Dual olfactory pathway in Hymenoptera: Evolutionary insights from comparative studies

Arthropod Structure & Development 40 (2011) 349e357

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Dual olfactory pathway in Hymenoptera: Evolutionary insights from comparative studies Wolfgang Rössler*, Christina Zube Department of Behavioral Physiology and Sociobiology, Biozentrum, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 October 2010 Accepted 3 December 2010

In the honeybee (Apis mellifera) and carpenter ant (Camponotus floridanus) the antennal lobe output is connected to higher brain centers by a dual olfactory pathway. Two major sets of uniglomerular projection neurons innervate glomeruli from two antennal-lobe hemispheres and project via a medial and a lateral antennal-lobe protocerebral tract in opposite sequence to the mushroom bodies and lateral horn. Comparison across insects suggests that the lateral projection neuron tract represents a special feature of Hymenoptera. We hypothesize that this promotes advanced olfactory processing associated with chemical communication, orientation and social interactions. To test whether a dual olfactory pathway is restricted to social Hymenoptera, we labeled the antennal lobe output tracts in selected species using fluorescent tracing and confocal imaging. Our results show that a dual pathway from the antennal lobe to the mushroom bodies is present in social bees, basal and advanced ants, solitary wasps, and in one of two investigated species of sawflies. This indicates that a dual olfactory pathway is not restricted to social species and may have evolved in basal Hymenoptera. We suggest that associated advances in olfactory processing represent a preadaptation for life styles with high demands on olfactory discrimination like parasitoism, central place foraging, and sociality. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Antennal lobe Olfactory glomerulus Projection neuron Tract Mushroom body Lateral horn

1. Introduction Olfactory discrimination, recognition and orientation play important roles for most insects. The complexity of olfactory systems in a given species may depend on its feeding ecology, diversity of pheromone communication and olfactory recognition, as well as utilizing chemical cues for orientation in the environment. Olfactory specializations, in general, may be reflected in the anatomical expression of certain traits within olfactory systems e for example the diversity of olfactory sensillum types on the antennae, the number and size of olfactory glomeruli (odor processing neuropil units in the antennal lobe, AL), and adaptations in higher olfactory centers such as the mushroom bodies (MBs) (Hildebrand and Shepherd, 1997; Hansson and Anton, 2000; Gronenberg, 2001; Kleineidam and Rössler, 2009; Strausfeld et al., 2009; Galizia and Rössler, 2010). Specializations for the detection of pheromonal odors at high accuracy were shown in the case of enlarged macroglomeruli typical for sex-pheromone communication systems, but also for trail-pheromone detection in leafcutter ants (e.g. Hildebrand and Shepherd, 1997; Kleineidam et al., 2005; * Corresponding author. Tel.: þ49 931 3184313; fax: þ49 931 3184309. E-mail address: [email protected] (W. Rössler). 1467-8039/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2010.12.001

Kuebler et al., 2010). However, correlation of olfactory traits with certain aspects in the behavioral ecology of different species may not always be simple as it was recently shown for differences in the overall numbers of antennal-lobe glomeruli and the feeding ecology in ants of the tribe Attini (Kelber et al., 2009). Previous studies have highlighted two special features or adaptations within the olfactory system of Hymenoptera: 1. The numbers of AL glomeruli across insects investigated so far were found highest in the Hymenoptera with up to 630 glomeruli in ants (Smid et al., 2003; Kirschner et al., 2006; Zube et al., 2008; Groh and Rössler, 2008; Kelber et al., 2009, 2010; Galizia and Rössler, 2010). 2. Two major sets of antennal-lobe projection neurons (PNs) project in opposite sequence to the MB and LH forming a dual (medial and lateral) antennal-lobe protocerebral tract. In this study we adopted the following nomenclature: antennal-lobe protocerebral tract (APT) for the tracts, and m-, ml-, and l-APT for medial, mediolateral, and lateral antennal-lobe protocerebral tract, respectively (Fig.1). As this nomenclature is purely based on position (innermost, intermediate and outermost tract positions) the tract names (e.g. m-APT or l-APT) do not necessarily imply homology. In Hymenoptera APTs are clearly subdivided structurally and have been well investigated in the honeybee, Apis mellifera and in the carpenter ant, Camponotus floridanus. Here the l-APT, m-APT and 3 ml-APTs connect the AL with

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Fig. 1. Schematic drawings of individual PNs from different antennal-lobe-protocerebral tracts (APTs) superimposed on a confocal image of a frontal view of the honeybee brain. Schematic drawings of one medial and lateral APT (m- and l-APT) uniglomerular projection neuron (uPN) are shown in the right half of the brain, respectively, and a schematic drawing of an example of a multiglomerular PN (mPN) is shown in the left half of the brain. Further abbreviations: AL, antennal lobe; CX, central complex; lCA and mCA, lateral and medial calyx of the mushroom body (MB); LH, lateral horn; OL, optic lobes. Scale bar ¼ 100 mm.

higher centers in the protocerebrum (Abel et al., 2001; Müller et al., 2002; Kirschner et al., 2006; Zube et al., 2008; Zube and Rössler, 2008) (Fig. 1). Whereas the m-APT and l-APT are mainly formed by uniglomerular PNs innervating single glomeruli, the ml-APTs comprise multiglomerular PNs with dendritic arborizations in many glomeruli (Abel et al., 2001; Müller et al., 2002; Krofczik et al., 2009) (Fig. 1 shows one example of a schematic drawing of an m-, l-, and ml-APT PN, respectively). Comparative studies of APTs across insects indicate that a dual olfactory pathway with a prominent l-APT is a unique feature of Hymenoptera (Kirschner et al., 2006; Zube et al., 2008; Galizia and Rössler, 2010). The glomerulus output tract association was investigated in detail in the honeybee (A. mellifera) and in the carpenter ant (C. floridanus) (Kirschner et al., 2006; Zube et al., 2008; Zube and Rössler, 2008). Regarding the association with output tracts to the MBs the AL is divided into two hemispheres with almost equal numbers of glomeruli. Each hemilobe is innervated by a set of uniglomerular projection neurons (uPNs) with axons forming the m- and l-APT to the MBs and LH. Axon terminals of PNs from the mand l-APT have largely segregated target regions in the MB calyx and LH indicating that olfactory information may be processed along two parallel streams (Müller et al., 2002; Kirschner et al., 2006; Zube et al., 2008; Krofczik et al., 2009; Yamagata et al., 2009). The significance of a dual uPN pathway for spatial and temporal aspects of olfactory coding and for olfactory guided behavior remained unclear and is in the focus of ongoing physiological and behavioral studies, mostly in the honeybee. Comparison of anatomical details of APTs across insect orders revealed that a genuine dual AL-MB pathway formed by two sets of PNs represents a special feature in Hymenoptera (Kirschner et al., 2006; Zube et al., 2008; Galizia and Rössler, 2010). As this feature, so far, has only been investigated in social Hymenoptera, this

triggers the question whether a dual olfactory pathway may be regarded as an adaptation to advanced olfactory processing associated with social life styles. A striking sexual dimorphism of the dual olfactory pathway found in the carpenter ant supports this view (Zube and Rössler, 2008). In C. floridanus males the number of AL glomeruli is reduced to w55% of the number found in the female castes, but only the m-APT proportion of AL glomeruli was smaller, and males engage less in social tasks and foraging (Zube and Rössler, 2008). A reduction of AL glomeruli was also found in honeybee drones (w106 in males compared to w164 in females; Sandoz, 2006), although the tract association of the missing glomeruli remains to be shown. Two recent studies indicate that the missing glomeruli may be associated with the m-APT as well. A study by Nishino et al. (2009) indicates that glomeruli of the T3 cluster are absent in drones, and Kirschner et al. (2006) demonstrated that the majority of T3 glomeruli in female workers are innervated by the m-APT. Studies in solitary Hymenoptera like female parasitoid wasps revealed glomerular numbers comparable to those found in the honeybee (Smid et al., 2003) indicating that high numbers of glomeruli (>100) may have evolved within the Hymenoptera, but at least initially independent from the acquisition of social life styles. This brings up the question whether a dual olfactory pathway is a general feature in Hymenoptera or whether it is exclusively present in the advanced eusocial Hymenoptera. To address this question, we compared the organization of APTs in selected social and solitary Apocrita, and in Symphyta, representatives of basal phytophagous Hymenoptera. Specifically, we addressed the following questions: 1. Is a dual olfactory pathway to the MBs restricted to social Hymenoptera?

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2. In case a dual pathway is present in advanced solitary Hymenoptera like parasitoid wasps, does it also occur in the basal Hymenoptera? 3. Is a dual olfactory pathway always associated with high numbers (>100) of AL glomeruli and a doubled MB calyx?

2. Materials and methods 2.1. Study animals Comparative analyses were carried out in eight species of Hymenoptera, one species belonging to the Ensifera, and one coleopteran species (the systematic classification and numbers of specimens are shown in Table 1.) All investigated samples were females. Samples of bushcrickets (Leptophyes punctatissima, Ensifera)) and dung beetles (Geotrupes auratus, Coleoptera) were collected in the vicinity of the Biocenter, University of Würzburg, Germany. Ponerine ants (Harpegnathos saltator, Ponerinae) were taken from laboratory colonies from J. Liebig (Arizona State University, USA), originally excavated at JogFalls, Karnataka State, Southern India in the years 1994, 1995 and 1999. Samples from a solitary digger wasp, the beewolf Philanthus triangulum (Apoidea, Crabronidae), were kindly provided by Martin Kaltenpoth (Max-Planck-Institute for Chemical Ecology, Jena, Germany). Two species of sawflies were kindly provided from laboratory colonies from Caroline Müller (University of Bielefeld, Germany) (Athalia rosae, Tenthredinidae), and Monika Hilker (Free University Berlin, Germany) (Diprion pini, Diprionidae). 2.2. Visualization of brain structures in whole mount preparations and immunocytochemistry For visualization and 3D-analysis of AL glomeruli and other brain neuropils in whole mount preparations we used tissue autofluorescence increased by glutaraldehyde fixation. After decapitation the head capsule was fixed in dental-wax coated dishes and opened by cutting a window between the compound eyes. Ice cold ringer solution (ant-ringer: solution A in 900 ml distilled water: 7.4 g NaCl, 0.5 g KCl, 0.22 g CaCl2; solution B in 100 ml distilled water: 0.11 g Na2HPO4; 0.05 g KH2PO4; solution A was added to solution B with 1.1 g TES and 1.2 g Trehalose, and the pH was adjusted to 7.0; bee ringer, also used for Leptophyes, Geotrupes, Philanthus and sawflies: 37 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.4 mM KH2PO4, pH ¼ 7.2) was instantly applied on the brain and glands and tracheae were gently removed. Brains were fixed immediately in cold 1% glutaraldehyde in phosphate-buffered saline (PBS, pH 7.2) for 4 days at 4  C. The brains were then washed in PBS (5  10 min) and dehydrated in an ascending series of ethanol (30%, 50%, 70%, 90%, 95%, 3  100%, 10 min each step). The brains were cleared in methylsalicylate (M-2047, Sigma Aldrich, Steinheim, Germany) and mounted on special aluminum slides with cover slips on both sides and stored at 20  C (Zube et al., 2008).

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For immunolabeling, brains were transferred in 4% formaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.2) overnight at 4  C. Brains were washed three times in PBS, embedded in 5% low melting point agarose (Agarose II, no. 210-815, Amresco, Solon, OH), and all sliced at 100 mm thickness (Vibratome, Leica VT 1000S, Nussloch, Germany), permeabilized in 0.2% Triton in PBS, and treated with 2% normal goat serum (NGS, 005-000-121, Jackson ImmunoResearch Laboratories, West Grove, PA) in 0.2% PBS-triton for 1 h at room temperature. Sections were simultaneously incubated in 0.2 units of Alexa Fluor 488 phalloidin (Molecular Probes, A-12379, Leiden, The Netherlands) in 500 ml 0.2% PBS-Tx with 2% NGS and a monoclonal antibody against the Drosophila synaptic-vesicle associated protein synapsin I (1:10; SYNORF1; kindly provided by Dr. E. Buchner, University of Würzburg, Germany) (Klagges et al., 1996) for 3 days at 4  C. After five washes in PBS, preparations were incubated in Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (1:250; Molecular Probes, A-11004) in 1% NGS-PBS for 2 h at room temperature (Rössler et al., 2002; Groh et al., 2006). Sections were washed at least five times in PBS, transferred into 60% glycerol/PBS overnight and then mounted on slides in 80% glycerol/PBS. 2.3. Neuronal tract tracing For labeling the trajectories of antennal-lobe protocerebral tracts (APTs) to higher centers in the protocerebrum, fluorescent dye (rhodamine dextran with biotin; Microruby, 3000 MW, lysinefixable, D 7162, Molecular Probes, Eugene, USA) was inserted directly into the AL neuropil according to the method described previously (Kirschner et al., 2006; Zube et al., 2008; Zube and Rössler, 2008). Individuals of the investigated species were fixed in dental-wax dishes. The head capsule was opened by cutting a window between the compound eyes, and glands and tracheae were gently removed. The AL tissue was then carefully perforated with a glass micropipette (laser-electrode puller, P2000, Sutter Instruments Co., Novato, USA) made from borosilicate capillaries (1B100F-3, Precision Instruments, Sarasota, USA). Dye was immediately applied into the perforated tissue using a dye coated broken glass micropipette. The pipette remained in the target area for w10 s until the dye had dissociated from the pipette tip. The pipette was removed and the brain was immediately rinsed with fresh antringer solution to remove excessive dye. The preparations were kept in a humid chamber for 5 h to let the dye diffuse. The brains were then dissected in ringer solution (see methods described above) and fixed in 4% formaldehyde in 0.1 M PBS overnight at 4  C. Thereafter, preparations were washed with 0.1 M PBS (3  10 min), dehydrated and cleared as described above. 2.4. Laser-scanning confocal microscopy and image processing All preparations were viewed with a laserescanning confocal microscope (Leica TCS SP2 AOBS; Leica Microsystems AG, Wetzlar, Germany) equipped with an argon/krypton laser and a heliumeneon

Table 1 List of species investigated for tract morphologies. The classification of species is according to Grimaldi and Engel (2005). Order

Suborder Infraorder

Superfamily

Family

Orthoptera Coleoptera Hymenopotera

Ensifera Polyphaga Symphyta

Tettigoinioidea Scarabaeoidea Tenthredinidoidea

Apocrita Aculeata

Vespoidea

Tettigoniidae Geotrupidae Tenthredinidae Diprionidae Formicidae

Apoidea

Crabronidae Apidae

Subfamily

Ponerinae Formicinae Myrmicinae

Species

Specimens

Leptophyes punctatissima Geotrupes stercorarius Athalia rosae Diprion pini Harpegnathos saltator Camponotus floridanus Atta vollenweideri Philanthus triangulum Apis mellifera

3 3 6 8 2 Zube et al., 2008 2, and pers. com. Kleineidam 8 Kirschner et al., 2006

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Fig. 2. Comparison of antennal-lobe protocerebral tracts (APTs) across different insect orders and between selected social and solitary Hymenoptera. A and B. Representative examples of anterograde mass stainings of the APTs in Leptophyes punctatissima (Ensifera) (A) (optical stack thickness 250 mm) and Geotrupes stercorarius (Coleoptera) (optical stack thickness 300 mm) (B). The arrows in A label different mediolateral APTs. C and D. Representative examples of anterograde mass stainings of the APTs in the ponerine ant Harpegnathos saltator (Ponerinae) (optical stack thickness 120 mm) and in the carpenter ant Camponotus floridanus (Formicinae) (optical stack thickness 250 mm). The arrow in C labels small ml-APTs. The inset shows a higher magnification of the lateral APT at the level of the lateral horn (LH) with fibers proceeding further to the mushroom bodies (MB). E and F. Representative examples of anterograde mass stainings of the APTs in a solitary parasitoid wasp, the beewolf Philanthus triangulus (Apoidea, Crabronidae) (optical stack thickness 120 mm) (E) and in the honeybee Apis mellifera (Apoidea, Apidae) (optical stack thickness 250 mm) (F). The ml-APTs are numbered (1e3) in both cases. Further abbreviations: mCA and lCA, medial and lateral calyx of the mushroom bodies. The spatial directions are shown in B with c, caudal; l, lateral; m, medial; and r, rostral. Scale bar in A ¼ 100 mm is also valid for BeF.

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laser. Excitation wavelength for Rhodamine Dextrane was 568 nm. Where needed, background fluorescence was enhanced and viewed with the customized settings. Two different HC PL APO objective lenses were used for image acquisition (10  0.4 NA imm and 20  0.7 NA imm). Optical sections were taken at distances between 1 and 10 mm. In certain cases a digital zoom of 2e3 was applied. Confocal-image stacks were viewed and processed with ImageJ (Wayne Rasband, NIH, Bethesda, MD) and further adjusted for brightness and contrast where needed with CorelPhotopaint and CorelDrawX3 (Corel Corporation, Ottawa, Ontario, Canada). For reconstruction of glomeruli and determination of glomerular numbers selected image stacks were viewed and processed with the 3D reconstruction software AMIRA 3.1 (Mercury Computer Systems, Berlin, Germany). Spatial directions are indicated according to Zube et al. (2008). 3. Results The most important aspect for the present study was the question whether a prominent lateral APT with collaterals to the LH first and continuing projections to the MB calyces is present in addition to a medial APT, which contains PNs sending off collaterals to the MBs first and continuing projections to the LH (see schematic drawing of the m- and l-APT in the honeybee brain shown in Fig. 1). 3.1. Comparison of APTs across insect orders and between advanced Hymenoptera Previous investigations of APTs in Orthoptera were mostly done in the locust (Leitch and Laurent, 1996; Hansson and Anton, 2000; Ignell et al., 2001; Anton et al., 2002). As the general organization of AL glomeruli in locusts (e.g. Locusta and Schistocerca belonging to the Caelifera) differs markedly from that in other insects investigated so far, we labeled the APTs in a species belonging to the Ensifera, as a further representative of Orthoptera, the bushcricket L. punctatissima (Tettoginiidae), to test whether they have a similarly simple organization of APTs like in the locust (Fig. 2A). Anterograde mass stainings revealed a prominent m-APT with projections to the MB calyx and processes proceeding further to the lateral protocerebrum or LH. Only very few, diffuse ml-APT-like projections were found that ended in the lateral protocerebrum. This APT configuration was very similar to those described in the locust (Ignell et al., 2001). Previous investigations in beetles indicate that a prominent l-APT is absent as well and the AL is mainly connected to the MB via a medial APT, a situation very similar to the one found in the locust (Wegerhoff, 1999; Farris, 2008; Strausfeld et al., 2009). To make sure that differences are not due to different staining techniques, we used the same technique as in the other species investigated in our present and previous studies to anterogradely label the APTs in the beetle Geotrupes stercorarius (Geotrupidae). The results confirmed the rather basal APT organization in Geotrupes (Fig. 2B). The stainings indicate that a prominent m-APT projects to the MBs with diffuse projections proceeding further laterally. In addition to the m-APT, only few ml-APT-like projections to other targets in the lateral protocerebrum were found. This confirms earlier observations in other members of the Coleoptera and indicates that the rather basal organization of APTs in beetles comes close to the conditions found in Orthoptera and Zygentoma (Strausfeld et al., 2009). Next we asked whether a dual PN pathway to the MBs is present in basal and advanced social and solitary aculeate Hymenoptera. The most important criterion for a dual PN pathway to the MB was the presence of an l-APT (in addition to the m-APT, which was present in all insects studied so far) that contains fibers originating

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in the AL and proceeding further to the MBs after sending collaterals to the LH. We selected species belonging to the superfamilies Vespoidea and Apoidea (Table 1; Fig. 2CeF). Within the ants (Formicidae), we compared socially divergent species: one ponerine ant species, Harpegnathos salator (Ponerinae), largely visually guided hunters with a very basal social organization, and C. floridanus (Formicinae) (data from Zube et al., 2008; Zube and Rössler, 2008) as well as Atta vollenweideri (Myrmicinae) with highly advanced social structures and pronounced trail-pheromone communication systems (Hölldobler and Wilson, 1990). Analyses of confocal-image stacks from anterograde mass stainings of the APTs in H. saltator (Ponerinae) and comparison with the conditions found in C. floridanus (Formicinae) revealed the presence of both a medial and lateral APT in H. saltator (Fig. 2C, D). In addition to the m- and l-APT, several very small ml-APTs were visible, although more difficult to detect in H. saltator compared to the conditions in C. floridanus. Similar results were found in the leafcutter ant, A. vollenweideri with distinct m- and l-APT projections similar to those in C. floridanus (data not shown; and personal communication Kleineidam and Kuebler). The results indicate that the APTs found in the ponerine ants which, in general, have retained more basal traits closely resemble those in the two species from two subfamilies of advanced ants. Since a dual m- and l-APT pathway was present in all investigated Formicidae, we conclude that a dual olfactory pathway may be present in most, if not all, species of ants. To address the question whether within the Apoidea a dual olfactory pathway is restricted to social species, we compared the APTs in a solitary predatory wasp, the European beewolf P. triangulum (Apoidea, Crabronidae) with those in the honeybee. The numbers of olfactory glomeruli in the honeybee (w164; Galizia et al., 1999; Kirschner et al., 2006) and glomerular numbers found in the European beewolf are in a similar range (J Rybak and C Kelber, pers. communication). Comparison of the APTs labeled in P. triangulum revealed a strikingly similar picture compared to the conditions in the honeybee (Fig. 2E, F). Careful analyses of confocalimage stacks revealed that prominent m- and l-APTs were present in the solitary wasp together with a set of three small ml-APTs, very close to the conditions of all APTs in the honeybee and in the carpenter ant (Kirschner et al., 2006; Zube et al., 2008). Close inspection at higher magnification showed that in close proximity to the LH, l-APT fibers proceeded further to the MBs. We therefore conclude that the presence of the l-APT and a dual olfactory pathway to the MBs and LH is not restricted to social Hymenoptera.

3.2. Comparison of APTs in basal Hymenoptera As we found a dual olfactory pathway to the MBs in samples from both social and solitary Hymenoptera, we asked whether a dual olfactory pathway to the MBs may have evolved within the basal Hymenoptera. As a first test of this hypothesis, we performed anterograde stainings of the APTs in two selected species of phytophagous sawflies belonging to two different families, the turnip sawfly A. rosae (Tenthredinidae), and the pine sawfly D. pini (Diprionidae) (Fig. 3; Table 1). Anterograde tract labeling in D. pini revealed multiple APTs including a prominent m-APT, several (at least three) small ml-APTs, and a tract with all features of an l-APT containing axons proceeding from the AL to LH and further to the MBs (Fig. 3A, C). Furthermore, the origin of the lateralmost tract in the AL was very similar to the l-APTs found in social Hymenoptera and in the beewolf (Fig. 2). In general, the organization of APTs in D. pini resembled the conditions we found in advanced Hymenoptera (Fig. 2CeF). The spatial organization of terminal arborizations of m- and l-APT PNs within the MB calyces indicates that the MB calyx in D. pini has a doubled structure (arrows in Fig. 3A).

Fig. 3. Comparison of antennal-lobe protocerebral tracts (APTs) in two species of sawflies (Symphyta) belonging to two different families, Athalia rosae (Tenthredinidae) and Diprion pini (Diprionidae). A and B. Overview of representative examples of anterograde mass staining of APTs in Diprion pini (A) and Athalia rosae (B) (optical stack thickness 120 mm in both cases). The arrows in A and B indicate the presence of two calyx compartments in both species. C and D. High magnification of projections from confocal substacks showing details in the branching pattern of lateral APT processes close to the lateral horn (LH) in Diprion pini (C) and Athalia rosae (D). The black arrow in C indicates m-APT fibers proceeding further to the MB calyx, the white arrow indicates two APT bundles in the upper region of the LH, most likely belonging to the m- and l-APT. E and F. Confocal section through the antennal lobe (AL) in Diprion pini (E) and Athalia rosae (F) with individual olfactory glomeruli (G) visible by tissue autofluorescence. G and H. Posterior view of a 3D-AMIRA-reconstruction (from the ALs shown in E and F) of glomeruli in the AL of Diprion pini (G) and Athalia rosae (H). Glomerular numbers were determined based on these 3D reconstructions. Further abbreviations: AN, antennal nerve; MB, mushroom body. The spatial directions for AeF are shown in F with c, caudal; l, lateral; m, medial; and r, rostral. Scale bar in B (also valid for A) ¼ 100 mm, scale bars in D, F, and H (also valid for C, E, and G) ¼ 50 mm.

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Anterograde mass labeling of the APTs in A. rosae revealed a different picture compared to the situation in D. pini. In addition to a prominent m-APT, we found ml-APT-like tracts that separate from the m-APT at two positions along its trajectory (Fig. 3B, D). We did not find a tract that leaves the AL at a position similar to the l-APT in D. pini. Only few diffuse additional fibers were stained that ended in areas close to the LH. This was very similar in three successful stainings we analyzed. In contrast to the situation in D. pini, we did not detect fibers proceeding from the LH to the MBs in A. rosae (Fig. 3D). This indicates that in A. rosae only axons from m-APT PNs project to the MB calyx and further to the LH. In summary, the total number of APTs was smaller in A. rosae, and the lateralmost APT did not contain fibers proceeding further to the MBs after sending off collaterals to the LH. Regarding their point of origin and target structure, all tracts in A. rosae found lateral to the m-APT closely resembled ml-APTs in the other Hymenoptera we investigated. These features indicate that none of the APTs in A. rosae combines all features of the l-APTs in the other Hymenoptera we investigated. As the organization of APTs in D. pini and A. rosae appeared different, we were interested whether the two species may differ in the number of olfactory glomeruli in the AL, and whether a higher number of AL glomeruli may correlate with an increase in the number of AL output tracts. We counted the numbers of olfactory glomeruli in the ALs of both sawfly species using confocal-image stack analyses of glomeruli by autofluorescence detection in glutaraldehyde fixed preparations (Fig. 3EeF) and AMIRA-reconstructions (Fig. 3G, H). The 3D reconstructions from confocal-image stacks revealed relatively small total numbers of relatively large glomeruli in both the AL of A. rosae females (48 and 47 AL glomeruli in two female samples) and D. pini (35, 37 and 38 AL glomeruli in three female samples). To further find out whether the presence of the l-APT is associated with a doubled calyx, we looked more closely at the organization of the MB calyx in both sawflies in autofluorescence preparations, tract stainings and immunolabeled preparations. The organization of terminal arborizations of m-APT PNs in the MB calyx (Fig. 3A, B; arrows) indicate that the MB calyx in both A. rosae and D. pini has a doubled structure. Both species of sawflies had two calyx compartments on each side. As A. rosae did not have a lateral APT, we further confirmed that the MB calyx was doubled by analyses of sections double-labeled with anti-synapsin antibody and f-actin-phallodin (Fig. 4). The results clearly demonstrate that the calyces and proximal part of the peduncles were doubled. This was further confirmed in confocal stacks of autofluorescence images showing that the initial part of the peduncle and the calyx was split into two parts in both A. rosae and D. pini (not shown). 4. Discussion This comparative study provides evidence that a dual olfactory pathway of PN projections from the AL to the MBs and LH (Kirschner et al., 2006; Zube et al., 2008; Zube and Rössler, 2008) is not restricted to social Hymenoptera. We demonstrate that an l-APT is present in a solitary hymenopteran species and in ponerine ants. Furthermore, our results indicate the presence of a dual olfactory pathway in one species of basal phytophagous Hymenoptera, whereas it is absent in another sawfly belonging to a different family. This supports the hypothesis that a lateral APT pathway from the AL via the LH to the MBs may have evolved in the basal Hymenoptera. The fact that both samples of sawflies, one with and one without an l-APT possess a low number of glomeruli and a doubled MB calyx suggests that a dual olfactory pathway and elaboration of AL output tracts may have evolved independently from an increase in the total number of glomeruli and a duplication of the MB calyx. We

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therefore hypothesize that a dual olfactory pathway may be associated with changes in AL PNs and the connectivity of AL glomeruli. The results further support the hypothesis that the l-APT pathway may represent a preadaptation in basal Hymenoptera. The division of the antennal lobe into two sets of glomeruli and associated PN output tracts may support advanced parallel processing of olfactory information. This may have promoted life styles associated with a more sophisticated feeding ecology and/or olfactory communication such as associated with parasitoid and/or social life styles. However, as both D. pini and A. rosae have been shown to employ quite elaborated forms of olfactory communication and discrimination tasks in the context of mating behavior (Amano et al., 1999; Hilker et al., 2000), we can only speculate about potential differences in the olfactory performance of both species. To comparatively test advantages that D. pini might gain over A. rosae, sophisticated olfactory discrimination or choice assays involving multi-component odor stimuli and/or olfactory learning paradigms are necessary in the future. The fact that a dual olfactory pathway does exist in advanced solitary Hymenoptera, like shown for the beewolf P. triangulum, is not entirely surprising as the detection and recognition of the host, a burrow, the maintenance of brood, and other specializations in the behavioral ecology of this species is highly dependent on sophisticated olfactory guided behavior. Furthermore, studies have shown that parasitoid wasps have similarly high numbers of glomeruli compared to the honeybee and ponerine ants (Smid et al., 2003; Hoyer et al., 2005; Galizia et al., 1999; Kirschner et al., 2006), and glomerular numbers in the beewolf appear to be in a similar range (J. Rybak and C. Kelber, personal communication). The same is true for ponerine ants as they show complicated colony level interactions based on the recognition of complex colony odors on the cuticle surface, as it was shown for the ponerine ant H. saltator (Liebig et al., 2000; Peeters et al., 2002). Olfactory recognition in ponerine ants may not be as sophisticated as in ants with a highly derived social structure like C. floridanus (Endler et al., 2004). Interestingly, the number of OR genes and olfactory glomeruli was shown to be smaller in H. saltator compared to C. floridanus (Hoyer et al., 2005; Zube et al., 2008; Bonasio et al., 2010). Despite this, H. saltator has an APT organization comparable to the one in C. floridanus indicating that the complexity of olfactory output tracts may, in general, be independent from the total number of glomeruli (178 glomeruli in H. saltator compared to 436 in C. floridanus) and from differences in the number of OR genes. In males of C. floridanus, AL glomeruli are reduced to about 55% of those found in female castes, and the reduction was shown to mostly affect the m-APT (Zube and Rössler, 2008). Glomeruli associated with the l-APT were almost unchanged in number compared to females. Similarly, male honeybees (drones) were shown to have a smaller number of glomeruli compared to workers and queens (Sandoz, 2006; Groh and Rössler, 2008). Whether the reduction of m-APT glomeruli may be linked to the lack of males to engage in social tasks (Hölldobler and Wilson, 1990) and requirements for processing of social odors, in particular colony odors (Liebig et al., 2000; Endler et al., 2004) or due to the absence of foraging behavior and related tasks in odor discrimination in males needs to be shown in future combinations of tract specific physiological recordings and behavior tests. Our study shows that in D. pini a dual olfactory pathway is present in combination with a relatively small number of glomeruli and a doubled calyx. In A. rosae, we found that the l-APT is absent. Comparison of the total number of glomeruli in the ALs of Diprion and Athalia revealed similarly small numbers (w35e38 in Diprion and w47e48 in Athalia), and PN arborizations and visual inspection of whole mount preparations showed that Athalia has a small doubled calyx, too. This was also described in a different sawfly (Thendredo)

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Fig. 4. Brain of the sawfly Athalia rosae double labeled with anti-synapsin antibody (magenta) and f-actin-phalloidin (green). The overview and the inset at higher magnification show that the mushroom bodies (MB) are doubled and divided into medial (mCA) and lateral calyx (lCA). AL, antennal lobe. Scale bar in the overview is 200 mm, and 50 mm in the inset.

shown in a comparative study by Riveros and Gronenberg (2009), earlier drawings by Jawlowski (1960), and an even earlier study by Alten (1910). This indicates that variations of AL output tracts may be independent from the number of AL glomeruli and MB calyx duplication. In the same line, a recent study by Dacks et al. (2010) showed substantial variation in innervations by histaminergic neurons in the AL of two members of the paraphyletic group of sawflies. Consequently, a dual PN pathway from the AL to the MBs may have evolved independently from changes in the number of AL glomeruli (and associated changes in OR gene numbers) and from the duplication of the MB calyx. As the phylogenetic relationships within the paraphyletic group of Symphyta are complicated, future studies of closely related species are necessary to further elucidate this aspect. The numbers of AL glomeruli we found in Diprion and Athalia were in a similar range compared to those in flies, moths and beetles (reviewed in Galizia and Rössler, 2010). One important question to follow up is at which point in the hymenopteran lineage AL glomeruli have increased. For the MBs, previous studies have shown that MB-calyx morphology does not show a simple correlation with the current understanding of insect phylogeny as single and doubled MB calyces were found across insect orders and phylogenetically widely divergent groups (reviewed in, e.g. Strausfeld et al., 1998, 2009; Farris, 2005). Multimodal input to the MB calyx, like visual input or a tritocerebral tract containing gustatory and/or mechanosensory input from the subesophageal ganglion were also shown to contribute to MB complexity (Schröter and Menzel, 2003; Farris and Roberts, 2005; Farris, 2008). The tritocerebral tract was shown to be present in many insect species, but the tract was always found in close vicinity to the m-APT, which excludes that a tritocerebral tract potentially contributed to the l-APT found in D. pini. We conclude that APT complexity, in particular the presence or absence of an l-APT, may be independent from both the number of AL glomeruli and a duplication of the MB calyx. We therefore hypothesize that an additional set of AL PNs may account for a lateral APT in Hymenoptera. This is supported by neurochemical differences between the two tracts in the honeybee (Kreissl and Bicker, 1989). Whereas the m-APT was positive for cholinergic transmission markers, the l-APT was not, and the putative neurotransmitter of the l-APT, so far, remains unknown (Galizia and Rössler, 2010).

What is the benefit of having multiple tracts from the AL to the MBs? Future comparative investigations on sensory orientation, olfactory performance, and differences in the feeding ecology of D. pini and A. rosae will help to answer this question. Most importantly, more samples from the two families of Symphyta and from other basal Hymenoptera like Xyelidae, the basal-most group of Hymenoptera and sister group to all other Hymenoptera, and other selected sawfly and parasitoid species have to be analyzed. Other social or semi-social insects like termites, cockroaches, or social beetles are highly interesting for future comparative studies. The present results provide a first framework for these future comparative analyses. What is the function of a hemilobe division of AL glomeruli and two separate uPN pathways to the MBs and LH? Galizia and Rössler (2010) proposed several scenarios for parallel processing of olfactory information based on morphological and physiological data from the honeybee and carpenter ant (e.g. Müller et al., 2002; Zube et al., 2008; Krofczik et al., 2009; Yamagata et al., 2009). The major question is how the two sets of glomeruli in the same antennal lobe differ regarding processing of olfactory information. If they process different sets of odors (differential processing) the two APTs most likely transfer different kinds of odor information. If they process similar sets of odors (parallel processing), the two APTs most likely extract different stimulus parameters from similar odors. To address this issue either simultaneous recordings from glomeruli in both AL hemilobes by optical imaging and/or simultaneous electrophysiological recordings from both APTs have to be performed. Furthermore, information about physiological and synaptic properties of the microcircuits postsynaptic to both APTs is important to address processing at the next synaptic level (Frambach et al., 2004; Groh et al., 2006; Zube et al., 2008; Stieb et al., 2010). Our present study revealed first insights in potential scenarios for the evolution of olfactory tract diversity and a possible basis of highly sophisticated coding strategies in the olfactory system of Hymenoptera. Acknowledgements We are grateful to Cornelia Grübel and Malu Obermayer for expert technical assistance in histological procedures and confocal

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microscopy, to Monika Hilker (Free University Berlin) and Caroline Müller (University of Bielefeld) for kindly providing D. pini and A. rosae and for sharing their knowledge about sawflies, to Martin Kalthenpoth (Max-Planck Institute for Chemical Ecology Jena) for kindly providing Philanthus triangulum, and to Sara Stieb for help with preparations of sawflies. We also thank Christina Kelber for excellent asscitance with confocal scans and 3D reconstructions of glomeruli in Symphyta. This work was supported by DFG SFB 554 (A8) and SPP 1392 (RO 1177/5-1).

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