Identification of (E)‐ and (Z)‐11‐tetradecenyl acetate as sex pheromone components of the currant pest Euhyponomeutoides albithoracellus

The currant bud moth Euhyponomeutoides albithoracellus is a destructive pest in black currant orchards in Northern Sweden and Finland. The larvae feed on the buds, and at high densities, the species can cause severe yield losses. Sex pheromone components of the bud moth were identified via solvent extraction of excised female pheromone glands, analyses by gas chromatography with electroantennographic detection and gas chromatography–mass spectrometry and field trapping experiments. Antennae of males responded strongly and consistently to two compounds in extracts, identified as (E)‐11‐tetradecenyl acetate and (Z)‐11‐tetradecenyl acetate. Weaker and less consistent responses were observed to the corresponding alcohols, (E)‐11‐tetradecenol and (Z)‐11‐tetradecenol, and tetradecyl acetate. Field tests showed strong attraction of bud moth males to a 1:1 blend of (E)‐11‐tetradecenyl acetate and (Z)‐11‐tetradecenyl acetate. Adding the alcohols to the binary acetate blend reduced trap catches drastically, whereas tetradecyl acetate had no statistically significant impact on male attraction when added to that binary blend. Finally, testing different compositions of the binary acetate blend revealed highest catch in traps baited with a 25:75 or 50:50 ratio of the E:Z acetate isomers. The identification of sex pheromone components of the bud moth contributes to developing sustainable control of this pest via monitoring and mating disruption with sex pheromone.

. The damage caused by E. albithoracellus is similar to that of L. capitella, and the two species are often found in the same orchard.
Pyrethroids have previously been used in early spring to kill young bud moth larvae when they disperse from their hibernation buds, but such insecticide application is difficult due to the wet soil conditions in the fields during this period. The routine applications with endosulfan or fenpropatrin, that formerly were carried out against the gall mite Cecidophyopsis ribis (Westwood) shortly before the flowering of black currant, also had some effect on larger larvae (Hellqvist, 1981). However, since 2010 all pyrethroids are banned within EU for use in black currant orchards, and there is thus an urgent need for alternative cost-effective and environmentally safe pest control methods. Targeting adults would be beneficial because it will limit the initial damage caused by young larvae. An optimized sex pheromone would facilitate monitoring of bud moths in currant orchards, and trap catch data could be a useful tool in integrated pest management (IPM) to get information about presence, flight phenology and abundance of the species, which will aid in decisions about optimal use of pesticides. The pheromone could also potentially be used for population control by mating disruption.
We here report the identification of sex pheromone components of E. albithoracellus by electrophysiological and chemical analyses of compounds produced from the terminal abdominal gland of female moths and field trapping experiments to demonstrate their behavioural activity.

| Collection and rearing of moths
Black currant twigs infested by E. albithoracellus were collected at Sikfors, Sweden (65°3′N, 21°11′E), in March-April and sent to Lund.
Twigs were placed in 500 mL glass jars filled with water to keep them fresh during the development of moth larvae. The twigs were transferred to transparent Plexiglass cages (30 × 30 × 60 cm) with a fine mesh net on the back side and placed in a climate room at 22°C, 65% r.h. and 20:4 L:D photoperiod. Larvae were allowed to feed on fresh twigs until pupation. The pupae were removed from their cocoons, separated by sex on the basis of genital characters, and kept in separate plastic boxes until adult emergence. Moths of 1-4 days of age were used in all analyses.

| Extraction of female pheromone glands
The terminal abdominal segments, including the ovipositor, were dissected ca. 2 h into the scotophase. Glands were placed in a micro vial including 5-10 μL of ultrapure (>99%) heptane (Merck, Darmstadt, Germany) and left for 30 min at room temperature, after which the extract was transferred to a new vial and stored at −18°C until used for electrophysiological or chemical analyses.

| Electrophysiological analyses
Gas chromatography coupled with electroantennographic detection (GC-EAD) was used to identify compounds in E. albithoracellus female gland extracts that elicited antennal response in conspecific males.
In these analyses, 1 μL of gland extract or a blend of synthetic candidate compounds including E11-14:OAc, Z11-14:OAc, E11-14:OH, Z11-14:OH and 14:OAc (1 ng/μL each) was injected into an Agilent 7890A gas chromatograph (Agilent Technologies), with hydrogen as carrier gas (velocity: 51 cm/s; flow rate: 1.8 mL/min) and an injector temperature set at 225°C. Columns used were either a mediumpolar HP-INNOWax (30 m × 0.25 mm ID, 0.25 μm film thickness) or a non-polar HP-5 (30 m × 0.32 mm ID, 0.25 μm film thickness) (J&W Scientific, Folsom, CA, USA). Column temperature was maintained at 80°C for 1 min and then increased to 210°C at a rate of 10°C/min and held for 10 min. The antennal preparation consisted of the head with both antennae and was mounted to a PRG-2 EAG probe (10× gain) (Syntech, Kirchzarten, Germany) using conductive gel (Blågel, Cefar, Malmö, Sweden). Charcoal-filtered and humidified air was blown over the antennae from a glass tube outlet positioned at 5 mm distance from the preparation. The effluent from a column was split 1:1 with half the sample going to the flame ionization detector (FID) and the other half to the antennal preparation after passing through a heated transfer line set at 230°C. In total, 20 antennal preparations gave reliable results in the GC-EAD recordings (each preparation was used only for 1-2 runs). Data were analysed using GC-EAD Pro Version 4.1 (Syntech, Kirchzarten, Germany).

| Chemical analyses
Analyses of pheromone gland extracts of E. albithoracellus and synthetic reference compounds were performed on an Agilent 5977B mass-selective detector coupled to an Agilent 8890 gas chromatograph equipped with an HP-INNOWax column (dimensions as above). Oven temperature was kept at 80°C for 1 min and then increased to 230°C at a rate of 10°C/min and held for 15 min. Injector and transfer line temperatures were 250°C and 280°C, respectively, and helium was used as the carrier gas. The compounds eliciting antennal responses in GC-EAD recordings were identified through comparison of their retention times with those of synthetic reference compounds (see above).

| Field trials
The first trapping experiment was performed 13th July -2nd August In each experiment, five replicates were used, separated by at least 20 m, and within a replicate, traps were randomized and set 5 m apart

| Statistical analyses
No males were trapped in control traps in the first experiment, and this treatment was excluded from the statistical analysis. Catches per trap were pooled and log (x + 1) transformed before applying oneway ANOVA, followed by multiple comparisons adjusted according to the Bonferroni post hoc test, to compare catches among treatments. All significance tests were performed using SPSS Version 27 (SPSS Inc., Chicago, IL, USA).

| Electrophysiological analyses
In the GC-EAD analyses of gland extracts using the INNOWax column, antennae of male E. albithoracellus showed strong and consistent response to two compounds eluting close to each other ( Figure 1a). Weaker and less consistent responses were observed to F I G U R E 1 Gas chromatography with flame ionization (FID) and electroantennographic detector (EAD: male Euhyponomeutoides albithoracellus antennae) responses to (a) an aliquot of a 0.2 female equivalent of pheromone gland extract or (b) synthetic reference compounds (tetradecyl acetate (1), (E)-11-tetradecenyl acetate (2), (Z)-11-tetradecenyl acetate (3), (E)-11-tetradecenol (4) and (Z)-11-tetradecenol (5); 1 ng per compound). The analyses were performed using HP-INNOWax column. two later eluting compounds with similar retention times. In addition, an antennal response was occasionally observed to a fifth compound eluting earlier than the other gland constituents. However, none of these compounds could be identified in the subsequent chemical analyses because the amounts present in the extracts were below the detection limit of the GC-MS. Based on the elution pattern of antennally active gland constituents, and the fact that male bud moths are attracted to the A. podana lure (Peltotalo & Touvinen, 1986), we hypothesized that the two compounds eliciting strongest antennal response were E11-14:OAc and Z11-14:OAc, that the later eluting compounds were the corresponding alcohols, E11-14:OH and Z11-14:OH and that the early eluting compound was 14:OAc, which has been reported as a sex pheromone component in other ermine moth species. Thus, additional GC-EAD analyses were performed using a synthetic blend including these five compounds as stimulus to confirm their activity.
All candidate compounds were shown to be electrophysiologically active, and their retention times matched the corresponding antennal responses observed in the analyses of gland extracts (Figure 1b).
When stimulated with the synthetic blend, the responses to the unsaturated acetates were consistently higher than the responses to the corresponding alcohols. In addition, 14:OAc was found to elicit a strong antennal response, although a similar response was only observed inconsistently when antennae were stimulated with gland extracts, indicating that the compound was not produced by females in amounts eliciting any EAD response (Figure 1a). The response amplitudes for E11-14:OAc and Z11-14:OAc were similar when antennae were stimulated with gland extract and synthetic compounds ( Figure 1a,b), indicating that these isomers were produced in similar amounts by female moths. Additional GC-EAD and GC-MS analyses using an HP-5 column showed similar results, although separation of the isomers of 11-14:OAc and 11-14:OH was very poor on this column (data not shown).

| Field trials
In the first experiment, we observed significant differences in catches among treatments (F = 18.72, df = 5, p < 0.001). High numbers of E. albithoracellus males were attracted to traps baited with a 1:1 blend of E11-and Z11-14:OAc, as well as to traps baited with this binary blend in combination with 14:OAc (Figure 2). Very few males were trapped when the lure contained only one of the ∆11 acetate isomers. In addition, trap catches were drastically reduced when the corresponding alcohols were present in a lure in the same amounts as the acetates. The average catch was numerically >60% higher when 14:OAc was added to the binary acetate blend (Figure 2), but the difference between that treatment and the binary blend alone was not statistically significant. A second trapping experiment was performed to further investigate the potential synergistic effect on male attraction when adding different amounts of 14:OAc to the binary acetate blend. Again, no significant effect of 14:OAc on trap catches was observed (F = 1.32, df = 4, p > 0.05, Figure 3). Finally, F I G U R E 2 Catch of male Euhyponomeutoides albithoracellus in traps baited with different blends of candidate pheromone compounds. The field trial was performed in 2004 in a black currant orchard at Sörfors, Sweden. Bars with different letters indicate significantly different catches (ANOVA on log(x + 1)-transformed data followed by multiple comparisons according to the Bonferroni post hoc test: p < 0.01).

F I G U R E 3
Catch of male Euhyponomeutoides albithoracellus in traps baited with different amounts of tetradecyl acetate in combination with a 1:1 blend of (E)-11-tetradecenyl acetate and (Z)-11-tetradecenyl acetate. The field trial was performed in 2005 in a black currant orchard at Sörfors, Sweden. No significant differences in catches among treatments were observed (ANOVA on log(x + 1)-transformed data followed by multiple comparisons according to the Bonferroni post hoc test: p > 0.05).
the third experiment revealed significant differences in attraction of males to different compositions of the binary acetate blend (F = 8.46, df = 4, p < 0.001). Large numbers of males were captured in traps baited with 25% or 50% of the E isomer, whereas the other ratios attracted significantly fewer males (Figure 4).

| DISCUSS ION
The results from our electrophysiological and chemical analyses, and trapping experiments, show that the main components of sex pheromone of E. albithoracellus are E11-14:OAc and Z11-14:OAc. Strong antennal responses to both acetate isomers were observed in GC-EAD analyses using female gland extracts and synthetic reference compounds (Figure 1). In addition, the first field trial revealed that both isomers are needed for attraction of conspecific males, and subtraction of either isomer resulted in a drastic trap catch reduction (Figure 2). Antennal responses to E11-14:OH and Z11-14:OH were also observed, but these were weaker and less consistent compared to the responses to E11-and Z11-14:OAc. Adding the alcohols to the binary acetate blend resulted in almost complete loss of attraction. We cannot, however, exclude the possibility that the alcohols are still part of the sex pheromone of E. albithoracellus and that the strong inhibitory effect observed was caused by using excessive amounts or skewed relative ratios of the alcohol isomers in relation to the acetate isomers.
Strong antennal response was also observed to 14:OAc during GC-EAD analyses with a blend of synthetic candidate compounds, but when antennae were exposed to gland extracts, a response at the retention time matching this compound was inconsistent. The second field test showed that adding different amounts of 14:OAc to the binary acetate blend did not cause any statistically significant differences in attraction of males to traps (Figure 3). An explanation for the strong antennal response, but lack of behavioural effect, to 14:OAc in male E. albithoracellus is thus unclear, but may be because of activation of the receptors for the unsaturated pheromone components on the male antenna, which has been observed in other ermine moth species (Löfstedt et al., 1990). Attraction of males to lures containing 14:OAc has not been reported for other species of the genus Euhyponomeutoides (www.phero base.com), and so far, reports of 14:OAc as a primary sex pheromone component or a synergistic secondary component are restricted to small ermine moths of the genus Yponomeuta (e.g. Löfstedt et al., 1986Löfstedt et al., , 1991. The third experiment testing different relative ratios of E11and Z11-14:OAc showed that male E. albithoracellus were highly attracted to lures containing 25% or 50% of the E isomer, whereas much fewer males were captured in traps baited with the other blends tested (Figure 4). The chemical analyses of gland extracts from individual females revealed that the E. albithoracellus sex pheromone is produced in minute amounts. Neither acetate isomer could be detected by FID, and the relative ratios of the compounds in the extracts could thus not be established. Our GC-EAD and catch data, however, suggest that females produce a pheromone that is indeed close to the 1:1 blend of E-and Z11-14:OAc used in lures for monitoring of A. podana (Persoons et al., 1974), which Tuovinen (1989) found useful also for trapping of E. albithoracellus.
Our identification of female-produced sex pheromone components of E. albithoracellus is the first such study from the genus Euhyponomeutoides. A screening study in Japan by Ando et al. (1981), testing attraction of moth species to various lures, reported catches of male Euhyponomeutoides trachydeltus (Meyrick) in traps baited with Z11-14:OAc, but data on pheromone or sex attractant composition for other congeners are lacking. In a broader phylogenetic context, E11-and Z11-14:OAc are common sex pheromone components in lepidopterans, and confirmed activity of these compounds in field tests has been reported from species in the families Cosmopterigidae (Bestmann et al., 1993), Crambidae (e.g. Klun et al., 1973), Noctuidae (e.g. Burns & Teal, 1989), Pyralidae (e.g. Wakamura et al., 1999), Tortricidae (e.g. Roelofs & Arn, 1968) and Yponomeutidae (e.g. Löfstedt & van der Pers, 1985).
Damage caused by bud moth larvae is a major problem for currant growers in northern Sweden and Finland, whereas recent monitoring suggests that the species is absent in currant orchards in Norway (O. Anderbrant, unpubl. observations). With stricter EU regulations on pesticide use, there is an urgent need for alternative control methods for currant pests, including E. albithoracellus.

F I G U R E 4
Catch of male Euhyponomeutoides albithoracellus in traps baited with different relative ratios of (E)-11-tetradecenyl acetate and (Z)-11-tetradecenyl acetate. The field trial was performed in 2022 in a black currant orchard at Rödupp, Sweden. Bars with different letters indicate significantly different catches (ANOVA on log(x + 1)-transformed data followed by multiple comparisons according to the Bonferroni post hoc test: p < 0.05).
Today, monitoring of the species is performed using lures aimed