Results

Sex pheromone identification

Analysis of green budworm moth H . nubiferana pheromone gland extracts by GC and GC-MS showed eight further compounds, in addition to the previously identified acetates (Frerot et al. 1979). The major compound E 8,E 10-12Ac was accompanied by the monounsaturated 8- and 10-dodecenyl acetates, its three geometric isomers (EZ , ZE , and Z 8,Z 10-12Ac) as well as the analogous alcohol codlemone, E 8,E 10-12OH (Table 1).
Field attraction of H. nubiferana males to compounds identified from the female gland confirms that the sex pheromone of H. nubiferana is a blend of E 8,E 10-12Ac and Z 8-12Ac (Table 2; Frerot et al. 1979). The main compound,E 8,E 10-12Ac by itself was not attractive, while addition of Z 8-12Ac had a strong synergistic effect (F(7,72)=61.95,P <0.0001). Addition of E 8-12Ac further increased male attraction in untreated apple orchards, but the difference was not significant. Blends of E 8,E 10-12Ac and the 10-12 monoenes or the analogous alcohol, codlemone, did not produce significant captures. Adding these compounds to the 3-component acetate blend slightly diminished trap catch (Table 2).
The gland compounds identified from female glands with no apparent effect on attraction may be biosynthetic by-products or precursors. A study of the female effluvium will show whether they are released at all, and at which ratio. The full blend of compounds may also carry information that cannot be revealed by a field trapping test.

Attraction to codlemone and pear ester

A trap test in an apple orchard adjacent to a pea field corroborates that codlemone acetate E 8,E 10-12Ac as a single compound does not attract green budworm moth. Attraction of pea moth confirms that the trap lures released E 8,E 10-12Ac at high isomeric purity (Table 3; Witzgall et al. 1993, 1996). In comparison, traps baited with codlemone alone regularly captured few green budworm moth males, in addition to codling moth. Blends of codlemone and codlemone acetate attract far fewer codling moths and no green budworm moths at all (Table 3).
Interestingly, a blend of codlemone and its three geometric isomers significantly increased green budworm moth captures over codlemone alone (Table 4; F(7,72)=2.62, P =0.04413). In contrast, this isomer blend captured fewer codling moth males (Table 4; F(7,72)=4.22,P =0.02135; El-Sayed et al . 1998).
Green budworm moth has also been reported to respond to pear ester (Schmidt et al. 2007; Jósvai et al. 2016). A further field test in Hungary confirmed this and showed that addition of codlemone to pear ester does not enhance attraction of either sex (Table 5).
Orchard mating disruption treatments with codlemone strongly diminished attraction of H. nubiferana males to pheromone traps (Table 2), corroborating a behavioural effect of codlemone via a dedicated olfactory channel. This supports the idea that communication disruption in moths may be achieved with single pheromonal compounds or incomplete pheromone blends (Cardé and Minks 1995; Porcel et al. 2015), which is of practical importance for the implementation of pheromonal control of codling moth and leafrollers in European orchards.

Phylogenetic analysis and antennal expression

Hedya nubiferana Haworth and Hedya dimidioalba Retzius are synonymous taxonomic names for green budworm moth. The National Center for Biotechnology Information (NCBI) lists OR sequences (including PRs) as ”HnubOR##”.
Predicted putative PRs from H. nubiferana clustered in 4 different subfamily clades when compared with PRs from other tortricid species (Figure 2A). Several of these displayed homology to receptors inCydia pomonella (CpomOR3, CpomOR6 and CpomOR22) and these relationships were supported by boostrap values above 95.
Notably, HnubOR6 was >50% similar to CpomOR6. Sequence comparison analysis revealed that CpomOR1 and HnubOR2 shared 49% amino acid identity and 66% similarity, while the OR3 orthologs of both species shared 64% and 76% identity and similarity, respectively. Amino acid differences between these putative PRs are observed across the entire length of the protein sequences (Figure 3).
Abundance estimation of the predicted sequences showed that the most highly expressed were HnubOR6 and HnubOR2. The other 3 putative PRs detected in male antennae were one or two orders of magnitude lower (Figure 2B).