BACKGROUND: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. RESULTS: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. CONCLUSIONS: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

Department of Biology Lund University SE 223 62 Lund Sweden

Department of Evolutionary Neuroethology Max Planck Institute for Chemical Ecology 07745 Jena Germany

Division of Biochemistry and Structural Biology Department of Chemistry Lund University SE 223 62 Lund Sweden

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Gilead Sciences and IOCB Research Center Flemingovo n 2 166 10 Prague 6 Czech Republic

Present address Faculty of Forestry and Wood Sci Excellent Team for Mitigation Czech University Life Sci Prague Kamycka 129 Prague 6 16521 Suchdol Czech Republic

Zobrazit více v PubMed

Hansson BS, Stensmyr MC. Evolution of insect olfaction. Neuron. 2011;72:698–711. doi: 10.1016/j.neuron.2011.11.003. PubMed DOI

Kandasamy D, Gershenzon J, Andersson MN, Hammerbacher A. Volatile organic compounds influence the interaction of the Eurasian spruce bark beetle ( PubMed PMC

Clyne PJ, Warr CG, Freeman MR, Lessing D, Kim J, Carlson JR. A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in PubMed

Kaupp UB. Olfactory signalling in vertebrates and insects: differences and commonalities. Nat Rev Neurosci. 2010;11:188–200. doi: 10.1038/nrn2789. PubMed DOI

Smart R, Kiely A, Beale M, Vargas E, Carraher C, Kralicek AV, et al. PubMed

Robertson HM, Warr CG, Carlson JR. Molecular evolution of the insect chemoreceptor gene superfamily in PubMed PMC

Andersson MN, Löfstedt C, Newcomb RD. Insect olfaction and the evolution of receptor tuning. Front Ecol Evol. 2015;3:53.

Nei M, Niimura Y, Nozawa M. The evolution of animal chemosensory receptor gene repertoires: roles of chance and necessity. Nat Rev Genet. 2008;9:951–963. doi: 10.1038/nrg2480. PubMed DOI

Brand P, Robertson HM, Lin W, Pothula R, Klingeman WE, Jurat-Fuentes JL, et al. The origin of the odorant receptor gene family in insects. eLife. 2018;7:e38340. doi: 10.7554/eLife.38340. PubMed DOI PMC

Butterwick JA, del Mármol J, Kim KH, Kahlson MA, Rogow JA, Walz T, et al. Cryo-EM structure of the insect olfactory receptor Orco. Nature. 2018;560:447–452. doi: 10.1038/s41586-018-0420-8. PubMed DOI PMC

Sato K, Pellegrino M, Nakagawa T, Vosshall LB, Touhara K. Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature. 2008;452:1002–1006. doi: 10.1038/nature06850. PubMed DOI

Wicher D, Schäfer R, Bauernfeind R, Stensmyr MC, Heller R, Heinemann SH, et al. PubMed

Andersson MN, Newcomb RD. Pest control compounds targeting insect chemoreceptors: another silent spring? Front Ecol Evol. 2017;5:5. doi: 10.3389/fevo.2017.00005. DOI

Murugathas T, Zheng HY, Colbert D, Kralicek AV, Carraher C, Plank NOV. Biosensing with insect odorant receptor nanodiscs and carbon nanotube field-effect transistors. ACS Appl Mater Interfaces. 2019;11:9530–9538. doi: 10.1021/acsami.8b19433. PubMed DOI

Khadka R, Aydemir N, Carraher C, Hamiaux C, Colbert D, Cheema J, et al. An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants. Biosens Bioelectron. 2019;126:207–213. doi: 10.1016/j.bios.2018.10.043. PubMed DOI

Khadka R, Carraher C, Hamiaux C, Travas-Sejdic J, Kralicek A. Synergistic improvement in the performance of insect odorant receptor based biosensors in the presence of Orco. Biosens Bioelectron 2020;153:112040. PubMed

Kurz WA, Dymond CC, Stinson G, Rampley GJ, Neilson ET, Carroll AL, et al. Mountain pine beetle and forest carbon feedback to climate change. Nature. 2008;452:987–990. doi: 10.1038/nature06777. PubMed DOI

Raffa KF, Andersson MN, Schlyter F. Chapter one-host selection by bark beetles: playing the odds in a high-stakes game. Adv Insect Physiol. 2016;50:1–74. doi: 10.1016/bs.aiip.2016.02.001. DOI

Biedermann PHW, Grégoire J-C, Gruppe A, Hagge J, Hammerbacher A, Hofstetter R, et al. Bark beetle population dynamics in the Anthropocene: challenges and solutions. Trends Ecol Evol. 2018;34:914–924. doi: 10.1016/j.tree.2019.06.002. PubMed DOI

Wermelinger B. Ecology and management of the spruce bark beetle

Bakke A, Frøyen P, Skattebøl LJN. Field response to a new pheromonal compound isolated from

Birgersson G, Schlyter F, Löfqvist J, Bergström G. Quantitative variation of pheromone components in the spruce bark beetle PubMed

Schlyter F, Birgersson G, Byers JA, Löfqvist J, Bergström G. Field response of spruce bark beetle, PubMed

Francke W, Sauerwein P, Vité JP, Klimetzek D. The pheromone bouquet of

Schlyter F, Birgersson G, Leufvén A. Inhibition of attraction to aggregation pheromone by verbenone and ipsenol. J Chem Ecol. 1989;15:2263–2277. doi: 10.1007/BF01014114. PubMed DOI

Binyameen M, Jankuvová J, Blaženec M, Jakuš R, Song L, Schlyter F, et al. Co-localization of insect olfactory sensory cells improves the discrimination of closely separated odour sources. Funct Ecol. 2014;28:1216–1223. doi: 10.1111/1365-2435.12252. DOI

Unelius RC, Schiebe C, Bohman B, Andersson MN, Schlyter F. Non-host volatile blend optimization for forest protection against the European spruce bark beetle, PubMed PMC

Byers J. Avoidance of competition by spruce bark beetles,

Andersson MN, Larsson MC, Schlyter F. Specificity and redundancy in the olfactory system of the bark beetle PubMed

Tømmerås BÅ. Specialization of the olfactory receptor cells in the bark beetle

Schiebe C, Unelius CR, Ganji S, Binyameen M, Birgersson G, Schlyter F. Styrene, (+)- PubMed PMC

Mustaparta H, Tømmerås BA, Baeckström P, Bakke JM, Ohloff G. Ipsdienol-specific receptor cells in bark beetles: structure-activity relationships of various analogues and of deuterium-labelled ipsdienol. J Comp Physiol A. 1984;154:591–596. doi: 10.1007/BF00610172. DOI

Tømmerås BA, Mustaparta H, Gregoire J-C. Receptor cells in PubMed

de Fouchier A, Walker WB, III, Montagné N, Steiner C, Binyameen M, Schlyter F, et al. Functional evolution of Lepidoptera olfactory receptors revealed by deorphanization of a moth repertoire. Nat Commun. 2017;8:15709. doi: 10.1038/ncomms15709. PubMed DOI PMC

Große-Wilde E, Gohl T, Bouché E, Breer H, Krieger J. Candidate pheromone receptors provide the basis for the response of distinct antennal neurons to pheromonal compounds. Eur J Neurosci. 2007;25:2364–2373. doi: 10.1111/j.1460-9568.2007.05512.x. PubMed DOI

Yuvaraj JK, Andersson MN, Corcoran JA, Anderbrant O, Löfstedt C. Functional characterization of odorant receptors from PubMed

Carey AF, Wang G, Su C-Y, Zwiebel LJ, Carlson JR. Odorant reception in the malaria mosquito PubMed PMC

Hallem EA, Carlson JR. Coding of odors by a receptor repertoire. Cell. 2006;125:143–160. doi: 10.1016/j.cell.2006.01.050. PubMed DOI

Mitchell RF, Hughes DT, Luetje CW, Millar JG, Soriano-Agatón F, Hanks LM, et al. Sequencing and characterizing odorant receptors of the cerambycid beetle PubMed PMC

Wang X, Wang S, Yi J, Li Y, Liu J, Wang J, et al. Three host plant volatiles, hexanal, lauric acid, and tetradecane, are detected by an antenna-biased expressed odorant receptor 27 in the dark black chafer PubMed

Antony B, Johny J, Montagné N, Jacquin-Joly E, Capoduro R, Cali K et al. Pheromone receptor of the globally invasive quarantine pest of the palm tree, the red palm weevil ( PubMed

Mitchell RF, Andersson MN. Olfactory genomics of the Coleoptera. In: Blomquist GJ, Vogt RG, editors. Insect pheromone biochemistry and molecular biology. 2. Oxford: Academic Press; 2020. pp. 547–590.

Andersson MN, Grosse-Wilde E, Keeling CI, Bengtsson JM, Yuen MM, Li M, et al. Antennal transcriptome analysis of the chemosensory gene families in the tree killing bark beetles, PubMed PMC

Mitchell RF, Schneider TM, Schwartz AM, Andersson MN, McKenna DD. The diversity and evolution of odorant receptors in beetles (Coleoptera) Insect Mol Biol. 2020;29:77–91. doi: 10.1111/imb.12611. PubMed DOI

Andersson MN, Keeling CI, Mitchell RF. Genomic content of chemosensory genes correlates with host range in wood-boring beetles ( PubMed PMC

Corcoran JA, Jordan MD, Carraher C, Newcomb RD. A novel method to study insect olfactory receptor function using HEK293 cells. Insect Biochem Mol Biol. 2014;54:22–32. doi: 10.1016/j.ibmb.2014.08.005. PubMed DOI

Hou X, Zhang D-D, Yuvaraj JK, Corcoran JA, Andersson MN, Löfstedt C. Functional characterization of odorant receptors from the moth PubMed

Brown HC, Randad RS. Chiral synthesis VIA organoboranes. 26. An efficient synthesis of isoprenyl derivatives of borane-valuable reagents for the isoprenylboration of aldehydes. A convenient route to both enantiomers of ipsenol and ipsdienol in high optical purity. Tetrahedron. 1990;46:4463–4472. doi: 10.1016/S0040-4020(01)85575-7. DOI

Erver F, Hilt G. Multi-component regio-and diastereoselective cobalt-catalyzed hydrovinylation/allylboration reaction sequence. Org Lett. 2011;13:5700–5703. doi: 10.1021/ol202481j. PubMed DOI

Klusener PAA, Hommes HH, Verkruijsse HD, Brandsma L. Direct metallation of isoprene. J Chem Soc Chem Comm. 1985;1985:1677–1678. doi: 10.1039/c39850001677. DOI

Nemoto H. A new alkenyl ether giving acetal with stereospecific manner. Tetrahedron Lett. 1994;35:7785–7788. doi: 10.1016/S0040-4039(00)77372-2. DOI

Nemoto H, Zhong W, Kawamura T, Kamiya M, Nakano Y, Sakamoto K. Synthesis of pptically active δ-dodecalactone via chiral resolution using CPF. Synlett. 2007;2007:2343–2346. doi: 10.1055/s-2007-985604. DOI

Corcoran JA, Sonntag Y, Andersson MN, Johanson U, Löfstedt C. Endogenous insensitivity to the Orco agonist VUAA1 reveals novel olfactory receptor complex properties in the specialist fly PubMed PMC

Hopf TA, Morinaga S, Ihara S, Touhara K, Marks DS, Benton R. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors. Nat Commun. 2015;6:6077. doi: 10.1038/ncomms7077. PubMed DOI PMC

Pellegrino M, Steinbach N, Stensmyr MC, Hansson BS, Vosshall LB. A natural polymorphism alters odour and DEET sensitivity in an insect odorant receptor. Nature. 2011;478:511–514. doi: 10.1038/nature10438. PubMed DOI PMC

Leary GP, Allen JE, Bunger PL, Luginbill JB, Linn CE, Macallister IE, et al. Single mutation to a sex pheromone receptor provides adaptive specificity between closely related moth species. Proc Natl Acad Sci U S A. 2012;109:14081–14086. doi: 10.1073/pnas.1204661109. PubMed DOI PMC

Nichols AS, Luetje CW. Transmembrane segment 3 of PubMed PMC

Hallberg E. Sensory organs in

Jones PL, Pask GM, Rinker DC, Zwiebel LJ. Functional agonism of insect odorant receptor ion channels. Proc Natl Acad Sci U S A. 2011;108:8821–8825. doi: 10.1073/pnas.1102425108. PubMed DOI PMC

Gu X-C, Zhang Y-N, Kang K, Dong S-L, Zhang L-W. Antennal transcriptome analysis of odorant reception genes in the red turpentine beetle (RTB), PubMed PMC

Antony B, Soffan A, Jakše J, Abdelazim MM, Aldosari SA, Aldawood AS, et al. Identification of the genes involved in odorant reception and detection in the palm weevil PubMed PMC

Hunt T, Bergsten J, Levkanicova Z, Papadopoulou A, St. John O, Wild R, et al. A comprehensive phylogeny of beetles reveals the evolutionary origins of a superradiation. Science. 2007;318:1913–1916. doi: 10.1126/science.1146954. PubMed DOI

Larsson MC, Leal WS, Hansson BS. Olfactory receptor neurons detecting plant odours and male volatiles in PubMed

Miazzi F, Schulze H-C, Zhang L, Kaltofen S, Hansson BS, Wicher D. Low Ca2+ levels in the culture media support the heterologous expression of insect odorant receptor proteins in HEK cells. J Neurosci Methods. 2018;312:122–125. doi: 10.1016/j.jneumeth.2018.11.021. PubMed DOI

Andersson MN, Corcoran JA, Zhang D-D, Hillbur Y, Newcomb RD, Löfstedt C. A sex pheromone receptor in the hessian fly PubMed PMC

Yuvaraj JK, Corcoran JA, Andersson MN, Newcomb RD, Anderbrant O, Löfstedt C. Characterization of odorant receptors from a non-ditrysian moth, PubMed PMC

Andersson MN. Mechanisms of odor coding in coniferous bark beetles: From neuron to behavior and application. Psyche J Entomol 2012;2012: Article ID 149572.

Schlyter F, Birgersson GA. Forest beetles. In: Hardie J, Minks AK, editors. Pheromones of non-Lepidopteran insects associated with agricultural plants. Oxford: CAB International; 1999. pp. 113–148.

Nichols AS, Chen S, Luetje CW. Subunit contributions to insect olfactory receptor function: channel block and odorant recognition. Chem Senses. 2011;36:781–790. doi: 10.1093/chemse/bjr053. PubMed DOI PMC

Kumar P, Wang Y, Zhang Z, Zhao Z, Cymes GD, Tajkhorshid E, et al. Cryo-EM structures of a lipid-sensitive pentameric ligand-gated ion channel embedded in a phosphatidylcholine-only bilayer. Proc Natl Acad Sci U S A. 2020;117:1788–1798. doi: 10.1073/pnas.1906823117. PubMed DOI PMC

Turner SL, Li N, Guda T, Githure J, Cardé RT, Ray A. Ultra-prolonged activation of CO2-sensing neurons disorients mosquitoes. Nature. 2011;474:87–91. doi: 10.1038/nature10081. PubMed DOI PMC

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644. doi: 10.1038/nbt.1883. PubMed DOI PMC

Waterhouse RM, Seppey M, Simão FA, Manni M, Ioannidis P, Klioutchnikov G, et al. BUSCO applications from quality assessments to gene prediction and phylogenomics. Mol Biol Evol. 2017;35:543–548. doi: 10.1093/molbev/msx319. PubMed DOI PMC

McKenna DD, Scully ED, Pauchet Y, Hoover K, Kirsch R, Geib SM, et al. Genome of the Asian longhorned beetle ( PubMed PMC

Schoville SD, Chen YH, Andersson MN, Benoit JB, Bhandari A, Bowsher JH, et al. A model species for agricultural pest genomics: the genome of the Colorado potato beetle, PubMed PMC

Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, et al. De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis. Nat Protoc. 2013;8:1494–512. PubMed PMC

Katoh K, Misawa K, Ki K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30:3059–3066. doi: 10.1093/nar/gkf436. PubMed DOI PMC

Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25:1972–1973. doi: 10.1093/bioinformatics/btp348. PubMed DOI PMC

Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B. PartitionFinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Mol Biol Evol. 2016;34:772–773. PubMed

Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–1313. doi: 10.1093/bioinformatics/btu033. PubMed DOI PMC

Miller MA, Pfeiffer W, Schwartz T. 2010 gateway computing environments workshop (GCE): 14 Nov. New Orleans: Ieee; 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees; pp. 1–8.

Zhang D-D, Löfstedt C. Functional evolution of a multigene family: orthologous and paralogous pheromone receptor genes in the turnip moth, PubMed PMC

Krieger J, Grosse-Wilde E, Gohl T, Dewer Y, Raming K, Breer H. Genes encoding candidate pheromone receptors in a moth ( PubMed PMC

Schultze A, Pregitzer P, Walter MF, Woods DF, Marinotti O, Breer H, et al. The co-expression pattern of odorant binding proteins and olfactory receptors identify distinct trichoid sensilla on the antenna of the malaria mosquito PubMed PMC

Zhang D-D, Wang H-L, Schultze A, Froß H, Francke W, Krieger J, et al. Receptor for detection of a type II sex pheromone in the winter moth PubMed PMC

Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46:W296–W303. doi: 10.1093/nar/gky427. PubMed DOI PMC

Hildebrand PW, Goede A, Bauer RA, Gruening B, Ismer J, Michalsky E, et al. SuperLooper—a prediction server for the modeling of loops in globular and membrane proteins. Nucleic Acids Res. 2009;37:W571–W574. doi: 10.1093/nar/gkp338. PubMed DOI PMC

Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, et al. Scalable molecular dynamics with NAMD. J Comput Chem. 2005;26:1781–1802. doi: 10.1002/jcc.20289. PubMed DOI PMC

Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31:455–461. PubMed PMC

Yuvaraj JK, Roberts RE, Sonntag Y, Hou X-Q, Grosse-Wilde E, Machara A, Zhang D-D, Hansson BS, Johanson U, Löfstedt C, Andersson MN. Putative ligand binding sites of two functionally characterized bark beetle odorant receptors. PubMed PMC

Yuvaraj JK, Roberts RE, Sonntag Y, Hou X-Q, Grosse-Wilde E, Machara A, Hansson BS, Johanson U, Löfstedt C, Andersson MN: Functional characterization of two bark beetle odorant receptors and their putative ligand binding site.

Identification of the trail-following pheromone receptor in termites

Complex Genomic Landscape of Inversion Polymorphism in Europe's Most Destructive Forest Pest

Antennal Transcriptome Screening and Identification of Chemosensory Proteins in the Double-Spine European Spruce Bark Beetle, Ips duplicatus (Coleoptera: Scolytinae)

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success

A highly-contiguous genome assembly of the Eurasian spruce bark beetle, Ips typographus, provides insight into a major forest pest

Interactions among Norway spruce, the bark beetle Ips typographus and its fungal symbionts in times of drought

Putative ligand binding sites of two functionally characterized bark beetle odorant receptors