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Topographically Distinct Visual and Olfactory Inputs to the Mushroom Body in the Swallowtail Butterfly, Papilio xuthus Michiyo Kinoshita,1* Miki Shimohigasshi,2 Yoshiya Tominaga,2 Kentaro Arikawa,1 and Uwe Homberg3 1

Laboratory of Neuroethology, Sokendai-Hayama (The Graduate University for Advanced Studies), Shonan Village, Hayama 240-0193, Japan 2 Department of Earth System of Science, Faculty of Science, Fukuoka University, Fukuoka 814-0180, Japan 3 Department of Biology, Animal Physiology, University of Marburg, D-35032, Marburg, Germany

ABSTRACT Papilio butterflies depend highly on visual information in their flower-foraging behavior. The retina of Papilio xuthus has been studied well, whereas the visual system in the brain is poorly understood. By investigating outputs from the optic lobe to the central brain, we found that the mushroom body of P. xuthus receives prominent direct inputs from the optic lobe in addition to olfactory inputs. The mushroom body consists of three components: the calyx, the pedunculus, and the lobes. The calyx is further subdivided into two cupshaped primary calyces and an accessory calyx. Each primary calyx consists of three concentric subareas, the inner zone, the outer zone, and the rim of the outer zone. Dextran injections into the optic lobe, the calyx, or the antennal lobe revealed three visual inputs and

one olfactory input into the calyx. The visual inputs originate from the medulla, the lobula, and a newly identified neuropil, the ventral lobe of the lobula. All visual inputs first innervate the accessory calyx, and the two lobula inputs further spread their processes through the inner zone and the rim of the outer zone of the primary calyces. Visual inputs from the medulla and the ventral lobe of the lobula collect light information from ventral eye regions, suggesting a role in visual target detection rather than sky compass orientation. In contrast to visual inputs, olfactory inputs innervate only the calycal outer zone. The multisensory inputs to the mushroom bodies in P. xuthus are probably related to their flower-foraging behavior. J. Comp. Neurol. 523:162–182, 2015. C 2014 Wiley Periodicals, Inc. V

INDEXING TERMS: insect brain; mushroom bodies; Lepidoptera; vision; olfaction

The mushroom bodies are prominent paired neuropils in the brain of insects. Their shape is based on the morphology of intrinsic neurons, termed Kenyon cells, whose dendritic ramifications form the calyx (CA), while their axonal fibers and terminals make up the pedunculus and lobes of the mushroom body. Various extrinsic neurons connect the CA, pedunculus, and lobes with other brain areas. Structural variations of the mushroom bodies among insects correlate with variations in the type and quantity of afferent input and the behavioral ecology of different species (Strausfeld et al., 1998; Farris, 2005; Farris and Roberts, 2005). In most insect species, the mushroom body CA receives massive olfactory input from antennal-lobe projection neurons and may, thus, be regarded as a secondary olfactory brain center. Extensive experimental work, largely on honeybees and fruit flies, revealed that the C 2014 Wiley Periodicals, Inc. V

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mushroom bodies are indispensable for long-term classical olfactory conditioning and are most likely the site of various forms of middle- and long-term olfactory memory (Menzel, 2001; Davis, 2011). In addition to olfactory input, visual and gustatory inputs to the CA have been reported in crickets (Honegger and Sch€urmann, 1975), cockroaches (Strausfeld and Li, 1999a; Nishino et al., 2012), bees (Schr€oter and Menzel, 2003), moths

Grant sponsor: Japan Society for the Promotion of Science (JSPS); Grant numbers: 17770062 (to M.K.) and 21247009 (to K.A.); Grant sponsor: Center for the Promotion of the Integrated Science (CPIS) in SOKENDAI (to M.K. in part). *CORRESPONDENCE TO: Michiyo Kinoshita, Laboratory of Neuroethology, Sokendai Hayama (Graduate University for Advanced Studies), Shonan Village, Hayama 240-0193, Japan. E-mail: [email protected] Received April 8, 2014; Revised September 5, 2014; Accepted September 5, 2014. DOI 10.1002/cne.23674 Published online September 10, 2014 in Wiley (wileyonlinelibrary.com)

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(Homberg et al., 1988a), and butterflies (Sj€oholm et al., 2005), suggesting that the mushroom body is a multimodal neuropil. Compared to olfactory input, however, visual or gustatory inputs are rather limited in most insects. Among the olfactory-dominant CA, hymenopteran insects have particularly prominent visual inputs to the CA, which are largely segregated from olfactory input (Mobbs, 1982; Gronenberg, 1986, 1999; Gronenberg and LopezRiquelme, 2004). In these species, the CA consists of medial and lateral calyces, which are composed of three regions, the lip, the collar, and the basal ring. The collar and a part of the basal ring receive visual input through two neural tracts from the optic lobe, whereas the lip and an olfactory-specific zone of the basal ring receive olfactory input. Visual inputs from the medulla (ME) and the lobula (LO) innervate largely segregated zones in the targeting regions in the CA (Ehmer and Gronenberg, 2002). Recently, a visually dominant CA was found in the whirligig beetle, an aquatic insect without antennal lobes (Lin and Strausfeld, 2012). Visual input in this species originates from dorsal eye regions and might, therefore, be related to a function of the mushroom body in place learning because the whirligig beetle is territorial on the water surface. Contribution of the mushroom body to learning and memory based on visual information has also been reported in the cockroach (Mizunami et al., 1998) and the fruit fly (Liu et al., 1999). Based on comparative studies, Farris (2013) discussed that capacities for spatial learning, visual cognition, and behavioral flexibility in feeding preference may be correlated with the evolution of large complex mushroom bodies receiving direct visual input from the optic lobes. Like bees, butterflies and moths are major pollinators and use both visual and olfactory cues for searching

nectar. Flower-foraging moths can learn visual and olfactory cues by association with nectar (Balkenius et al., 2006; Goyret et al., 2007, 2009). In comparison to the nocturnal hawkmoth (Deilephila elpenor), the diurnal hawkmoth (Macroglossum stellatarum) favors visual stimuli over olfactory cues (Balkenius et al., 2006). Similarly, flower-foraging butterflies, Luehdorfia japonica (Omura et al., 1999), Vanessa indica (Omura and Honda, 2005), and Heliconius melpomene (Andersson and Dobson, 2003) depend on visual cues for foraging and can discriminate and learn floral scents only when presented with visual cues. These findings suggest that lepidopteran insects integrate visual and olfactory cues and learn those sensory modalities by association with nectar when foraging. Visual inputs to the CA have been found in the moths Sphinx ligustri (Pearson, 1971), Manduca sexta (Homberg et al., 1988a; Balkenius et al., 2009), and Spodoptera littoralis (Sj€oholm et al., 2005), whose mushroom bodies are olfactory-dominant. Their lateral and medial calyces are further divided into two layers, a thick outer layer and a small inner layer. In addition to the primary CA, an accessory calyx (ACA) has been described in M. sexta (Homberg et al., 1988a). In S. littoralis, dye injection into the antennal lobe and the optic lobe revealed that the outer layer receives olfactory input, while visual input innervates the inner layer (Sj€oholm et al., 2005). Similarly, the CA in M. sexta has no olfactory input in the inner layer and the ACA (Homberg et al., 1988a). Recently, Heinze and Reppert (2012) provided a 3D reconstruction of the mushroom body of the monarch butterfly, Danaus plexippus. The monarch mushroom body consists of three main components: the CA, the pedunculus, and the lobes, as in other insects. The CA

Abbreviations ACA AL AME AMMC AN AOTU AVLP CA CB CV d DL ES GNG iz l LA LAL LC LH l-l L LCA LO LOP

accessory calyx antennal lobe accessory medulla antennal mechanosensory and motor center antennal nerve anterior optic tubercle anterior ventrolateral protocerebrum calyx central body cervical connective dorsal dorsal lobe of the mushroom body esophagus gnathal ganglia inner zone of thecalyx lateral lamina lateral accessory lobe lateral cell cluster of the antennal lobe lateral horn lateral lobelet of the Y lobe lobes of the mushroom body lateral calyx of the mushroom body lobula lobula plate

mALT MC MCA ME m-l mlALT NO oz p PCT PED PENP PLP PB r-oz OCH1 OCH2 SEZ SLP SNP VL vLO YL YT

medial antennal lobe tract medial cell cluster of the antennal lobe medial calyx of the mushroom body medulla medial lobelet of the Y lobe mediolateral antennal lobe tract noduli outer zone of the calyx posterior postocerbro-calycal tract pedunculus periesophageal neuropils posterior lateral protocerebrum protocerebral bridge rim of outer zone of the calyces first optic chiasma second optic chiasma subesophageal zone superior lateral protocerebrum superior neuropil ventral lobe of the mushroom body ventral lobe of the lobula Y lobe Y tract

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consists of two main cup-shaped calyces and a small ACA. The main calyces are further divided into three concentric regions: the inner zone, the outer zone, and the basal zone. In addition to the pedunculus, the secondary pedunculus, the Y tract, connects the CA to the Y lobes as in the mushroom body of moths. In contrast to clearly separated vertical and medial lobes as in moths, however, only a single spherical lobe is divided into two to three subregions. Another nymphalid butterfly, Heliconius charitonius (Farris, 2013) has a mushroom body with a large CA, whose subdivisions are not clear. In a pierid butterfly, Pieris brassicae, the mushroom body is composed of two cup-shaped calyces, a thin pedunculus, a small vertical lobe, and a large medial lobe (Pearson, 1971). Pearson (1971) also reported visual input from the optic lobe to the CA in P. brassicae, which was recently confirmed in Pieris rapae (Snell-Rood et al., 2009). The Japanese yellow swallowtail butterfly, Papilio xuthus, searches for flowers using sophisticated color vision (Kinoshita et al., 1999, 2008; Kinoshita and Arikawa., 2000). Its retina has been studied particularly well among insects regarding spectral receptor types and their distribution (Arikawa, 2003). Among six classes of spectral receptor in the retina, four spectral receptor classes (UV, blue, green, and red) appear to contribute to tetrachromatic color vision in foraging (Koshitaka et al., 2008). In contrast to detailed knowledge of visual behavior and peripheral visual processing, visual pathways and mechanisms in the central brain are still largely unexplored. Although the olfactory system of P. xuthus is poorly understood, olfactory cues probably play a role in flower detection, as in other flower-foraging butterflies (Omura et al., 1999; Anderson, 2003; Omura and Honda, 2005). Based on previous reports on visual inputs to butterfly mushroom bodies together with the foraging behavior of P. xuthus, we hypothesize that mushroom bodies of P. xuthus play a role in multisensory integration, in particular of visual and olfactory information. Because there are no studies on brain organization of P. xuthus, to the best of our knowledge, we first analyzed the overall structure of the mushroom body together with a general overview of neuropil organization in the brain. We then investigated visual inputs to the mushroom body in comparison to olfactory inputs. The data show that the mushroom bodies of P. xuthus receive prominent visual inputs, which are clearly segregated from olfactory inputs.

MATERIAL AND METHODS Animals We used both sexes of Japanese yellow swallowtail butterflies, Papilio xuthus. We collected eggs laid by

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females caught in the field around the laboratory in Kangawa, Japan. The hatched larvae were reared with fresh citrus leaves at 25–27 C under a photoperiod of 10L:14D producing diapausing pupae or 14L:10D producing nondiapausing pupae. The diapausing pupae were kept at 4 C for at least 3 months and then allowed to emerge as spring-form adults in a Styrofoam box at 26 6 2 C. The nondiapausing pupae were kept in a Styrofoam box at 30 6 2 C without cold treatment to obtain summer form adults.

Bodian staining Head capsules of butterflies were partly opened in FAA fixative solution (85% ethanol, 3.7% formaldehyde, 5% glacial acetic acid) on ice. The heads were kept in the fixative for 1 hour at room temperature. The brains were carefully isolated from the head capsule in 70% ethanol and kept in 70% ethanol at 4 C overnight. On the next day, the brains were dehydrated in an ethanol series and cleared in terpineol before embedding in Paraplast Plus (Oxford Labware, St. Louis, MO). Serial sections of 8 lm thickness were deparaffinized, rehydrated, and silverimpregnated with Bodian’s original procedure (Bodian, 1936), using 2 g protargol (Roques, Paris), 150 ml distilled water containing 4 g of clean copper shots at 65 C for 4 hours. The sections were then developed in 1% hydroquinone with 2% sodium sulfate for 5 minutes. After washing in distilled water for 15 seconds, the sections were toned in 1% gold chloride under strong light, washed in distilled water, and then differentiated in 2% oxalic acid. Afterwards the sections were treated for fixation with 5% sodium thiosulphate for 10 minutes. Stained sections were dehydrated and mounted under coverslips with Mountquick (Daido Sangyo, Tokyo, Japan).

Dextran tracing Butterflies were immobilized on a small plate with a mixture of beeswax and resin. A small window was made in the head capsule, and large tracheae were removed in the area where dextran was applied. For tracing neural connections with the optic lobe, the window was made behind the compound eye. The dorsal area of the head capsule was removed for applying dextran into the CA of the mushroom body or the antennal lobe. After exposing the respective brain area, the neural sheath was cut with a small knife. A few small crystals of biotinylated dextran (3000 MW, lysine fixable, Molecular Probes, Eugene, OR) were picked up with the tip of a glass micropipette covered with Vaseline and then were manually inserted into the target area of the brain under a dissection microscope. Sharp tweezers were also used when applying dextran into the optic lobe. Subsequently, remaining dextran on the brain surface was rinsed off

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with Lepidoptera saline (147 mM NaCl, 1.3 mM KCl, 4 mM CaCl2, 10 mM NaHCO3, pH 7.3). After applying dextran, the window of the head capsule was covered by putting back the removed piece of cuticle. The butterflies were kept overnight in a humid box at 4 C to allow dextran to be taken up and distributed. On the following day, the head capsule was opened again and washed with saline to remove excessive dye from the inserted area and hemolymph. The brain was carefully dissected out on ice from the head capsule in fixative solution containing 4% paraformaldehyde, 0.25% glutaraldehyde, and 0.2% saturated picric acid in 0.1 M phosphate buffer (PB) pH 7.4 and was kept in the same fixative at room temperature for 4 hours or at 4 C overnight. Brains were subsequently washed in 0.1 M PB for 15 minutes and then embedded in gelatin/albumin. Blocks containing the brains were postfixed in 8% formaldehyde in 0.1 M PB at 4 C overnight. The brains were sectioned either frontally or horizontally at a thickness of 35 or 40 lm with a vibrating blade microtome (VT 1000S; Leica, Wetzlar, Germany). Floating brain sections were collected and washed in 0.1 M PB for the following histochemical reaction. The sections were incubated with streptavidin conjugated with horseradish-peroxidase at a dilution of 1:200 in 0.01 M PB with 0.1 M NaCl containing 0.5% Triton X-100 (PBST) at room temperature for at least 18 hours with gentle agitation. After washing in PBST several times, the staining was developed in a solution of 3,30 diaminobenzidine tetrahydrochloride (0.3 mg/ml) in 0.05 M Tris-HCl buffer, pH 7.4, with 0.3% nickel ammonium sulfate and 0.015% H2O2. Then the sections were washed in Tris-HCl buffer and mounted on chrome alum/ gelatin coated slides under a coverslip with Mountquick.

Image acquisition and processing All sections were observed with an upright light microscope (Leica DM2500) equipped with Apo 103 or 403 oil immersion objectives. Photomicrographs were taken with a digital camera (Leica DFC420). Images at different focal points were automatically stacked and aligned by e-Tiling software (v. 3.8, Mitani, Tokyo, Japan). Some images from several serial sections were imported in Adobe Photoshop (v. CS4, Adobe Systems, San Jose, CA) and superimposed for tracing neural tracts. In some cases, brightness, contrast, and color balance were adjusted in Photoshop.

Definition of axes and terminology of neuropils Positional information is given with respect to the body axis of the butterfly. Brains were sectioned either

horizontally or frontally. The terms ipsilateral and contralateral are used with reference to the site of tracer injection. In all images, the right hemisphere of the Papilio brain is presented in horizontal (Fig. 1B–D) and frontal sections (Fig. 1E–G). The terminology for brain areas and fiber tracts is based on the systematic nomenclature system for the insect brain (Ito et al., 2014) and, for specific terms, on terminology used for the brain of the sphinx moth Manduca sexta (Homberg et al., 1988a; Huetteroth et al., 2010) and the monarch butterfly, Danaus plexippus, (Heinze and Reppert, 2012).

RESULTS General layout of neuropils in the brain of Papilio xuthus The brain of P. xuthus consists of a central brain connected to a large optic lobe on each side (Fig. 1A) as in other insects. The central brain is a fused structure consisting of the cerebrum and the gnathal ganglia below the esophagus running through the esophageal foramen in the center of the central brain (Fig. 1A). We first studied the general layout of neuropils and the structure of the mushroom body on serial Bodianstained sections. Cell bodies are distributed in the cell body rind near the surface of the brain, and neuropils are segregated by fiber bundles and glial sheaths, as in other insects. The neuropil organization of the brain of P. xuthus is similar to that of Danaus plexippus and other lepidopteran insects. Figure 1B shows the arrangement of major neuropils in the brain in dorsal view based on horizontal (Fig. 1C,D) and frontal sections (Fig. 1E–G). The mushroom body, the anterior optic tubercle, the antennal lobe, the lateral horn, and the lateral accessory lobe are present in each hemisphere of the brain, whereas the central body and the protocerebral bridge occupy central midline-spanning positions. The mushroom body of P. xuthus is composed of calyx (CA), pedunculus, and lobes. The CA faces the posterior surface of the brain (Fig. 1C,D,G). The pedunculus extends from the center of the CA in anterior direction (Fig. 1D,F,G) and gives rise to the lobes that appear as a single compressed spheroidal structure (Fig. 1D, arrowhead). The lobes extend toward the midline of the brain (Fig. 1C,D) and come close to the dorsal and anterior surface of the brain (Fig. 1E). The anterior optic tubercle is an ovoid-shaped neuropil anterior-lateral from the lobes of the mushroom body (Fig. 1C,E). The anterior optic tubercle is connected to the optic lobe via the anterior optic tract (Fig. 1E, arrowhead) and is a major target site of visual inputs to the central brain. The antennal lobe occupies the

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Figure 1. Brain structure and neuropil organization of Papilio xuthus. A: Schematic diagram of the brain. SEZ, subesophageal zone. B: Schematic diagram of the main neuropils in the right brain hemisphere, dorsal view. AL, antennal lobe; AOTU, anterior optic tubercle; CA, calyx of the mushroom body; CB, central body; L, lobes of the mushroom body; LAL, lateral accessory lobe; LH, lateral horn; NO, noduli; PB, protocerebral bridge; PED, pedunculus; C,D: Horizontal sections of the brain at dorsal (C) and intermediate (D) levels. AN, antennal nerve; AVLP, anterior ventrolateral protocerebrum; l, lateral; p, posterior; SLP, superior lateral protocerebrum; SMP, superior medial protocerebrum; SNP, superior neuropils; arrowhead, connection between the PED and the L. E–G: Frontal sections from anterior (E) to posterior (G). Dashed line shows midline of the brain in C–G. Numbers at left bottom in each panel show depths (lm) from the dorsal surface (C,D) and from the anterior surface, respectively (E–G). AMMC, antennal mechanosensory and motor center; CV, cervical connective; d, dorsal; ES, esophagus; GNG, gnathal ganglia; PENP, periesophageal neuropils; PLP, posterior lateral protocerebrum. Scale bars 5 100 lm.

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anterior position in the brain and comprises numerous glomeruli (Fig. 1C,D). The antennal nerve enters the brain dorsally (Fig. 1A) and innervates the antennal lobe from its posterior-lateral side (Fig. 1D,E). The lateral horn of the protocerebrum lies most dorsolaterally and approximately halfway along the posterior–anterior axis (Fig. 1C,F). Its inner surface is fused with the superior neuropil (Fig. 1C,D,F). The central complex consists of the protocerebral bridge, the central body, and a pair of noduli and is flanked by the lateral accessory lobes (Fig. 1B,D). The two hemispheres of the protocerebral bridge are separated across the brain midline by a fiber fascicle (Fig. 1D,G). The central body spans the brain midline anteriorly from the protocerebral bridge; it is most prominently divided into a posterior upper division and a smaller anterior lower division (Fig. 1D,F). Laterally and ventrally around the esophagus, periesopageal neuropils, the antennal mechanosensory and motor center, and the neuropil of the gnathal ganglia can be distinguished (Fig. 1E,F). Several nerves provide connections to neck muscles and mouthparts, and the posteriorly extending cervical connectives provide connections to the thoracic ganglia (Fig. 1A,G). From distal to proximal the optic lobe is composed of the lamina (LA), the medulla (ME), and the lobula complex (Fig. 2A,B). The lobula complex is further divided into two regions: an anterior neuropil, the lobula (LO), and a flat posterior neuropil, the lobula plate (LOP). The accessory medulla is a small neuropil at the anterior medial edge of the medulla (Fig. 2A). In the first optic chiasma between the LA and the ME and the second optic chiasma between the ME and LO (Fig. 2A,B), nerve fibers cross over between anterior and posterior positions maintaining retinotopy. A cartridge structure corresponding to ommatidial units is maintained in the LA, the ME, and the distal LO. The ME and the LO have a layered organization corresponding to differences in ramification patterns and synaptic densities. As established in previous work (Hamanaka et al., 2013), the ME is divided into eight layers based on the pattern of synapsin immunostaining (Fig. 2C). ME layers 4 and 5 are further divided into two and three sublayers, respectively. ME layer 5 corresponds to the so-called serpentine layer containing large tangentially projecting fibers. The LO is divided into three layers, and layers 2 and 3 are further divided into two sublayers (Fig. 2D). In the LO, a small area ventroanteriorly and most proximally is distinct from the rest of the LO and is named here the ventral lobe of the LO (vLO; Fig. 2B, double arrowhead). The vLO has an ovoid shape in frontal sections (Fig. 2B) but, as shown in horizontal sections, is further divided into an anterior and a posterior subunit (Fig. 2E, double arrowhead, arrowhead).

Mushroom body As in other insects, the CA of the mushroom body of P. xuthus receives sensory inputs. It is surrounded by many small cell bodies of intrinsic Kenyon cells with diameters of about 4 lm (Fig. 3A). The CA consists of three components: two cup-shaped structures, the medial and lateral calyces, and an ovoid accessory calyx (ACA). Whereas the medial and lateral calyces lie next to each other horizontally and are partly fused, the ACA is ventrally attached to the medial and lateral calyces. The medial and lateral calyces are divided into two largely concentric ring-like subareas, the outer zone and the inner zone. The outer zones of both calyces are fused. Within the outer zone, a small rim (rim of outer zone) can be distinguished (Fig. 3A, brackets). It appears as a thin band, which is fused with the outer zone and lies adjacent to the inner zone (Fig. 3B). Transverse sections through the pedunculus anterior from the CA reveal that it consists of at least three areas (Fig. 3C). Figure 3D shows a schematic superimposed diagram of the CA, based on serial horizontal sections. The three subareas of the primary calyces, the outer zone, the rim of outer zone and the inner zone, and the ACA are clearly distinguished on horizontal sections (Fig. 3E–H). In addition to the pedunculus, fibers enter or exit the CA via two more neural tracts (Fig. 3E,F). The first tract is the Y tract, whose diameter is about 20 lm. It exits the outer zone between the lateral and medial calyx and extends anteriorly (Fig. 3E, arrowhead). The second tract, the protocerebro-calycal tract, is connected to the CA via the base of the outer and inner zone, immediately above the pedunculus (Fig. 3F, double arrowheads). It corresponds to the protocerebro-calycal tract in Manduca sexta, connecting the lobes and the calyces of the mushroom body with cell bodies in the lateral protocerebrum (Homberg et al., 1987). The pedunculus bifurcates near the CA and both hemipedunculi are connected to the bases of the inner zones in the centers of the lateral and medial calyx, respectively (Fig. 3G). At this depth, the accessory calyx emerges between the two inner zones and the pedunculus. Ventrally from the pedunculus, the ACA increases in size, and many fibers extend toward the lateral protocerebrum (Fig. 3H). The lobes in the anterior dorsal protocerebrum are the output sites of the mushroom body. In P. xuthus, the lobes are compressed to a single spheroidal neuropil mass, as in D. plexippus (Heinze and Reppert, 2012). Detailed analysis reveals three subdivisions, termed here the dorsal lobe, the ventral lobe, and the Y lobe in accordance with the relative position and shape of each subdivision (Fig. 4). The ventral lobe is a large

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Figure 2. Organization of the optic lobe. A: Horizontal section through the optic lobe. The optic lobe contains four main neuropils, the lamina (LA), medulla (ME), lobula (LO), and lobula plate (LOP) and a small neuropil at the anterior edge of the ME, the accessory medulla (AME). OCH1, first optic chiasma; OCH2, second optic chiasma. l, lateral; p, posterior. B: Frontal section through the optic lobe. Within the LO, a distinct subregion, the ventral lobe of the lobula (vLO, double arrowhead) can be identified. d, dorsal. C: Layering of the ME. There are 11 layers in total including sublayers in layers 4 and 5. D: Layering of the LO. There are five layers in total, including sublayers in the layers 2 and 3. E: Horizontal section through the LO at a level ventrally from the optic stalk. The ventral lobe is divided into two subregions (arrowhead and double arrowhead). Scale bars 5 100 lm in A,B; 25 lm in C,D; 50 lm in E.

homogeneous structure. It has a bulbous appearance and extends toward the midline of the brain, whereas the dorsal lobe is elongated running along the anteriordorsal side of the ventral lobe (Fig. 4A-C). The dorsal lobe may be further subdivided into two sublobes (Fig. 4A,B). The ventral and dorsal lobes correspond to the proposed vertical and medial lobes in D. plexippus, respectively. The Y lobe is embedded between the ven-

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tral and dorsal lobes (Fig. 4A–C). It appears dumbbell– shaped with two lobelets (Fig. 4D,G), which are obvious in horizontal sections (Fig. 4E–H). The medial lobelet is located at the posterior-medial side of the lobes, whereas the lateral lobelet lies anterior-laterally. The medial and lateral lobelets correspond to the dorsal and ventral lobelets in D. plexippus, respectively (Heinze and Reppert, 2012).

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Figure 3. Subregions of the mushroom-body calyx. A: Frontal section of the calyces at a depth of 48 lm from the posterior brain surface. Three subregions are defined: the outer zone (oz), the inner zone (iz), and the accessory calyx (ACA). The white brackets indicate the rim of the outer zone. d, dorsal; l, lateral. B: Enlarged image of the white box in A. A rim of the oz (r-oz, white bracket) exists between the iz and the oz. C: Three regions (white dotted lines) on a transverse section of the pedunculus. D: Schematic diagram of the calyces (LCA: lateral calyx, MCA: medial calyx, ACA: accessory calyx) with the Y tract, the protocerebro-calycal tract (PCT), and the pedunculus (PED). p, posterior. E–H: Horizontal sections from dorsal (E) to ventral (G). The Y tract emerges from the outer zone (arrowhead in E), the PCT from between the calyces (arrowhead in F), and the PED, from the bases of each calyx (in G). The accessory calyx becomes large at ventral levels (H). The white brackets indicate the rim of the outer zone. Numbers at bottom left show depths (lm) from the dorsal surface of the calyces. Scale bars 5 50 lm in A,E–H 20 lm in B; 25mm in C.

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Figure 4. Structure of the mushroom body lobes. A–C: Frontal sections of the lobes from anterior (A) to posterior (C). A: The ventral lobe (VL) dorsally covers the dorsal lobe (DL). The lateral lobelet (l-l) of the Y lobe (YL) occupies a dorsolateral area in the lobes. B: A thin fiber bundle (arrowhead) traverses the VL. C: The medial lobelet (m-l) of the YL has a dorsomedial position in the lobes. d, dorsal; l, lateral. D: Schematic diagram of the lobes, dorsal view. The YL with its two lobelets is embedded between the VL and the DL (shown in gray). PCT, protocerebro-calycal tract; PED, pedunculus; YT, Y tract. p, posterior. E–H: Horizontal sections through the lobes from dorsal (E) to ventral (H). E: The VL runs from lateral to medial. F: The protocerebro-calycal tract is posteriorly connected to the VL (double arrowhead). G: The fascicle connecting the two lobelets of the YL passes through the VL. H: The YT enters the lobes at the medial edge of the VL (arrowhead) from posterior. Numbers at bottom left show distance (lm) from the anterior surface of the lobes in A–C and distance (lm) from the dorsal brain surface in E–H. Scale bars 5 50 lm.

Visual inputs in butterfly mushroom body

Three pathways connecting the CA and lobes were observed. The pedunculus connects the CA ventrolaterally to the lobes (Fig. 4D). The protocerebro-calycal tract, connected to the CA, invades the lobes posteriordorsally (Fig. 4F, double arrowhead). As proposed in honeybees (Gronenberg, 1987; Gr€unewald, 1999), the protocerebro-calycal tract likely contains recurrent processes from the lobes to the CA. Finally, immediately ventral to the protocerebro-calycal tract, the Y tract (Fig. 4D,H, arrowhead) is connected to the ventrolateral side of the medial lobelet in the Y lobe (Fig. 4D,G).

Distribution of visual inputs in the calyx Visual inputs into the CA were found in 34 out of 70 preparations after dextran injection into the optic lobe. Dye injection into the optic lobe (mainly the ME) labeled numerous neurons with projections into the central brain including the CA of the mushroom body (Fig. 5A). The CA in the ipsilateral brain hemisphere was constantly stained, while the CA in the contralateral hemisphere was never invaded by stained fibers (Fig. 5A). As seen in horizontal (Fig. 5A–C) and frontal sections (Fig. 5E,F), the medial and lateral calyces were sparsely invaded by widely projecting processes, whereas the ACA was densely stained, preventing the identification and tracing of individual neurites. Stained fibers in the CA had small swellings and varicosities widely spread in the inner zone and the rim of outer zone of the CA, whereas no fiber innervated the outer zone (Fig. 5D). The rim of outer zone always contained stained neurons when the inner zone was stained. In the ACA, a layering of staining was observed in some preparations, with particularly dense staining in the center surrounded by a sparser supply of processes at the periphery (Fig. 5G blacket). The ACA was stained in all cases when dextran was injected into wide areas of the optic lobe or into the ME, while the inner zone and the rim of outer zone were not stained in some cases (n 5 11/34). Interestingly, when dextran was injected into the LO, but not the vLO, fibers exclusively passed through the ACA and spread to the rest of CA (Fig. 5H,I). We did not find any staining in the vLO when dye was injected into the dorsal ME (data not shown).

Neurons connecting the optic lobe to the calyx The origin and cell body positions of neurons projecting from the optic lobe to the mushroom body were characterized by dextran injection into the CA (Fig. 6, n 5 23). Stained neurons were found in two neuropils in the optic lobe, the LO and the ME (Fig. 6A,C). In the ventral two-thirds of the ME, the serpentine layer and

distal layers contained stained neurons (Fig. 6A,C). Numerous neurons with small cell bodies were stained at the anterior edge of the ME (Fig. 6B). In the LO, stained neurons extended ramifications throughout the LO and the vLO (Fig. 6A,C). The LO and vLO were stained in most cases (n 5 21/23), while the ME was not stained in some preparations (n 5 9/23). Between the central brain and the optic lobe, three fiber tracts were identified (Fig. 6D). These tracts consisted of fibers with different thickness and connected the CA with the LO (Fig. 6D). The most dorsal tract containing neurons with 3–5 lm diameter originated from the LO (Fig. 6D, arrow), and two other tracts originated in the vLO. The middle tract consisted of relatively thick fibers (Fig. 6D, double arrowhead), whereas the ventral tract contained very fine fibers only, which could not be resolved individually (Fig. 6D, arrowhead). Numerous small cell bodies (163–581) with diameters of a few micrometers were distributed around the anterior ventral edge of the ME in the optic lobe (Fig. 6B,E, arrowheads). Their primary neurites passed through the entire ME (arrows in Figs. 6B, 7A) and gave rise to main fibers connecting the vLO to the anterior edge of the ME (Fig. 6A, arrowheads; Fig. 6B, double arrowhead). These fibers passed anterior-ventrally through the second optic chiasma toward the ME (Fig. 6D, arrowheads). The fibers entering the anterior ME formed a narrow sheath extending posteriorly throughout the ventral two-thirds of the ME in the center of layer 5 (layer 5b; Fig. 7A,B). Side branches projected distally and invaded layer 5, and fine processes continued to distal parts of layer 4a (Fig. 7B). In the LO, thick fibers spread through layer 3 and entered the most dorsal tract to the CA (Figs. 6C,D, 7C). Fine fibers extended toward layer 2 in some preparations (Fig. 7C, arrow). Both subunits of the vLO contained stained neurons. In the posterior subunit of the vLO, fibers were too fine to be identified individually (Fig. 7C). The tract with fine fibers extending from the posterior side of the vLO entered medulla layer 5 via the anterior edge of the ME (arrows in Figs. 6A, 7C). To identify the cell bodies of neurons from the LO, the region around the CA and the fiber tracts were carefully examined. At least two clusters of cell bodies were found near the tracts entering the root of the pedunculus and the CA (Fig. 7D,E). The lateral cluster (black dotted line in Fig. 7D,E) connecting to the dorsal tract contained at least 41 cell bodies (white arrow in Fig. 7D,E). Neurons of this cluster provided a connection with layer 3 of the LO (Fig. 6D, arrow). In the ventrolateral cluster (white dotted line in Fig. 7E) 22–58 cell bodies were counted and connected to the middle

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Figure 5. Visual inputs in the calyx. A: Overview of staining pattern in the mushroom body after dye application into the optic lobe. The calyx (CA) of the mushroom body and the anterior optic tubercle (AOTU) contain stained neurons, and several fibers are stained. CB, central body; L, lobes of the mushroom body; l, lateral; p, posterior; PED, pedunculus. B,C: Horizontal sections dorsally (B) and ventrally (C) through the calyx illustrating visual input. The inner zone (iz) and part of the outer zone (oz) contain stained fibers (B). The accessory calyx (ACA) is heavily stained (C). D: Enlarged image of the box in A. The outer zone (oz) does not contain stained fibers, whereas the inner zone (iz) and rim of the oz (r-oz; bracket) contain stained fibers with small swellings (arrowheads). E,F: Visual input illustrated on frontal sections at posterior (E) and anterior (F) levels. d, dorsal. G: Enlarged image of the box in F. The ACA is divided into two layers (white bracket). Stained fibers in the inner zone have small swellings (arrowheads). H,I: Horizontal sections showing neural fibers from the lobula in the calyx at the level of the PED (H) and ventral from the pedunculus (I). Arrows indicate fibers passing the accessory calyx (ACA). iz, inner zone of the CA; oz, outer zone of the CA. Scale bars 5 100 lm in A; 50 lm in B,C,E,F,H,I; 25 lm in D,G.

Visual inputs in butterfly mushroom body

Figure 6. Optic lobe neurons connecting to the calyx. A: Horizontal section showing staining pattern obtained by dye injection into the calyx. The medulla (ME) and the lobula (LO) contain stained neurons. Some fibers (arrowheads) run from the posterior LO to the second optic chiasma (OCH2). l, lateral; LA, lamina; OCH1, first optic chiasma; p, posterior. B: The cell bodies of the ME neurons (arrowheads) are distributed in the cell body rind near the anterior edge of the ME. Primary neurites from the cell bodies (arrow) pass through the ME. Main fibers of the ME neurons (double arrow) run in the center of layer 5. C: Overview of staining pattern on a frontal section. Neurons with arborizations in the ventral two-thirds of the medulla (ME, dotted line), the proximal lobula (LO), and the ventral lobe of the lobula (vLO) are stained. d, dorsal; l, lateral; LOP, lobula plate; OCH1, first optic chiasma; OCH2, second optic chiasma. D: Three tracts with fibers of different diameter connecting the calyx and the LO (arrow, double arrowhead and arrowhead). E: The cell bodies are distributed at the anterior ventral area of the ME (arrows) on the frontal section. Scale bars 5 100 lm in A,C; 50 lm in B,D,E.

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Figure 7. Detailed staining patterns in the medulla and lobula (horizontal sections) and the cell clusters around the calyx (frontal sections) after dye injection into the calyx. A: Thin neurites from the cell bodies in the first optic chiasma (arrow) pass through the medulla (ME, arrows) and connect to main fibers in the second optic chiasma (OCH2). B: Many side branches extend from fibers in ME layer 5 to layer 4. C: Staining pattern in the lobula (LO). A fascicle of fibers (arrowhead) invades the ventral lobe of the lobula (vLO). Thin fibers extend to layer 2 (arrow). D,E: Cell body clusters of visual inputs near the calyx (CA) on sections from anterior (D) to posterior (E). The two clusters around the CA are surrounded by black and white dotted lines. A tract (arrow) is connected to the cell cluster lateral to the CA (black dotted line in D,E), and the other tract (arrowhead in E) is connected to the cell cluster at the ventrolateral side of the CA (white dotted line in E). Scale bars 5 50 lm.

tract passing to vLO (Fig. 6D double arrowhead) and the ventral tract (Fig. 7E arrowhead).

Connections between the calyx and other areas Dextran injection into the CA revealed connections with mainly three regions in the central brain: the lobes of the mushroom body, the lateral horn, and the antennal lobe (Fig. 8) in the ipsilateral hemisphere of the

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brain. We could not observe any stained neurons innervating the contralateral CA. Dense staining was consistently observed in the pedunculus (Fig. 8B). In addition, the protocerebro-calycal tract, entering the CA dorsally, provided a connection with the dorsoposterior side of the ventral lobe (Fig. 8A, arrows). About halfway between the CA and the lobes a fascicle of cell body fibers branched off and revealed a cluster of 29–109 cell bodies in the anterior dorsolateral cell body rind of the protocerebrum (arrowheads in Fig. 8A,B). The Y

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Visual inputs in butterfly mushroom body

Figure 8. Neural connections with the calyx in the central brain. A,B: Horizontal sections showing connections with the calyx (CA) at dorsal (A) and medium-depth levels (B). The protocerebro-calycal tract (PCT) innervates posterior regions of the lobes (A: arrow). A fiber bundle branching off laterally from the PCT (A,B: arrowhead) extends to a cell body cluster (dashed line). Some fibers (A: double headed arrow) innervate the medial lobelet of the Y lobe (m-l). The medial antennal lobe tract (mALT) projects to the lateral horn (LH) via the CA (double arrowheads). AOTU, anterior optic tubercle; CB, central body; l, lateral; L; lobes of the mushroom body; p, posterior; PED, pedunculus. C,D: Projection neurons from the antennal lobe (AL) with fibers to the CA shown in horizontal (C) and frontal section (D). The mALT originates from two separate roots in the AL that fuse posterior to the AL (C). In the AL a medial (MC) and a lateral (LC) cluster of cell bodies can be distinguished (circles of dotted lines). AN, antennal nerve; d, dorsal. Scale bars 5 50 lm

tract and Y lobe were not stained in most preparations (Fig. 8A). In a few cases some fibers innervating the medial lobelet of the Y lobe from its lateral side were faintly stained (Fig. 8A, double headed arrow). Two other tracts were connected to the basal region of the CA anteriorly and laterally (Fig. 8B double arrowhead). The anteriorly projecting tract could be traced to the antennal lobe and the lateral tract to the lateral horn. The former one, the medial antennal lobe tract,

bifurcated near the antennal lobe (Fig. 8C) and innervated the glomeruli of the antennal lobe. Two cell clusters, a medial cell cluster and a lateral cell cluster, were labeled dorsomedially and ventrolaterally in the antennal lobe, respectively (Fig. 8C,D). Each cell cluster consisted of at least 40–50 stained cell bodies. Dextran injection into the antennal lobe (n 5 2) revealed specific target areas of antennal lobe tracts in the mushroom body, the superior lateral protocerebrum,

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and the lateral horn (Fig. 9A). The medial antennal lobe tract projected into the base of the medial calyx, passed dorsally along the pedunculus, and exited the root of the lateral calyx (Fig. 9B, double headed arrow). In the medial and lateral calyces, only the outer zones were strongly stained (Fig. 9C). The tract continued in anterior lateral direction, gave rise to side branches in the superior lateral protocerebrum, and finally reached the lateral horn (Fig. 9A). In addition, the lateral horn also received direct projections from the antennal lobe by the mediolateral antennal lobe tract bypassing the CA anteriorly through the superior protocerebrum (Fig. 9A, arrows).

DISCUSSION We describe the structure of the mushroom body and sensory inputs to the CA of the mushroom body in Papilio xuthus. The mushroom body of P. xuthus is composed of a fused double CA and an ACA as the main sensory input sites, a pedunculus, a secondary pedunculus, the Y tract, and fused lobes. The CA receives prominent visual input, which is segregated from olfactory input. Visual inputs originate in the ME, the LO, and the vLO. The topographically separated visual and olfactory inputs to the mushroom body are illustrated in Figure 10. The multisensory inputs to the CA may relate to the ecological niche of foraging flowers and finding citrus trees for egg laying.

Specific features of neuropil organization in P. xuthus The organization of lepidopteran brains as studied in P. xuthus, Danaus plexippus (Heinze and Reppert, 2012), Manduca sexta (Homberg et al., 1988a; Huetteroth et al., 2010), and Sphinx ligustri (Pearson, 1971) is quite similar. Nevertheless, two neuropils, the LO in the optic lobe and the mushroom body in the central brain, show specific features in P. xuthus. The LO is the third processing center in the optic lobe and is responsible for extracting information about shape, color, and small object movement, while the lobula plate is involved in processing motion (Strausfeld and Lee, 1991; O’Carroll, 1993; Yang et al., 2004; Paulk et al., 2008, 2009; Nordstr€om and O’Carroll, 2009). The LO in P. xuthus contains a specific vLO (Fig. 2B,E), which is described here for the first time. The vLO is prominently connected with the mushroom body of the central brain. It may be related to the optic glomerular complex in D. plexippus, which is located between the LO and the mushroom body CA and seems to send fibers to the CA (Heinze and Reppert, 2012). Among the three components of the mushroom body, the CA of P. xuthus is different from the CA in D.

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Figure 9. Antennal lobe inputs to the mushroom body. A: Overview of projections from the antennal lobe (AL) in horizontal section. The medial antennal lobe tract (mALT) runs into specific areas of the calyx (CA) (arrowhead) and terminates in the lateral horn (LH) via the superior lateral protocerebrum (SLP, double arrowheads). The mediolateral antennal lobe tract (mlALT) passes dorsally from the pedunculus through the central brain and directly terminates in the LH. CB, central body; iz, inner zone of the mushroom body calyx; l, lateral; L, lobes of the mushroom body; p, posterior; PED, pedunculus. B: Olfactory inputs to dorsal areas of the CA. Neural fibers (double headed arrow) innervate the outer zone of the calyces (oz indicated by arrowheads). C: Enlarged image of the olfactory inputs in the calyx. Only the outer zone contains stained neurons. ACA, accessory calyx; r-oz, rim of the outer zone. Scale bars 5 100 lm in A,B; 25 lm in C.

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Figure 10. Schematic diagram of neural connections with the mushroom-body calyx in dorsal (A) and frontal view (B,C). The three visual input tracts from the medulla (ME), the lobula (LO), and the ventral lobe of the lobula (vLO) indicated in red, yellow, and orange, respectively, innervate the accessory calyx and extend through the calyx (CA) as shown in orange. Inputs from the antennal lobe (AL) terminate in the lateral horn (LH) by way of the outer zone of the CA (shown in blue). The protocerebro-calycal tract (PCT) and the Y tract (YT) and Y lobe (YL) are shown in yellow green and blue green, respectively. Defined cell clusters of neurons in each neural tract are shown in the same colors as the corresponding neural tracts. d, dorsal; CB, central body; l, lateral; L, lobes of the mushroom body; p, posterior; PED, pedunculus.

plexippus (Fig. 3). Both the medial and lateral cupshaped calyces are composed of two main concentric regions: the inner zone and the outer zone, which is rather similar to the CA of the moths S. littoralis (Sj€ oholm et al., 2005) and M. sexta (Homberg et al., 1988b). In P. xuthus a particular subdivision of the outer zone, the rim of the outer zone, can be distinguished, which has not been recognized in moth species. Subdivisions of the calyces seem to vary among butterfly species, because the calyces of D. plexippus are rather similar to the calyces in bees and consist of

three concentric regions: the basal zone, the outer zone, and the inner zone (Heinze et al., 2012). The correspondence of calycal subdivisions in D. plexippus and P. xuthus should be revealed in future experiments on sensory inputs to the CA in D. plexippus. In comparison with D. plexippus, P. xuthus has a relatively large ACA located ventrally from the main CA (Heinze et al., 2012). An ACA has been reported in many insect taxa (Farris, 2005): cockroaches (Farris and Strausfeld, 2003), locusts (Kurylas et al., 2008), crickets (Frambach and Sch€urmann, 2004), and moths (Homberg

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et al., 1988a). Locusts and crickets have particularly large ACA (Kurylas et al., 2008). On the other hand, the large mushroom body CA in hymenopteran insects completely lacks an ACA. Judged by the differences in sensory supply, i.e., gustatory input in other insects, it is questionable, whether ACAs across insects are in fact homologous structures. Differences in the structure of CAs and ACAs among lepidopteran insects may, nevertheless, be related to differences in foraging strategies and other ecological aspects. Compressed medially projecting lobes as found in P. xuthus (Fig. 4) have, likewise, been reported in D. plexippus (Heinze and Reppert, 2012), P. brassicae (Pearson, 1971), and other species (Kinoshita, unpubl.). The compressed lobes can be divided into at least two lobes in P. xuthus and D. plexippus. Distinct bifurcated lobes corresponding to bifurcated morphology of Kenyon cells, the medial and the vertical lobes, are present in S. ligustri (Pearson, 1971), Huebneriana trifolii (Strausfeld et al., 1998), and most other insect taxa (Sj€oholm et al., 2005; Farris and Roberts, 2005; Fukushima and Kanzaki, 2009; Strausfeld et al., 2009). The medial and vertical lobes in the moths are further divided into three layers, the b-, the b’-, and the g-lobe or the a-, the a’-, and the g-lobe. Among these three layers, the g lobe occupies the most anterior and dorsal position in moths (Sj€oholm et al., 2005; Fukushima and Kanzaki, 2009). These layers correspond to different morphological types of Kenyon cell identified by single cell staining or Golgi staining. What type of Kenyon cell constructs the dorsal and the ventral lobe of the mushroom body in P. xuthus? This question will be addressed by future experiments demonstrating the morphology of single Kenyon cells. We identified three layers in the pedunculus of P. xuthus (Fig. 3C), which is different from the five layers identified in D. plexippus. This difference in the number of layers between the two species, however, may be due to different staining techniques. The secondary pedunculus, the Y tract, giving rise to a specific Y lobe, is usually present in lepidopteran insects (Pearson, 1971; Homberg et al., 1988b; Sj€oholm et al., 2005; Fukushima and Kanzaki, 2009). In addition to the pedunculus and Y tract, the protocerebro-calycal tract provides a third connection between the CA and the lobes (Figs. 3D, 4D). As shown in the sphinx moth (Homberg et al., 1987) and honeybee (Bicker et al., 1985), the protocerebro-calycal tract consists of GABAergic neurons providing negative feedback from the lobes to the CA (Gronenberg, 1987; Gr€unewald, 1999). GABAergic neurons in the mushroom body have also been reported in other insects and have been studied intensely in fruit flies and bees with respect to a function in olfactory learning and memory (Yamazaki

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et al., 1998; El Hassani et al., 2008; Busto et al., 2010; Kahsai and Zars, 2011; Boumghar et al., 2012). The transmitter of the protocerebro-calycal tract in P. xuthus remains to be identified.

Visual inputs into the mushroom body Dextran injection into the optic lobe revealed that the mushroom body CA of P. xuthus receives substantial direct visual inputs (Fig. 5A). We identified at least three independent visual inputs from the optic lobe (Figs. 6G, 7C). The first one is the ME input (red in Fig. 10A,B). These neurons have small cell bodies at the anterior edge of the ME, fine arborizations in layer 4a of the ventral two-thirds of the ME, then innervate the vLO and finally project fibers to the ACA. The second and third visual inputs are from the LO and vLO, respectively (yellow and orange respectively in Fig. 10A,B). These neurons have cell bodies laterally and ventrally from the CA, respectively, and send fibers to the primary calyces. The visual inputs of different origin in the optic lobe are also segregated in the CA. The ACA receives the ME input, whereas the primary calyces mainly receive two types of LO input (Fig. 5A–G). The ME projection neurons to the CA may receive input from ME interneurons but not directly from photoreceptors or lamina neurons. This is because lamina monopolar cells and long visual fibers terminate in layer 1–3 and layer 4b of the outer ME, respectively (Hamanaka et al., 2013). The ME inputs probably consist of two classes of neurons innervating either the ACA or the main calyces, because in some preparations dextran application into the ME (n 5 7/15) stained not only the ACA but also the rest of the CA (Fig. 5A). Further classification of these neurons by mass dextran injection is difficult. Instead, single-cell dye injections would be more appropriate to reveal the subclasses of ME neurons connected to the CA. Visual information into the ACA of P. xuthus seems to be conveyed by ME neurons because the ACA was stained in most cases of dextran injection into the optic lobe or the ME, while the ACA was not stained when dextran was applied only to the LO (Fig. 5I). In addition, the vLO and the LO were stained in all cases when dextran was applied into the CA, but the ME neurons were not always stained. The likely reason for this is that dextran did not have access to the ACA in all cases because of its position. As described above, the ACA is dominated by visual inputs in P. xuthus, although we cannot completely exclude the existence of other sensory inputs such as mechanosensory and gustatory afferents from the subesophageal ganglion reported in the ACA of cockroaches, crickets, and locusts (Farris and Strausfeld, 2003; Farris, 2008).

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Visual inputs in butterfly mushroom body

The visual inputs from the LO are segregated from those from the ME in the CA (Fig. 5I). The LO neurons innervate the primary CA through the ACA. Segregation between the LO neurons and the vLO neurons has not been observed in the CA. Likewise, the origin of visual inputs to the rim of the outer zone has not been uncovered. These questions can be addressed by identifying projections of single neurons from the LO and the vLO. The inner zone of the CA is spacious but was only moderately innervated by visual fibers after dextran injection into the OL (Fig. 5A–G). Its supply by visual fibers may actually be much more substantial, because the LO and vLO, whose positions are deep in the optic lobe, were hardly reached by the dextran injections. Strong visual input to the CA has been demonstrated in hymenopteran insects (Gronenberg, 1999, 2001; Ehmer and Gronenberg, 2002; Gronenberg and Lopez-Riquelme, 2004; Paulk and Gronenberg, 2008) and the whirligig beetle (Lin and Strausfeld, 2012). In bees and ants the CA receives visual inputs from the ME and the LO, while visual inputs in the whirligig beetle only comes from dorsal parts of the ME corresponding to dorsal eye regions. Visual inputs in bees are as complex as those in P. xuthus. There are at least four types of visual input in the honeybee: three from the ME and one from the LO (Ehmer and Gronenberg, 2002), while in the bumblebee three types were identified by single-cell dye injection: one ME and two LO inputs (Paulk and Gronenberg, 2008). The visual inputs from the ME and the LO are segregated from each other and also separated from olfactory inputs in the CA. In honeybees, the basal ring receives both visual and olfactory input, even though both inputs target different regions in the basal ring, suggesting that the basal ring is more multimodal than the collar and lip regions receiving visual and olfactory input, respectively. In P. xuthus, visual and olfactory inputs are, likewise, segregated in the CA, but here no subareas receive both sensory modalities. Visual inputs into the CA have been also reported in other insects. The mushroom body of the cockroach receives visual input from the ME, which targets an inner layer of the CA, but not the ACA, where mechanosensory and gustatory afferents from the subesophageal ganglion terminate (Farris and Strausfeld, 2003). In M. sexta and S. littoralis, the inner zone of the primary calyces receives visual inputs from the optic lobe (Homberg et al., 1988b; Sj€oholm et al., 2005). In P. rapae, a multitude of visual inputs (one or two ME inputs and possibly an LO input) innervate the CA, whose compartments are still unknown (Snell-Rood et al., 2009). The data suggest substantial variation in the organization of visual inputs even within Lepidoptera.

Dorsal and ventral asymmetry of visual inputs to the mushroom body Judging from the arborization pattern of three visual inputs to the CA in the optic lobe, the mushroom body of P. xuthus appears to be more strongly involved in processing information from ventral areas of the compound eye. The ME input neurons have many small processes in layer 4a only in the ventral two-thirds of the ME and project to the CA via the vLO (Figs. 6A,C, 7B, 10A,B). In addition, the input from the vLO is connected with ventral areas of the ME (Figs. 6D, 10B). In contrast, the LO input consists of neurons with wide arborizations extending throughout the dorsal and ventral regions, suggesting that only the LO input to the CA collects information from the entire visual field (Fig. 6C,D). The inputs from different eye regions, however, are not clearly segregated in the CA, which is different from the situation in honeybees. Visual inputs from ventral areas of the optic lobe in P. xuthus suggest that the mushroom body is required for neural processing of object-related tasks such as nectar feeding. In contrast to P. xuthus, the CA in honeybees receives inputs from the dorsal eye region by two types of visual input (Ehmer and Gronenberg, 2002). One is an ME input with arborizations in the entire ME. The other is an LO input with small dendritic fields in dorsal regions of the LO. In addition, ME inputs from dorsal and ventral areas clearly segregate and form a layered organization in the collar of the calyces. This layered organization may be related to distinct functions of different eye regions. Visual information from dorsal eye regions may have a role in sky compass navigation, which is a prominent behavior in bees.

Segregation of sensory modalities in the calyx In P. xuthus, visual inputs from the optic lobe are segregated in the CA from olfactory input (blue in Fig. 10A,C). In Hymenoptera, the lip and the basal ring of the calyces receive olfactory input (Gronenberg, 2001; Gronenberg and Lopez-Riquelme, 2004). Although the basal ring in honeybees receives both visual and olfactory inputs, the two modalities are separated in two different subareas (Ehmer and Gronenberg, 2002). In cockroaches, there are at least three visual inputs from the LO and the ME projecting to the inner layer of the calyces. These visual inputs are separated from the olfactory inputs in the outer layer, which is dominant in the calyces (Strausfeld and Li, 1999b; Nishino et al., 2012). In most cases, calyces dominated by olfactory input do not receive direct visual inputs from the optic lobe. The mushroom body of the fruit fly contributes to

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visual learning and memory but direct pathways from the optic lobe have not been found, presuming that visual inputs innervate the CA indirectly from the central brain (Li and Strausfeld, 1999; Otsuna and Ito, 2006; Tanaka et al., 2008).

bodies was influenced by a combination of odor and color stimuli in M. sexta, indicating that the two modalities were integrated there (Balkenius et al., 2009). Prominent visual inputs are, therefore, likely to be integrated with olfactory inputs in the CA in P. xuthus during its visually dominated foraging behavior.

Functional role of the mushroom bodies in P. xuthus

ACKNOWLEDGMENTS

Diurnal butterfly species strongly depend on visual information. In fact, P. xuthus can easily learn and discriminate color targets associated with nectar (Kinoshita et al., 1999). The butterflies also use other visual features, such as brightness and polarized light for detecting targets (Kinoshita et al., 2011; Koshitaka et al., 2011). Butterflies whose dorsal eyes are covered with black paint cannot fly at all, whereas animals prevented from seeing in their ventral visual field can fly but cannot detect targets on the floor (Kinoshita, unpubl.). This indicates that ventral eye regions are crucial for detecting visual features of targets, whereas dorsal eye regions are important for controlling flight behavior itself. Considering that three visual inputs to the CA collect information from the ventral area of the compound eye, each input might transfer different visual modalities such as color, shape, and movement of objects. Visual information sent to the CA has been studied in honeybees (Gronenberg, 1986), bumblebees (Paulk and Gronenberg, 2008), and cockroaches (Strausfeld and Li, 1999a; Nishino et al., 2012). In bumblebees, ME and LO input neurons into the CA are spectrally sensitive, including color opponency, broad and narrow spectral tuning, and show motion sensitivity and specific temporal responses to repeated stimuli (Paulk and Gronenberg, 2008). Visual inputs in cockroaches were not only excited by light stimuli but were also inhibited by olfactory stimuli (Nishino et al., 2012). We have no evidence that distinct visual information is processed separately by the three visual inputs to the CA in P. xuthus. This point is of particular interest in future studies Lepidopteran insects use several sensory modalities for nectar foraging, finding host plants for oviposition, and mating and show associative learning and memory between different modalities (Daly and Smith, 2000; Omura and Honda, 2005; Costanzo and Monteiro, 2007; Goyret et al., 2009). In addition, diurnal species foraging on flowers, like P. xuthus, rely more heavily on visual than olfactory information for foraging (Andersson and Dobson, 2003; Balkenius et al., 2006). The mushroom body may contribute to integrate these multisensory channels. In fact, the activity of the mushroom

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We thank Dr. Hisaharu Koshitaka for technical assistance and Ms. Yumi Saito for raising butterflies and assistance in anatomical experiments.

CONFLICT OF INTEREST The authors declare that there is no conflict of interest associated with study.

ROLE OF AUTHORS Study concept and design: M.K.; acquisition of data: M.K., M.S., and Y.T.; analysis and interpretation of data: M.K. and U.H.; drafting of the article: M.K., U.H. and K.A.

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