2020-02-11 20:35:40
David Ruel, Esther Yakir and Jonathan D. Bohbot
INTRODUCTION
Aromatic and heterocyclic compounds play an important role in the ecology of adult mosquitoes as indicated by the odor space of the Anopheles gambiae odorant receptor (OR) repertoire (Carey et al., 2010; Wang et al., 2010). Specifically, indole (IUPAC name, 1H-indole) and skatole (IUPAC name, 3-methylindole) are respectively detected by the narrowly tuned Or2 and Or10 paralogous genes found in Culex quinquefasciatus (Hughes et al., 2010; Pelletier et al., 2010), Aedes aegypti and An. gambiae (Carey et al., 2010; Wang et al., 2010) reflecting their ancestral origin (Bohbot et al., 2011). Due to the sensitive and selective nature of the OR2-indole and OR10-skatole interactions, they have been referred to as the "indolergic” receptors (Bohbot and Pitts, 2015).
Indole and skatole are released by a wide variety of organisms but are mainly synthesized by bacteria (Elgaali et al., 2002; Schulz and Dickschat, 2007; Lindh et al., 2008; Hubbard et al., 2015), fungi (Chen et al., 2014; Tomberlin et al., 2017) and plants (Turlings et al., 1991; Frey et al., 2000; Ober, 2005). In adult mosquitoes, both compounds have been proposed to mediate oviposition site (Blackwell and Johnson, 2000) and host-locating behaviors (Cork, 1996). However, their exact ecological role(s) remain complex since indoles are major constituents of floral (Knudsen et al., 2006) and animal scents (Meijerink et al., 2001; Lee et al., 2015). Interestingly, indolic compounds play additional ecological roles in mosquito larvae (Xia et al., 2008; Scialo et al., 2012).
Or2 is expressed in the adult and larval stages of Ae. aegypti (Bohbot et al., 2007) and An. gambiae (Hill et al., 2002; Xia et al., 2008). Or10 expression is more complex: in An. gambiae, Or10 is expressed both in larvae and adults. In Ae. Aegypti, Or10 is only expressed in adults, while a third paralog named Or9, is expressed in the larval antenna (Bohbot et al., 2007). Based on pharmacological studies, we have suggested that receptor sensitivity towards odorants in the nanomolar concentration range is a predictor of OR-semiochemical relationships (Bohbot and Pitts, 2015). The activation of AaegOR9 by indole in the low micromolar concentration range (Bohbot et al., 2011) indicated that a more potent indolic cognate ligand selectively activates this receptor.
Using a reverse chemical ecology approach, we set out to identify a potential cognate ligand for this larval-expressed Or9 gene (Supplementary Table 1). First, we used a panel of 31 indole derivatives from plants and microbes to identify a potent activator of AaegOR9, then we showed that AaegOR9 is narrowly tuned to skatole in the low nanomolar concentration range. Our findings suggest that Culicinae have developed a supersensitive skatole receptor that operates in water where this compound exhibits low solubility. The occurrence of two skatole receptors, each assigned to a different developmental stage indicates the central role of this odorant in the Ae. aegypti life cycle. The deorphanization of AaegOR9: (i) provides a molecular target for future larval behavioral disruption studies;
(ii)improves our understanding of insect OR coding; and
(iii)raises questions on the possible ecological roles of mosquito indolergic receptors.
MATERIALS AND METHODS
Chemical Reagents
The chemicals (Supplementary Table 1) used for the deorphanization of AaegOR9 were obtained from Sigma- Aldrich (Milwaukee, WI, USA), ChemCruz (Dallas, TX, USA), Glentham Life Sciences (Corsham, UK), FluoroChem (Hadfield, UK), SL Moran (Jerusalem, Israel), Holland Moran (Yehun, Israel), Alfa Aesar (Ward Hill, MA, USA) and from the generous contribution of the Dr. Kolodkin-Gal Lab (Weizmann Institute of Science, Israel).
Two-Electrode Voltage Clamp of Xenopus Oocytes Expressing ORs
The methodologies and protocols have been described in details elsewhere (Bohbot and Dickens, 2009). AaegOr9 and Aaeg- ORco cRNAs (Bohbot et al., 2011) were synthesized from linearized pSP64DV expression vectors using the mMESSAGE mMACHINE⑥ SP6 kit (Life Technologies). Stage V-VII oocytes were harvested from Xenopus laevis females, mechanically separated, treated with collagenase (8 mg/mL, 30 min, 18°C) and rinsed in washing solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl2 and 5 mM HEPES, pH 7.6). Oocytes were microinjected with 27.6 ng AaegOr9 and AaegORco cRNAs, incubated at 18° C for 3-4 days in ND96 solution (96 mM NaCl, 2 mM KCl, 5 mM MgCl?, 0.8 mM CaCl? and 5 mM HEPES, pH 7.6), supplemented with 5% dialyzed horse serum, 50 Mg/mL tetracycline, 100 jig/mL streptomycin and 550 Mg/mL sodium pyruvate. Whole-cell currents were recorded using the two-microelectrode voltage-clamp technique. During recording sessions, the holding potential was maintained at -80 mV using an OC-725C oocyte clamp (Warner Instruments, LLC, Hamden, CT, USA). Oocytes placed in a RC-3Z oocyte recording chamber (Warner Instruments, LLC, Hamden, CT, USA) were exposed to odorants for 8 s. Current was allowed to return to baseline between odorant applications. Data acquisition and concentration-response analyses were carried out with a Digidata 1550A and the pCLAMP10 software (Molecular Devices, Sunnyvale, CA, USA), and analyzed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). Stock concentration of odorants (10-2 M) were dissolved in ringer solution containing 2% dimethyl sulfoxide (DMSO) in order to solubilize the hydrophobic indolic compounds.
Pharmacological Characterization
The response profile was established using multiple sessions, each including six compounds at a time and indole as an internal reference. The order in which these compounds were administered was reversed within a session to mitigate against any potential sequence effects between compounds (none were observed). All the response values were normalized to the indole reference in each recording session (Supplementary Figure 1).
For the establishment of the concentration-response curves, oocytes were exposed to increasing concentrations of indole, skatole and indole-3-carboxaldehyde (I3C; Supplementary Figure 2). Quantitative characterization of OR sensitivity was estimated using the averaged effective concentration at 50% of the maximal response (EC50) over the sample population. The data to establish the concentration response curve and EC50 of AaegOR10-skatole was extracted from a previous study (Bohbot and Dickens, 2012).
Phylogeny OR Intron-Exon Structure and Phylogeny
All the sequences used in our phylogenic analysis were obtained from the VectorBase and NCBI databases using AaegOr2/9/10 as query (for accession numbers, see Supplementary Table 3). DNA sequences for Toxorhynchites Or2 and Or10 can be accessed here: http://dx.doi.org/10.6084/m9.figshare.1092617. MAFFT version 7 (Nakamura et al., 2018) was used for multiple amino-acid sequence alignment. The phylogenic software IQ-TREE (Nguyen et al., 2015; Kalyaanamoorthy et al., 2017; Hoang et al., 2018) and the FigTree phylogenic tree based on the maximum likelihood method (Model: JC, UFbootstrap: 5,000). Using MAFFT (default parameters) and the Vectorbase database, we located the intron positions on the indolergic receptor genes.
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