Antennal-Lobe Organization in Desert Ants of the Genus Cataglyphis

Desert ants of the genus Cataglyphishave become model systems for the study of animal navigation in general, and visually guided behavior in particular [Wehner, 2003]. While foraging they employ a path-integrating system [for a review, see Wehner and Srinivasan, 2003] including a skylight-based visual compass [Wehner and Müller, 2006] and a stride-integrating odometer [Wittlinger et al., 2006] as their main navigational means. In addition, they use landmarks to define places and routes [Wehner and Räber, 1979; Collett et al., 1992; Bisch-Knaden and Wehner, 2003; Collett and Collett, 2009]. Even though they establish quite pronounced individual sector fidelities, i.e. they leave the nest and return to it along familiar vectors or landmark-defined routes, during their foraging lives [Wehner et al., 2004], they do not use trail pheromones [Schmid-Hempel, 1983; Wehner et al., 1983], as it is the case in many other ant species [Hölldobler and Wilson, 1990]. However, they rely on chemical cues for pinpointing food sources [Wolf and Wehner, 2000]. Furthermore, in the vicinity of the nest, they can use olfactory landmarks for homing [Steck et al., 2009, 2010]. Ants, in general, employ a variety of olfactory signals in the context of sexual communication, nestmate recognition and caste discrimination, alarm or recruitment behavior, or trail marking. In fact, chemical communication is the most prominent type of social interactions among ants [Hölldobler and Wilson, 1990; Kleineidam and Rössler, 2009]. Therefore, the question arises whether the olfactory system of the visual navigator Cataglyphis is specialized and/or adapted to the particular means of foraging and homing under the extreme ecological conditions of desert environments inhabited by Cataglyphis species.

In ants as in all insects, odor information is received via olfactory receptor neurons (ORNs) located in the olfactory sensilla on the antennae. The axons of ORNs project to the first olfactory neuropil, the antennal lobe (AL), and terminate in the functional units of the AL, the glomeruli [for reviews, see Homberg et al., 1989; Mustaparta, 1990; Hildebrand and Shepherd, 1997; Hansson and Anton, 2000; Kleineidam and Rössler, 2009; Galizia and Rössler, 2010]. In social Hymenoptera, the incoming axons of ORNs are sorted into several sensory tracts, which innervate different clusters of glomeruli [Kirschner et al., 2006; Zube et al., 2008; Kelber et al., 2010]. Excitation of ORNs by odors results in a spatial activation pattern of different glomeruli, e.g. in honeybees [Joerges et al., 1997; Galizia et al., 1999b; Sachse et al., 1999], Drosophila [Fishilevich and Vosshall, 2005], and in ants [Zube et al., 2008; Kuebler et al., 2010]. In several insect species, sex-pheromone-sensitive ORNs in males of moths [Anton and Homberg, 1999] or honeybee drones [Arnold et al., 1985; Sandoz, 2006], for example, send their projections to a single or to several enlarged glomeruli, the so-called macroglomeruli (MGs) or MG complexes (MGCs). In non-sexual individuals of leaf-cutting ants (Atta and Acromyrmex), an MG was found in large workers [Kleineidam et al., 2005; Kelber et al., 2009], where it is involved in the detection and processing of trail pheromone components [Kuebler et al., 2010].

As in Cataglyphisbehavioral studies have shown that olfactory cues are used during various phases of the ants’ foraging journeys [Wolf and Wehner, 2000, 2005; Steck et al., 2009, 2010], we inquired about potential adaptations in the primary olfactory centers of Cataglyphis. In particular, we investigated the glomerular organization of the AL of 5 species of Cataglyphisby focusing on the number, size, and spatial arrangement of the olfactory glomeruli. We addressed the question of whether differences in microhabitats and resulting variances in foraging strategies (e.g. lengths of outbound runs) have led to adaptations in the olfactory system of different Cataglyphis species inhabiting different desert environments (steppe-like, sandy, or salt pan areas). In particular, C. fortis is endemic to the salt flats of western North Africa, which are completely avoided by other Cataglyphisspecies [Wehner, 1983]. The utilization of such a specialized habitat results in outstanding traits, e.g extremely large foraging ranges [Wehner, 1987] and high nest-site stabilities [Dillier and Wehner, 2004], compared to other Cataglyphisspecies. In fact, C. fortis shows an enlarged glomerulus near the AL entrance that is absent in other Cataglyphis species.Hence, the AL structure of C. fortis workers is analyzed in more detail. Furthermore, we compare the number and size of glomeruli in different C. fortis castes (workers, queens, and males). Finally, we include 2 species of the wood ant Formica, which inhabits mesic environments and employs trail pheromone recruitment. Even though ecologically different from Cataglyphis, Formica is phylogenetically very closely related to Cataglyphis, as both genera belong to the same subfamily of ants (Formicinae) and even to the same tribe (Formicini).

In total 5 Cataglyphis species and 2 Formica species were compared regarding their AL anatomy: C. fortis(Forel, 1902), C.albicans(Roger, 1859), C. bicolor (Fabricius, 1793), C.rubra(Forel, 1903), C.noda(Brullé, 1832), F. sanguinea(Latreille, 1798), and F.rufibarbis (Fabricius, 1793). Workers and males/females of C. fortis were collected in a salt pan near Menzel Chaker (348580N/108250E), and workers of C.albicans, C.rubra,and C.bicolor were obtained from shrubby habitats around the salt pan as well as near Mahares (343149N/102949E), Tunisia, in July 2007 and July 2010. Workers of C. nodacame from the southern Peloponnese (370166N/222639E), Greece (collected in June 2007). All Cataglyphis ants were either transferred alive to the Biocenter of the University of Würzburg, or dissection and fixation of the brains were conducted on-site. The phylogenetic relationships between the Cataglyphis species examined in the present account are depicted in figure 1.

Workers of F. rufibarbis and F.sanguinea were collected in the surroundings of the Biocenter of the University of Würzburg (494644N, 95817E) in April 2010.

For 3D reconstruction of single glomeruli in the AL, all brains were treated as described in the following: the ants were anesthetized with CO₂, decapitated, and the head capsules were fixed in dental-wax-coated dishes. The head capsule was covered with fresh ant saline solution (127 mM NaCl, 7 mM KCl, 1.5 mM CaCl₂, 0.8 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 4.8 mM TES, and 3.2 mM trehalose, pH 7.0) and opened by cutting a square window in-between the compound eyes. Glands and tracheae were gently removed, and the brains were dissected and fixed immediately in ice-cold Fix-Mix (2% paraformaldehyde/2% glutaraldehyde) in phosphate-buffered saline (PBS, pH 7.2) and stored for 3–30 days in the fridge. This fixation leads to increased autofluorescence which, compared to formaldehyde fixation only, allowed us to better identify the outlines of glomeruli [Kelber et al., 2009]. The brains were then rinsed in PBS (3×, 10 min), dehydrated in an ascending series of ethanol (50, 70, 80, 90, 95, and 3× 100%, 10 min each), and finally transferred into methylsalicylic acid (M-2047; Sigma-Aldrich, Steinheim, Germany).

To trace the sensory tracts of antennal ORN axons subdividing the AL glomeruli into clusters and later localize these clusters, anterograde stainings of the antennal nerve were conducted in C. fortis workers (n = 2). The ants were immobilized in a plexiglass holder, and 1 antenna was cut off at the base of the scapus. A droplet of tetramethylrhodamine dextran with biotin (‘micro-ruby’, D-7162; Molecular Probes, Eugene, Oreg., USA) dissolved in distilled water was applied in a ring of wax on the cut base and left in place for 1 h. After staining, workers were allowed to move freely for 4 h before the brains were dissected in saline solution followed by fixation with 4% paraformaldehyde in PBS (pH 7.2) and dehydration as described above.

To visualize the synaptic neuropil in individual glomeruli in more detail at higher magnification, preparations were doubly labeled with an antibody to synapsin and f-actin-phalloidin staining (methods according to Rössler et al. [2002]). Brains were dissected, fixed in cold 4% formaldehyde in PBS (pH 7.2) overnight, and sectioned at 100 µm thickness with a vibrating microtome (Leica VT 1000S; Leica, Nussloch, Germany). Afterwards, they were simultaneously incubated in Alexa Fluor 488 phalloidin (A12379; Molecular Probes) and in monoclonal antibody to the Drosophila synaptic-vesicle-associated protein synapsin I (1:50, SYNORF1, kindly provided by E. Buchner, University of Würzburg) followed by incubation in Alexa-Fluor-568-conjugated goat anti-mouse secondary antibody (1:250, A11004; Molecular Probes); precise descriptions have been published by Rössler et al. [2002]; Groh et al. [2004], and Stieb et al. [2010].

Confocal images and image stacks were taken at a resolution of 1,024 × 1,024 pixels and an optical thickness of 1 µm from the ALs in whole-mount preparations using a laser-scanning confocal microscope (Leica TCS SP2 AOBS, 20 × 0.7 NA and 63 × 1.20 NA lens; Leica Microsystems AG, Wetzlar, Germany). 3D software (AMIRA 3.1.1; Mercury Computer Systems, Berlin, Germany) was used for reconstruction of individual glomeruli within the AL, sensory tracts, and the antennal nerve (AN). The total number and volume of all glomeruli were assessed by complete AL reconstructions in 17 ants in total (C. fortis: workers n = 4; males n = 2, and queens n = 2; C. albicans: workers n = 3; C. bicolor: workers n = 1; C. noda: workers n = 1; C. rubra: workers n = 1; F. sanguinea: workers n = 2, and F. rufibarbis: workers n = 1; table 1). Additional Cataglyphis specimens were analyzed by visual inspection.

In order to compare the size of glomeruli between different ALs and due to the fact that the number and size of glomeruli in the AL varied considerably between the investigated species, we used a relative measurement of the glomerular volume. Therefore, the median (all data were not normally distributed: Kolmogorov-Smirnov procedure, p < 0.05) of all glomerular volumes was calculated for each AL to subsequently compute the ratio of the volume of each glomerulus and the median volume (method modified after Kelber et al. [2009]). This normalized volume describes how many times bigger a single glomerulus is compared to the median size of all glomeruli. Boxplots were used to illustrate the normalized volume values of all glomeruli within 1 AL (fig. 2). Outliers were defined as cases with values that are 1.5–3 box lengths, extreme values (with the largest glomerulus termed ‘enlarged glomerulus’) as cases with values >3 box lengths distant from the upper and lower margins of the box, whereas the length of the box equals 1 interquartile range. All descriptive statistical analyses were performed with SPSS 18.0 software (SPSS, Chicago, Ill., USA).

In the non-sexual caste of the 5 Cataglyphis species tested in this account, the average number of glomeruli in the AL was lowest in C. fortis (198 ± 7 glomeruli; table 1). In workers of C. albicans and C. noda, the 3D reconstructions revealed about 226 glomeruli, while C. bicolor and C. rubra workers had maximal numbers, namely 249 glomeruli. Compared to workers, males of C. fortis had a reduced number of 150 ± 2 glomeruli, whereas C. fortis queens had a slightly higher number of 219 ± 3 glomeruli. In comparison to the 2 Formica species (about 370 glomeruli), all tested Cataglyphis species had a smaller number of glomeruli.

In workers of all Cataglyphis species, the size of the glomeruli ranged from 424 to 42,683 µm³. The difference between the smallest and the largest glomeruli (table 1) was the highest in C. fortis(see numbers given above). With respect to the normalized glomerular volumes, all workers of the Cataglyphis and Formica species showed several outliers. Extreme values were found in workers of all species with the exception of C. noda. Among the Cataglyphis workers, C. fortis possessed the biggest singular glomerulus (up to 7.2 times bigger) compared with the median size of all glomeruli within 1 AL (fig. 2).

In the sexual castes of C. fortis, the size ranges of glomerular volumes were smaller for queens (1,027–21,302 µm³) and largest for males (672–90,342 µm³; table 1). Regarding the normalized volumes, C. fortis queens exhibited extreme values (besides several outliers) that lay within the range of those of the worker caste (fig. 2). In contrast, the extreme values of C. fortis males were up to 21.6 times larger than the median size of all glomeruli (again besides several outliers; fig. 2). These severely enlarged male-specific glomeruli are termed MGs in the following (using the criteria described by Kelber et al. [2009]).

The largest glomerulus within 1 AL (as defined by the criteria mentioned in the Materials and Methods section and termed ‘enlarged glomerulus’; fig. 2) could be well localized in the 3D reconstructions (fig. 3). All enlarged glomeruli in workers of C. rubra and C. bicolor as well as the enlarged glomeruli of 2 C. albicans workers were located in a group of glomeruli occupying the dorsalmost part of the AL. In the 3rd C. albicans worker, the enlarged glomerulus was located near, but not directly at, the entrance of the AN. In all C. fortis specimens, the largest glomeruli of the workers were located at the same position close to the entrance of the AN (fig. 3a; enlarged glomerulus; yellow). F-actin phalloidin and anti-synapsin labeling confirmed the existence of a glomerulus containing very large synaptic neuropil located directly adjacent to the AN (fig. 4b). Anterograde mass stainings of ORN axons demonstrated that the enlarged glomerulus at the AN entrance is innervated by a high number of ORN axons (fig. 4b, c). Figure 4c clearly shows that all other glomeruli in the same focal plane are less innervated than the enlarged glomerulus. The remaining glomeruli of C. fortis workers with extreme values were located either in the already mentioned dorsalmost part of the AL or more laterally.

In C. fortis queens, the largest glomerulus was located in a position corresponding to that of C. fortis workers, i.e. close to the AN entrance (fig. 3b). In both C. fortis males, the largest glomerulus was found medial to the AN with 1 normal-sized glomerulus in between the AN entrance and the largest glomerulus (fig. 3c). The other glomeruli identified as extreme values of both males and queens were located in different positions in the AL. We would also like to mention 2 glomeruli identified as extreme values which in males are located at the dorsalmost part of the AL, near the dorsal lobe (DL).

In both Formica species, all enlarged glomeruli were found in different locations in the AL even within the same species.

Mass labeling of ORN axonal projections allowed the classification of sensory-tract-specific glomerular clusters. The cluster classification was done for 2 C. fortis workers: 1 by using 3D reconstruction and 1 by performing careful visual inspection. Figure 4c shows a confocal image with the enlarged glomeruli clearly visible, while figure 4d provides the corresponding 3D reconstruction. In C. fortis workers, the ORN axons form 4 distinct sensory tracts (T1–T4) innervating 4 characteristic clusters of glomeruli within the AL. In the following, these glomerular subregions are termed T1–T4 clusters (fig. 4d). T1 proceeds at the ventral surface of the AL and innervates 37 glomeruli of the T1 cluster (blue glomeruli in fig. 4d) located on the ventral part of the AL flanked laterally by the T2 (green glomeruli in fig. 4d) and medially by the T3 (red glomeruli in fig. 4d) cluster. The T2 cluster comprises 69 glomeruli spreading along the lateral side of the AL from ventral to dorsal. Similarly, the medial-situated T3 cluster disperses from ventral to dorsal and comprises the highest number of glomeruli (85 glomeruli). Next to the AN, this cluster contains the enlarged glomerulus, whereas the dorsal part of this cluster consists of a high number of small glomeruli. Only 18 glomeruli form the T4 cluster (yellow glomeruli in fig. 4d) located on the dorsalmost AL region near the DL. In contrast to the ORNs of sensory tracts T1–T3, the ORNs of the T4 subpopulation innervate not only the cortical layer but also the entire glomerulus, being similar to findings in the honeybee [Arnold et al., 1985]. One tract, from which the T4 tract is emerging, projects toward the DL.

Many insect species exhibit caste- and sex-specific variations in general features of the ALs. The behavioral repertoires of hymenopteran males are in general restricted, which in turn results in a reduced number of glomeruli compared to females (Apis mellifera [Arnold et al., 1985; Brockmann and Brückner, 2001; Sandoz, 2006]; Harpegnathos saltator [Hoyer et al., 2005]; Camponotus floridanus [Zube and Rössler, 2008]; Camponotus japonicus [Nishikawa et al., 2008],and Atta vollenweideri [Kuebler et al., 2010]). Analyses of the number of glomeruli in C. fortis males confirm these findings. Within the female caste, C. fortis queens have a higher number of glomeruli than workers and males. Other studies in ants showed that the number of glomeruli in queens exceeded that in males, but was still lower or comparable to the numbers found in workers (A. vollenweideri [Kuebler et al., 2010]; A. mellifera [Groh and Rössler, 2008]; C. japonicus [Nishikawa et al., 2008], and C. floridanus [Zube and Rössler, 2008]). That this ratio is reversed in C. fortis might most likely be due to the especially low number of glomeruli in the workers rather than to a relatively larger number of glomeruli in the queens.

With a mean of 198 glomeruli, C. fortis has by far the lowest glomerular number among all Cataglyphis species tested in this account (see table 1: from 198 glomeruli in C. fortis to 249 glomeruli in C. rubra and C.bicolor). Compared to other hymenopteran species (156–166 glomeruli in the honeybee [Arnold et al., 1985; Flanagan and Mercer, 1989; Galizia et al., 1999a; Kirschner et al., 2006] and 186–198 glomeruli in 2 parasitoid wasps [Smid et al., 2003]), ant workers in general have a higher number of glomeruli per AL: the highly olfactory fungus-growing ant species possess 257–630 glomeruli [Kelber et al., 2009; Kuebler et al., 2010]; likewise, the group-recruiting and trail-following carpenter ants [Haak et al., 1996] also have high glomerular numbers(430 glomeruli in C. japonicus [Nishikawa et al., 2008] and 434 glomeruli in C. floridanus [Zube and Rössler, 2008]). Formica species, which have been tested in this account, perform chemical recruitment [Hölldobler and Wilson, 1990] which nicely correlates with high numbers of about 370 glomeruli in the 2 tested species (table 1). In contrast to all the ant species mentioned above, Cataglyphis ants forage solitarily and do not use trail pheromones, but rely primarily on visual guidance mechanisms [Wehner, 2003].

The interspecific variations in glomerular numbers indicate that the number of glomeruli appears to be generally correlated with the amount of chemically mediated behaviors. Ant species with a pronounced olfactory-guided behavior like the use of a trail pheromone have a high glomerular number [Kleineidam and Rössler, 2009]. The results in Cataglyphis indicate that species relying more on visual cues may possess a reduced glomerular number. This hypothesis is further supported by a small number of glomeruli in the Namibian desert ant (223 glomeruli [Wenzler, pers. commun.]), which in contrast to Cataglyphisoccasionally exhibits a particular kind of recruitment behavior [Marsh, 1985; Wehner, 1987]. In the same way, Gigantiops destructor, which is characterized by huge compound eyes and impressive capabilities of visually detecting moving objects and navigating by visual means [Hölldobler and Wilson, 1990, pp. 259; Gronenberg and Hölldobler, 1999; Beugnon et al., 2001], as well as the mostly visually guided ponerine ant H. saltator have low numbers of glomeruli (250 glomeruli in G. destructor [Kelber, pers. commun.]; 200 glomeruli in Formica pratensis [Goll, 1967], and around 177 glomeruli in H. saltator [Hoyer et al., 2005]).

The assumed correlation between a reduced glomerular number in C. fortis and the largely visually guided behavior of this species goes along with comparative studies among different ant species showing that Cataglyphishas not only relatively large optic lobes, but also possesses an exceedingly large collar – the visual input region of the learning- and memory-associated mushroom bodies [Gronenberg and Hölldobler, 1999; Kühn-Bühlmann and Wehner, 2006].

In C. fortisworkers, the AN is divided into several sensory tracts of which 4 innervate the AL glomeruli. The grouping of glomeruli into a variable number of clusters is a common feature in Hymenoptera and allows for interspecific comparisons and potential phylogenetic inferences. In the honeybee, there are 4 glomerular clusters as in C. fortis [Arnold et al. 1985; Flanagan and Mercer, 1989; Galizia et al., 1999a; Kirschner et al., 2006], but 6 and 7 sensory tracts innervate the AL glomeruli in the ants A. vollenweideri[Kelber et al., 2010] and C. floridanus[Zube et al., 2008] as well as C. japonicus[Nakanishi et al., 2010], respectively. The reduced number of glomerular clusters in C. fortis compared to other ant species correlates with the small number of glomeruli and might be indicative of the importance of vision rather than olfaction in this species.

A sexual dimorphism in the AL structure based on sex pheromone communication is reported for a number of insect species [for review, see Rospars, 1988; Galizia and Rössler, 2010]. The occurrence of an MG or an MGC always located near the AN entrance and exclusively receiving input from sex-pheromone-sensitive ORNs has been described for males of several insect species, e.g. moths [Hansson et al., 1991; Anton and Homberg, 1999; Rössler et al., 1999] and honeybees [Arnold et al., 1985; Sandoz, 2006]. The increased size of MGs results from a high number of terminating ORNs [Vickers and Baker, 1997; Berg et al., 1998]. Similarly, in males of several ant species, enlarged glomeruli have been described (H. saltator [Hoyer et al., 2005]; A. vollenweideri [Kuebler et al., 2010], and C. japonicus [Nakanishi et al., 2010]), but their specific involvement in sex pheromone communication remains to be elucidated.

An MG near the AN entrance was also found in the males of C. fortis. The MG location differs from that of the enlarged glomeruli found in the female castes. Due to the fact that this MG was only found in C. fortis males, it is likely that sex pheromone detection plays a prominent role for the male during courtship and mating behavior. In some Cataglyphisspecies, it has been shown that the males respond to the sexual ‘calling behavior’ of potentially pheromone-emitting females (C. iberica [Cerdá, 1988]; C. cursor [Lenoir et al., 1988], and C. savignyi [Wehner and Wehner, unpubl. data]).

With only one exception, all enlarged glomeruli in the workers of C. rubra, C. bicolor and C. albicans are located in a group of glomeruli constituting the dorsalmost part of the AL nearest to the DL. Analyses of the sensory tracts in C. fortis revealed that these glomeruli belong to the T4 cluster. Based on the conspicuous position of this dorsalmost cluster of glomeruli and the fact that some of these glomeruli are enlarged in all tested Cataglyphis species, we assume that the corresponding glomeruli in all Cataglyphis workers belong to the T4 cluster and might be involved in the processing of CO₂-, thermo- or hygroreceptive information. In a corresponding region of the AL, a CO₂-sensitive glomerulus has been described for several moth species [Bogner et al., 1986; Kent et al., 1986; Lee and Altner, 1986; Kent et al., 1999; Guerenstein et al., 2004], and thermo- and hygroreceptive glomeruli were identified in cockroaches, honeybees, and carpenter ants [Nishikawa et al., 1995; Nishino et al., 2003, 2009; Nakanishi et al., 2010].

In C. fortis workers, the largest glomerulus is located next to the AN entrance. An MG not involved in the processing of sex pheromone information was discovered for the first time in leaf-cutting ants [Kleineidam et al., 2005]. There the MG is found only in large workers and is located close to the AN. Physiological as well as anatomical studies indicate that this MG is involved in the detection and processing of trail pheromone components [Kleineidam et al., 2005; Kelber et al., 2009, 2010; Kuebler et al., 2010]. These studies clearly demonstrate that the occurrence of enlarged glomeruli or even MGs may vary substantially across ant species and even within the same species, and may correlate with certain behavioral requirements of odor processing. Despite the fact that the enlarged glomerulus of C. fortis workers does not fulfill all the criteria of an MG (if we use the criteria used for defining an MG by Kelber et al. [2009]), its size, stable location among tested workers, and its absence in all other tested Cataglyphis species indicate a specialized function of this glomerulus. As the backfill stainings clearly show that the enlarged glomerulus receives an increased input from ORN axons (fig. 4c), it is very likely that this glomerulus serves a particular and unique function that may be related to the extreme habitat in this species.

If we now recall that C. fortis possesses the AL with the lowest number of glomeruli among all tested Cataglyphis species and assume that the number of glomeruli reflects the complexity of olfactory-guided behavior, why is it that of all Cataglyphisspecies just C. fortishas an enlarged glomerulus at its AN entrance? Physiological (in particular functional imaging) studies are needed to characterize the odor specificity of the enlarged glomerulus. The fact that the enlarged glomerulus in the AL entrance of C. fortis is absent in the AL of the most closely related C. albicans(fig. 1) may indeed indicate that this glomerulus represents an adaptation to the special behavioral ecology of C. fortis,which among all its congeners takes an outstanding position regarding its preferred habitat, the food-impoverished salt pan flats of the Algerian and Tunisian chotts and sebkhas [Wehner, 1981, 1983; Wehner et al., 1994]. The North African C. bicolor, C. albicans and C. rubrainhabit the nutritionally richer low-shrub semi-deserts surrounding the salt pans [Dillier and Wehner, 2004]. To unravel which of the many differences in the behavioral ecology between C. fortis and its congeners [Schmid-Hempel, 1983; Wehner, 1983, 1987; Dillier and Wehner, 2004] are functionally related to the presence of the remarkably enlarged glomerulus in C. fortis remains a challenging task to accomplish.

We thank Nils Becker, Anna Hellwig, and Nadine Wenzler for providing valuable assistance during the field work in Tunisia and together with Linda Kuebler, Marieluise Obermayer and Anne-Kristin Wagner for their help with brain preparations and/or 3D reconstructions. We also thank Markus Knaden, Kathrin Steck and Sebastian Schwarz for providing us with Cataglyphis specimens. We thank the Graduate School of Life Sciences, University of Würzburg, for support (S.M.S. is an associated member). Funding was obtained from the German Research Foundation [DFG SFB 554 (A8) to W.R.], a woman grant (to S.M.S.), and the Humboldt Foundation (to R.W.).