fphys-09-00777 June 25, 2018 Time: 11:57 # 1 ORIGINAL RESEARCH published: 25 June 2018 doi: 10.3389/fphys.2018.00777 Sensilla Morphology and Complex Expression Pattern of Odorant Binding Proteins in the Vetch Aphid Megoura viciae (Hemiptera: Aphididae) Daniele Bruno1†, Gerarda Grossi2†, Rosanna Salvia2†, Andrea Scala2†, Donatella Farina2, Annalisa Grimaldi1, Jing-Jiang Zhou3, Sabino A. Bufo2, Heiko Vogel4, Ewald Grosse-Wilde5*, Bill S. Hansson5 and Patrizia Falabella2* 1 Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy, 2 Department of Sciences, University of Basilicata, Potenza, Italy, 3 Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, United Kingdom, 4 Department of Entomology, Max Planck Institute for Chemical Ecology, Jena, Germany, 5 Department of Edited by: Evolutionary Neuroethology, Max Planck Institute for Chemical Ecology, Jena, Germany Nicolas Durand, Université Pierre et Marie Curie, France Chemoreception in insects is mediated by several components interacting at Reviewed by: different levels and including odorant-binding proteins (OBPs). Although recent studies Andrew Dacks, demonstrate that the function of OBPs cannot be restricted to an exclusively olfactory West Virginia University, United States Julian Chen, role, and that OBPs have been found also in organs generally not related to Institute of Plant Protection (CAS), chemoreception, their feature of binding molecules remains undisputed. Studying China the vetch aphid Megoura viciae (Buckton), we used a transcriptomic approach to *Correspondence: Ewald Grosse-Wilde identify ten OBPs in the antennae and we examined the ultrastructural morphology [email protected] of sensilla and their distribution on the antennae, legs, mouthparts and cauda of Patrizia Falabella wingless and winged adults by scanning electron microscopy (SEM). Three types [email protected] of sensilla, trichoid, coeloconic and placoid, differently localized and distributed on †These authors have contributed equally to this work. antennae, mouthparts, legs and cauda, were described. The expression analysis of the ten OBPs was performed by RT-qPCR in the antennae and other body parts of Specialty section: This article was submitted to the wingless adults and at different developmental stages and morphs. Five of the Invertebrate Physiology, ten OBPs (MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7, and MvicOBP8), whose a section of the journal antibodies were already available, were selected for experiments of whole-mount Frontiers in Physiology immunolocalization on antennae, mouthparts, cornicles and cauda of adult aphids. Most Received: 19 December 2017 Accepted: 04 June 2018 of the ten OBPs were more expressed in antennae than in other body parts; MvicOBP1, Published: 25 June 2018 MvicOBP3, MvicOBP6, MvicOBP7 were also immunolocalized in the sensilla on the Citation: antennae, suggesting a possible involvement of these proteins in chemoreception. Bruno D, Grossi G, Salvia R, Scala A, Farina D, Grimaldi A, Zhou J-J, MvicOBP6, MvicOBP7, MvicOBP8, MvicOBP9 were highly expressed in the heads Bufo SA, Vogel H, Grosse-Wilde E, and three of them (MvicOBP6, MvicOBP7, MvicOBP8) were immunolocalized in the Hansson BS and Falabella P (2018) sensilla on the mouthparts, supporting the hypothesis that also mouthparts may be Sensilla Morphology and Complex Expression Pattern of Odorant involved in chemoreception. MvicOBP2, MvicOBP3, MvicOBP5, MvicOBP8 were highly Binding Proteins in the Vetch Aphid expressed in the cornicles-cauda and two of them (MvicOBP3, MvicOBP8) were Megoura viciae (Hemiptera: Aphididae). Front. Physiol. 9:777. immunolocalized in cornicles and in cauda, suggesting a possible new function not doi: 10.3389/fphys.2018.00777 related to chemoreception. Moreover, the response of M. viciae to different components Frontiers in Physiology| www.frontiersin.org 1 June 2018| Volume 9| Article 777 fphys-09-00777 June 25, 2018 Time: 11:57 # 2 Bruno et al. Megoura viciae Odorant Binding Proteins of the alarm pheromone was assessed by behavioral assays on wingless adult morph; (−)-a-pinene and (C)-limonene were found to be the components mainly eliciting an alarm response. Taken together, our results represent a road map for subsequent in- depth analyses of the OBPs involved in several physiological functions in M. viciae, including chemoreception. Keywords: vetch aphid, chemoreception, odorant-binding proteins, RT-qPCR, immunolocalization, behavioral assays INTRODUCTION only basiconic sensilla contain pore tubules (Shanbhag et al., 2000), the authors do not exclude that OBP28a expressed in Chemical perception in insects is known to be mediated by other sensilla type may play different roles including the classical molecules belonging to the classes of olfactory, gustatory and function of odorants carrier (Larter et al., 2016). ionotropic receptors and to the classes of soluble olfactory Alternatively, different studies suggest that a sensible proteins, odorant-binding proteins (OBPs) and chemosensory reduction in olfactory function is related to the reduced levels of proteins (CSPs); however, what these proteins do and how they certain OBPs (Xu et al., 2005; Biessmann et al., 2010; Pelletier interact is still not completely clear (Fan et al., 2010; Leal, et al., 2010; Swarup et al., 2011). Within the processes relying 2013; Pelosi et al., 2017). In particular, OBPs have long been on chemoreception, it has been proposed that OBPs also play a thought to act exclusively as carriers of chemicals that, once role in removing chemicals, both those bound to the ORs and solubilized, were transported to the olfactory receptors (Pelosi those located in the sensory lymph, in order to speed up nervous et al., 2006; Brito et al., 2016). The generally hydrophobic stimulus termination (Vogt and Riddiford, 1981; Ziegelberger, odorants need to reach the specific receptors bound to the 1995). That the role of OBPs is related to their binding task plasma membrane of sensory neuron dendrites, overcoming the is apparent from their multifunctional features, which are not hydrophilic barrier that is the sensillar lymph (Pelosi, 1996; confined to chemical perception (Smartt and Erickson, 2009; Jeong et al., 2013). Several studies performed in vivo indicate Sun Y.L. et al., 2012; Ishida et al., 2013; Pelosi et al., 2017). that OBPs play a key role in chemoreception. RNAi was used Indeed, OBPs are expressed in organs that are not connected to reduce the expression of OBPs in Anopheles gambiae and to chemoreception. In some cases, the same OBP is expressed Culex quinquefasciatus (Biessmann et al., 2010; Pelletier et al., in chemoreceptive and non-chemoreceptive tissues, suggesting 2010), in Drosophila melanogaster (Swarup et al., 2011) and in that one type may have multiple roles (Calvello et al., 2003; Li Acyrthosiphon pisum (Zhang et al., 2017). Results demonstrated et al., 2008; Sirot et al., 2008; Vogel et al., 2010; Dani et al., 2011; that OBPs play a specific role in olfactory perception, suggesting Iovinella et al., 2011; Sun Y.L. et al., 2012; De Biasio et al., 2015). there is a direct correlation between the expression level of For example, since the same OBPs are expressed in antennae and OBPs and the ability of insects to perceive odors. Previous reproductive organs (Sun Y.L. et al., 2012; Ban et al., 2013), or studies found that Drosophila OBP76a (LUSH) mutants, played in antennae and in pheromone glands (Jacquin-Joly et al., 2001; an essential role in binding and mediating the recognition of the Strandh et al., 2009; Gu et al., 2013; Zhang et al., 2013, 2015; sex pheromone 11-cis-vaccenyl acetate (c-VA) (Xu et al., 2005; Xia et al., 2015), they may both mediate the recognition of and Ha and Smith, 2006; Laughlin et al., 2008). These preliminary assisting with the release of the same chemical message. In both results should be partially reconsidered in light of more recent cases, the role of OBPs is to solubilize hydrophobic pheromones, research demonstrating that, at sufficiently high concentrations, binding them in a hydrophilic environment where OBPs are c-VA is able to activate neuronal stimuli without LUSH (Gomez- present in high concentration (Nagnan-Le Meillour et al., 2000; Diaz et al., 2013; Li et al., 2014). However, LUSH is still considered Jacquin-Joly et al., 2001; Pelosi et al., 2017). a protein that can increase the sensitivity of the c-VA receptor, The different functions imputed to OBPs are in any case also protecting pheromone molecules from degradation by ODEs linked to the ability of these proteins to bind small hydrophobic (Gomez-Diaz et al., 2013). Moreover, the capability of LUSH to molecules, signals of different types originating from different bind c-VA has been further demonstrated by in vitro experiments sources. However, the expression of soluble olfactory proteins (Kruse et al., 2003). in chemosensory structures (mainly antennae and mouthparts) It has been demonstrated that deleting OBP28a in Drosophila indicates that they play a role in chemoreception (Pelosi et al., melanogaster basiconic sensilla did not reduce the insect’s 2017). ability to respond to olfactory stimuli (Larter et al., 2016), Chemoreception is just one of the roles that OBPs play suggesting that OBP28a is not required for odorant transport. in aphids (Hemiptera: Aphididae), a group of insects that Larter and colleagues hypothesize a novel role for OBP, namely, includes major crop pest in the world. Aphids cause damage that it modulates