Hansson, B.

Harzsch, S.

Sombke, A.

English

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Brain structure of Scutigera coleoptrata: New insights into theevolution of mandibulate olfactory centres – short communication –Andy Sombke1*, Steffen Harzsch1,2 & Bill S. Hansson11 Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology. Hans-Knöll-Straße 8, 07745Jena, Germany; e-mail: asombke@ice.mpg.de2 Ernst Moritz Arndt Universität Greifswald, Zoologisches Institut. Cytologie und Evolutionsbiologie. Johann-Sebastian-Bach-Straße 11/12. 17497 Greifswald, Germany*Corresponding authorAbstractMyriapods represent an arthropod lineage, originating from a marine arthropod ancestor that mostlikely conquered land independently from hexapods. The successful transition from marine to terrestriallife requires a number of physiological adaptations important for survival out of water. The sensoryorgans of terrestrial species must be able to function in air rather than in water. In chemoreception,establishing aerial olfaction requires molecules to be detected in gas phase instead of in water solution.In general, the neuroethology of myriapods and the architecture of their central nervous systems arepoorly understood. In a set of preliminary experiments with the centipede Scutigera coleoptrata, weanalysed the central olfactory pathway with serial semi-thin sectioning combined with 3Dreconstruction, antennal backfilling with neuronal tracers, and immunofluorescence combined withconfocal laser-scanning microscopy. These experiments indicate that S. coleoptrata possess the neuronalsubstrate for a good sense of aerial olfaction. However, the architecture of its olfactory system is clearlydistinct from hexapods and also from terrestrial crustaceans, indicating independent evolution of itsolfactory sense.Keywords: Chilopoda, nervous system, olfaction1. IntroductionAs descendants from marine ancestors, Hexapoda and Myriapoda represent two cladesthat, considering the latest molecular analyses as well as neurophylogenetic studies (e.g.Harzsch 2006, 2007), conquered land independently from each other. The successfultransition from marine to terrestrial life requires a number of physiological adaptations e.g.gas exchange, salt and water balance, nitrogenous excretion, thermoregulation, moulting,and reproduction (Powers & Bliss 1983, Burggren & McMahon 1988). Concerning thenervous system, the sensory organs of terrestrial species must be able to function in air ratherthan in water. For olfaction, a transition from sea to land requires molecules to be detectedin gas phase instead of in water solution. The odour stimulus also changes from mainlyhydrophilic molecules in aqueous solution to mainly hydrophobic in the gaseous phase. Inaddition, the olfactory system is, like the rest of the organism, very prone to desiccation andSOIL ORGANISMS Volume 81 (3) 2009 pp. 319–325ISSN: 1864 - 6417mechanical abrasion in the terrestrial environment. All of these new selection pressures mayparticipate in reshaping the sense of smell (see e. g. Harzsch & Hansson 2008 for an exampleon terrestrial Crustacea). Furthermore, the structure of the central nervous system may alsomirror functional adaptations of the olfactory system to the terrestrial life style. Studying theolfactory system in Myriapoda and comparing it to that of Hexapoda may therefore provideinsights into how the arthropod nervous system evolved in response to new environmentaland ecological challenges.A wide variety of structures in the arthropod brain such as the central complex and the opticneuropils as well as eye development have been analysed against a phylogenetic background(Harzsch 2002, Loesel et al. 2002, Sinakevitch et al. 2003, Strausfeld 2005, Harzsch et al.2007). However, the comparative anatomy of the olfactory pathway has received lessattention, and conflicting hypotheses on the evolution of structures involved have beenbrought forward (Strausfeld & Hildebrand 1999 versus Schachtner et al. 2005). We havechosen the centipede Scutigera coleoptrata (Linnaeus 1758) as a model for terrestrialolfactory pathways. S. coleoptrata is a raptorial animal that preys on live arthropods and isamong the fastest-moving terrestrial arthropods (Rosenberg 2009). According to phylogeneticanalyses, the Scutigeromorpha are the most basal myriapod taxon (Edgecombe & Giribet2007) and therefore, their sensory systems may have retained aspects that are plesiomorphicfor the Myriapoda. In the present preliminary report, we provide a first overview overmorphological studies on the central olfactory pathway of this animal.2. Materials & methodsSpecimens were collected on the Balearic island Ibiza and kept in plastic boxes. For sectionseries, several individuals were decapitated and prefixed for 24h in a solution of 80 % ethanol,37 % formaldehyde and 100 % acetic acid. After washing in sodium hydrogen phosphatebuffer (PBS, pH 7.4) specimens were postfixed for 1h in 2 % OsO4 solution (same buffer) atroom temperature and, following dehydration in a graded series of acetone, embedded inAraldite. Serial semi-thin sections (1.5 µm) were prepared with a Microm HM 355 S andstained using 1 % toluidine blue and Pyronin G in a solution of 1 % sodium tetraborate.Overall, section series of 5 specimens were investigated. The alignment and 3-dimensionalreconstruction was made using AMIRA. For immunohistochemistry, specimens were fixedfor 4h at room temperature in 4 % parafomaldehyde in sodium hydrogen phosphate buffer(PBS, pH 7.4). The brains were dissected, washed 4 x 30 min in PBS, embedded in agarose,and subsequently sectioned (80 µm) with a vibratome (Microm HM 650 V). Permeabilisationof the brains in PBS-TX (PBS, 0.3 % Triton X-100) for 1 h was followed by incubation inprimary antibodies against synaptic proteins (Synapsins, Klagges et al. 1996), theneuropeptide FMRF-amide (compare Harzsch and Hansson 2008), Allatostatin (generouslyprovided by Prof. Dr H. Agricola, Vitzthum et al. 1996), and Tyrosine-Tubulin (Sigma), aswell as in phalloidin (a probe against actin), and the HOECHST stain (nuclear marker). Brainswere incubated over night at 4 °C, washed 4 x 30 min in PBS and incubated for 1 h in acocktail of secondary antibodies conjugated with Alexa 488 or Alexa 564 (compare Harzschand Hansson 2008 for a detailed description of the methods). After incubation, the sectionswere washed 4 x 30 min in PBS and mounted. For antenna-backfills, the left antenna was cutleaving app. 3 mm. Then, a capillary filled with Dextranbiotin (Molecular Probes) dissolvedin aqua dest. was placed over the cut tip of the antenna. After incubation for 4 hours, theAndy Sombke et al.320specimen were decapitated and fixed in 4 % paraformaldehyde. Following dissection, brainswere washed 4 x 30 min in PBS and subsequently sectioned (80 µm) with a vibratome.Specimens were incubated in streptavidin conjugated to Alexa 488 (1:2500 in PBS) over nightat 4 °C. After washing in PBS sections were subsequently mounted in Mowiol.3. Results and discussionStudies on the anatomy and microarchitecture of the nervous system of Scutigeracoleoptrata have been conducted by Saint-Remy (1889) and Hörberg (1931). Fahlander(1938) described the central nervous system of Thereuopoda clunifera (Wood, 1862)(Scutigeromorpha). The deutocerebrum of Scutigeromorpha is filled with two glomerularmasses. The structure of the anterior mass was described by Saint-Remy (1889) and Hörberg(1931) as irregular, ‘sausage-like’, or as convoluted ribbons. In T. clunifera, Fahlander(1938) specified the deutocerebral lobe neuropils as numerous, dense glomerular masses thatare circular in diameter. Our neuroanatomical data show that the deutocerebral lobes of S.coleoptrata are filled with dense neuropils that take on the shape of elongate cylinders (Figs1A, B, C). In histological sections, the neuropils are highly contrasted. Because of theirelongate shape we suggest they be referred as olfactory neuropils rather than gomeruli orglomerular masses. The olfactory neuropils are bilaterally symmetrically arranged, so that itis possible to match the corresponding neuropils in the left and right deutocerebral lobe(Fig. 2C). In three histological section series, we found 34 individually identifiable neuropilsin each lobe (Fig. 2D). All of them had a more or less cylindrical form, while the distal endwas thickened or bent posteriorly. At least one neuropil extended a contralateral connection(Figs 1D clc, 2C) as described by Fahlander (1938) as anterior antennal commissure. Incontrast to crustacean olfactory neuropils (Harzsch & Hansson 2008), in all preparations,both core and periphery of all observed neuropils were uniformly stained. No evidence forsubcompartments was found.Immunolocalisation of synaptic proteins and phalloidin histochemistry confirmed thecylindrical form of the neuropils (Figs 1B, C, E). The immunolocalisation of FMRF-amidein the deutocerebral lobes showed a diffuse distribution pattern and an absence of labellingin the olfactory neuropils (Fig. 1E). Instead, only fibres surrounding the olfactory neuropilswere stained. Projecting tracts were not visible. Allatostatin-immunoreactivity was presentthroughout the deutocerebral lobes. The olfactory neuropils were filled with numerous fineimmunolabeled profiles (Fig. 1F).By backfilling the antennal nerve with the anterograde axonal marker Dextranbiotin, weobtained reliable information concerning the innervation pattern of the neuropils (Fig. 1D).Based on these preparations, we were able to verify that antennal input does in fact target alldeutocerebral lobe neuropils. The olfactory neuropils and the contralaterral connection wereclearly stained. Filled tracts also project to the dorsal protocerebrum and deep into thesuboesophageal ganglion. These experiments suggest that S. coleoptrata possesses theneuronal substrate for a good sense of aerial olfaction.Brain structure of Scutigera coleoptrata 321Andy Sombke et al.322Fig. 1 A: Histological cross-section of the head of Scutigerea coleoptrata. dc: deutocerebrum; fg:frontal ganglion; na: nervus antennalis; on: olfactory neuropils; pc: protocerebrum; B:Immunolocalisation of synaptic proteins (red) and phalloidin labelling (green) in the rightantennal lobe; C: Immunolocalisation of synaptic proteins (green) and phalloidin labelling(red) in the left antennal lobe (horizontal section); D: Dextranbiotin backfill of the leftantennal lobe. cl: corpus lamellosum; clc: contralateral connection; tr: tracts; E:Immunolocalisation of synaptic proteins (red) and FMRF-amide (green) in the left antennallobe; F: Immunolocalisation of Tubulin (red) and Allatostatin (green) (sagittal section).Arrows mark colocalisation in single olfactory neuropils.All scale bars 100 µm. Arrow abbreviations: d: dorsal; m: median; f: frontal; c: caudal; v:ventral.The posterior deutocerebral mass is innervated by the ventrocaudal part of the antennalnerve and is called corpus lamellosum or masse lamelleuse (after Saint-Remy 1889). Hörberg(1931) described it as glomerular structure composed of parallel tracts. Immunolocalisationof synaptic proteins and phalloidin histochemistry as well as antenna backfill experimentsrevealed a composition of approx. ten lamellae (Figs 1D, E, 2A, B). The lamellae can bedivided into two different types: (1) outer lamellae that form a loop (not shown) and (2) innerlamellae that project dorsomedially (Fig. 2B). Fahlander (1938) observed that the corpora areconnected over a tract within the posterior antennal comissure. In contrast to observations inScolopendromorpha (Sombke, unpublished), in S. coleoptrata this contralateral connectionwas not present in any of our preparations.The function of the corpus lamellosum is unknown.We speculate that it may process mechanosensory input from the antennae comparable to thelateral neuropil in Crustacea and the mechanosensory neuropil in Hexapoda.Brain structure of Scutigera coleoptrata 323Fig. 2 A: Histological cross-section. cl corpus lamellosum, dc deutocerebrum, pc protocerebrum,tc tritocerebrum; B: Immunolocalisation of synaptic proteins (red) and phalloidin labelling(green) in the right antennal lobe; C: 3D-reconstruction of the brain. cl corpus lamellosum(yellow), on olfactory neuropils (blue); D: 3D-reconstruction of the neuropils in the leftdeutocerebral lobe.All scale bars 100 µm. Arrow abbreviations: d: dorsal; m: median.In hexapods and malacostracan crustaceans, the axons of the olfactory receptor neuronstarget the primary chemosensory neuropil where the receptor axons synapse with localolfactory interneurons and projection neurons (reviewed in Schachtner et al. 2005). Theneuropils in the deutocerebral lobes are organised in numerous spherical glomeruli in mostinsects and in elongate columnar glomeruli in crustaceans. In-depth comparison of specieswithin and across tetraconate taxa, however, demonstrates that many characters of theorganisation of tetraconate olfactory centres are shared among distantly related clades, buthave been modified in various taxon-specific ways (reviewed in Schachtner et al. 2005). Incontrast, Strausfeld & Hildebrand (1999) are doubtful concerning a homology of the primaryolfactory neuropils in insects and crustaceans. Our results show that, clearly, the shape of theolfactory neuropils in S. coleoptrata is different from both that in hexapods and malacostracancrustaceans. This suggests that upon conquering land, the Myriapoda followed their owndistinct pathway in evolving an olfactory system that is suited for aerial olfaction.Nevertheless, in our view the possession of distinct neuropils for chemosensory andmechanosensory procession in Scutigera coleoptrata, malacostracan Crustacea and Insectacould indicate a common architectural principle within the Mandibulata.4. AcknowledgementsWe wish to thank Prof. Dr E. Buchner (Würzburg) and Prof. Dr H. Agricola (Jena) forgenerously providing antibody samples. Our special thanks go to Dr C. H. G. Müller(Greifswald) for providing experimental animals, Dr J. Rosenberg (Essen/Duisburg) and DrShannon Olsson for stimulating discussions.5. ReferencesBurggren, W. W. & B. R. McMahon (1988): Biology of the land crabs. – Cambridge University Press:492 pp.Edgecombe, G. & G. Giribet (2007): Evolutionary Biology of Centipedes (Myriapoda: Chilopoda). –Annual Review of Entomology 52:151–70.Fahlander, K. (1938): Beiträge zur Anatomie und systematischen Einteilung der Chilopoden. –Zoologiska Didraf fran Uppsala 17: 1–148.Harzsch, S. (2002): Phylogenetic significance of the crustacean optic neuropils and chiasmata: a re-examination. – Journal of Comparative Neurology 453: 10–21.Harzsch, S. (2006): Neurophylogeny: Architecture of the nervous system and a fresh view on arthropodphylogeny. – Integrative and Comparative Biology 46: 162–194.Harzsch, S. (2007): Architecture of the nervous system as a character for phylogenetic reconstructions:examples from the Arthropoda. – Species, Phylogeny and Evolution 1: 33–57.Harzsch, S. & B. Hansson (2008): Brain architecture in the terrestrial hermit crab Coenobita clypeatus(Anomura, Coenobitidae): neuroanatomical evidence for a superb aerial sense of smell. – BMCNeuroscience 9: 1–35.Harzsch, S., R. R. Melzer & C. H. G. Müller (2007): Mechanisms of eye development and evolution ofthe arthropod visual system: the lateral eyes of Myriapoda are not modified insect ommatidia. –Organism Diversity & Evolution 7: 20–32.Hörberg, T. (1931): Studien über den komparativen Bau des Gehirns von Scutigera coleoptrata L. –Lunds universiets absskrift 27(19): 1–24.Andy Sombke et al.324Klagges, B. R. E., G. Heimbeck, T. A. Godenschwege, A. Hofbauer, G. O. Pfulfelder, R. Refegerste, D.Reisch, M. Schaupp, S. Buchner & E. Buchner (1996): Invertebrate synapsins: a single gene code forseveral isoforms in Drosophila. – The Journal of Neuroscience 16: 3154–3165.Loesel, R., D. R. Nässel, & N. J. 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Strausfeld (2003): Conserved andconvergent organization in the optic lobes of insects and isopods, with reference to other crustaceantaxa. – Journal of Comparative Neurology 467: 150–172.Strausfeld, N. J. (2005): The evolution of crustacean and insect optic lobes and the origin of chiasmata.– Arthropod, Structure and Development 34: 235–256.Strausfeld, N. J. & J. G. Hildebrand (1999): Olfactory systems: common design, uncommon origins? –Current Opinion in Neurobiology 9: 634–639.Vitzthum, H., U. Homberg, & H. Agricola (1996): Distribution of Dip-Allatostatin I-likeimmunoreactivity in the brain of the Locust Schistocerca gregaria with detailed analysis ofimmunostaining in the central complex. – Journal of Comparative Neurology 369: 419–437.Accepted 26 February 2009Brain structure of Scutigera coleoptrata 325

Brain structure of Scutigera coleoptrata: New insights into the evolution of mandibulate olfactory centers - short communication -

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Brain structure of Scutigera coleoptrata: New insights into the evolution of mandibulate olfactory centers - short communication -

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