New Monoterpenoid as the Sex Pheromone of Spanish Populations of the Longtailed Mealybug Pseudococcus Longispinus (Hemiptera: Pseudococcidae)

Pseudococcus longispinus (Targioni-Tozzetti) (Hemiptera: Coccoidea: Pseudococcidae), a polyphagous and cosmopolitan pest native to Australia, is a highly damaging pest for numerous crops of economic importance. The sex pheromone of this species (2-(1,5,5-trimethylcyclopent-2-en-1-yl)ethyl acetate), currently used for pest monitoring purposes, was not attractive to males in field experiments conducted in Spanish persimmon orchards infested with this mealybug. The virgin and mated female volatile profiles of these P. longispinus populations were studied by the volatile collection of effluvia in Porapak-Q. The resulting extracts were analyzed by gas chromatography coupled to mass spectrometry (GC–MS), revealing a new compound specific to virgin females and different from the previously described sex pheromone. Based on GC–MS data and nuclear magnetic resonance experiments, we envisaged monoterpene 2-(1,5-dimethyl-4-methylenecyclopent-2-en-1-yl)ethyl acetate as the new sex pheromone candidate, which was synthesized and shown to be attractive in the field to P. longispinus males of the Spanish population.


■ INTRODUCTION
The long-tailed mealybug, Pseudococcus longispinus (Targioni-Tozzetti; Hemiptera: Coccoidea: Pseudococcidae), is a polyphagous and cosmopolitan pest that attacks a wide variety of plants (fruits, vegetables, and ornamental plants), causing serious damage to leaves, bark, branches, fruits, and roots. 1 It has been described on 209 genera of 98 families of plants. 2 Specifically, it is known as a very damaging pest for numerous crops of economic importance, among which are apple, pear, persimmon, grape, citrus, avocado, banana, and other tropical fruits. 3Although this species is native to Australia, 4,5 it has spread throughout the world, 6 reaching the Mediterranean region in the 19th century (1887 in Italy). 7It is currently present in 22 countries in continental Europe and the Mediterranean basin. 8s a mealybug, P. longispinus feeds on sap and produces honeydew that causes the proliferation of saprophytic fungi, as well as a decrease in the photosynthetic rate, loss of plant vigor, premature ripening, and fruit drop.In addition, due to its feeding habits, P. longispinus is capable of acquiring and transmitting viruses, being known for the effective transmission of some belonging to the families Betaflexiviridae 9 and Closteroviridae, 10 among others.In fact, the species P. longispinus is the only known vector of the grapevine leafrollassociated viruses GLRaV-5. 11he control of P. longispinus is currently based on the application of conventional chemical treatments, but their cryptic habits and the existence of overlapping generations make it difficult to manage.Moreover, since the ban by the European Commission on the use of methyl chlorpyrifos in January 2020, only a few active materials are currently available with limited efficacy, such as mineral oil and spirotetramat, 12 the latter with an upcoming prohibition order in Europe.In this context, the lack of effective methods for controlling P. longispinus is evident, and farmers and producer associations need both direct control measures and tools for detection and population monitoring.These tools are of vital importance to improving mealybug control both in agricultural and ornamental ecosystems.However, monitoring usually consists of a laborious visual inspection of the plant material in search of the stages of the insect.Sticky traps baited with sex pheromones are a great tool for monitoring the flight of males in a more comfortable and sensitive way than visual inspection. 13Many of the economically important pseudococcid species reproduce sexually, with females producing a sex pheromone to attract conspecific males.So far, the chemical structure of the sex pheromones of 32 species of coccoid insects belonging to the families Diaspididae, Matsucoccidae, Margarodidae, and Pseudococcidae is known, 14 and some of them are used for the detection and monitoring of their populations.The sex pheromone of the species P. longispinus was described by Millar et al. 15 as the compound 2-(1,5,5trimethylcyclopent-2-en-1-yl)ethyl acetate 1 and further tested green lemons, which were previously covered with paraffin wax around the midsection to delay their desiccation and prolong their useful life.Green lemons were preferred because of their size and the ease of detecting male pupae on their surface.Gravid females from pumpkins were gently transferred to green lemons.Then, newly hatched individuals established themselves on the surface of lemons and followed the developmental cycle.After the second instar stage, males produce a distinguishable cottony cocoon to pupate and transform into winged adults (Figure 1).Groups of lemons were visually inspected every 2−3 days for the presence of cocoons, which were manually removed with an entomological needle to leave lemons infested only with virgin females for volatile collection and profiling purposes.Other groups of lemons were left undisturbed to sample mated females.Lemons with virgin females were maintained in separated rooms under the same climate conditions.
Collection of Volatiles.Groups of 5−6 lemons infested with approximately 300 P. longispinus females (virgin or mated separately) were placed in 5 L glass containers (25 cm high ×17.5 cm diameter flask), with a 10 cm open mouth and a ground glass flange to fit the cover with a clamp.The cover had a 29/32 neck on top to fit the head of a gas washing bottle to connect downstream a glass cartridge to trap effluents in 3 g of Porapak-Q (Supelco Inc., Torrance, CA, USA) adsorbent.Samples were collected continuously for 7−8 days (collection round) by using an ultrapurified-air stream, provided by an air compressor (Jun-air Intl.A/S, Norresundby, Denmark) coupled with an AZ 2020 air purifier system (Claind Srl, Lenno, Italy) to provide ultrapure air (amount of total hydrocarbons <0.1 ppm).In front of each glass container, an ELL-FLOW digital flowmeter (Bronkhorst High-Tech BV, Ruurlo, The Netherlands) was fitted to provide an air push flow of 300 mL/min during sampling.Trapped volatiles were eluted with 20 mL of pentane (Chromasolv, Sigma-Aldrich, Madrid, Spain), and the resulting extracts were concentrated to 500 μL under a gentle nitrogen stream prior to the chromatographic analysis.Lemons were replaced after each collection round (7−8 days), and ten rounds of virgin female collections were performed to obtain approximately 21,000 female-day equivalents (FDE), a sufficient quantity for the nuclear magnetic resonance (NMR) analysis.
All the resulting pentane extracts were analyzed by GC−MS in a Clarus 600 GC−MS (PerkinElmer Inc., Waltham, MA), equipped with a ZB-5MS fused silica capillary column (30 m × 0.25 mm i.d.× 0.25 μm; Phenomenex Inc., Torrance, CA).The oven was held at 40 °C for 2 min and then programmed at 5 °C/min to 180 °C before being raised to 280 °C at 10 °C/min and maintained at 280 °C for 1 min.Helium was used as a carrier gas with a flow of 1 mL/min.Detection was performed in the electron impact (EI) mode (70 eV) with the ionization source set at 200 °C.Spectrum acquisition was carried out in full scan mode [mass range m/z 35−500 atomic mass units (amu)], and chromatograms and spectra were recorded by means of GC−MS TurboMass software v. 5.4 (PerkinElmer Inc., Waltham, MA).
Isolation of the Candidate Compound.After comparing the GC−MS volatile profiles of the virgin and mated samples (lemons infested with virgin and mated females, respectively), the virginspecific compound was isolated by fractionation of the Porapak-Q pentane extracts by gravity column (300 × 15 mm i.d.) using the whole sample of ca.21,000 FDE in 1 mL pentane.This sample was loaded into the column, and fractionation was performed using 12.5 g of silica gel as the stationary phase (40−60 μm) and eluents of pentane/diethyl ether mixtures (100:0, 95:5, 80:20, 50:50, and 0:100; 20 mL each).Forty fractions were collected, and they were all analyzed by GC−MS using the methods described in the previous section to identify those that exclusively contained the candidate compound.These fractions were gathered to collect the required quantity for the NMR analysis (ca. 60 μg).Then, the 1 H NMR spectrum of the natural isolated compound was recorded by a Bruker 600 Ultrashield Plus spectrometer (Bruker, Billerica, MA) at a frequency of 600 MHz using C 6 D 6 as the solvent with tetramethylsilane (TMS) as the internal standard.
Microreaction: Hydrolysis of the Natural 2-(1,5-Dimethyl-4methylenecyclopent-2-en-1-yl)ethyl Acetate (3).The pentane extract of a volatile collection sample of ca.200 FDE (∼500 ng of pheromone) was hydrolyzed following related procedures. 20The extract was dried under a gentle nitrogen stream in a 2 mL GC glass vial.The residue was treated with a 0.5 M solution of KOH in methanol (150 μL) and stirred for 45 min at room temperature.After this time, water (0.5 mL) was added, and the solution was extracted with dichloromethane (0.5 mL twice).The combined organic phases were concentrated under nitrogen to ca. 100 μL volume before being submitted to GC−MS analysis.
Synthesis of 2-(1,5-Dimethyl-4-methylenecyclopent-2-en-1-yl)ethyl Acetate (3).General Procedures.The 1 H and 13 C spectra were recorded on a Bruker AC-300 spectrometer (Bruker, Billerica, MA) using CDCl 3 or C 6 D 6 as the solvent and TMS as the internal standard.Chemical shift values in 1 H and 13 C NMR are reported at δ (ppm) relative to chloroform (7.26/77.0ppm) or benzene (7.16/128.4ppm).High-resolution mass spectra (ESI-HRMS) were measured on a Waters Xevo Q-TOF spectrometer (Waters Corp., Milford, MA) coupled with an Acquity UPLC-PDA system (Waters Corp., Milford, MA) using ionization by electrospray (ESI).The ESI source operated in the positive ionization mode using leucine-enkephalin as the reference mass ([M + H] + ion m/z 556.2771).The sample (2 μL) was injected into a Waters Acquity BEH column (50 × 2.1 mm i.d., 1.7 μm) using MeOH as an isocratic eluent.The GC−MS analyses were performed with the aforementioned equipment (apparatus and column) and the following oven temperature program: 55 °C for 3 min, raised at 15 °C/min up to 180 °C and then at 35 °C/min up to 280 °C, held for 6 min.A helium flow of 1 mL/min and an injection volume of 1 μL were employed.Detection and spectral acquisition were performed as indicated above.
All reagents and solvents (reagent grade) were purchased from Sigma-Aldrich (Madrid, Spain) and employed with no further purification unless otherwise stated.In the case of reactions requiring anhydrous conditions, the solvents used were dried with the appropriate drying agents and distilled before use.Unless otherwise stated, all of the reactions sensitive to moisture and/or air were carried out under a nitrogen atmosphere.The solvent extracts of the reaction mixtures were dried over anhydrous MgSO 4 or Na 2 SO 4 and concentrated by rotary evaporation under reduced pressure.Crude products were purified by flash column chromatography using silica gel Merck 9385 (230−400 mesh).Thin-layer chromatography (TLC) was performed using Macherey-Nagel silica gel 60 F254 plates with a fluorescent indicator and UV light of 254 nm wavelength as the visualizing agent.Ceric ammonium molybdate and p-anisaldehyde were used as stains.

2-(4-(Hydroxymethyl)-1,5-dimethylcyclopent-2-en-1-yl)ethyl 4-Nitrobenzoate
The number of captured males with each substance was compared using generalized linear mixed models (GLMM).For this purpose, the glmer function from the lme4 package was employed by assuming the poison error distribution.Models were constructed with the fortnightly captures as the dependent variables, substance and time (week of the study period) as fixed factors, and the block (experimental replicate) as the random factor.The significance of substance effects was assessed by removing them from each model and comparing models with likelihood ratio tests.The glht function in the multcomp package was then used to perform Tukey HSD tests for post hoc pairwise comparisons (P < 0.05).

■ RESULTS
Chemical Analysis and Structure Elucidation.The chromatographic volatile profiles of lemons infested with females, either virgin or mated, revealed an unknown, major, virgin-specific compound at 20.69 min, as well as another minor one at 20.26 min (1% regarding the area of the major compound) (Figure 2).The latter was identical to 2-(1,5,5trimethylcyclopent-2-en-1-yl)ethyl acetate 1, described by Millar et al., 15 in its GC retention time and MS spectra (Figure 2A).By sampling 21,000 FDE, ca.60 μg of the major compound was collected, which suggests that a single P. longispinus female emitted approximately 2.86 ng on average of this substance.The yield of 1 was approximately 100 times lower.Neither of the two substances mentioned was found in the samples of mated females.
At first, our main hypothesis was to find a related structure to 2-(1,5,5-trimethylcyclopent-2-en-1-yl)ethyl acetate 1, originally described by Millar et al. 15 (Figure 3).Assuming the peak at m/z 194 to be the molecular ion (Figure 2B), the mass spectrum of this compound shows its base peak at m/z 119 and main fragments at m/z 134, 107, 91, and 43.The characteristic loss of 60 amu to give m/z 134, together with the presence of the ion at m/z 43, suggested the presence of an acetate ester.As expected from other sex pheromones of related species, the fragmentation pattern seemed to be related to a monoterpenoid structure.A micro saponification of a sample of the isolated compound was performed, giving a new compound with a molecular ion of m/z 152, and main  fragments at m/z 137, 121, 119, 107, and 91 (Figure S25), confirming the presence of the acetate moiety.Interestingly, the mass loss fragments of this alcohol followed a very close fragmentation pattern when compared with the substituted cyclopentane alcohol obtained by reduction of the aldehyde pheromone 2 (Figure 3) of the pineapple mealybug Dysmicoccus brevipes (Cockerell), 22 showing mass fragments at m/z 139, 121, 109, and 93.This fact suggested a structural similarity of both compounds, but with an extra unsaturation (four in total) in the case of the newly isolated compound, with a possible molecular formula of C 12 H 18 O 2 .
Analysis of the 1 H NMR, 13 C NMR, and multiplicity-edited HSQC spectra of the natural isolated compound (Figure S26) showed characteristic signals for an unsaturated monoterpene.In particular, two coupled protons were clearly defined at δ 6.02 and 5.70 (each d, J = 5.8 Hz), which correlate with olefinic CH carbons at δ 145.2 and 132.6 ppm, respectively, clearly indicating the presence of a cis olefinic moiety.On the other hand, the protons at δ 4.87 (d, J = 2.2 Hz) and 4.68 (bs) ppm showed a single correlation with a carbon signal in the alkene region (δ 102.9 ppm), indicative of the presence of an exo methylene moiety.The only additional signal in the most deshielded region of the proton spectrum is a multiplet centered at δ 4.03 ppm, correlated with the signal of a methylene carbon atom at about δ 62 ppm, which is compatible with a methylene group attached to an acetoxy moiety, whose methyl group is also observed in the proton and carbon NMR spectra at δ 1.67 and 20.6 ppm, respectively.The rest of signals of the proton spectrum appear in the resonance region of aliphatic protons, highlighting a multiplet centered at δ 2.25 ppm, correlated with the signal of a methyne carbon atom at about δ 49 ppm, compatible with the presence of an allylic CH group.The most shielded signals in the proton spectrum correspond to two methyl groups, one at δ 0.95 ppm (d, J = 7.2 Hz), correlated with the signal of a carbon atom at δ 11.8 ppm, compatible with a CH 3 group attached to the methyne carbon atom, and another at δ 0.88 ppm (s), correlated with the carbon signal at δ 25.6 ppm, compatible with a CH 3 group on a quaternary center.
The mass spectra and NMR data discussed above allowed us to tentatively suggest that the chemical structure of the new pheromone is based on a 3-methylene-cyclopentene framework bearing a methyl group at position C−4 and a methyl and a 2-acetoxyethyl groups at position C−5.We envisage a structure such as 3 [2-(1,5-dimethyl-4-methylenecyclopent-2en-1-yl)ethyl acetate], initially assuming a trans relationship between the methyl groups at C−4 and C−5 as in the previously mentioned sex pheromone of the pineapple mealybug.A confirmatory synthesis of the proposed structure was carried out in order to confirm the identity of this new sex pheromone.
Synthesis and Identification of the Pheromone.The synthetic strategy followed for the preparation of compound 3 is outlined in the retrosynthetic analysis depicted in Figure 4.In principle, compound 3 could be synthesized from an intermediate such as i using a base-mediated elimination reaction upon transformation of the protected hydroxyl group into a suitable leaving group.This intermediate could be synthesized from allylic acetate ii using a Claisen-Ireland [3,3]sigmatropic rearrangement of the silyl enol ether derivative of the acetate moiety as key step, giving thus access to the quaternary carbon atom present in the target structure, a  strategy previously used by Millar et al. 15 for the synthesis of the related skeleton of the P. longispinus sex pheromone 1. Acetate ii could be achieved from readily available cyclopentenone 4 21 via hydroxymethylation, followed by appropriate protection of the introduced hydroxyl group, reduction of the carbonyl group, and acetylation of the resulting allylic alcohol.
The synthesis of 3 commences with the hydroxymethylation of cyclopentenone 4 (Figure 5) that was undertaken following the procedure previously described for related substrates. 23hus, treatment of the enolate generated from the reaction of 4 with LDA with paraformaldehyde afforded a 95:5 racemic mixture of anti/syn diastereoisomeric 5-hydroxymethylated cyclopentenones, which could be separated by column chromatography to afford the pure anti diastereomer 5 in 40% yield.Protection of the hydroxyl group of 5 as tetrahydropyranyl ether under standard conditions with DHP and catalytic PPTS gave 6 in 65% yield.Regioselective reduction of enone 6 with LiALH 4 in Et 2 O gave the secondary alcohol 7 as a 95:5 mixture of epimers at the carbinol center, as deduced from the analysis of the 1 H NMR spectra (for clarity, only the major β-epimer is drawn in Figure 5).This high stereoselectivity is in concordance with the result observed in the reduction of structurally related cyclopentenones 24,25 and could be probably rationalized in terms of the steric effect exerted by the methyl group at C−4 and the potential coordination of the reducing agent to the tetrahydropyranyl ether moiety.In our hands, no separation of this mixture of epimeric alcohols using conventional silica gel chromatography was possible.Conversion of 7 into its corresponding acetate afforded 8 in a 70% yield.Ireland-Claisen rearrangement of the trimethylsilyl enol ether derived from acetate 8, followed by acid treatment of the reaction mixture, gave carboxylic acid 9 in a modest 55% yield, but that is in agreement with the results obtained in rearrangements of structurally related substrates. 19he trans relative disposition of the methyl groups in the rearranged product 9 was inferred from NOE experiments that showed that irradiation of both the AB system contiguous to the carboxylic group (δ 2.26 ppm), and the methyl group at the quaternary C−1 position (δ 1.21 ppm) caused a weak but significant increase in the intensity of the methyl group (δ 1.04 ppm) and the methyne proton (δ 1.67 ppm) at C−5, respectively.The determination of the relative stereochemistry of acid 9 also served to confirm the stereochemistry initially assigned for the major epimeric alcohol formed in the reduction reaction of enone 6 since the Ireland-Claisen Once the carbon framework of compound 3 was completed, the rest of the steps to complete its synthesis involved only modifications of the functional groups present in 9.The first functional transformation carried out was the reduction of the carboxylic group to the corresponding alcohol, a reduction that was carried out by treating acid 9 with LiAlH 4 in Et 2 O to afford the primary alcohol 10 in a 95% yield which was acylated using acetic anhydride, Et 3 N, and DMAP under usual conditions to obtain the corresponding acetate 11 in 66% yield.Next, the tetrahydropyranyl group was removed by treatment of 11 with MeOH and catalytic PTSA to give alcohol 12 in 90% yield, whose hydroxyl group was replaced by an iodine atom using the iodine−triphenylphosphine−imidazole reagent.Finally, iodide-obtained 13 was subjected to a bimolecular elimination process to generate the exocyclic double bond using DBU as a base and relatively mild temperature conditions.The E2 reaction proceeded quite efficiently to provide the racemic form of cyclopentadiene 3 in 91% yield.
The 1 H NMR spectroscopic data of synthetic diene 3 in C 6 D 6 afforded a set of main signals, which were fully coincident with those observed for the natural isolated compound from virgin female volatile emissions (Figure 6).The identity of both compounds was further corroborated by GC and the mass fragmentation pattern which was virtually identical for the synthetic and natural samples.
To unequivocally confirm the relative stereochemistry assigned to the generated stereogenic centers based on NMR data, we performed an X-ray diffraction analysis of a crystalline derivative of alcohol 10.The derivative chosen was the corresponding p-nitrobenzoyl ester 15 (Figure 7) that was prepared in two steps from alcohol 10.First, treatment of 10 with p-nitrobenzoyl chloride and Et 3 N to give ester 14 with a 90% yield, followed by the removal of the THP group with PTSA in MeOH to afford ester-alcohol 15 in 85% yield.An appropriate sample for single-crystal X-ray diffraction analysis was obtained when the solid p-nitrobenzoyl ester was crystallized from cold hexane, whose ORTEP diagram is shown in Figure S28, confirming the relative configuration initially proposed for all the stereogenic centers of the cyclopentene skeleton.
Field Activity.In the field, blank traps and traps baited with compound 1 captured only 1 and 26 males, respectively, throughout the trial, whereas a total of 4178 males were captured in traps baited with compound 3 (Figure 8).This confirms the identity of the natural sex pheromone compound previously determined by the analytical procedures.

■ DISCUSSION
Compound 1 (2-(1,5,5-trimethylcyclopent-2-en-1-yl)ethyl acetate), originally identified as the sex pheromone of the longtailed mealybug by Millar et al., 15 has been recently employed in field trials conducted in New Zealand, 26 effectively capturing males and finding an economic optimal dose of 20 μg when employing the racemic mixture of 1 for monitoring P. longispinus.They also found that the non-natural enantiomer did not affect the attraction of the natural (S)-(+)-enantiomer detrimentally.−18 However, traps baited with the reported synthetic pheromone 1 captured only a few males in those persimmon orchards, whereas traps baited with virgin females of the local populations captured 100 times more males than compound 1.This result strongly suggested that the sex pheromone blend of P. longispinus Spanish populations was different from that previously reported.Accordingly, our study on the volatile profile of P. longispinus females collected in Spanish persimmon orchards revealed a new monoterpenoid, 2-(−1,5-dimethyl-4-methylenecyclopent-2-en-1-yl)ethyl acetate (compound 3), as the major sex pheromone component of these insect populations with a powerful attractant activity.Interestingly, a minor (ca.1%) quantity of compound 1 was also detected in virgin female effluvia but demonstrated very poor field attraction by itself.
Following the structural patterns observed in mealybug sex pheromones identified until now, compounds 1 and 3 belong to the monoterpenes.However, the described new compound possesses a 1,2,3 trimethyl cyclopentane skeleton, similar to the one described by Tabata et al. 22 for the sex pheromone of D. brevipes.The biosynthetic origin of compound 3 could be rationalized from a regular head-to-tail 4′−3 linkages between the isoprene units, followed by a 3′-3 connection and Wagner−Meerwein rearrangements.The first insaturation could be explained via elimination reaction and, presumably, as in the case of the pheromone of Delottococcus aberiae (De Lotto), 20 a desaturase is necessary to introduce second unsaturation into the cyclopentane framework (Figure 9).
It is well established that the already reported structural diversity of regular and irregular monoterpenoids of Coccoidea sex pheromones does not always follow the phylogenetic  relationship between closely related mealybug species, such as in the case of D. brevipes and D. neobrevipes Beardsley, the former having a cyclopentane aldehyde and the last an acyclic acetate. 22Moreover, intraspecies pherotype blend modulation or divergence is not uncommon and has been also described for moths (E/Z isomeric ratio changes in Ostrinia nubilalis (Hubner) 27 ) or mealybug species, such as Planococcus ficus (Signoret), whose Californian populations produce a singlecomponent sex pheromone, while the Israeli ones produce an additional compound. 28Isolation of populations during more than a century may explain such phenotype changes.In the case of P. longispinus, Californian and European populations were reported in 1918 29 and 1866, 7 respectively, although Australia was concluded to be the native habitat of the species, on the basis of its low population density and high parasitism rates. 5Indeed, it is important to mention that other factors, such as the host plant, may influence the species-specific sex pheromone evolution, being more plastic than previously assumed. 30Considering that P. longispinus is a polyphagous species, this fact could support the existence of, at least, two different pherotypes pointing to a different cyclization process of the isoprene units in the sex pheromone production that is operating in each population.This fact should be studied by using molecular techniques, trying to identify the mutations that produce these pheromone blend modulations in the P. longispinus populations originating from Australia, America, and Europe.
Mealybug sex pheromones could be powerful tools for controlling pest populations, and some examples of their use have been recently described.In particular, the mating disruption technique has been developed and tested for P. ficus, 31 Pseudococcus calceolariae (Maskell), 32 or Planococcus kraunhiae (Kuwana), 33 but only for P. ficus there is a commercially available mating disruption treatment for controlling the pest. 34,35This is probably due to a more cost-effective synthetic pheromone according to their structures. 36Compound 1, the first reported sex pheromone of P. longispinus, has been shown to be useful for population monitoring purposes; 37−39 however, no direct control method based on this substance has been described yet, probably due to its complicated and expensive synthetic process.
The absolute configuration of the natural compound 3 is not yet established, but the racemic mixture has proven effective to capture P. longispinus males in our field experiments.The use of this new monoterpenoid to monitor pest populations can be immediately implemented, improving the sensitivity of the current visual inspection procedures.However, further studies are needed to evaluate the activity of the combination of compounds 3 and 1, as detected in virgin female effluvia, as well as the potential of direct control techniques based on this new sex pheromone, such as mating disruption or attract and kill.

Figure 1 .
Figure 1.Pictures of female and male Pseudococcus longispinus collected in persimmon orchards.

Figure 6 .
Figure 6. 1 H NMR spectra of the natural and synthetic compound 3 samples were fully coincident.
The authors would like to thank Elytra Agroscience Services S.L. (Valencia, Spain) for the taxonomic identification of P. longispinus specimens and CANSO COOP (L'Alcudia, Valencia, Spain) for providing trial orchards.Funding for open access charge: CRUE-Universitat Politecnica de Valeǹcia.