evolution, discovery, and interpretations of arthropod

Evolution, Discovery, and Interpretationsof Arthropod Mushroom BodiesNicholas J. Strausfeld,1,2,5 Lars Hansen,1 Yongsheng Li,1 Robert S. Gomez,1

and Kei Ito3,4

1Arizona Research Laboratories Division of NeurobiologyUniversity of ArizonaTucson, Arizona 85721 USA2Department of Ecology and Evolutionary BiologyUniversity of ArizonaTucson, Arizona 85721 USA3Yamamoto Behavior Genes ProjectERATO (Exploratory Research for Advanced Technology)Japan Science and Technology Corporation (JST)at Mitsubishi Kasei Institute of Life Sciences194 Machida-shi, Tokyo, Japan

Abstract

Mushroom bodies are prominentneuropils found in annelids and in allarthropod groups except crustaceans. Firstexplicitly identified in 1850, the mushroombodies differ in size and complexity betweentaxa, as well as between different castes of asingle species of social insect. Thesedifferences led some early biologists tosuggest that the mushroom bodies endow anarthropod with intelligence or the ability toexecute voluntary actions, as opposed toinnate behaviors. Recent physiologicalstudies and mutant analyses have led todivergent interpretations. One interpretationis that the mushroom bodies conditionallyrelay to higher protocerebral centersinformation about sensory stimuli and thecontext in which they occur. Anotherinterpretation is that they play a central rolein learning and memory. Anatomical studiessuggest that arthropod mushroom bodiesare predominately associated with olfactorypathways except in phylogenetically basalinsects. The prominent olfactory input tothe mushroom body calyces in more recent

insect orders is an acquired character. Anoverview of the history of research on themushroom bodies, as well as comparativeand evolutionary considerations, provides aconceptual framework for discussing theroles of these neuropils.

Introduction

Mushroom bodies are lobed neuropils thatcomprise long and approximately parallel axonsoriginating from clusters of minute basophilic cellslocated dorsally in the most anterior neuromere ofthe central nervous system. Structures with thesemorphological properties are found in many ma-rine annelids (e.g., scale worms, sabellid worms,nereid worms) and almost all the arthropodgroups, except crustaceans. The most primitive lo-bopods, the Onychophora (velvet worms), possessthese structures as well as the most advanced so-cial insects.

Like all other parts of an organism, mushroombodies are the products of evolution. Their struc-ture and functions reflect their evolutionary historyand the specific sensory and behavioral adapta-tions that characterize a particular taxon. Presentunderstanding and interpretations of mushroombody function also reflect the evolution of researchon insect brain and behavior, which has resulted indisparate views of mushroom body function. Oneview holds that mushroom bodies are the site ofolfactory learning and memory. Another view is

4Present address: National Institute for Basic Biology,Myodaiji-cho, Japan.5Corresponding author.

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that they support a variety of other functions, notall necessarily represented in the same species.Such functions include sensory discrimination andintegration with other modalities, the control ofcomplex behavioral repertoires, and spatial orien-tation.

EARLY HISTORY OF MUSHROOM BODY RESEARCH

Mushroom bodies were discovered in 1850 bythe French biologist Felix Dujardin, who calledthese structures corps pedoncules, likening theirappearance to the fruiting bodies of lichens. Manyrecent accounts have co-opted Dujardin’s paper insupport of the widely held belief that mushroombodies are learning and memory centers. However,Dujardin did not suggest this, but proposed thatthese centers endowed an insect with a degree offree will or intelligent control over instinctive ac-tions. He supported this idea from comparativestudies of the brains of ichneumons, solitary bees,and honeybees, showing that advancing socialitywas correlated with the possession of enlargedmushroom bodies (Dujardin 1850). He observedthat insects with small mushroom bodies showedgreater coordination of thoracic motor actions af-ter decapitation than did insects with large mush-room bodies and proposed that the smaller themushroom body, the more automatic or instinctivethat insect’s behavior. Dujardin also carried out ex-periments on homing abilities in ants (Dujardin1853) to underpin his ideas of insect intelligence.Two other French biologists, Faivre (1857) and Bi-net (1894), furthered Dujardin’s ideas, performingsophisticated ablation experiments to demonstratethat although the suboesophageal ganglion is nec-essary for maintaining synchronized movements ofthe limbs (Faivre showed that, if fed, a dytiscidbeetle can survive for months without its supra-esophageal ganglion), it was insufficient for provid-ing complex and varying patterns of motor activity.Faivre, in particular, demonstrated these complexmovements to be under the control of the supra-esophageal ganglion (the brain proper). Severalother 19th century studies supported the idea thatmushroom bodies of insects mediate intelligentversus innate behavior on the basis of comparativeanatomy, with particular emphasis on the socialHymenoptera and the differences between thebrains of different castes (Leydig 1864; Forel 1874;Flogel 1876, 1878). Like Dujardin’s, none of thesestudies explicitly suggested that the mushroombodies underlie learning and memory.

Flogel (1876) was the first to define criteria foridentifying mushroom bodies across insect species:the presence in the supraoesophageal mass ofpaired groups of several hundred to several hun-dred thousand minute cells (termed globuli cellsbut now known as Kenyon cells; Strausfeld 1976)that surmount lobed neuropils that Flogel pro-posed were composed of parallel fibers. Kenyon(1896a,b), who was the first to use Golgi methodson insect brains, confirmed Flogel’s ideas about thefibrous nature of globuli cell morphology. Kenyonshowed that the dendritic branches of globuli cellsinvade the head of the mushroom body to form astructure called the calyx. He described the parallelaxons of globuli cells forming a pedunculus thatextends to the front of the brain where axons thenbranch to provide a vertical and a medial lobe(Kenyon 1896a,b). Kenyon identified afferents tothe calyces and suggested that these carried olfac-tory, visual, and tactile information. He proposedthat mushroom bodies provided a center for sen-sory-motor integration, quite separate from directsensory-motor relays that characterize other brainareas or thoracic or abdominal ganglia (Kenyon1896a). Kenyon did not suggest that mushroombodies are involved in learning and memory.

Golgi studies on the mushroom bodies of Blat-todea (cockroaches: Sanchez 1933), Hemiptera(true bugs: Pflugfelder 1937), and Hymenoptera(ants, wasps, bees: Goll 1967) all confirmedKenyon’s (1896a,b) findings, as have descriptionsof these neuropils in the cricket Acheta domesti-cus (Schurmann 1973, 1974), the sphingid mothsSphinx ligustri (Pearson 1971) and Manducasexta (Homberg et al. 1989), the house fly Muscadomestica (Strausfeld 1976), and the honeybeeApis mellifera (Mobbs 1982, 1984).

FIRST EVOLUTIONARY STUDIES

Even before the advent in the early 1900s ofmethods that selectively reveal neural architecture(Cajal and de Castro 1933), early anatomists pro-vided reasonably accurate descriptions of themushroom bodies. Viallanes (1893), for example,was the first to recognize the enormous number ofmushroom body globuli cells in the horseshoe crabLimulus, a feature that led the Swedish neurologistHolmgren (1916) to wonder why this animal re-quired such a huge center when, in his view, it soobviously lacked behavioral sophistication. Vi-allanes (1887a,b) also identified mushroom bodiesin dragonflies, wasps, and crickets, thereby provid-

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ing early comparative descriptions of this neuropilfrom sectioned material. Bretschneider (1913,1914, 1918, 1924) published a series of studies oninsect brains, including that of the cockroach Peri-planeta orientalis (Bretschneider 1914) in whichhe identified the characteristic striations in themushroom body lobes, now known to be causedby the layered arrangements of Kenyon cell axons(Li and Strausfeld 1997; Mizunami et al. 1997;Strausfeld 1998a,c). Like Flogel (1876), Bret-schneider (1918) also attempted to use brain fea-tures for inferring insect relationships. However,the first attempt at constructing phylogenetictrees, on the basis of neural features, was Holm-gren’s (1916) elegant and systematic study ofarthropod phylogeny from comparative brainanatomy. Significantly, Holmgren used Flogel’s andKenyon’s anatomical criteria for identifying mush-room body-like neuropils in onychophorans, myria-pods (centipedes and millipedes), chelicerates(e.g., horseshoe crabs, scorpions, whip spiders),and insects. His failure to find any correlation be-tween caste status and the size of the mushroom

bodies in termites (Holmgren 1909) reinforcedHolmgren’s opposition to the idea that mushroombodies endowed an arthropod with intelligence.He found that cockroaches, near relatives of ter-mites, had mushroom bodies almost as advanced(in cell number and gross morphology) as those ofHymenoptera. But not a keen observer of animalbehavior, he wrote that ‘‘the psychic ability of theroach is hardly worth comparing with that of thetermite.’’

The second serious attempt to reconstructarthropod phylogeny from brain anatomy wasby Holmgren’s student Bertil Hanstrom who, in1926 and 1928, based his theory of arthropodmonophyly exclusively on features of the opticlobes and the occurrence of mushroom bodies(Fig. 1). Again, Hanstrom used mainly Flogel’s andKenyon’s criteria but he also made a leap of faithregarding layered neuropils, called hemiellipsoidbodies, in the crustacean eye stalks, which he be-lieved to be highly modified mushroom bodies (seebelow). A recent study (Strausfeld et al. 1995) re-iterated this point of view, noting that hemiellip-

Figure 1: (Top) Hanstrom’s classic1926 paper claimed arthropod mono-phyly on the basis of observed similari-ties among visual systems. To accommo-date mushroom bodies into this view(bottom) Hanstrom (1928) accordedthem a primary function in both olfactionand vision. This allowed their seamlessdemonstration from annelids (A) throughto the araneans (C, bottom). Annelids,however, show no evidence of connec-tions between visual neuropil and mush-room bodies. In Hanstrom’s figure, thepanel labeled arthropods (B) depicts onlytwo optic neuropils suggesting either abranchiopod crustacean or a thysanuran,either equipped with an insect-likemushroom body. However, mushroombodies have not been identified in non-malacostracans, and thysanurans lack ol-factory glomeruli and calyces.

MUSHROOM BODY EVOLUTION


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soid bodies are supplied by first order olfactoryneuropils, the antennule lobes (Mellon et al. 1992).However, studies on thalassinid crustaceans, inwhich the eyestalks are reduced or absent (e.g.,the burrowing shrimp Callianassa; Strausfeld1998b) have failed to resolve morphologies at allreminiscent of mushroom bodies even though inCallianassa the hemiellipsoid bodies reside withinthe brain proper.

HISTORICAL ORIGIN OF THE CONCEPTOF MUSHROOM BODIES AS LEARNINGAND MEMORY CENTERS

Originating with Dujardin’s 1850 paper, nu-merous early investigators attributed to the mush-room bodies a role in intelligent behavior. Particu-lar significance was drawn from comparative stud-ies of social Hymenoptera which were based onclaims that the relatively large mushroom bodies ofworkers and queens served the broadest range ofbehaviors, as compared with, say, drones, whichhave relatively small mushroom bodies (Forel1874; Jonescu 1909; von Alten 1910).

Suggestions that mushroom bodies play crucialroles in learning and memory are comparativelyrecent, deriving from lesioning experiments on antmushroom bodies that perturbed the animal’s abil-ity to negotiate a maze using olfactory cues(Vowles 1964a). Ablations of the cockroach (Peri-planeta) pedunculus and medial lobes, combinedwith place memory tests (Mizunami et al. 1993),have reinforced the possibility that the mushroombodies may play a role in spatial orientation. Com-parisons of mushroom body dimensions, foragingranges, and behaviors in butterflies have also beenused to suggest that these neuropils may be in-volved in spatial learning (Sivinski 1989).

The idea that mushroom bodies may harborthe cellular basis for associative memory originallyderives from studies on honeybees by Menzel et al.(1974; see also Erber et al. 1980) and on Dro-sophila by Heisenberg (1980). In studies on hon-eybees, abolition of short-term olfactory memorywas shown to be induced by cooling the verticallobes and, along with them, the surrounding pro-tocerebral neuropils (Erber et al. 1980). Studies onDrosophila involved the mutagenization of flies toisolate strains that are defective in odorant-drivenbehavior. Defective lines were subsequently exam-ined for neural and molecular correlates (Quinn etal. 1974; for review, see Heisenberg 1998). A sec-ond strategy involved the mutagenization of flies to

isolate structural brain mutants (Heisenberg 1980).Structurally defective lines were then screened forbehavioral defects. Two of these mutations, mush-room bodies deranged and mushroom bodies re-duced, have earned special attention (for review,see Heisenberg 1998) because structural defects ofthe mushroom bodies correlate with defects in ol-factory conditioning (Heisenberg 1980, 1994; Hei-senberg et al. 1985). Genetic and experimental in-duction of structural or biochemical defects, cor-related with learning and memory deficits, havebeen invoked many times to support the possiblerole of the mushroom bodies in olfactory condi-tioning (Heisenberg et al. 1985; Nighorn et al.1991; de Belle and Heisenberg 1994; Connolly etal. 1996), and the intellectual momentum in learn-ing and memory research on insects during the last25 years has largely been from such studies. Theidentification of substances in the mushroom bod-ies thought to be crucial in memory formation (forreview, see Davis 1993) is germane to any discus-sion about cellular events underlying learning andmemory. The significance of these works are re-viewed by Heisenberg (1998) and discussed by Itoet al. (1998).

COMPARISONS BETWEEN MUSHROOM BODIESAND VERTEBRATE BRAIN CENTERS

Dujardin (1850) was intrigued by the mush-room bodies because they reminded him of foldsand gyri in the cerebral cortex. This comparisonwas taken up by subsequent investigators in thelate 1800s and early 1900s, among them Hanstrom(1928) who suggested that mushroom bodies areanalogous to the thalamus of fish. Comparisonswith the vertebrate hippocampus have been pro-posed, because both the hippocampus and mush-room bodies may play roles in similar types oflearning and memory, such as place memory inmammals (for review, see Muller 1996) and incockroaches (Mizunami et al. 1993). It has beendemonstrated that both hippocampus and Dro-sophila mushroom bodies show apparent eleva-tion of expression of various learning-related mol-ecules (for review, see Kandel and Abel 1995).

The characteristic cellular organization of themushroom bodies, and their position in the olfac-tory pathway, have stimulated three other analo-gies: with the olfactory cortex, with the cerebel-lum, and with the striate cortex (Mizunami et al.1997). The morphological and functional similaritybetween antennal glomeruli and glomeruli of ver-

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tebrate olfactory bulbs has been emphasized byShepherd and Greer (1998). In mammals, the nextstage of processing is in the olfactory cortex,which, like the hippocampus, is a phylogeneticallyancient and conserved part of the brain. Its posi-tion in the olfactory pathway would be seriallyequivalent to the calyx of the insect mushroombody.

Schurmann (1974) has suggested that the se-quential organization of narrow dendritic fields ofoutput neurons across parallel arrays of Kenyoncell axons is comparable to the arrangements be-tween Purkinje cells and parallel axons of granulecells in the cerebellum. Schurmann (1974) pro-poses that the arrangement of parallel fibers andefferent neurons could serve to detect temporalevents in olfactory stimulation similar to the timerhypothesis for the cerebellum (Braitenberg 1967).Recent studies on synchronized activity in the ol-factory pathway suggest that timing among neuralassemblies plays a crucial role in odor discrimina-tion (for review, see Laurent 1997).

Laminae in the cockroach mushroom body(see below) have been analogized with columns inmammalian striate cortex (Mizunami et al. 1997),but this comparison would be difficult to reconcilewith the mode of mushroom body development.Whereas cortical columns, each with a distinct setof afferents, can be potentially established duringneurogenesis (Kuljis and Rakic 1990), their matu-ration and final arrangements depend strongly onsensory experience (Chapman and Stryker 1992).The laminae of the cockroach mushroom bodies,however, represent annular arrangements ofKenyon cell dendrites in the calyces, which sharethe same afferents but increase in number at eachdevelopmental instar (Weiss 1974). Possibly, thisarrangement is hard wired. Experiments that denyantennal input to the antennal glomeruli through-out post embryonic development and thereby dras-tically reduce the size of the antennal lobes andtheir connections to the calyces do not reduce thenumber of laminae (N.J. Strausfeld, unpubl.).

Considering that some genes control the de-velopment of comparable structures across verydiverse phyla, as in the case of the role of the Pax-6gene in eye development (Callaerts et al. 1997),there are obviously certain commonalities be-tween arthropod and vertebrate brains. However,given the gross structural differences betweenthem, the probability seems low that the mush-room bodies and certain vertebrate brain regionscould be derived from the same ancestral neural

network and develop under the control of homolo-gous genes.

MUSHROOM BODIES IN ODOR-SENSITIVE INSECTS

This section outlines the organization of insectmushroom bodies. These can be usefully com-pared with mushroom body-like neuropils in dis-tantly related groups, such as the annelids (worms;Fig. 2A) and the cheliceriformes (e.g., solpugids,pycnogonids; see Fig. 6, below) described in thenext sections.

PEDUNCULUS AND LOBES

Insect mushroom bodies usually have two ormore sets of lobes arising from the pedunculus atthe front of the brain: the vertical lobe assemblage,the medial lobe assemblage, and, in some species,additional frontal or recurrent lobes (Figs. 2–4).However, Jawlowski (1959b) reports that in cer-tain vespids (wasps) the pedunculus is undividedand does not form a vertical lobe. In Jawlowski’sdescriptions, the vespid pedunculus is greatly wid-ened beneath the calyces, tapering to a small me-dial lobe.

Comparisons between insect groups suggestthat within an order there are highly conservedfeatures of mushroom body shape and lobe ar-rangements (Figs. 2–4). In Drosophila and otherbrachyceran Diptera (Fig. 4D), the medial and ver-tical lobes are bipartite, each divided into two par-allel components called, respectively, b, g, and a,a8 (Ito et al. 1998). Kenyon cells providing an axonto the medial b generally provide a branch into thevertical a. Kenyon cells supplying g usually supplya tributary to a8. The segregation between the ver-tical a and a8 is less clear than between the medialb and g. In some Diptera, such as the horse flyTabanus, divisions of the medial lobes are almostcompletely segregated into two entities (Fig. 4E,F).Undivided medial and vertical lobes occur in manyHymenoptera (e.g., honeybees, ichneumon wasps,ants; Jawlowski 1959a,b, 1960; Goll 1967) and Blat-todea (e.g., cockroaches; Fig. 2A). In Dermaptera(earwigs), the pedunculus and lobes are clearly di-vided into three parallel components that carrythrough the tripartite arrangement of their calycalneuropil (Fig. 2D). Coleopteran mushroom bodies(see also, Jawlowski 1936) generally appear sim-pler than those of many other groups. Their verti-cal lobes comprise a single shaft as does the mediallobe (Fig. 2E,G). But both are subtly divided intoconcentric longitudinal components. These struc-



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tures are all highly conserved, even between odor-sensitive species with large calyces (Figs. 2E and4H) and almost anosmic species of the same orderthat have reduced calyces, such as diving beetles(Figs. 2G and 4I).

The Lepidoptera provide another example oforder-specific arrangements (Bretschneider 1924).Pearson’s (1971) description of the hawk mothSphinx ligustri describes a ‘‘g lobe,’’ disposed par-allel to the b component of the medial lobe, that issupplied by a bundle of Kenyon cell axons project-ing separately from the pedunculus. This organiza-tion has also been identified in various other lepi-dopterans, such as the cinnabar moth Huebneri-ana trifolii (Fig. 2F), the wood nymph butterflyCercyonis pegala, and the hummingbird hawkmoth Hemaris thisbe (Fig. 4C). In all three, a sepa-rate bundle of Kenyon cell axons supplies a lobethat is satellite to a complicated arrangement ofmedial and vertical lobes (Figs. 2F and 4C). Addi-tional features shared by Lepidoptera are the un-usually large and relatively sparse Kenyon cell bod-ies supplying a large cap-like calyx (Fig. 2F).

INTERNAL ARCHITECTURE OF THE PEDUNCULUS AND LOBES

A variety of internal architectures distinguish

the medial and vertical lobes of different genera.Vowles (1955) noted that striations in ant mush-room bodies extend throughout the pedunculusand lobes. Goll (1967) related three concentriczones in the calyces of the ant Formica to threediscrete subsets of globuli cells and to three lami-nae that extend through the pedunculus and lobes.Mobbs (1982) also suggested that the concentricarrangements of the lip, collar, and basal ring neu-ropils of the honeybee calyx were transformed intothree parallel layers that extend through the pe-dunculus and lobes. In the Blattodea (e.g., Peripla-neta americana), alternating dark and pale lami-nae were first identified by Bretschneider (1914)using basic staining methods and have since beenconfirmed by Bodian staining (Li and Strausfeld1997; Mizunami et al. 1997; Strausfeld 1998c). InPeriplaneta, synaptic specializations from efferentneuron dendrites and afferent terminals coincidewith alternate laminae (Fig. 5A), which themselvesare continuous throughout the pedunculus andlobes (Fig. 5B,C). Laminae have also been identi-fied in orthopteran mushroom bodies (Barytettixpsolus; Acheta sp.). Immunocytology of the hon-eybee mushroom bodies demonstrates, however,many more longitudinal subdivisions than origi-nally suggested by Mobbs (1982, 1984). Antibodies

Figure 2: Mushroom body variation ininsects. Organization of globuli cellgroups (dotted outline enclosing darkgray areas), calyces (light gray), pedunculiand lobes (open profiles) in odorant-sen-sitive (A–F) and anosmic (G–I ) insects. (A)Periplaneta americana (cockroach; Blat-todea); (B) Barytettix psolus (horse lubber,Acrididae); (C) Acheta domesticus (cricket;Acrididae); (D) Labidura riparia (earwig;Dermaptera); (E) Calosoma scrutator (cat-erpillar hunter beetle; Coleoptera); (F )Huebnerniana trifolii (cinnabar moth;Lepidoptera); (G) Dytiscus marginalis(diving beetle; Coleoptera); (H) Noto-necta undulata (backswimmer; Hemip-tera); (I ) Argia sp. (damselfly; Odonata). Insome species, the calyx is divided intoinner, middle, and outer components (i,m, o); (V,M,F,R) vertical, medial, frontal,and recurrent lobes (in some species, lobesubdivisions represent inner, middle, andouter calyces); (S) spur. sat, satellite neu-ropil. Scale bars, 100 µm.

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raised against neuropeptides exquisitely demon-strate Kenyon cell laminae. These are variously de-fined by the presence of taurine (Bicker 1991),FMRFamide (Schurmann and Erber 1990), and gas-trin-cholecystokinin, or combinations of these (Fig.5G; also Strausfeld 1998a,c).

Longitudinal subdivisions of the lobes suggestthat in many species mushroom bodies may com-prise several parallel and isolated networks thatmay support different computational functions(Strausfeld 1998a). Therefore, it is significant thatefferent neurons are not themselves responsiblefor the lamination within the mushroom bodylobes. Instead, their branches are restricted within

specific pre-existing laminae (Fig. 5A,E,F). Each ef-ferent arborization occupies only a short distanceof the length of the lobe and the patterning ofdendritic arborizations from successive efferentneurons changes from one position to the nextalong the lobes. If extrinsic neurons would sub-stantially contribute to one or another lamina, ashas been suggested (Rybak and Menzel 1993), thenmany identical dendritic trees would be requiredto provide an isomorphic structure (lamina) ex-tending from the calyces to the distal ends of thelobes. This is not the case.

A number of insect orders show columnar orconcentric subdivisions through the pedunculusand lobes, as in the blow fly Calliphora, in whichantibodies against a GABA receptor protein revealfour columns in the pedunculus (Brotz et al. 1997).Staining for nitric oxide synthase has revealed aquadripartite arrangement of columns through thelocust’s vertical lobes (Elphick et al. 1995). Prod-ucts of gene expression also show discrete subdi-visions throughout the lobes of Drosophila thatcan be ascribed to different populations of Kenyoncells (Yang et al. 1995; Ito et al. 1997). Judgingfrom all these studies (with the possible exceptionof that by Elphick et al. 1995), it is most likely thatKenyon cells are responsible for all of these longi-tudinal divisions. Direct confirmation has been ob-tained by retrograde dye injection into laminae ofcockroach mushroom bodies revealing them to bederived from an annular arrangement of Kenyoncell dendrites in the calyces (N.J. Strausfeld, L. Han-sen, and Y.-S. Li, unpubl.).

Do the different types of parallel subdivisionsamong Kenyon cells share a common organiza-tional plan? In other words, can the relativelysimple subdivisions among Kenyon cells in Dro-sophila, for example (Yang et al. 1995), be recon-ciled with the isomorphic laminar arrangements ofKenyon cell axons in the cockroach and can thisarrangement, in turn, be reconciled with the un-equal laminations observed in the honeybee lobes?A possible answer is suggested by comparisons be-tween hemimetabolic insects that develop throughseveral instars, each providing an immature versionof the adult, and holometabolic insects that un-dergo a more or less complete metamorphosisfrom larva to adult through postembryonic pupaldevelopment.

In the cockroach, which is a hemimetabole,the adult mushroom body possesses between 24and 30 laminae, alternating as pale and darkerstructures (Fig. 5C) and reflecting the successive

Figure 3: Surface-tessellated reconstructions of mush-room bodies. (A) Primitive calyxless condition in thesilverfish Lepisma. (B) Single calyx in Schistocerca (lo-cust). (C) Double calyces of the honeybee, Apis mellif-era. (cb) Globuli cell bodies; (V,M) vertical and mediallobes, respectively.



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growth of annular arrangements of Kenyon cells inthe calyx (Weiss 1974). An intriguing aspect aboutthis lamination is that first-instar Periplaneta mush-room bodies have only two longitudinal divisionsand their medial lobes look remarkably similar tothose of adult Drosophila and other Diptera (e.g.,Tabanus, Sarcophaga; Fig. 4D–F). The mushroombodies of a third- to fourth-instar cockroach nymph(Fig. 5H) possess only eight laminae of unequalwidth, which, together, look like the arrangement

of laminae in the adult honeybee (Fig. 5F,G). Thus,early stages of the hemimetabolous mushroombodies appear to be representative of mushroombodies of adult holometabolous insects. This obser-vation raises the possibility that mushroom bodiesin the Holometabola are, to various degrees, neo-tenic: the mushroom body in one species beingsimilar to an evolutionarily basal cockroach mush-room body at a specific stage of its development.

In addition to longitudinal subdivisions, there

Figure 4: (See facing page for legend.)

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are also obvious transverse divisions across thelobes defined by segment-like arrangements of den-drites belonging to efferent neurons (Fig. 5D; seealso Li and Strausfeld 1997) and terminal domainsof certain afferents that reach the lobes from otherbrain areas (Li and Strausfeld 1997, and unpubl.).Histochemical staining with the Falck-Hillarpmethod also reveals segmentation across Kenyoncell axons (Frontali and Norberg 1966; Frontali andMancini 1970; Schurmann and Klemm 1973;Klemm 1983) suggesting that certain afferents tothe lobes are rich in catecholamines.

CALYCES

In all insects, Kenyon cells originate from clus-ters of cell bodies (globuli cells) over the dorsalanterior surface of the lateral protocerebra. Neu-rites (cell body fibers) of globuli cells prolongateanteriorly to give rise to the pedunculus and lobes.In insects equipped with antennal lobes, Kenyoncells provide dendrites that form a cap- or cuplikeregion, called the calyx, at the head of the poste-rior end of the pedunculus. Generally, Kenyon celldendrites in the calyx are visited by varicose spe-cializations from collaterals of antennal lobe pro-jection neurons, en route to their termination areain the protocerebrum’s lateral horn (Strausfeld1976; Homberg et al. 1989; Malun et al. 1993; Ito etal. 1998).

There is considerable variation of calyx mor-phology in different genera (Figs. 2–4). Whereasprimitive apterygotes and palaeopteran insectslack calyces (Fig. 3A), neopteran insects, such asorthopterans (grasshoppers, lubbers, crickets, Figs.

2B,C and 3B) have mushroom bodies possessing asingle calyx that is characteristically subdividedinto a central hillock and an outer ring (Figs. 2B,C;see also Jawlowski 1954; Weiss 1981). Other neop-terans, such as the Blattodea (cockroaches), havetwo cuplike calyces for each mushroom body (Fig.2A). Each calyx of a pair receives essentially iden-tical inputs and its Kenyon cell organization andprojections are indistinguishable (Weiss 1974;Strausfeld 1998a). The Hymenoptera also have twocalyces for each mushroom body (Fig. 3C). Theseare simplest in the Symphyta (Jawlowski 1960) inwhich each calyx consists of a knoblike neuropilon a short peduncular stalk that merges with theother stalk to form the pedunculus proper, as oc-curs in the Blattodea. Coleoptera have a pair ofcaplike calyces for each mushroom body(Jawlowski 1936), though in some species theseare fused (Fig. 2E). In Diptera and Lepidoptera (Fig.2F), each mushroom body might also be consid-ered as having two calyces that are secondarilyfused, each providing a short outer stalk that issupplied by two bundles of axons, the two stalksthen merging to form the pedunculus. Even in an-osmic neopteran species that secondarily lack ca-lyces, globuli cells provide four strands of cell bodyfibers that contribute to two bundles that convergeinto a thin pedunculus (Fig. 2I).

As first described by Flogel (1878), and againshown by Pflugfelder’s (1937) study of the Hemip-tera, the size of the calyx is often thought to beproportional to the number of antennal lobe glo-meruli, although this relationship may be more dif-ficult to assess in certain Hymenoptera in whichcalyx size may combinatorially reflect the size of

Figure 4: Mushroom body lobes of an annelid compared with those of insects. (A) Mushroom body of the scale wormArctenoe vittata has its pedunculus (ped) capped by many thousands of globuli cells. Its pedunculus and single lobereceive inputs from the olfactory lobe (olf lob). (B) Apterygote Thermobia (firebrat) has a divided vertical lobe (V1, V2) andfive glomerular medial lobes (1–5) flanking a smaller lobe in the middle (M). (C) Medial lobes of the hummingbird mothHemaris thisbe are elaborately subdivided with the g lobe, as originally defined by Pearson (1971), lying alongside thevertical lobe (V). Like in other Lepidoptera, the medial lobe is subdivided into many components (b, d, u), with satelliteneuropil (sat) provided by a small bundle of Kenyon cell axons (not in plane of section). (D) Medial lobe of the fleshflySarcophaga carnaria, like Drosophila (Ito et al. 1998), does not show obvious division into separated g and b components.(E,F ) In the horsefly Tabanus, the medial lobes show complete terminal separation of the b and g components and thevertical lobe F is deeply divided into two components (a and a8). The spur (s) is an outgrowth of the junction of thepedunculus with the vertical and medial lobes. In this tabanid, the spur is divided into three components. (G) Vertical andmedial lobes of the tettigonid Scudderia furcata, like those of many other orthopterans, show striking longitudinal zona-tions. A dense band of Kenyon cells (arrow) is flanked by two parallel divisions M1, M2, corresponding to V1, V2 of thevertical lobe. (H,I ) Vertical (V) and medial lobes (M) of a predatory tiger beetle (H, Cicindelidae) are proportionally as largeas those of the water beetle Dytiscus marginalis (I), although the latter has a greatly reduced calyx. Note the extrinsicneuron axons (arrow) leaving the distal end of the vertical lobe. The pedunculus (ped) of each is sectioned obliquely toshow two parallel divisions (broken lines in H, I ) comprising thick and thin Kenyon cell axons. Scales in A, G, H, I, 50µm; scales in B–F, 100 µm. The midline in B and F is indicated by an arrow (m).



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Figure 5: Internal organization of the mushroom bodies in hemi- and holometabolous insects (Periplaneta; A–D,Hand Apis; E–G). (A) Laminar organization of efferent neuron dendrites matches Kenyon cell laminae. (B) Oblique sectionsthrough the tips of the left and right medial lobes reveal the alternating pale and dark Kenyon cell laminae, which, in anyindividual, are symmetrical about the midline (double arrows). (C) Oblique section, through the base of the pedunculus(ped), the origin of the vertical lobe (V), and the medial lobe (M) shows the unbroken continuity of Kenyon cell laminae.Profiles at right angles to the laminae belong to efferent dendrites arranged as palisades, as shown in frontal sections(bracketed in D). (E) In honeybees (Apis mellifera), processes of extrinsic neurons invade specific laminae. The equivalentlevels in E–G are indicated by double-headed arrows. In Apis, Kenyon cell laminae are of unequal width (F) and havedifferent affinities to antibodies raised against peptides (e.g., anti-gastrin staining, shown in G). (H) Immature fourth instarpedunculus of Periplaneta showing laminae of unequal width and different staining affinities, reminiscent of the adulthoneybee. Scale bars in A, 20 µm; B–G, 50 µm; H, 10 µm.

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the antennal lobes, the amount of optic lobe input,the caste status, and the sex of the individual. How-ever, although Dujardin suggested that the calycesof social Hymenoptera are the largest, Jawlowski(1959a) claims that certain Ichneumonidae possessthe largest calyces relative to the rest of the brain’svolume. In ichneumonid and aculeate Hymenop-tera (e.g., honeybees), calyces comprise three (inhoneybees four) concentric zones, termed I, II, III,and IIIa (Jawlowski 1959b), or the lip, collar, andbasal ring (III and IIIa) of Mobbs (1982). Olfactoryafferents supply zones I and III (Mobbs 1982,1984), whereas projections from the medulla andlobula of the optic lobe invade zone II (Jawlowski1958; Gronenberg 1986, 1998). Massive conver-gence in the calyces of two modalities from periph-eral sensory neuropils is, however, unusual. Inother insect orders, calyces have meager, if any,afferents from the optic lobes and instead serve asa specialized distal region of the mushroom bodiesassociated with olfactory inputs from the antennallobes. As will be discussed later, the lobes alsoreceive major afferent supply.

MUSHROOM BODIES IN ANOSMIC INSECTS

In secondarily anosmic insects, such as the div-ing beetle Dytiscus, the back-swimmer Notonectaundulata, and cicadas, antennae are sometimesgreatly reduced or may serve mechanosensoryfunctions exclusively (as in Notonecta; Rabe 1953).Such species lack antennal lobes. They also eitherlack calyces (cicadas, Notonecta; Fig. 2H) or thecalyces are greatly reduced (as in Dytiscus; Fig. 2G)compared with an odor-sensitive species of thesame order (e.g., the caterpillar hunter beetle,Calosoma scrutator; Fig. 2E). In such calyxlessspecies, thin cell body fibers from the globuli cellsform a narrow pedunculus that increases in diam-eter anteriorly only where it provides the lobes.Because there are no Kenyon cell dendrites dis-tally, as there are in the calyces of odor-detectingspecies (see below), Kenyon cells can only contrib-ute to local circuits in the lobes between afferentneurons supplying the lobes and efferent neuronsleaving them.

MUSHROOM BODY-LIKE STRUCTURESIN CHELICERIFORMES

The relationship between mushroom body-likestructures and first order olfactory neuropils is ex-quisitely shown in the Cheliceriformes.

All Cheliceriformes possess mushroom body-like neuropils within the anterior neuromere (pro-tocerebrum) of the supraoesophageal (prosomal)ganglion. Chelicerate mushroom bodies accordwith Flogel’s and Kenyon’s criteria for mushroombodies of insects. They comprise many hundreds,or in some species, hundreds of thousands, of par-allel fibers that originate from dorsal clusters ofbasophilic globuli cells. Golgi impregnations dem-onstrate that parallel fibers give rise to dendriteseither proximate to the globuli cell clusters, orthey give rise to groups of dendrites at specificpositions along the lengths of the lobes. Except inthe case of araneans (spiders), in which mushroombody-like centers are visual neuropils (Strausfeldand Barth 1993), Kenyon cells are supplied by af-ferents that relay from olfactory glomeruli. The lat-ter are not situated within the chelicerate brain. Noarthropod interpreted as a chelicerate (includingthe Mid-Cambrian species Sanctacaris; Briggs andCollins 1988) possesses antennae. Instead, olfac-tory glomeruli are situated in segmental neuro-meres associated with olfactory appendages thatarise from body (opisthosomal) segments.

The cheliceriform mushroom bodies can reachvarying degrees of elaboration. In scorpions, mush-room bodies are relatively small, being supplied bya few thousand globuli cells (in Centruroidessculpturatus). In the amblypygids, or whip-spiders(amblypygids are not true spiders), the lobes arehuge, richly convoluted (Babu, cited on p. 1256 ofBullock and Horridge 1965), and are supplied bytwo pairs of globuli cell clusters (∼300,000 neu-rons in Tarantula sp) that form a roof over theprotocerebrum. Limulus polyphemus possessessome millions of globuli cells (Viallanes 1893; Fahr-enbach 1979), which give rise to neurons that bearclose resemblance to insect Kenyon cells exceptthat their axons are described as relatively short.(Fahrenbach 1979). Our own studies suggest thatsome axons extend either side of the oesophagusto form lobes that extend posteriorly and mediallytoward ventral neuromeres where they are possi-bly confluent with tracts of ascending axons ofsegmental olfactory interneurons. Limulus mush-room body afferents (assumed to be olfactory pro-jection neuron endings) that reach Kenyon celldendrites are described (see Fig. 15, in Fahrenbach1979).

In different chelicerate orders, olfactory glo-meruli are associated either with specialized ab-dominal (metasomal) appendages, modified walk-ing limbs of the thorax (mesosoma) or, in one case,



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with the palps. For example, in scorpions, a pair ofchemosensory organs (called pectines; Gaffin andBrownell 1997) extends from the first abdominalsegment. Pectines send their chemosensory axonsinto a small cluster of large olfactory glomeruli situ-ated in the first metasomal and in the last meso-somal neuromere. Yet, in scorpions as in all che-licerates, the mushroom bodies are situated withinthe protocerebrum, receiving relays from olfactoryglomeruli via tracts that carry ascending axons ofprojection neurons.

The typical relationships between the size ofchelicerate mushroom bodies and the number ofolfactory glomeruli is shown in Figure 6. In sol-pugids (sun spiders but, again, not true spiders),specialized chemoreceptor organs called malleoli(Brownell and Farley 1974) extend from the lastleg pair. The enlarged palps of solpugids are alsospecialized as olfactory receptor organs and dem-onstrate an interesting example of evolutionaryconvergence: the modification of anterior append-ages to antenna-like structures (Fig. 6A). In sol-pugids, olfactory glomeruli are situated medially inthe first and second mesosomal ganglia, in whichthey receive ascending receptor axons from themalleoli and receptor axons from the palps (Fig.6B). In uropygids (vinegaroons) and amblypygids(whip spiders; Fig. 6C), it is the first leg pair thathas evolved into an antennoform chemoreceptororgan, again providing a wonderful example ofconvergent evolution of a frontal antenna. The firstleg pair is grotesquely elongated in the amblypy-gids in which the first, second, and third meso-somal ganglia are packed with small olfactory glo-meruli that anteriorly invade cephalic (prosomal)neuromeres (Fig. 6D). The amblypygid mushroombody lobes are so large and so convoluted that theyappear to have miniaturized other brain neuropils.Reconstructions of these mushroom bodies showgyri and folds that are reminiscent of the gyri seenin some mammalian cortices (Fig. 6D). In sol-pugids, which have relatively few olfactory glo-meruli, the mushroom bodies are about one-fiftiethof the size of those of amblypygids (Fig. 6B).

The most basal organization of the cheliceri-form olfactory pathway is in the Pycnogonidae(Fig. 6E,F), in which each leg supplies a group of afew glomeruli that are associated with interseg-mental pathways ascending to a small mushroombody in the brain. Several morphological featuressuggest the primitive nature of this system. Meta-meric thoracic ganglia are unfused, a conditionseen in no other cheliceriform; the olfactory sen-

sillae repeat on each segmental appendage; and themushroom body lobes, like those of onychopho-

Figure 6: Chelicerate mushroom bodies showing therelationship between glomeruli number and mushroombody size and elaboration. Olfactory receptor organs areshown in black in the solpugid Eremboates pallipes (A),the amblypygid Tarantula sp. (C), and the pycnogonidLecythorhyncus hilgendorfii (E ). Irrespective of the loca-tion of glomeruli, mushroom bodies (solid black profiles,B,D,F) are located within the protocerebrum. In sol-pugids (B), malleoli provide afferents to glomeruli(shaded in B,D,F) in the first and second opisthosomal(postoral) ganglion. In amblypygids (D), a grotesquelyelongated first leg pair supplies afferents to hundreds ofsmall glomeruli in the first opisthosomal ganglion thatspread into supraoesophageal neuropil. In pycnogonids(F ), glomeruli are arranged segmentally in ganglia asso-ciated with the legs. In this class of Cheliceriformes, theleft and right mushroom body lobes are confluent at theprotocerebrum’s midline.

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rans and diplopods (Holmgren 1916; Schurmann1995; Strausfeld et al. 1995), are confluent acrossthe midline.

MUSHROOM BODY-LIKE STRUCTURES IN OTHERSEGMENTED INVERTEBRATES

Neuroanatomical evidence suggests that mush-room bodies in chelicerates are second-order neu-ropils of the olfactory pathway. The same seems tobe true of annelids, onychophorans, centipedes,and millipedes, all of which have specialized olfac-tory cerebral appendages. Comparisons betweenthese different groups also show that olfactory glo-meruli are associated with the mushroom bodies(Strausfeld et al. 1995). For example, in the preda-tory and highly territorial scale worm Arctenoe vit-tata, olfactory glomeruli are associated with enor-mous mushroom bodies (Fig. 4A). In the errantpolychaete Nereis vexillosa, olfactory receptorendings terminate as small glomeruli near the headof the mushroom body pedunculus, in which theyappear to be grasped by dendrites of globuli cells.In the onychophoran Euperipatoides rowellii, re-ceptors from the olfactory appendages (tentacles)terminate in olfactory glomeruli, which are them-selves a contiguous outgrowth of the mushroombodies (Holmgren 1916; Schurmann 1995; Straus-feld et al. 1995). In diplopods (millipedes), the an-tennae supply receptor axons to a glomerular an-tennal lobe that apposes the head of the mush-room bodies. In chilopods (centipedes), there areprominent antennal (olfactory) glomeruli in thesubesophageal ganglion from which ascending fi-bers reach the mushroom body pedunculus andlobes (Strausfeld et al. 1995).

In diplopods and onychophorans, the mush-room body pedunculus divides into several parallelmedial lobes. In both orders, medial lobes fusewith their contralateral counterparts at the midlineas they do in pycnogonids. In centipedes (Chi-lopoda), the medial lobes are lateralized and do notfuse at the midline but divide into swellings remi-niscent of the swellings at the head of the g lobesin Drosophila. There is no obvious calyx, however,in any of the above groups (Holmgren 1916; Straus-feld et al. 1995).

Crustaceans are the only group in which mush-room bodies, structurally defined according to Flo-gel and Kenyon’s criteria, have not yet been re-solved. In decapod crustaceans (e.g., shrimps,crabs), olfactory glomeruli are supplied by the an-tennules (the main olfactory receptor organs). Sec-

ond-order interneurons with dendrites in the (ol-factory) antennule lobes give rise to bifurcatingaxons whose two tributaries project out into theeyestalks via a pair of tracts reminiscent of the in-ner antennocerebral tract of insects except that, ininsects, axons linking olfactory glomeruli to proto-cerebral neuropils are strictly homolateral. In mostdecapod crustaceans, the tracts terminate in adense neuropil, called the hemiellipsoid body, situ-ated just proximal to the optic lobes (Mellon et al.1992). Although Hanstrom (1928; see also, Nasseland Elofsson 1987; Strausfeld et al. 1995) believedthat the hemiellipsoid body is homologous to themushroom body (Fig. 1, bottom), hemiellipsoidbodies neither provide a lobed structure nor par-allel fibers. Instead, the neuropils are usually ar-ranged as strata. However, like the mushroom bod-ies, hemiellipsoid bodies are associated with thou-sands of minute basophilic cell bodies. Thehemiellipsoid bodies might be more easily com-pared with mushroom bodies in decapods such asthe burrowing shrimp Callianassa californiensesin which the eyestalks are reduced or absent andthe hemiellipsoid bodies are incorporated into themidbrain proper. However, even in this species,the hemiellipsoid bodies do not provide parallelfibers but, instead, are compact glomerular neuro-pils (Strausfeld 1998b).

One group of decapods lacks any neuropileven vaguely reminiscent of a mushroom body or ahemiellipsoid body. This group is the isopods (sowbugs, sea slaters, pill bugs) whose antennules sup-ply large glomerular neuropils that are similar inappearance to the antennal lobes of flies. Yet iso-pods entirely lack obvious higher order olfactoryneuropils, including the accessory lobes, which, inthe brains of many other decapods, are glomerularneuropils in the midbrain linked to the antennulelobes (Sandeman et al. 1993). Nevertheless, iso-pods are a remarkably successful group, occupyinghabitats ranging from Antarctic Ocean to SaharanDesert. Despite their lack of mushroom bodies orpossible analogues, isopods can learn olfactorycues and use olfactory communication for kin rec-ognition and maintaining territory (Linsenmair1987).

EVOLUTION OF ARTHROPOD MUSHROOM BODIES

The phylogenetic affinities among insects andother arthropods is still much debated, with some-times conflicting results derived from sequenceanalysis of rRNA (Ballard et al. 1992) and DNA



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(Wheeler et al. 1993). In contrast, the classic ap-proach of Holmgren (1916) and Hanstrom (1926),which relied on comparative anatomy of brain cen-ters to reconstruct evolutionary relationships, sug-gests today that invariant cerebral architecturescould be as useful in reconstructing relational treesas are other highly conserved pheno- or genotypicfeatures.

A recent study (Strausfeld 1998b) reconstructsarthropod phylogenies on the basis of the presenceor absence of 100 brain characters in 26 taxa, byuse of computational tools designed for investigat-ing closest affinities among taxa according to thedegree to which they share derived characters(Hennig 1966; Swofford 1992). Raw data for theneural analysis (Strausfeld 1998b) treats any char-acter as wholly independent of any other. Thereare no conditional characters (e.g., antennal lobes,and then calyces). Thus, globuli cells are scored aspresent or absent independent of the presence orabsence of parallel fibers. This eliminates assump-tions about synapomorphy and, hence, tautology.Mutually exclusive character states are scored aspresent or absent and characters are treated as un-ordered and unweighted. The resulting tree(Strausfeld 1998b) places Onychophora basal tothe Arthropoda (see also Budd 1996) with Dip-lopoda as a sister group, closer to them than to theChilopoda (centipedes). Crustaceans and insectsemerge as sister clades, agreeing with recent 18sRNA sequence analysis (e.g., Ballard et al. 1992;Friedrich and Tautz 1995) and developmental stud-ies (Whitington et al. 1991).

The occurrence of characters within the neu-ral tree has been traced by use of MacClade (Mad-dison and Maddison 1992). Characters treated asindependent entities, but that nevertheless appearto originate together and that apparently contrib-ute to a defined neuropil, probably have mutualfunctional relevance, as is the case for architecturalentities that, in insects, together comprise the cen-tral complex (Strausfeld 1998b). Character tracing(Fig. 7) reveals the deep occurrence of a characterassemblage comprising globuli cells, parallel fibers,and lobed neuropils, all of which are preciselythose features identified originally by Flogel andKenyon to characterize insect mushroom bodies.These features are plesiomorphic to millipedes,onychophorans, and annelids as well as to chilo-pods and chelicerates. A derived loss of globulicells, parallel fibers, and lobes (at event 13, Fig. 7)is proposed to account for their absence in thestem group leading to the branchiopod crusta-

ceans and basal insects (archaeognathans; Laban-deira and Beal 1990), both of which are hypoth-esized, on the basis of shared features of their vi-sual neuropils, to derive from a common ancestor(Strausfeld 1998b). Globuli cells, and parallel fibersorganized as lobes, reappear again in basal aptery-gotes and palaeopterans. Globuli cells occur againin the malacostracan Crustacea concomittant withthe first appearance of hemiellipsoid bodies. Thesededuced events (Fig. 7) can be compared with theoccurrence and segmental locations in the chelic-erate assemblage of glomeruli and head append-ages, and the occurrence among the Insecta of an-tennal glomeruli and calyces (see also Fig. 6).

The absence of mushroom bodies in Crusta-cea, but their apparent reappearance in the in-sects, suggests at least two possible evolutionaryscenarios.

1. Mushroom body-like structures arose only onceand have been highly modified as hemiellipsoidbodies in the malacostracan Crustacea (Hans-trom 1928) because of transformational homol-ogy (Patterson 1982). This would imply thatgenes for globuli cells and parallel fibers werenever lost from the genome, but that a develop-mental pathway leading to the formation ofmushroom bodies was transformationally sup-pressed with subsequent reversion to the origi-nal (and hence plesiomorphic) structure in in-sects and the appearance of a characteristicallydifferent architecture in crustaceans

2. Mushroom bodies in different groups are ho-moplastic and have independently evolved sev-eral times to serve a variety of sensory functions:in polyclad Platyhelminthes, in the annelid-ony-chophoran-diplopod-chelicerate-chilopod assem-blage, and in insects subsequent to the archaeog-nathans.

There are two problems with the first sce-nario. First, mushroom bodies may have been lostprior to the emergence of the first crustaceans, inwhich case stem taxa of the crustacean/insect as-semblage would have lacked mushroom bodies.This seems quite well supported by comparisonsbetween the archaeognathan Machilis, possiblyrepresenting the most primitive insects (Laban-deira et al. 1988), and the basal branchiopod crus-tacean Triops. Preliminary observations of thesegenera show their brains lack mushroom bodies(and globuli cells) or any other structure that could

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be interpreted as possibly homologous to them(N.J. Strausfeld, unpubl.). Second, if mushroombodies are plesiomorphic, then one might expectthat crustacean stem taxa (e.g., cephalocarids;Brusca and Brusca 1990) should possess mush-room bodies as defined by Flogel and Kenyon. Ex-tremely tenuous support for this could be sug-gested from studies on the cephalocarid Hutchi-soniella macrocantha, which Elofsson and Hessler(1990) describe from gross morphology and elec-tron microscopy as possessing a mushroom body.However, their figures are difficult to interpret asthey show only either surface features of the brainor high-resolution electron micrographs. Neither

adequately resolves neural architectures and,hence, there is no clear definition of a mushroombody in this species.

If mushroom body-like structures in varioustaxa are the product of convergent evolution, thisthen raises the question of whether mushroombodies are analogous only with respect to theirmorphology or whether they are analogous bio-chemically and functionally. This line of inquirywould strengthen the rationale for performingcomparative molecular biology, such as generatingcDNA libraries from globuli cells of various arthro-pod species with the aim of identifying mushroombody-specific proteins and thence homologous se-

Figure 7: Strict parsimony tree, derivedfrom an analysis of 100 neural charactersand 26 taxa. (outgroups omitted; seeStrausfeld 1998b). The tree widely sepa-rates chilopods from diplopods (oftencombined in other trees as a group calledthe Myriapods). Diplopods emerge as sis-ter to the Onychophorans. Pycnogonids(L. hilgendorfii) are unambiguouslyplaced into the clade Cheliceriformes.Platyhelminthes here shown basal to coe-lomates. Nonmalacostracan crustaceans(branchiopods) are more closely related toarchaeognath insects than to any arthro-pod group, making insects and crusta-ceans sister groups (Strausfeld 1998b).Taxa possessing mushroom bodies, as de-fined by the Flogel-Kenyon criteria, areconnected by heavy lines. Crustacea, thebasal archaeognathan insects, and theCollembola stand apart (see text). Charac-ter mapping: (1) Globuli cells; (2) discretelobes comprising parallel fibers; (3) che-mosensory afferents ending in glomeruli;(4) lateralization of lobes; (5 ) metamericrepetition of glomeruli; (6) postoral ap-

pendage (the antenna; secondarily preoral in crustaceans; Brusca and Brusca 1990); (7 ) postoral appendage chemosen-sory; (8) segmental glomeruli retained in first postoral ganglion; (9) glomeruli lost in all ganglia; (10) glomeruli retained infirst abdominal/last thoracic neuromere; (11) glomeruli retained in first two postoral ganglia; (12) glomeruli retained insecond postoral neuromere; (13) loss of characters 1–5; (14) preoral appendage (antennule); (15) wedge-shaped glomeruli;(16) homoplastic origin (re-expression?) of globuli cells; (17) dense, layered, nonretinotopic neuropil in eye stalk (he-miellipsoid body); (18) second glomerular neuropil (termed accessory lobe); (19) hemiellipsoid body in midbrain; (20)homoplastic re-expression of globuli cells; (21) parallel fibers comprising bilateral lobed neuropils; (22) antennae acquireolactor receptors; (23) glomerular neuropil supplied by antenna; (24) calyces; (25) absence of features 22–24. Characters8–11 show a general trend for the reduction of segmental glomeruli (but these basal characters are retained in Limulus andpycnogonids). Character assemblages 1 with 2, and 20 with 21, accord with the Flogel-Kenyon criteria for mushroombodies. Mushroom bodies, sensu Flogel-Kenyon, are shared by the annelid-onychophoran-diplopod-cheliceriform-chilo-pod (AODCC) assemblage but are absent in crustaceans and archaeognathan insects. Their reappearance in thysanuraninsects suggests homoplasy and convergent evolution with the AODCC assemblage. The character assemblage 22, 23, 24is unique to neopteran insects. The character assemblage 14–18 is unique to malacostracan crustaceans. Boxed character(event) 13 presumes character loss. Circled characters (16,20) indicate two possible homoplastic origins of globuli cells.



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quences in different taxa. Such an analysis wouldalso allow a search for possible mushroom bodyanalogs in species with few structural similarities,such as in chordates.

An ancient origin of mushroom body-likestructures is supported by morphological similari-ties between modern scale worms such as Arc-tenoe, which possesses large mushroom bodies(Fig. 4A), and Mid-Cambrian annelids, such as Ca-nadia spinosa (Conway Morris 1979). The samecomparison can be made between the externalmorphology of extant onychophorans, which alsopossess mushroom bodies, and the morphology ofMid-Cambrian Onychophora, such as Aysheaia(Whittington 1978).

Further studies on cephalocarids and on bran-chiopod Crustacea are urgently needed to fill gapswhere there is not enough data for character analy-sis. So too, are comparative studies of other terres-trial hexapods (Collembola, Protura, Diplura) asthese may indicate whether mushroom bodies, asdefined, are homoplastic among the hexapods. Forexample, neanurinid Collembola (which are para-insects) do not appear to have mushroom bodies,yet they possess an insect-like central complex(N.J. Strausfeld, unpubl.).

Comparison between geologically distant gen-era would also be important to determine whetherspecies within the same group, but separated bygeological time, have evolved at similar rates. Forexample, amblypygids from West Africa and East-ern America should not have a common ancestormore recent than when the continents split apart.Do their mushroom bodies share the same charac-teristic elaboration of the lobes, for example, orhave they diverged by some measurable set of char-acters? And, studies of linked characters will beuseful because they may suggest emergent mush-room body functions among insect groups inwhich the calyces are secondarily reduced or ab-sent.

EARLIEST INSECT MUSHROOM BODIES DID NOTSERVE OLFACTION

The evolutionary history of the mushroombodies within the Insecta suggests that these cen-ters did not originate as olfactory neuropils (Fig. 7).Of crucial significance are the mushroom bodies ofpalaeopteran insects, such as the Ephemoptera andOdonata, (mayflies, dragonflies, damselflies), aswell as primitive apterygotes such as the Thys-anura. The fossil record suggests that these are the

earliest orders for which extant representativesstill exist (Labandeira and Sepkoski 1993) and thatthey arose some 100 million years before the ad-vent of neopteran taxa, such as the Blattodea,whose modern representatives possess antennallobes and calyces.

Thysanura, Ephemoptera, and Odonata areprobably all primarily anosmic with respect to air-borne odors. This conclusion derives from neuro-anatomical studies that demonstrate that in Ther-mobia and Lepisma (firebrat, silverfish) the an-tennae supply a mechanosensory neuropil the ar-chitecture of which is almost identical to striatemechanosensory neuropil of crustacean brains,which also receives afferents from the antennae(Strausfeld 1998b). A crucial feature is that theseapterygotes, as well as palaeopteran insects, alllack the glomerular antennal lobes typical of Neop-tera whose ancestors first appeared in the Late Car-boniferous. Another important feature of themushroom bodies of primitive anosmic insects isthat they lack calyces. However, their neuropilsderive from thousands (in odonates, hundreds ofthousands) of globuli cells that provide cell bodyfibers forming a thin pedunculus, which, anteri-orly, gives rise to elaborately subdivided and swol-len lobes (Figs. 2I and 4B).

Judging from their modern representatives,the mushroom body lobes of these earliest insectsthus seem to serve mainly mechano- and optosen-sory integration rather than olfaction. Neverthe-less, it is important to note that this evolutionarylegacy has been maintained in neopteran speciesthat are sensitive to odors and that it is crucial toour understanding of how mushroom bodies work.In Periplaneta, for example, which by most ac-counts represents an evolutionarily basal species(Kukalova-Peck 1991; Kambhampati 1996), as wellas in orthopterans and in the more recentlyevolved brachyceran Diptera (e.g., Drosophila; seeMacAlpine 1989), mushroom bodies receive affer-ents to their medial lobes in addition to the olfac-tory supply to their calyces (Schurmann 1970a,b,1971; Li and Strausfeld 1997; Ito et al. 1998). Af-ferents have also been identified in the verticallobes of Apis (Strausfeld 1998a). Intracellular re-cordings from Periplaneta demonstrate that affer-ents to the lobes carry multimodal information(Fig. 8A). If this is a general feature across taxa,then it would account for the range of modalitiesthat can be recorded from efferent neurons carry-ing information from the orthopteran and blattoidmushroom bodies to other areas of the protocere-

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Figure 8: Multimodal and context-modified responses in the efferent neurons of Periplaneta mushroom bodies relate toafferent identities. (A) In addition to their supply from the antennal lobes (ant lob; shown in C), the calyces (ca) are suppliedby afferent neurons originating in superior lateral protocerebrum (s l pr) and responding to nonolfactory modalities (visualand tactile; top traces, inset A). Another afferent is shown originating in the dorsal lobes (d lob) and terminating at the tipof the medial lobe (arrow). (B) Combinations are more effective than unimodal stimuli in eliciting a response from thisefferent neuron linking the medial lobe to the inferior lateral protocerebrum (i l pr). There is no response to light ON, aweak response to acoustic stimulation, and vigorous activation by both combined (inset B). (C) An efferent neuron fromthe medial lobe to the inferior medial protocerebrum was inhibited by acoustic stimuli after presentation of visual andolfactory cues (top trace, inset C) but excited by sound after flicker and tactile cues (lower trace, inset C).



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brum (Schildberger 1981, 1984; Li and Strausfeld1997).

It may seem curious that the earliest insectswere ill equipped for olfaction. But consider theLate Silurian to Early Devonian landscape, devoidof any but the most primitive vegetation. The firstinsects colonizing the shoreline were presumablylittoral detritus feeders, subject to relentless preda-tion by chilopods and chelicerates. The selectivepressure for retaining vibration and tactile organsmust therefore have been greater than that for theevolution of appendages specialized for detectingdistant cues, such as air-borne odorants. The earlyrole of antennae in mechanoreception is even sup-ported by evidence from Drosophila genetics sug-gesting that the protoinsect possessed leglike headappendages. In Drosophila, alleles of the Hox geneAntennapedia transform the olfactory appendage,the antenna, into the atavistic default appendage, aleg, by inhibiting the expression of genes that pro-gram the development of an antenna (Casares andMann 1998). One hypothesis that can be derivedfrom the neural phylogenetic tree shown in Figure7 is that a crustaceomorph ancestor to insects andcrustaceans possessed a single pair of uniramousleglike appendages equipped for mechanorecep-tion (Strausfeld 1998b). Observations of primitiveapterygotes suggest that their antennae retain char-acters of this crustacean-like ancestor: equippedwith sensors typical of a leg for mechanoreceptionand contact chemoreception but not for far-fieldolfactory perception.

Interestingly, the transformation of an odor-sensitive antenna back to a default mechanosen-sory appendage has occurred without human in-tervention. The result of this transformation is seenin certain anosmic terrestrial and freshwater neop-terans, particularly among the Hemiptera in whicha reduced mechanosensory antenna is accompa-nied by the absence of olfactory glomeruli and adrastic reduction or even absence of the mush-room body calyces, as in the backswimmer Noto-necta (see Fig. 2H). This adaptation provides addi-tional evidence that the calyx is not fundamental tomushroom body design but is a specialized neuro-pil associated with the ability to detect distant air-borne odors.

MUSHROOM BODIES AS OLFACTORYAND MULTIMODAL INTEGRATORS

Until Vowles’s experiments with mushroombody lesioning, the general view of mushroom

body function was that they played a major role inintegrating olfactory and visual signals and wereimportant for controlling complex behaviors.Their involvement in the integration of several sen-sory modalities was supported by Jawlowski’s(1958, 1960) studies on Hymenoptera describingessential features of the axonal pathways from theoptic and antennal lobes to the calyces. Weiss(1981) demonstrated that orthopteran mushroombodies are supplied by the antennal lobes as well asby a parallel tract of projection neurons from thelobus glomerulatus, which is itself supplied by af-ferents from chemoreceptors of the mouth parts(Ernst et al. 1977).

Schurmann (1970a,b, 1971) demonstrated inthe cricket Achaeta that the dendrites of Kenyoncells can be postsynaptic to olfactory interneuronterminals in the calyces and that Kenyon cell axonsare presynaptic to efferent neurons in the lobes.Afferents to the calyces synapse onto at least a siz-able subset of Kenyon cell dendrites, althoughthere are more dendrites offering postsynapticsites than antennal lobe terminals providing pre-synaptic ones. Together, these publications pro-vide a broad consensus regarding the organizationof Kenyon cell dendrites and support the idea thata major role for the mushroom bodies in odor-sen-sitive neopteran insects is in olfactory processing.This has been demonstrated many times by elec-trophysiology (e.g., see Burrows et al. 1982; Kan-zaki et al. 1989; Laurent and Naraghi 1994). Recentstudies, for example, demonstrate that olfactory in-terneurons show synchronized activity, inter-preted as activated neural assemblies, when theyencode specific odors (MacLeod and Laurent1996). Conversely, pharmacologically induced de-synchronization impairs the discrimination of simi-lar odorants, suggesting that oscillation synchrony,possibly mediated by the mushroom bodies, is es-sential for fine detail discrimination (Stopfer et al.1997).

In addition to their role in odor discrimination,extra- and intracellular recordings have shown thatmushroom bodies have another important role:that of integrating different sensory modalities.These include visual (Gronenberg 1986; Homberg1984), tactile (Schildberger 1984) and acousticstimuli (Li and Strausfeld 1997, and unpubl.). Howdo mushroom bodies process different modalitieswhen, as exemplified by the cockroach andcricket, the calyces appear to receive predomi-nantly olfactory inputs? The answer comes fromstudies of the mushroom body lobes in which syn-

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aptic relationships are much more complex thanmere connections between Kenyon cell axons andefferent neuron dendrites. Kenyon cell axons inthe lobes have both pre- and postsynaptic special-izations (Li and Strausfeld 1997) confirming elec-tron microscopical studies showing Kenyon cellaxons pre- and postsynaptic to each other, post-synaptic to afferent profiles (Schurmann 1970a,b;Strausfeld 1998a), and having synaptic relation-ships with fibers that contain dense core vesiclesindicative of neuromodulator elements (Frontaliand Mancini 1970; Schurmann and Erber 1990;Strausfeld 1998a). There is now abundant evidencethat various sensory modalities reach the mush-room body lobes (and calyces) indirectly throughother protocerebral neuropils, such as the inferiorand superior medial protocerebra. In cockroaches,for example, visual, tactile, and acoustic modalitiesare carried by afferents that terminate in the verti-cal and medial lobes (Fig. 8A; see also, Li andStrausfeld 1997, and unpubl.).

The cited studies suggest that mushroom bod-ies play important roles in multimodal sensory in-tegration, possibly processing many types of sen-sory signals in conjunction with, or independentof, olfactory inputs (Fig. 8). However, dependingon their internal architectures and lobed divisions,mushroom bodies in different insects may mediatea variety of functions. In certain species, such asDrosophila (Ito et al. 1998), the comparativelysimple mushroom bodies might serve functionsmainly associated with olfactory perception. Inphylogenetically basal species, such as Peripla-neta, mushroom bodies may serve functions asso-ciated with olfaction and, because they receive somany other modalities, they may also play essentialroles in a variety of other sensory and motor path-ways.

The range of functions is suggested from ex-tracellular recordings. For example, units in thecockroach medial and vertical lobes appear to dis-tinguish mechanical self-stimulation from imposedtactile stimulation. Other units reflect motor ac-tions and are modulated by changes in direction(Mizunami et al. 1993). Lesions of the medial lobesadversely affect place memory (Mizunami et al.1993). Intracellular recordings (Li and Strausfeld1997; see also Fig. 8) reveal responses to a largerange of sensory stimuli and modalities.

The role of mushroom bodies in motor controlhas been suggested previously by Erber et al.(1987) who stressed the importance of experi-ments that use focal stimulation or ablation of the

mushroom bodies to elicit or abolish specific mo-tor actions. This strategy, introduced by van derKloot and Williams (1954) and by Huber (1955; seealso Wadepuhl 1983), indicates that mushroombodies might play a pivotal role in coordinatingbehavioral programs. Likewise, electrical stimula-tion and lesion experiments suggest the impor-tance of mushroom bodies in specific behavioralrepertoires such as courtship (Huber 1959, 1960)and other motor actions (Maynard 1967). An in-volvement by the mushroom bodies in meeting avariety of behavioral demands has been proposedfrom studies on honeybees and ants that show en-largement of mushroom body neuropils that re-sults from multitasking by ants (Gronenberg et al.1996) or, in honeybees, that coincides with hor-monally induced changes of the behavioral reper-toire (Withers et al. 1993, 1995). In crickets, hor-mone-dependent increase in the number ofKenyon cells at sexual maturity (Cayre et al. 1994)may also be linked to the expression of new be-haviors.

Erber et al. (1987) proposed that mushroombodies perform at least five discrete computationson sensory inputs and relay the results to distrib-uted areas in the protocerebrum, some of whichcompare ongoing and past stimuli or form olfac-tory memory. The five functions summarized fromelectrophysiological data are: the generation of af-ter-effects in output neurons (Vowles 1964b),which can persist for minutes after the stimulus(Schildberger 1981, 1984; Gronenberg 1987); en-hancement of the signal-to-noise ratio of the olfac-tory stimulus; detection of stimulus combinations;detection of temporal events in an olfactory stimu-lus; and detection of stimulus sequences.

There is compelling evidence that efferentneurons from the mushroom bodies have context-specific responses, meaning that the activity of anefferent neuron depends on the accompanying orthe immediately preceding sensory stimuli. In thecricket Acheta domesticus, the rate of discharge,or the level of inhibition or excitation of an effer-ent neuron, can depend on what modalities pre-cede the test stimulus (Schildberger 1984). An ef-ferent neuron can increase its discharge rate fromresting when the cercus is stimulated after re-peated mechanical stimulation of the antenna. Thesame neuron decreases its discharge rate from rest-ing when repeated stimulation of the cercus is fol-lowed by stimulation of the antenna (Schildberger1981). In Periplaneta, the activity of efferent neu-rons reacting to one stimulus alone is modified



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Figure 9: Relationships between themushroom body and other parts of thenervous system. (Round-cornered boxes)Sensory organs grouped according to mo-dality. (Shaded boxes) Neuropil regionsrelatively well investigated. (Whiteboxes) Regions essentially uninvesti-gated. (Thick shaded lines) Major tractsconnecting neuropil regions. (Arrows)Supposed direction of information (omit-ted where evidence for polarity is lack-ing). Information about ambient light in-tensity is received by the ocelli, prepro-cessed in the ocellar neuropil and sent tothe caudal part of the deutocerebrum(posterior slope) via the ocellar nerve(ocellar n). Visual information is receivedby the compound eyes and processed innested retinotopic optic lobe neuropils(lamina, medulla, and lobula/lobulaplate) before entering the central brain.The ventro-lateral protocerebrum (v l pr)receives visual signals almost exclusively,and thus can be considered to contain theprimary optic foci. Other areas [e.g., lat-eral horn (l ho); superior lateral protoce-rebrum (s l pr); inferior lateral protocere-brum (i l pr)] are also supplied by theoptic lobes, but they also receive inputsfrom other sensory neuropils. A directconnection from the medulla and lobulato the calyces is observed in certain Hy-menoptera (dotted arrow). Certain effer-ents from the lobula/lobula plate projectdirectly to premotor neuropil of the pos-terior slope. Mechanosensory receptorson the antennae (including Johnston’s or-gan supplying acoustic information inflies and mosquitoes) enter the brain viathe antennal nerve (ant n) terminating inthe antennal mechanosensory neuropil (dorsal lobe) of the deutocerebrum. Mechanosensory axons from the head andproboscis project to specific neuropils in suboesophageal ganglion (sog) via labial (lb n), pharyngeal (phy n), and accessorypharyngeal (ac phy n) nerves. Mechanosensory axons from the body and extremities (wings, legs, genitalia) project todefined regions in the respective thoracic or abdominal neuromere, (vnc) ventral nerve cord. Connections with somaticmotor circuits provide appropriate local computations for reflexive motor actions (see Burrows 1992). Tactile and acousticrelays (in crickets, grasshoppers, certain Diptera) reach the brain via the cervical connective (cv con) and median bundle(m bdl) to reach superior medial protocerebrum (s m pr). Airborne (olfactory) chemical stimuli are detected by the thirdantennal segment and maxillary palps. The former sends axons through the antennal nerve (ant n) to the antennal lobe.Axons from the latter enter the suboesophageal ganglion via the labial nerve (lb n) and project to the antennal lobe, viathe antenno-suboesophageal tract (AST). Olfactory receptor terminals have odortypic segregation to specific glomeruli(Rodriguez and Buchner 1984; Rodriguez and Pinto 1989; Stocker 1994). Contact (gustatory or taste) chemosensoryneurons in the labial palps project via the labial nerve (lb n) to a gustatory center in the sog, which, unlike the antennallobe, does not show prominent glomerular structures. Gustatory neurons from the ventral cibarial sense organ (VCSO) andthe labral sense organ (LSO) project to the gustatory center via the accessory pharyngeal nerve (ac phy n), and those fromthe dorsal cibarial sense organ (DCSO) via the pharyngeal nerve (phy n). In Blattodea and Orthoptera, a second glomerularneuropil, the lobus glomerulatus, receives inputs from the mouthparts (Ernst et al. 1977) and provides axons to the calycesvia a parallel strand of the i ACT (Weiss 1981). Gustatory neurons in the fore, middle and hind legs, wings, and femalegenitalia project to the thoracic and abdominal ganglia (vnc) via respective segmental nerves. It is likely that the somatic

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when the stimulus is given with a second stimulusof a different modality (Fig. 8B). Other efferentsresponding to one modality can be conditionallyinhibited or excited to that modality depending onwhat stimulus combinations immediately precedethe test stimulus (Fig. 8C). Experience-associatedchanges of neuronal activity have also been shownfrom intracellular recordings from one type of ex-trinsic neuron in the honeybee a lobe (Pe-1),which has been shown to change its electrophysi-ological signature to olfactory stimulation duringassociative conditioning (Mauelshagen 1993). It isnot known, however, whether such modificationsare a consequence of computational events occur-ring within the mushroom body or at some as yetundefined location between the antennal receptorand the efferent neuron’s dendrites.

CONCLUSION: INSECT MUSHROOM BODIESAND THE BRAIN

Figure 9 summarizes the relationship betweenthe brain and a mushroom body of a generic insect,that is, one endowed with features of both recent(e.g., Apis, Drosophila) and basal taxa (e.g., Peri-planeta, Orthoptera, Odonata, Zygoentomata).Shown in Figure 9 are the known pathways thatrelate mushroom bodies to their afferent supplyand to other major brain regions. The four classesof afferents received by the mushroom bodies are

1. Afferents to the mushroom body lobes. Thesereflect the primitive condition in which mush-room bodies without calyces are supplied byafferents from protocerebral neuropils. For ex-ample, the medial lobes of cockroaches are sup-plied with visual, tactile, and acoustic modali-ties carried by afferents terminating in them(Fig. 8A; also, Li and Strausfeld 1997, and un-publ.). Comparable extrinsic neurons havebeen morphologically identified in Drosophila(Ito et al. 1998) and honeybees (Strausfeld1998a).

2. Afferents to the calyces, originating in theproto- and deutocerebrum, also carry multimo-dal information (Fig. 8A). Thus, the absence ofdirect inputs to the calyces from the optic lobesin certain species does not preclude their mush-room bodies from integrating visual informa-tion.

3. Afferents to the calyces from the antennal lobesand the lobi glomerulati. Exemplified by Peri-planeta, each calyx receives olfactory interneu-rons from the ipsilateral antennal lobe (Fig. 8C)via the inner antennocerebral tract. Some in-terneurons projecting from the dorsal lobes(mechanosensory receptor neuropil) send col-laterals to the calyx en route to the superiorlateral protocerebrum. The ipsilateral lobus glo-merulatus in acridids (Ernst et al. 1977) suppliesthe calyx via the globularis–cerebral tract(Weiss 1981). In Lepidoptera (Kent et al. 1986),

Figure 9: (Continued) gustatory signal is conveyed to the brain via the cervical connective (cv con). Projection neuronsfrom the antennal lobe, lobus glomerulatus, and sog gustatory centers contribute to inner antennocerebral tracts (iACT).These project to the lateral horn (l ho) sending collaterals to the mushroom body calyx (a). Projection neurons in the middleantennocerebral tract (mACT) project directly to the l ho with a small subset of mACT entering the pedunculus andterminating in the calyx. The outer antennocerebral tract (oACT) contains fewer fibers connecting the ant lob and inferiorlateral protocerebrum (i l pr). The broad root (Power 1946) is supposed to contain a few fibers that project posteriorly fromthe ant lob. Vertical lobes of the mushroom bodies are connected with the anterior region of the superior medial and lateralprotocerebra (s m pr and s l pr). Medial lobes are connected to the anterior part of the inferior medial protocerebrum (im pr). The mushroom bodies receive multimodal sensory information from protocerebral regions to their lobes and sendoutput back to the same neuropil regions. The central complex is connected to many protocerebral regions but has nodirect connection with the mushroom bodies nor receives direct information from any primary sensory neuropils. Theventral body is connected to various regions of the protocerebrum and has major connections with the central complex.Mushroom bodies and the central complex possess easily identifiable structures. The surrounding neuropils (lateral horn,ventro-lateral protocerebrum, superior and inferior medial/lateral protocerebra) show a much more ambiguous organi-zation. Although often (erroneously) referred to as diffuse neuropils, these areas do have characteristic but complexfibroarchitectures whose neural networks within and between them are scarcely known. Behavior is accomplished by theorganized contraction of muscles resulting in the further stimulation of sensory organs (indicated by feedback loopbehavior). Muscles are innervated by motor neurons originating in the sog and in thoracic and abdominal ganglia. In thebrain (three pre-oral supraoesophageal ganglia) only the deutocerebrum contains motor neurons, which control antennalmovements. The brain’s deutocerebrum, and parts of the protocerebrum, possess premotor neuropils that contain den-drites of descending neurons that supply somatic motor circuits. Details of connections between most brain regions anddescending neurons are not yet known.



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Blattodea, Diptera, and possibly also in Hyme-noptera, gustatory glomeruli appear to have be-come integrated into the antennal lobe, withtheir projection neurons sharing the inner an-tennocerebral tract.

4. Afferents from the medulla and lobula of theoptic lobes of certain Hymenoptera (honey-bees, ant: Gronenberg 1986, 1998) are directlyconnected to the collar zone of the calyces.Mushroom bodies of Blattodea, Diptera, Lepi-doptera, Coleoptera, and Orthoptera appear tolack this annular region suggesting it might bean evolutionary innovation of the Hymenoptera.However, as is discussed above, visual informa-tion reaches the calyces and lobes of theseother groups indirectly via other protocerebralareas.

An important aspect, when discussing the roles ofmushroom bodies, is to determine the target areasof their outputs. Figure 9 proposes indirect path-ways between the mushroom bodies and descend-ing premotor pathways. Two efferent neurons inthe blow fly Calliphora have been reported as pro-jecting from the mushroom bodies directly to de-scending pathways (Strausfeld et al. 1984). In thecockroach Periplaneta one efferent neuron ap-pears to visit a descending neuron (Li and Straus-feld 1987a) and one descending neuron sends ashort collateral into one lamina at the level of thepedunculus (N.J. Strausfeld, unpubl.). Direct con-nections between the mushroom bodies and de-scending neurons have not yet been identified inother species and claims that efferent neurons gen-erally terminate at descending pathways are as yetunsubstantiated. Studies on fruit flies (Ito et al.1998) suggest that the mushroom bodies are notdirectly connected to descending neurons, asKenyon (1896a,b) originally thought, but insteadsupply higher order regions of the protocerebrum(Fig. 9). Such an organization supports the thesis ofErber et al. (1987) that associative processing, suchas learning and memory, does not necessarily haveto occur within the mushroom bodies themselves,but could be performed within distributed regionsof the protocerebrum that may or may not includethe mushroom bodies proper. Reduced to its basiccomponents, the mushroom body is a system ofbifurcated neurons that form elaborate intercon-nections and networks with one another (Schur-mann 1970a,b, 1971; Kaulen et al. 1984), but as aself-contained assemblage it cannot perform anyrole without close cooperation with other ele-

ments of the surrounding neuropils. It seems pru-dent, therefore, to study the mushroom bodies inthe context of the broader landscape of the proto-cerebrum in which they are but one pair of neu-ropils connected to many others.

AcknowledgmentsEvolutionary studies were supported by fellowships to

N.J.S. from the Simon J. Guggenheim Memorial Foundationand the John D. and Catherine T. MacArthur Foundation.Studies on mushroom bodies are supported by a grant toN.J.S. from the National Science Foundation (IBN 9726957).Peter Kloppenberg provided the preparation for Figure 5G.John S. Edwards supplied living Machilis, and Bruno Bernetsupplied living Arctenoe. We are grateful to CamillaStrausfeld, Reinhard Stocker, Martin Heisenberg, and UweHomberg for helpful discussion.

The publication costs of this article were defrayed inpart by payment of page charges. This article must thereforebe hereby marked ‘‘advertisement’’ in accordance with 18USC section 1734 solely to indicate this fact.

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