell clus- ters supplying them are magenta. Among the six midbrains shown lobus glomerulatus, a neuropil that receives mechanosensory here, only that of the tiger beetle Cicindela ocellata possesses anten- and gustatory input from the maxillary palp (Farris, 2008a). nal lobes (orange) and an ascending tract supplying calyces. The Lobus glomerulatus projection neurons ascend to the proto- mushroom body of basal palaeopterans (exemplified by the damsel fly cerebrum, where they invade two enlarged microglomeruli in Agrion virgo and mayfly Potamanthus luteus) possesses lobes; those of the odonate are substantial. Secondarily aquatic neopterans, here the ventral calyx. In this respect, the inputs to the zygentoman the sunburst beetle Thermonectus marmoratus and the hemipteran mushroom body are in essence similar to those of the or- Notonecta undulata (back-swimmer), have calyxless mushroom bod- thopteran mushroom body, exemplified by crickets and lo- ies, the latter showing tuberous outgrowths from the base of the custs (Frambach and Schurmann, 2004), in which the smaller pedunculus that are reminiscent of those in Potamanthus (see Fig. 6A). The terrestrial homopteran, the cicada Diceroprocta semicincta, of two calyces (the accessory calyx) receives afferents from lacks antennal lobes but possesses calyxless mushroom bodies. the lobus glomerulatus, whereas the larger calyx (the primary calyx) is supplied from the antennal lobe (Fig. 13). Bowman et al., 2006; Siller et al., 2006). The mechanisms by Do the Zygentoma represent the earliest appearance of which morphologically and functionally distinct Kenyon cell mushroom bodies? Here we have to be cautious. In addition subpopulations have arisen from an ancestrally uniform pop- to the absence of a deep fossil record for the Zygentoma (the ulation remain elusive in large part because the molecular earliest known fossils are relatively recent, from the Lower The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 285 Functional implications of the mushroom body ground plan Neuroanatomical observations support a mushroom body ground plan in the terrestrial Dicondylia comprising parallel arrangement of Kenyon cells that possess distal postsynaptic sites in a specialized component called the “calyx” and whose axon-like prolongations provide pre- and postsynaptic sites along the length of the lobes. However, if we take into con- sideration taxa that have secondarily lost all vestiges of their calyces, including the complete loss of microglomeruli at the level where a calyx is present in other taxa, then the ground plan that would apply to all dicondylic insects is one that comprises the pre- and postsynaptic axon-like processes of Kenyon cell divested of their distal dendrites (Fig. 14). What unites all neopteran taxa is that these axon-like processes contribute to circumscribed domains down the length of the lobes, each of which comprises a system of local circuits that integrate afferent information projected into the lobes from protocerebral neuropils and providing inputs onto dendrites of efferent neurons that extend back into the protocerebrum (Strausfeld, 2001). In neopterans, the lobes alone can inte- Figure 13. grate sensory information, as demonstrated by intracellular Similarities between flightless and winged dicondylian insects, here the zygentoman apterygote Lepisma saccharina (“silverfish”, left) recordings showing that multimodal information is relayed by compared with the orthopteran Schistocerca gregaria (“American input neurons to the lobes and that context-specific informa- desert locust”, right). In both taxa, mechanosensory terminals from tion is relayed from the lobes by efferent neurons extending the antennae (AN) supply the tritocerebrum, and olfactory receptors from the antennae supply axons to the antennal lobe in the deutoce- back out to the protocerebrum. Crucially, these efferent neu- rebrum. In both, the lobus glomerulatus receives gustatory inputs and, rons are driven by modalities, such as acoustic, tactile, and like the antennal lobe, provides a separate stream of projection neu- visual parameters, that are not represented in the calyces (Li ron axons to a discrete region of the calyx (Ca I, accessory calyx). and Strausfeld, 1997, 1999). Gustatory and olfactory projection neuron axons appear to extend beyond their specific calyx regions (Ca I, Ca II) into the lateral horn Relatively few changes are required for a transition from (LH) of the superior lateral protocerebrum. gc, Globuli cells; M, medial such a simple ground plan (Fig. 14, upper left) to that of a lobe; V, vertical lobe. mushroom body equipped with a calyx serving two or more modalities (Fig. 14, lower left). Successive duplication of an original complement of one neuroblast provides a quadripar- Cretaceous; Sturm, 1998), several neurological features speak tite mushroom body and hence four identical sets of circuits against this group being primitive. Its central complex is operating on the same afferent supply (Ito et al., 1997). In- equipped with a fan-shaped body, superior arch, and ellipsoid cluded in the neopteran ground plan is the potential for body, along with a protocerebral bridge. These features typify Kenyon cell numbers to increase and for their processes in the the central complex of crown palaeopterans and neopterans lobes to be subdivided lengthwise into discrete parallel com- and differ from the simple bilayered central body of machilids ponents each of which represents a defined region in the and ephemeropterans. Another curious aspect of zygento- calyx. The latter is nothing more than an enfolding of one or mans is exemplified by the silverfish Ctenolepisma longicau- more planar subdivisions, which in the mushroom bodies of data, in which the developing meso- and metathoracic ganglia some species, such as coleopterans, form a structure of con- have precisely the same numbers and locations of neuro- blasts as in a neopteran insect such as the locust. Moreover, centric cylinders, whereas, in others, such as the honey bee, the sequence in which they segregate from the neuroecto- they maintain their planar arrangements. Elaboration of the derm is the same in both taxa (Truman and Ball, 1998). Either calyx into discrete zones, each receiving a different set of this suggests that the entire complement of thoracic neuro- sensory interneurons, as in many species of Hymenoptera, blasts was present in the insect ventral nerve cord prior to the imposes nothing new on the original ground plan of parallel evolution of flight, as argued by Truman and Ball, or it may intrinsic neurons and local regions of inputs and outputs in the suggest the retention of a developmental program associated lobes. However, the acquisition of new sensory inputs to the with flight in a secondarily flightless insect allied to the calyx has been accompanied by an overall increase in the Neoptera. Although the suggestion that zygentoman insects number of Kenyon cells resulting from sequential proliferation are secondarily flightless conflicts with molecular sequence of many descendent neuroblasts from the original quartet. data (Regier et al., 2004), the neuroanatomical observations The consequent expansion of the calyx, and thus the number summarized above could support the divergence of the Zy- of divisions through the lobes, is comparable to an evolution- gentoma from within the neopteran stem lineage. Despite the ary trend described from studies of the cortex of mammals in drastic reduction of the visual system, the zygentoman brain which additional modalities, combinations of modalities, or shares more similarities with the brain of Neoptera than with accentuation of a sensory modality can impose novel cortical the other group of apterygotes, the Archaeognatha, or with territories or expansions of existing ones (Kaas, 1995; Catania, the winged Ephemeroptera. 2005; Krubitzer, 2007). The Journal of Comparative Neurology 286 N.J. STRAUSFELD ET AL.

Figure 14. Elaboration of the mushroom body ground plan as evidenced by the apterygote Zygentoma [upper left panel: calyces receive olfactory inputs (red)]. In all mushroom bodies, with or without a calyx [the latter as in the Ephemeroptera (upper middle) and certain Hemiptera], parallel fibers contribute to local networks that receive inputs (purple) and provide outputs (black). In odonates (upper right), the distal part of the pedunculus reveals a remnant calyx equipped with microglomeruli receiving inputs (blue) from visual interneurons. Calyces are most elaborated when they serve several modalities, as in many species of Hymenoptera (olfactory input red, visual input blue). Modality-specific zones of the calyces supply longitudinally partitioned lobes (lower left). In some Neoptera, the entire calyx is dominated by a modality other than olfaction, as in the “visual” calyces of the Gyrinidae (lower middle). Irrespective of how many modalities supply the calyces, the lobes of most mushroom bodies are divided into parallel or concentric divisions (lower right). These are usually represented by a concentric organization of Kenyon cell populations in the calyces.

The presence of a calyx imposes a distinctive property on a mean that, in any local circuit in which a Kenyon cell partici- system that can also operate without one. A calyx does not pates, its synaptic activity will be subject to modulation by the alter the basic circuitry of the lobes, but it does impose phys- ambient sensory signal relayed to its distal dendrites. A spe- iological constraints. Although the basic functional organiza- cific odor relayed by antennal lobe projection neurons to the tion of the mushroom body lobes is the same in neopterans as calyces that results in a change of the excitability of a cohort in palaeopteran insects, sensory input to the calyx would of Kenyon cells (Jortner et al. 2007; Finelli et al., 2008) must The Journal of Comparative Neurology GROUND PLAN OF THE INSECT MUSHROOM BODY 287

Figure 15. Evolution of mushroom bodies. Glomerular olfactory lobes supplied by the first antennae (character 1, see Note on Terminology) are resolved in malacostracan Crustacea and in the Archaeognatha. Projection neurons from the glomerular lobes extend to the lateral protocerebrum of both malacostracan crustaceans and archaeognathans (character 2). Mushroom bodies with calyces and lobes (3), supplied by olfactory glomeruli and gustatory inputs (4) are resolved in the Zygentoma and are thus basal to Palaeoptera and Neoptera. Double calyces (5) and parallel subdivisions of the lobes (6) are resolved in pterygote insects. The representation of modalities in addition to olfaction and taste (visual afferents (7) is resolved in the Odonata and higher Neoptera. Modality substitution (8; vision substituting olfaction) in the calyces or their rudiments has occurred independently in some Coleoptera and Odonata. Secondary loss of antennal lobe glomeruli and the reduction or complete loss of calyces (9, 10) has occurred several times independently within the Palaeoptera and Neoptera. Aquatic species are shown in boldface. The phylogeny on which characters 1–10 are mapped is based on Wheeler et al. (2001). The Journal of Comparative Neurology 288 N.J. STRAUSFELD ET AL. accordingly modify their participation in local computations in unlikely to be related to olfaction, might the general functional the lobes. This conclusion means that odor encoding by Ke- significance of the lobes might be better resolved from the nyon cells should not be regarded as the primary function of study of such anosmic insects than, say, from study of an a mushroom body. Rather, information about an odor pro- olfactory species such as Drosophila, where certain subdivi- vides the context in which multimodal integration is performed sions of the lobes are required for different aspects of olfac- at discrete loci within the mushroom body lobes. In species tory memory (Pascual and Pre´ at, 2001; Yu et al., 2007). How- such as the honey bee, those divisions of the mushroom body ever, such roles need not be universal, nor the sole functional calyces that receive visual inputs (Paulk and Gronenberg, attributes. As demonstrated in a preliminary study (Strausfeld 2008) would likewise modulate local circuits in the relevant et al., 2006), circuitry within the lobes is comparable across divisions of the lobes in the context of the ambient visual widely divergent species, including those that lack antennal signal. lobes. In short, the basic organization of the mushroom body lobes is the same with or without a calyx. It is the functional Elaboration through time relevance of the calyxless ground plan that, once understood, Sequential elaboration of the mushroom body, originating may reveal the real significance of the mushroom body. from a zygentoman-like ground plan and culminating in a neopteran mushroom body typified by Apis or Drosophila, can be mapped onto a “standard” insect phylogeny (Fig. 15). In ACKNOWLEDGMENTS this tree, the zygentoman mushroom body is close to repre- Our insights into the mushroom bodies have profited from senting the basal ground plan. various discussions with colleagues. We thank Daniel Kal- A pronounced calyx, present in most terrestrial neopteran deron (Department of Biological Sciences, Columbia Univer- insects, might suggest an adaptation to the evolution of high- sity) for the gift of anti-DCO antibodies. We are grateful to Kei growth flora that lead to ecosystems in which foraging and Ito (University of Tokyo), Martin Heisenberg (Theodor Boveri intraspecific recognition required the detection of airborne Institute, University of Wu¨ rzburg), and Gilles Laurent (Califor- plant odors. However, rudimentary calyces probably also nia Institute of Technology) for their views. We especially equipped the earliest mushroom body. Assuming that the first thank Camilla Strausfeld (University of Arizona) for detailed dicondylic insects were detritovores, then Thermobia and comments on the paper and for extensive editing of the text, Lepisma may be good modern representatives of a life style and Pavel Masek for the photograph of the machilid shown in similar to that of ancient insects. Both species have small Fig. 11A. Partial support for the evolutionary analyses derives antennal lobes as well as a large columnar mechanosensory from a fellowship to N.J.S. from the John D. and Catherine T. neuropil. The latter does not provide an input into the calyces, MacArthur Foundation. but the former does. Gustatory inputs also target the calyx. It is thus likely that the mushroom bodies of ancient dicondylic LITERATURE CITED insects played a role in chemosensory processing. This leaves open the question: what do the lobes do that causes them to Akalal D-BJ, Wilson CF, Zong L, Tanaka NK, Ito K, Davis RL. 2006. Roles of Drosophila mushroom body neurons in olfactory learning and mem- be retained when olfaction is lost? They are obviously capable ory. Learn Mem 13:659–668. of participating in local circuits supplied by multimodal inputs Anton S, Ignell R, Hansson BS. 2002. 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