Dippel et al. BMC Biology (2016) 14:90 DOI 10.1186/s12915-016-0304-z RESEARCH ARTICLE Open Access Morphological and Transcriptomic Analysis of a Beetle Chemosensory System Reveals a Gnathal Olfactory Center Stefan Dippel1†, Martin Kollmann2†, Georg Oberhofer3, Alice Montino1, Carolin Knoll2, Milosz Krala2, Karl-Heinz Rexer4, Sergius Frank2, Robert Kumpf5, Joachim Schachtner2* and Ernst A. Wimmer1* Abstract Background: The red flour beetle Tribolium castaneum is an emerging insect model organism representing the largest insect order, Coleoptera, which encompasses several serious agricultural and forest pests. Despite the ecological and economic importance of beetles, most insect olfaction studies have so far focused on dipteran, lepidopteran, or hymenopteran systems. Results: Here, we present the first detailed morphological description of a coleopteran olfactory pathway in combination with genome-wide expression analysis of the relevant gene families involved in chemoreception. Our study revealed that besides the antennae, also the mouthparts are highly involved in olfaction and that their respective contribution is processed separately. In this beetle, olfactory sensory neurons from the mouthparts project to the lobus glomerulatus, a structure so far only characterized in hemimetabolous insects, as well as to a so far non-described unpaired glomerularly organized olfactory neuropil in the gnathal ganglion, which we term the gnathal olfactory center. The high number of functional odorant receptor genes expressed in the mouthparts also supports the importance of the maxillary and labial palps in olfaction of this beetle. Moreover, gustatory perception seems equally distributed between antenna and mouthparts, since the number of expressed gustatory receptors is similar for both organs. Conclusions: Our analysis of the T. castaneum chemosensory system confirms that olfactory and gustatory perception are not organotopically separated to the antennae and mouthparts, respectively. The identification of additional olfactory processing centers, the lobus glomerulatus and the gnathal olfactory center, is in contrast to the current picture that in holometabolous insects all olfactory inputs allegedly converge in the antennal lobe. These findings indicate that Holometabola have evolved a wider variety of solutions to chemoreception than previously assumed. Keywords: Tribolium castaneum, olfaction, insect, chemoreception, gustation, neuroanatomy, lobus glomerulatus * Correspondence: [email protected]; [email protected] †Equal contributors 2Department of Biology – Neurobiology/Ethology, Philipps-University Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany 1Department of Developmental Biology, Göttingen Center for Molecular Biosciences (GZMB), Georg-August-University Goettingen, Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany Full list of author information is available at the end of the article © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dippel et al. BMC Biology (2016) 14:90 Page 2 of 31 Background receptor CD36 [48–50]. Besides the perireceptor events Insects use chemical cues for most tasks they encounter involved in effective activation, the high temporal during their life history. Over long distances, airborne resolution of olfactory reception probably also requires chemical stimuli guide insects to food sources, mates, signal termination, which is supposedly mediated by se- and places for oviposition [1–6]. Within close range, creted or membrane-bound odorant-degrading enzymes olfaction as well as gustation are used to discriminate (ODEs) [51–55]. between different food qualities, to avoid toxins or Activation of the described chemoreceptors elicits ac- harmful microbes, to communicate intra- or interspecifi- tion potentials in the CSNs that are further transmitted cally, to identify suitable mating partners, and to find via the antennal nerve to the antennal lobe (AL), the first appropriate egg-laying sites [6–15]. Because of insects’ integration center of the olfactory pathway in the brain, or devastating impact on agriculture and stored food for GSNs, to the primary gustatory center of the gnathal products, as well as their ability to serve as vectors for ganglion (GNG) [56]. The AL of insects consists typically detrimental diseases, insect olfaction has become an im- of spherical sub-compartments, the olfactory glomeruli portant research field in biology [4]. [57]. Usually OSNs express only one typical (specific) OR Chemical signals are typically perceived within special- gene and all antennal OSNs expressing the same typical ized antennal and palpal cuticular structures, the olfac- OR converge into the same olfactory glomerulus, creating tory or gustatory sensilla. These chemosensory sensilla a chemotropic map-like representation of chemical coding form a hollow structure filled with aqueous lymph and in the AL [58–60], known as the central dogma of olfac- harbor the dendritic branches of the chemosensory neu- tion [61, 62]. In Drosophila melanogaster, the OR/Orco rons (CSNs), namely the olfactory (OSNs) or gustatory and IR derived sensory information from the antennae sensory neurons (GSNs) [16, 17]. They are divided into and the maxillary palps is processed in the AL [63], several sub-types according to their different morph- whereas in several hemimetabolous insects, CSNs from ology [16]. The volatile molecules enter the cavity the palps converge typically in the lobus glomerulatus through wall pores finally to reach and activate the che- (LG), next to but outside the AL [57, 64–66]. In the AL, moreceptors on the dendrites of the OSNs. To enhance olfactory information from the OSNs, is processed by a olfactory sensitivity and specificity, odorant binding complex network of local interneurons [57, 67–69]. The proteins (OBPs) or potentially chemosensory proteins processed odor information is further relayed by distinct (CSPs) facilitate the translocation of many, mostly antennal lobe tracts (ALTs) formed by the projection neu- hydrophobic, chemicals through the aqueous lymph rons (PNs) to the mushroom body (MB) and the lateral [18]. In insects, typically three different receptor families horn (LH) [57, 70]. The MBs are higher-order integration are involved in chemoreception [4]: the ionotropic centers for multiple processed sensory information and are glutamate-like receptors (IRs) [19, 20], the gustatory re- responsible for odor discrimination, associative learning, as ceptors (GRs) [13, 21], and the odorant receptors (ORs) well as memory storage and retrieval. The LHs receive odor [22–24]. The IRs are evolutionarily highly conserved input directly from the ALs or indirectly from the MBs, de- chemoreceptors involved in protostome olfaction [19]. code the quality and intensity of the information, and finally They contain three transmembrane domains and form trigger immediate odor-driven behavior [71–77]. functional heteromers between an odor-specific IR and a Despite the evolutionary success and ecological as well co-receptor (IR8a and IR25a) The GRs are seven trans- as economic importance of beetles [78, 79], little is membrane receptors found across arthropods [25–28] known on the neuroanatomy, genetics, or biochemistry whose quaternary structure [13, 29–31], as well as the of their olfactory pathway. Within the Coleoptera, the signal transduction mechanism [32, 33], are still under red flour beetle Tribolium castaneum has become the debate. The typical ORs are seven transmembrane re- prime model organism for developmental biology and ceptors found in pterygote insects [24] that form func- pest management [80]. With its fully annotated genome tional heteromers with the atypical (general) odorant [81–83] and the multiple powerful genetic tools – such receptor co-receptor (Orco) [22, 23, 34–36]. Their signal as systemic RNA interference [84, 85], insertional muta- transduction mechanism is currently discussed and they genesis [86], and transgene-based misexpression systems may either form an ionotropic receptor complex that is [87, 88] – T. castaneum represents an eligible beetle regulated by second messengers or be functional metab- model organism for olfaction. In the current study, we otropic receptors [22, 23, 34, 37–40]. The described in- present a substantial overview of the olfactory pathway fluence of G-proteins and affiliated second messengers in T. castaneum, covering the morphology of the sensilla on insect olfaction supports both mechanisms [41–47]. and the antenna, all major neuropils including AL, MB, Moreover, sensitive pheromone detection requires the LH, LG, and the gnathal olfactory center (GOC), a previ- OR/Orco complex to interact with a sensory neuron ously undescribed glomerularly organized neuropil in membrane protein (SNMP) related to the scavenger the GNG. Additional support for the importance of the Dippel et al. BMC Biology