Functional Evolution of a Bark Beetle Odorant Receptor Clade Detecting Monoterpenoids of Different Ecological Origins

Abstract

Insects detect odors using an array of odorant receptors (ORs), which may expand through gene duplication. How and which new functions may evolve among related ORs within a species remain poorly investigated. We addressed this question by functionally characterizing ORs from the Eurasian spruce bark beetle Ips typographus, in which physiological and behavioral responses to pheromones, volatiles from host and non-host trees, and fungal symbionts are well described. In contrast, knowledge of OR function is restricted to two receptors detecting the pheromone compounds (S)-(–)-ipsenol (ItypOR46) and (R)-(–)-ipsdienol (ItypOR49). These receptors belong to an Ips-specific OR-lineage comprising seven ItypORs. To gain insight into the functional evolution of related ORs, we characterized the five remaining ORs in this clade using Xenopus oocytes. Two receptors responded primarily to the host tree monoterpenes (+)-3-carene (ItypOR25) and p-cymene (ItypOR27). Two receptors responded to oxygenated monoterpenoids produced in larger relative amounts by the beetle-associated fungi, with ItypOR23 specific for (+)-trans-(1R, 4S)-4-thujanol, and ItypOR29 responding to (+)-isopinocamphone and similar ketones. ItypOR28 responded to the pheromone E-myrcenol from the competitor Ips duplicatus. Overall, the OR responses match well with those of previously characterized olfactory sensory neuron classes except that neurons detecting E-myrcenol have not been identified. The characterized ORs are under strong purifying selection and demonstrate a shared functional property in that they all primarily respond to monoterpenoids. The variation in functional groups among OR ligands and their diverse ecological origins suggest that neofunctionalization has occurred early in the evolution of this OR-lineage following gene duplication.

Codon Optimization of Insect Odorant Receptor Genes May Increase Their Stable Expression for Functional Characterization in HEK293 Cells

“…ItypOR25 WT was recently shown to respond primarily to the conifer/host compound (+)-3-carene and secondarily to several additional compounds when tested in Xenopus oocytes (Hou et al, 2021). Here, HEK cells transfected with the ItypOR25 WT gene did not respond to any stimulus, which correlates to its low protein levels in cells (Figure 4A).…”

“…We previously functionally characterized ItypOR23, 25, 27, 28, and 29 from non-codon optimized (wildtype, WT) genes using X. laevis oocytes, and putative key ligands were identified for all five ORs (Hou et al, 2021). Non-codon optimized genes of these five ORs were transfected into ItypOrco WT -expressing HEK cells for characterization also in this system.…”

“…ItypOR29 WT previously responded best to (+)isopinocamphone (produced by symbiotic fungi and the conifer host tree) when tested in Xenopus oocytes (Hou et al, 2021). Secondary, yet strong, responses were triggered by the structurally similar ketones (−)-isopinocamphone, (+)-pinocamphone, (−)-pinocamphone, (−)-pinocarvone, and (±)-camphor.…”

“…Neither wildtype nor codon optimized versions of ItypOR23, ItypOR27 and ItypOR28 showed any response to their proposed key ligands (trans-4-thujanol, p-cymene, and E-myrcenol, respectively; from Xenopus oocytes) (Hou et al, 2021), or any other tested compound (Supplementary Data 1), although cells with codon optimized genes displayed increased OR protein levels in the Western blot analysis.…”

“…The aim of the present study was to investigate whether codon optimization could increase the protein levels and hence facilitate functional expression of insect ORs and Orcos in HEK293 cells with stable and inducible expression. We tested the Orco proteins from I. typographus and the mountain pine beetle Dendroctonus ponderosae ("Dpon"; Scolytinae) as well as six ItypORs previously de-orphanized in HEK cells and/or Xenopus oocytes (Hou et al, 2021;Yuvaraj et al, 2021). Our results show that codon optimization led to increases in HEK cell protein levels for seven of the eight receptors and increased ligand-induced responses in four of them, including two ORs that responded to ligands only when expressed from codon-optimized genes.…”