Mushroom bodies are paired nervous centerslocated in the protocerebrum of the insect brain. Theytake part in performing types of nervous activity suchas processing olfactory information and learning bylong�term and short�term memory storage of incom�ing signals (Fahrbach, 2006). The complexity anddiversity of functions of mushroom bodies arereflected in their complex organization. The cellularunit of mushroom bodies is a unipolar neuron called aglobuli cell or Kenyon cell. These cells usually have asmall pericaryon with reduced cytoplasm and a moreor less developed dendrite and axon, which is usuallydivided into two collaterals. The sum of the dendritesof Kenyon cells together with the incoming externalfibers forms the calyx of the mushroom body, with theaxons proximal of their branching forming the pedun�cle, or stalk, and collaterals, in the simplest case,assembled into two lobes: vertical and medial.
The complexity of the general organization isreached in several ways, two of them being especiallywell known. The first of these is the complication ofthe composition of Kenyon cells in the course ofontogeny. Kenyon cells are descendants of single neu�roblasts segregating in early embryogeny from the neu�roectoderm (Korotneff, 1885) and functioning in thecourse of embryonic, larval, and pupal periods, and, insome cases, also in adults (Cayre et al., 1998). A singleneuroblast divides asymmetrically into a daughterneuroblast and a so�called ganglion mother cell,which, in turn, divides evenly into two cells that differ�entiate into Kenyon cells. In some insects, mushroombodies contain, instead of single neuroblasts, groups ofneuroblasts, which include neuroblasts of two catego�ries: asymmetrically dividing, as described above, andsymmetrically dividing, forming two daughter neuro�blasts.
A number of studies have shown that the neuro�blasts dividing asymmetrically are multipotent, since
in different periods of the insect development they giverise to Kenyon cells differing in a number of character�istics. For example, the long�known division of beeKenyon cells into three categories (Barendrecht,1931) is related to their gradual production by neuro�blasts, as was shown for the honeybee (Panov, 1957;Farris et al., 1999). The variety of Kenyon cells in thehoneybee manifests itself not only in their differentlocalization and size of cell bodies, but also in thetopography, structure, and immunohistochemicalparameters of the processes incorporated in theircalyx, peduncle, and lobes (Vowles, 1955; Strausfeldet al., 2000; Strausfeld, 2002). A variety of Kenyoncells emerging in the course of ontogeny has also beenrecorded in dipterans (Lee et al., 1999) and coleopter�ans (Zhao et al., 2008). Single neuroblasts in otherparts of the brain are also multipotent (Boyan et al.,2010).
The second way of complicating the structure ofmushroom bodies is their multiplication by division ofeach mushroom body into two or three principal sub�units, joined with each other to some degree. Themechanism of this multiplication is usually an increasein the number of mushroom body primordia, repre�sented by single neuroblasts or groups of neuroblasts,each of these neuroblast giving rise to a group of itsdescendants, Kenyon cells. As a result, in the dorsalcell cortex of each half of the brain, instead of onegroup of Kenyon cells, two or three such groups form,more or less isolated from each other. The sum of thesedifferentiating cells forms their neuropil parts (calyx,peduncle, lobes), also segregated from each other todifferent degrees. It is traditionally believed that thepurpose of this primordium multiplication is only toincrease the number of Kenyon cells (Farris et al.,1999). However, in some cases it was found that thedescendants of different neuroblasts form nonidenti�
BRIEF COMMUNICATIONS
The Mushroom Bodies of the Lower Nematocera: A Link between Those of the Higher Diptera and Other Mecopteroids
A. A. PanovSevertsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 119071 Russia
Abstract—Nematoceran Diptera are nonuniform in the structure of their mushroom bodies. Members of themore basal families (Ptychopteridae, Pediciidae, and Tipulidae) have bipartite mushroom bodies, character�istic of members of the other mecopteroid complex orders. In members of Bibionomorpha (Bibionidae andAnisopodidae), tripartite mushroom bodies have been found characteristic of Brachycera Orthorrhapha.
DOI: 10.1134/S1062359012040097
BIOLOGY BULLETIN Vol. 39 No. 4 2012
THE MUSHROOM BODIES OF THE LOWER NEMATOCERA 383
cal structures included in mushroom bodies (Panov,2009a, 2010).
The number of mushroom body primordia ininsects is probably quite a fundamental character, typ�ical of particular taxa. A certain number of mushroombody primordia (such as single neuroblasts or groupsof neuroblasts) can characterize a taxon of the orderrank, or even of the superorder rank (Panov, 1957,1966). Of the groups studied, only coleopterans anddipterans do not follow this rule. Among coleopterans,the number of mushroom body primordia differs inmembers of the suborders Adephaga and Polyphaga.Members of the former suborder have three mush�room body primordia, as do those neuropteroids inwhich this character has been studied, while membersof the latter suborder have only two such primordia, asdo many other Oligoneoptera.
Dipterans strongly differ from other insects in theconsiderable differences in the number of mushroombody primordia found within this order. Initially, inMusca domestica and Calliphora vicina, from one totwo tens of single neuroblasts were found in eachmushroom body of mature larvae and young pupae.Each of these neuroblasts gave rise to a group of neu�rons the processes of which entered the outwardly sin�gle calyx as a separate bundle (Panov, 1957). Laterstudies have shown, however, that this set of mush�room body primordia is not characteristic of all dipter�ans. In Drosophila melanogaster, only four single neu�roblasts were found in the larval mushroom body (Itoand Hotta, 1992). As a result, its outwardly single calyxappears to consist of four equal parts (Ito et al., 1997).The quadripartite structure of the Drosophila mush�room body was confirmed immunocytochemically,and discovered also in the calliphorid Phaenicia seri�cata (Strausfeld et al., 2003). The number of parts thatconstitute the calyx could be determined not onlyfrom the number of neuroblasts, but also from thenumber of bundles of the Kenyon cell processes thatenter the calyx and, especially, those that exit from thecalyx and form its peduncle.
Using as the parameter of mushroom body “par�titeness” mainly the number of fiber bundles that enterthe calyx and exit from it, we found that BrachyceraOrthorrhapha typically have tripartite mushroom bod�ies (Panov, 2009b). Judging by published descriptions,tripartite mushroom bodies are also found in membersof Anisopodidae (Groth, 1971) among Nematoceraand members of Syrphidae (Jarnicka, 1959) amongBrachycera Cyclorrhapha.
Finally, it has been found that the quadripartitestructure of mushroom bodies is probably widespreadamong Brachycera Cyclorrhapha. Multiple mush�room body primordia in some members of this groupcould have emerged as a result of changes in the pro�gram of cell divisions in single neuroblasts, whichstarted dividing evenly at early stages of development,forming a group of neuroblasts, and only subsequently,having diverged within the group of Kenyon cells,
Thus, according to the data that were availableprior to this study, the number of mushroom body pri�mordia, and, thus, the “partiteness” of the mushroombody structure in the dipterans studied differ fromthose found in other members of the superorderMecopteroidea, which includes dipterans. Indeed,bipartite mushroom bodies are characteristic of Pan�orpa (Bierbrodt, 1942) and many lepidopterans (Han�ström, 1928; Schrader, 1938; Norlander and Edwards,1968). In many species of Lepidoptera, two singleneuroblasts (Schrader, 1938; Panov, 1957) or twoassemblages of neuroblasts (Norlander and Edwards,1968) were found in each mushroom body. Two singleneuroblasts were found in embryonic mushroom bod�ies of the caddis fly Stenopsyche griseipennis (Mi�yakawa, 1974).
Taking into account the existence of these differ�ences, as well as the fact that the general structure ofmushroom bodies was studied in nematoceran Dipteraonly once (Groth, 1971), we performed this study,choosing as its objects members of families belongingto different evolutionary lineages of lower dipterans(Wiegmann et al., 2011).
MATERIALS AND METHODS
The mushroom bodies were studied in adults ofPtychoptera scutellaris Mg. (Ptychopteridae), adults ofPedicia rivosa L. (Pediciidae), pupae of Tipula peliostigmaSchumm. (Tipulidae), adults of Sylvicola fenestralis L.(Anisopodidae), and adults and larvae of Bibio marci F.(Bibionidae). Paraffin sections and digital images wereprocessed as in previous works (Panov, 2009a, 2009b).
RESULTS AND DISCUSSION
The mushroom body of P. scutellaris is quite similarin its general structure to the weakly developed mush�room bodies of Xylophagus (Panov, 2009b). The calyxis pillow�shaped, deeply embedded in the protocere�bral neuropil, and subdivided into the anterior glom�erular part, through which the fibers of the antennal�globular tract run, and the posterior part, where glom�erules are absent, and through which bundles of axialprocesses of Kenyon cells run (Fig. 1a). Kenyon cellsare assembled in a single group, and clustering ofKenyon cell processes into bundles prior to theirentrance into the calyx is not pronounced. However,in sections of the calyx approximately perpendicularto the direction of Kenyon cell processes, only theirclustering in two bundles is distinctly visible, instead ofthree, as recorded in Xylophagus (Fig. 1b). At the levelof the peduncle, the bundles fuse into a single bundle.
In the mushroom body of adult P. rivosa, Kenyoncells are also assembled in a rather small single group,and their processes do not cluster into a single groupprior to their entrance into the calyx. However, closer
384
BIOLOGY BULLETIN Vol. 39 No. 4 2012
PANOV
(а) (b)
(d)(c)
(e) (f)
Ca
P
Cp
KK
KK
Ca
CpCp
CpCa
C
PB
KK
KK
Fig. 1. Structure of mushroom bodies in Nematocera. Adult Ptychoptera scutellaris: sagittal section of a mushroom body (a) andsection of the calyx perpendicular to the direction of the axons of Kenyon cells (b). Adult Pedicia rivosa: oblique longitudinal sec�tions of the posterior part of the calyx and beginning of the peduncle (c); arrows indicate two bundles of processes of Kenyon cellsthat exit from the calyx. Pupa of Tipula peliostigma: oblique longitudinal section through the posterior part of the calyx (d); arrowsindicate two tufts of processes of Kenyon cells that enter the calyx. Adult Sylvicola fenestralis: frontal horizontal section of a groupof Kenyon cells and calyx (e); arrows indicate three groups of newly formed Kenyon cells with three corresponding bundles ofprocesses that enter the calyx. Larva of Bibio marci: frontal horizontal section of the right calyx (f); arrows indicate three tufts ofprocesses of Kenyon cells that enter the calyx. C, calyx; Ca, anterior glomerular part of calyx; Cp, posterior part of calyx; KK, bod�ies of Kenyon cells; P, peduncle; PB, protocerebral bridge. Scale bar: 20 µm.
to the base of the calyx, two bundles with a moreintensely stained core are segregated out of the generalmass of the neuropil (Fig. 1c). As in Ptychoptera, thesebundles fully fuse in the peduncle.
In pupae of T. peliostigma strongly advanced in theirdevelopment (compound eyes fully differentiated),neuroblasts in the mushroom bodies are already absent,and Kenyon cells, as in the two species discussed above,
BIOLOGY BULLETIN Vol. 39 No. 4 2012
THE MUSHROOM BODIES OF THE LOWER NEMATOCERA 385
form a single rather small group, from which, however,two thick bundles of fibers stem fused into a single bun�dle as early as the base of the calyx (Fig. 1d).
Quite a different pattern has been found in twoother nematocerans. In the restudied S. fenestralis,following Groth (1971), clear evidence of tripartitemushroom bodies has also been found. In spite of thegeneral unity of the group of Kenyon cells, there werethree rather small distinct groups of darkly stainedcells near the calyx, and at the apices of some of thesegroups, larger cells were located, one cell per group,strongly resembling single neuroblasts. These threegroups of darkly stained cells correspond to three bun�dles of cell processes entering the neuropil of the calyx(Fig. 1e). On the whole, the pattern observed was quitesimilar to that found earlier in tabanids (Panov,2009b).
In adults of B. marci, mushroom bodies are consid�erably less strongly developed, compared to Sylvicola,and no traces of calyx differentiation could be found inthe calyx area. However, in the larva, the entrance ofthree bundles of Kenyon cell processes into the calyxwas found (Fig. 1f). Interestingly, a group of darklystained cells was present only near the middle bundle;the existence of these cells gives evidence of the pro�cess of new formation of Kenyon cells. It probablysuggests nonsimultaneous termination of proliferationprocesses in different subgroups of Kenyon cells.
It follows from the above�said that nematoceranDiptera are nonuniform in the number of their mush�room body primordia and, thus, in the complexity of thegeneral organization of their mushroom bodies. In mem�bers of families especially close to the base of the phylo�genetic tree of dipterans (Ptychopteridae, Pediciidae,Tipulidae) (Wiegmann et al., 2011), mushroom bodiesbipartite in their general structure have been found. Inaddition, two single neuroblasts were found earlier in themushroom bodies of the larva of Culex pipiens (Panov,1966), a member of Culicomorpha, currently consideredby some authors to be closely related to ptychopteroids(Yeates et al., 2007). Thus, a bridge is thrown betweendipterans and other mecopteroids typically characterizedby bipartite mushroom bodies.
On the other hand, in members of two families ofBibionomorpha, including Anispodidae, consideredby many authors to be closely related to Brachycera(Krivosheina, 1988; Woodley, 1989; Oosterbroek andCourtney, 1995; Shcherbakov et al., 1995), tripartiteorganization of mushroom bodies is found, which istypical of Brachycera Orthorrhapha.
Thus, in spite of the limited number of taxa studied,the evolution of the composition of mushroom bodies inDiptera can, apparently, be described as follows. Thelower Nematocera have inherited their bipartite mush�room bodies from their ancestors. However, alreadywithin Nematocera, tripartite mushroom bodiesemerged, typical of Brachycera Orthorrhapha. The tri�partite composition of mushroom bodies is also retainedby some Brachycera Cyclorrhapha (Syrphidae), but
quadripartite mushroom bodies are characteristic ofmost studied members of this group. Finally, in some spe�cies single neuroblasts of mushroom bodies multiply;their multiplication could have emerged as a result of adouble shift in the proliferation program of neuroblasts: ashift from asymmetrical division to symmetrical and sub�sequent reestablishment of asymmetrical division.
REFERENCES
Barendrecht, G., Die Corpora Pedunculata bei den Gat�tungen Bombus und Psithyrus, Acta Zool., 1931, vol. 12,pp. 123–152.Bierbrodt, E., Der Larvenkopf von Panorpa communis undseine Verwandlung, mit besonderer Berücksichtigung desGehirns und der Augen, Zool. Jb. Anat., 1942, vol. 68, no. 1,pp. 49–136.Boyan, G., Williams, L., and Herbert, Z., MultipotentNeuroblasts Generate a Biochemical Neuroarchitecture inthe Central Complex of the Grasshopper Schistocerca gre�garia, Cell Tissue Res., 2010, vol. 340, pp. 13–28.Cayre, M., Strambi, C., Charpin, P., et al., Neurogenesis inthe Adult Insect Mushroom Bodies, J. Comp. Neurol., 1998,vol. 371, part 2, pp. 300–310.Fahrbach, S.E., Structure of the Mushroom Bodies of theInsect Brain, Ann. Rev. Entomol., 2006, vol. 51, pp. 209–232.Farris, S.M., Robinson, G.E., Davis, R.L., and Fahrbach, S.E.,Larval and Pupal Development of the Mushroom Bodies inthe Honey Bee, Apis mellifera, J. Comp. Neurol., 1999,vol. 414, pp. 97–113.Groth, U., Vergleichende Untersuchungen über die Topog�raphie und Histologie des Gehirns der Dipteren, Zool. Jb.Anat., 1971, vol. 88, pp. 203–319.Hanström, B., Vergleichende Anatomie des Nervensystemsder wirbellosen Tiere unter Berücksichtigung seiner Funktion,Berlin: Springer Verlag, 1928.Ito, K. and Hotta, Y., Proliferation Pattern of Postembry�onic Neuroblasts in the Brain of Drosophila melanogaster,Dev. Biol., 1992, vol. 149, pp. 134–148.Ito, K., Awano, W., Suzuki, K., et al., The DrosophilaMushroom Body Is a Quadruple Structure of Clonal UnitsEach of Which Contains a Virtually Identical Set of Neu�rones and Glial Cells, Development, 1997, vol. 124,pp. 761–771.Jarnicka, H., On the Structure of the Brain in SomeDiptera, Ann. Univ. Marie Curie�Sklodowska. Sect. C. Biol.,1959, vol. 14, pp. 161–167.Korotneff, A., Die Embryologie der Gryllotalpa, Z. Wiss.Zool., 1885, vol. 41, no. 4, pp. 570–604.Krivosheina, N.P., Approaches to Solving the Problems ofthe System of Diptera, Entomol. Obozr., 1988, vol. 67, no. 2,pp. 378–390.Lee, T., Lee, A., and Luo, L., Development of the Droso�phila Mushroom Bodies: Sequential Generation of ThreeDistinct Types of Neurons from a Neuroblast, Development,1999, vol. 126, pp. 4065–4076.Miyakawa, K., The Embryology of the Caddisfly Stenop�syche griseipennis Maclachlan (Trichoptera, Stenopsy�chidae): III. Organogenesis: Ectodermal Derivatives, Jap.J. Entomol., 1974, vol. 42, no. 3, pp. 305–324.
386
BIOLOGY BULLETIN Vol. 39 No. 4 2012
PANOV
Norlander, R.H. and Edwards, J.B., Morphology of theLarval and Adult Brains of the Monarch Butterfly, Danausplexippus plexippus L., J. Morphol., 1968, vol. 126, pp. 67–94.Oosterbroek, P. and Courtney, G.W., Phylogeny of Nema�tocerous Diptera (Insecta), Zool. J. Linn. Soc., 1995,vol. 115, pp. 267–311.Panov, A.A., The Structure of the Insect Brain at SuccessiveStages of Postembryonic Development, Entomol. Obozr.,1957, vol. 36, no. 2, pp. 269–284.Panov, A.A., The Ratio of the Ontogenetic Development ofthe Central Nervous System of House Crickets Gryllusdomesticus L. and the European Mole Cricket Gryllotalpagryllotalpa L. (Orthoptera, Grylloidea), Entomol. Obozr.,1966, vol. 45, no. 2, pp. 326–340.Panov, A.A., Some Cetoniinae (Coleoptera, Scarabaeidae)Have Structurally Distinct Medial and Lateral Calyces ofMushroom Bodies, Zool. Zh., 2009a, vol. 88, no. 3,pp. 320–325.Panov, A.A., General Structure of the Mushroom BodyCalyx in Brachycera Orthorrhapha Flies (Diptera,Brachycera Orthorrhapha), Biol. Bull., 2009b, vol. 36,no. 3, pp. 267–276.Panov, A.A., Structure of the Mushroom Bodies in Scara�baeoidea (Coleoptera): 2. Phytophagous Scarabaeidae andGeneral Discussion, Biol. Bull., 2010, vol. 37, no. 6,pp. 585–595.Panov, A.A., Diversity in Neuroblast Number FormingMushroom Bodies of the Higher Dipterans (Insecta,Diptera, Brachycera Cyclorrhapha), Biol. Bull., 2011,vol. 38, no. 1, pp. 77–81.Schrader, K., Untersuchungen über die Normalentwick�lung des Gehirns und Gehirnexplantationen bei der Mehl�motte Ephestia kühniella Zeller nebst einigen Bemerkungenüber das Corpus Allatum, Biol. Zbl., 1938, vol. 58, pp. 52–90.
Shcherbakov, D.E., Lukashevich, E.D., and Blagoderov, V.A.,Triassic Diptera and Initial Radiation of the Order, Int. J.Dipt. Res., 1995, vol. 6, no. 2, pp. 75–115.
Strausfeld, N.J., Homberg, U., and Kloppenberg, P., Paral�lel Organization in Honey Bee Mushroom Bodies by Pepti�dergic Kenyon Cells, J. Comp. Neurol., 2000, vol. 424,pp. 179–195.
Strausfeld, N.J., Organization of the Honey Bee MushroomBody: Representation of the Calyx within the Vertical andGamma Lobes, J. Comp. Neurol., 2002, vol. 450, pp. 4–33.
Strausfeld, N.J., Sinakevitch, I., and Vilinsky, I., TheMushroom Bodies of Drosophila melanogaster: An Immu�nocytological and Golgi Study of Kenyon Cell Organiza�tion in the Calyces and Lobes, Microsc. Res. Techn., 2003,vol. 62, pp. 151–169.
Vowles, D.M., The Structure and Connections of the Cor�pora Pedunculata in Bees and Ants, Quart. J. Micr. Sci.,1955, vol. 96, pp. 239–255.
Wiegmann, B.M., Trautwein, M.D., Winkler, I.S., et al.,Episodic Radiations in the Fly Tree of Life, Proc. Natl.Acad. Sci. USA, 2011, vol. 108, no. 14, pp. 5690–5695.
Woodley, N.E., Phylogeny and Classification of the “Orth�orrhaphous” Brachycera, in Manual of Nearctic Diptera,McAlpine, J.E., Wood, D.M., Eds., Ottawa: ResearchBranch, Agriculture Canada, 1989. V. 3. P. 1371–1395
Yeates, D.K., Wiegmann, B.M., Courtney, G.W., et al.,Phylogeny and Systematics of Diptera: Two Decades ofProgress and Prospects, Zootaxa, 2007, no. 1668, pp. 565–590.
Zhao, X., Coptis, V., and Farris, S.M., Metamorphosis andAdult Development of the Mushroom Bodies of the RedFlour Beetle, Tribolium castaneum, Dev. Neurobiol., 2008,vol. 68, no. 13, pp. 1487–1502.
