Discussion

Green budworm moth response to codlemone and pear ester

The empirical finding that green budworm moth H. nubiferana males respond to codling moth C. pomonella sex pheromone and kairomone, codlemone and pear ester, correlates with the antennal transcriptomes of these two species. Molecular phylogenetics of olfactory receptors, informed by behavioural and functional data, generates sound hypotheses for the identification of semiochemicals driving olfactory behaviour.
Food and mate finding, the essential components of insect reproductive behaviour, depend on a finite number of ORs encoding relevant odour signals. Peripheral olfactory perception employs 39 ORs in the fruit flyDrosophila melanogaster (Menuz et al. 2014, Grabe et al. 2015), 58 ORs in codling moth C . pomonella (Walker et al. 2016) and a similar number of ORs has been found in other tortricids (Corcoran et al. 2015, Steinwender et al. 2015, Rochas et al. 2018). Insect OR genes are under strong selection (McBride and Arguello 2007, Sanchez-Gracia et al. 2009; Arguello et al. 2016, Robertson 2019) and orthologous receptors descending from shared ancestral genes, which are conserved across taxonomic clades, such as CpomOR3 and HnubOR3 (Figure 2), likely play adapative roles.
Functional characterization of CpomOR3, a codling moth OR, has established pear ester as its principal ligand. This was achieved through heterologous expression of CpomOR3 in olfactory sensory neurons of ab3 and T1 antennal sensilla in Drosophila melanogaster , followed by single sensillum electrophysiological recordings (SSR) (Bengtsson et al. 2014; Gonzalez et al. 2016) and has meanwhile been corroborated by luminescence assays after expression in human embryonic kidney cells and Xenopus oocytes (Cattaneo et al. 2017, Wan et al. 2019). CpomOR3, albeit tuned to a plant volaile compound, is part of the lepidopteran pheromone receptor (PR) clade (Bengtsson et al. 2012, 2014; Walker et al. 2016).
The hypothesis that H . nubiferana perceives pear ester via HnubOR3 is parsimonious: a PR phylogeny of tortricid moths (Figure 2A), together with sequence similarity analysis (Figure 3) show that CpomOR3 and HnubOR3 are close. In addition, HnubOR3 and CpomOR3 are among the most abundant transcripts in the male antenna (Figure 2B; Walker et al. 2016). This compares to the receptor orthologs CpomOR19 and SlitOR19 (Spodoptera littoralis ). Following functional characterization of SlitOR19, ligand affinity of CpomOR19 was predicted on the basis of amino acid sequence similarity (Gonzalez et al. 2015).
Oriental fruit moth Grapholita molesta , although taxonomically closer to C . pomonella than to H . nubiferana(Bradley et al. 1979; Regier et al. 2012), is not known to respond to dienic pheromone compounds or pear ester, which is corroborated by our PR phylogeny (Figure 2). The broad host range of G. molestaoverlaps only partially with C . pomonella and H .nubiferana food plants.

Attraction to sex pheromone and codlemone employs distinct olfactory channels

Attraction of green budworm moth H. nubiferana to its multicomponent sex pheromone and to codling moth pheromone employs separate olfactory channels. Codlemone E 8,E 10-12OH does not mimic the H. nubiferana main pheromone compound codlemone acetate E 8,E 10-12Ac, since codlemone is active as single compound, while codlemone acetate is not (Tables 2, 3). Tortricid moths differentiate analogous alcohol from acetate pheromone compounds at high resolution (Witzgall et al. 1991, 1993, 1996, 2010b), probably due to steric differences of the functional groups (Bengtsson et al. 1990). PR phylogeny, together with expression levels in H. nubiferana andC. pomonella (Figure 2; Walker et al. 2016), suggests that CpomOR1 and HnubOR2 are tuned to codlemone, and CpomOR6 and HnubOR6 to codlemone acetate (Catteaneo et al. 2016). In codling moth, codlemone acetate is a pheromone synergist or antagonist, when added to the main pheromone compound codlemone at small and large amounts, respectively (Hathaway et al. 1974; Witzgall et al. 2001).
Presence of two pheromone channels in H. nubiferana males is reminiscent of the ”hopeful monster” (Baker 2002, Dietrich 2003) and ”asymmetric tracking” (Phelan 1992) concepts, suggesting that new communication channels arise through saltational shifts in female pheromone production, which are subsequently tracked by the male sex. Such shifts are facilitated by redundancies in the PR repertoire.
Three related species, H . ochroleucana , H. prunianaand H. salicella are best attracted to the Z ,Eisomers of codlemone and codlemone acetate, andZ ,E -codlemone is active in codling moth (El-Sayed et al. 1998; Witzgall et al. 2010b). A candidate PR forZ ,E -codlemone is HnubOR1 (Figure 2). Regarding HnubOR7a and HnubOR7b, which are close to GmolOR1 and GmolOR11, we hypothesize that they respond to the minor acetate pheromone components (Z )- and (E )-8-dodecenyl acetate (Tables 2, 3), which are main pheromone compounds of Oriental fruit moth G. molesta (Carde et al. 1979).

In silico identification of semiochemicals for the development of insect control

Semiochemicals are efficient tools for insect control, by air-permeation and mass trapping (El-Sayed et al. 2009; Witzgall et al. 2010a). The know-how of behaviour-modifying chemicals can also be brought to application through push-pull techniques or plants with modified metabolite release profile (Khan et al. 2014; Stenberg et al. 2015; Tamiru et al. 2015). A current bottleneck and urgent research challenge for further advancement is our understanding of which plant volatile metabolites mediate host recognition in phytophagous insects.
Availability of plant volatiles which attract insects to mating sites, elicit oviposition or feeding in adults and larvae leads to multiple applications. Pear ester, for example, is efficient for monitoring codling moth males and females, it can be used to supplement pheromone-based communication disruption and is a stand-alone tool for disruption of larval host-finding and feeding (Schmidt et al. 2008; Light and Knight 2011, Light and Beck 2012; Knight et al. 2012, Knight and Light 2013, Kovanci 2015, Light 2016). Another example is an efficient kairomone lure for apple fruit moth Argyresthia conjugella , based on characteristic host plant compounds (Bengtsson et al. 2006; Knudsen and Tasin 2015; Knudsen et al. 2008, 2017).
Current research on behaviourally active plant metabolites relies on electroantennogram recordings, coupled to a gas chromatograph. The GC-EAD method was originally conceived for sex pheromone identification (Arn et al. 1975). The conundrum, when working with plant volatiles, is that antennal recordings do not provide information on behavioural activity. GC-EAD recordings are also biased by the most abundant compounds, which invariably produce an antennal response. Recordings from single sensilla provide a solution, but in vivo recordings from sensilla other than s . trichodea , containing pheromone-sensitive neurons, are delicate. In codling moth, SSR recordings produced conclusive results when investigating pheromones, not plant volatiles (Bäckman et al. 2000; Ansebo et al. 2005).
In silico identification of OR ligands now emerges as an opportune advancement for plant semiochemical research. Phylogenetic analysis of OR gene sequences, in combination with functional characterization of selected ORs in model species, affords powerful predictions about behaviour-modifying plant volatiles. Identification of ORs, following antennal RNA sequencing, and hypotheses concerning their putative ligands is facilitated by the rapidly accumulating database of insect ORs. Antennal transcriptomes highlight highly expressed or sex-specific ORs, which become prime research targets. Furthermore, heterologous expression of ORs in select sensory neurons inDrosophila enables single sensillum recordings as a convenient and reliable approach for unambiguous identification of OR ligands (Dobritsa et al. 2003; Hallem et al. 2004; Gonzalez et al. 2016).
Promising targets for future work include, for example, tephritid fruit flies, in view of our thorough knowledge of Drosophila ORs (Liuet al . 2016; Muench and Galizia 2016) or moths from several families, aided by a rapidly accumulating database of lepidopteran antennal transcriptomes (e.g. Zhang et al. 2013; Cao et al. 2014; Jiang et al. 2014; Zhang et al. 2014, 2017; Corcoran et al. 2015; Koenig et al. 2015; Li et al. 2015; Park et al. 2015; Steinwender et al. 2015; Zeng et al. 2015; Zhang et al. 2015; Dong et al. 2016; Jia et al. 2016, 2018; Chang et al. 2017; Feng et al. 2017; Yang et al. 2017; Du et al. 2018; Rochas et al. 2018; Tian et al. 2018)

Interaction of plant volatiles and pheromones

Green budworm moth attraction to pear ester and codlemone is intriguing, because it provides further evidence for the association of olfactory channels dedicated to social and environmental signals in phytophagous insects.
Transcriptome data and phylogenetic context confirm this association. CpomOR3 is tuned to the plant volatile pear ester, while it belongs to the pheromone receptor clade (Figure 2A, 3; Bengtsson et al . 2012, 2014; Walker et al . 2016). That PRs respond to pheromones and plant volatiles has even physiological consequences: OR genes with highest sequence similarity tend to be expressed in OSNs that project to neighbouring glomeruli in the antennal lobe, facilitating interactions between the circuits encoding these signals (Couto et al. 2005, Krieger et al. 2009, Ramdya and Benton 2010). This has indeed been confirmed in codling moth, by intracellular recordings from olfactory projection neurons and functional imaging of the antennal lobe, showing a powerful synergistic interaction between codlemone and pear ester (Trona et al. 2010, 2013).
HnubOR3 has not been deorphaned, but the recent discovery that CpomOR3 responds to pear ester and to a lesser extent also to codlemone (Wan et al. 2019) provides an explanation for consistent attraction of H .nubiferana to codlemone (Tables 3, 4; Arn et al. 1974). Codling moth C. pomonella and H. nubiferana both feed on apple, but belong to different tortricid tribes (Bradley et al. 1979; Regier et al. 2012). Occurrence of conserved olfactory genes contributing to mate finding and host plant attraction lends further support to the concept that host plant recognition and sexual communication are interlinked (Borrero-Echeverry et al. 2018) and that a combination of natural and sexual selection gives rise to reproductive isolation in insect herbivores (Paterson 1978, Boughman 2002, Rosenthal 2017). A more complete analysis of olfactory genes and their behavioural and ecological functions will contribute to the study of phylogenetic divergence in phytophagous insects. Equally rewarding is the perspective that this research also drives the development of semiochemicals for efficient and sustainable insect control.