Female semiochemicals stimulate male courtship but dampen female sexual receptivity.

Chemical communication plays a vital role in mate attraction and discrimination among many insect species. Here, we document a unique example of semiochemical parsimony, where four chemicals act as both aphrodisiacs and anti-aphrodisiacs in different contexts in Bactrocera dorsalis. Specifically, we identified four female-specific semiochemicals, ethyl laurate, ethyl myristate, ethyl cis-9-hexadecenoate, and ethyl palmitate, which serve as aphrodisiacs to attract male flies and arouse male courtship. Interestingly, these semiochemicals, when sexually transferred to males during mating, can function as anti-aphrodisiacs, inhibiting the receptivity of subsequent female mates. We further showed that the expression of elongase11, a key enzyme involved in the biosynthesis of these semiochemicals, is under the control of doublesex, facilitating the exclusive biosynthesis of these four semiochemicals in females and guaranteeing effective chemical communication. The dual roles of these semiochemicals not only ensure the attractiveness of mature females but also provide a simple yet reliable mechanism for female mate discrimination. These findings provide insights into chemical communication in B. dorsalis and add elements for the design of pest control programs.

Fungal Volatile Organic Compounds: More Than Just a Funky Smell?

Many volatile organic compounds (VOCs) associated with industry cause adverse health effects, but less is known about the physiological effects of biologically produced volatiles. This review focuses on the VOCs emitted by fungi, which often have characteristic moldy or "mushroomy" odors. One of the most common fungal VOCs, 1-octen-3-ol, is a semiochemical for many arthropod species and also serves as a developmental hormone for several fungal groups. Other fungal VOCs are flavor components of foods and spirits or are assayed in indirect methods for detecting the presence of mold in stored agricultural produce and water-damaged buildings. Fungal VOCs function as antibiotics as well as defense and plant-growth-promoting agents and have been implicated in a controversial medical condition known as sick building syndrome. In this review, we draw attention to the ubiquity, diversity, and toxicological significance of fungal VOCs as well as some of their ecological roles.

Solvent Extraction of PDMS Tubing as a New Method for the Capture of Volatile Organic Compounds from Headspace.

Polydimethylsiloxane (PDMS) tubing is increasingly being used to collect volatile organic compounds (VOCs) from static biological headspace. However, analysis of VOCs collected using PDMS tubing often deploys thermal desorption, where samples are considered as 'one-offs' and cannot be used in multiple experiments. In this study, we developed a static headspace VOC collection method using PDMS tubing which is solvent-based, meaning that VOC extracts can be used multiple times and can be linked to biological activity. Using a synthetic blend containing a range of known semiochemicals (allyl isothiocyanate, (Z)-3-hexen-1-ol, 1-octen-3-one, nonanal, (E)-anethol, (S)-bornyl acetate, (E)-caryophyllene and pentadecane) with differing chemical and physicochemical properties, VOCs were collected in static headspace by exposure to PDMS tubing with differing doses, sampling times and lengths. In a second experiment, VOCs from oranges were collected using PDMS sampling of static headspace versus dynamic headspace collection. VOCs were eluted with diethyl ether and analysed using gas chromatography - flame ionization detector (GC-FID) and coupled GC - mass spectrometry. GC-FID analysis of collected samples showed that longer PDMS tubes captured significantly greater quantities of compounds than shorter tubes, and that sampling duration significantly altered the recovery of all tested compounds. Moreover, greater quantities of compounds were recovered from closed compared to open systems. Finally, analysis of orange headspace VOCs showed no qualitative differences in VOCs recovered compared to dynamic headspace collections, although quantities sampled using PDMS tubing were lower. In summary, extraction of PDMS tubing with diethyl ether solvent captures VOCs from the headspace of synthetic blends and biological samples, and the resulting extracts can be used for multiple experiments linking VOC content to biological activity.

Confirmation that Monochamol is a Male Produced Aggregation-Sex Pheromone for Monochamus maculosus Haldeman (Coleoptera: Cerambycidae).

The recognition of cerambycids as frequent and damaging invaders led to an increase in the interest in the chemical ecology of the group with the identification of pheromones and pheromone-like attractants for well over 100 species. Pheromone components of the Cerambycidae are often phylogenetically conserved, with a single compound serving as a pheromone component for several related species. In the subfamily Lamiinae, the compound 2-(undecyloxy)ethanol (monochamol) has been identified as an aggregation-sex pheromone for several species of the genus Monochamus. In other species, including Monochamus maculosus Haldeman, field trials have demonstrated that monochamol is a pheromone attractant, but at that point it was still unknown as to whether it was a pheromone for this species. Here we report the identification, and laboratory and field trials of a pheromone component produced by adult male M. maculosus. Chemical analyses of headspace volatile collections sampled from field collected beetles of both sexes revealed the presence of one male-specific compound that was identified as 2-(undecyloxy)ethanol. Electroantennography analyses showed that monochamol elicited responses from the antennae of female beetles. Traps baited with monochamol in the field captured M. maculosus adults of both sexes corroborating the identification of monochamol as the sex-aggregation pheromone of this species. The attractivity of monochamol to adult M. maculosus in our field trapping experiment was synergized by the addition of the host volatile α-pinene.

A semiochemical view of the ecology of the seed beetle Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae, Bruchinae).

The dried bean beetle, Acanthoscelides obtectus, is an economically important pest of stored legumes worldwide. Tracking the human-aided dispersion of its primary hosts, the Phaseolus vulgaris beans, it is now widespread in most bean-growing areas of the tropics and subtropics. In temperate regions where it can only occasionally overwinter in the field, A. obtectus proliferates in granaries, having multiple generations a year. Despite its negative impact on food production, no sensitive detection or monitoring tools exist, and the reduction of local populations still relies primarily on inorganic insecticides as fumigating agents. However, in the quest to produce more nutritious food more sustainably and healthily, the development of environmentally benign crop protection methods is vital against A. obtectus. For this, knowledge of the biology and chemistry of both the host plant and its herbivore will underpin the development of, among others, chemical ecology-based approaches to form an essential part of the toolkit of integrated bruchid management. We review the semiochemistry of the mate- and host-finding behaviour of A. obtectus and provide new information about the effect of seed chemistry on the sensory and behavioural ecology of host acceptance and larval development.

Black blow fly (Diptera: Calliphoridae) bacterial symbionts inform oviposition site selection by stable flies (Diptera: Muscidae).

Larval habitats of blood-feeding stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae), overlap with foraging sites of black blow flies, Phormia regina (Meigen) (Diptera: Calliphoridae). We tested the hypothesis that bacteria in blow fly excreta inform oviposition decisions by female stable flies. In laboratory 2-choice bioassays, we offered gravid female stable flies fabric-covered agar plates as oviposition sites that were kept sterile or inoculated with either a blend of 7 bacterial strains isolated from blow fly excreta (7-isolate-blend) or individual bacterial isolates from that blend. The 7-isolate-blend deterred oviposition by female stable flies, as did either of 2 strains of Morganella morganii subsp. sibonii. Conversely, Exiguobacterium sp. and Serratia marcescens each prompted oviposition by flies. The flies' oviposition decisions appear to be guided by bacteria-derived semiochemicals as the bacteria could not be physically accessed. Oviposition deterrence caused by semiochemicals of the 7-isolate-blend may help stable flies avoid competition with blow flies. The semiochemicals of bioactive bacterial strains could be developed as trap lures to attract and capture flies and deter their oviposition in select larval habitats.

(1R,4R)-Quercivorol, a Male-Produced Aggregation-Sex Pheromone of the Beetle Elytrimitatrix undata (F.) (Coleoptera: Disteniidae).

The beetle family Disteniidae is currently considered to be closely related to the much larger family Cerambycidae, the longhorned beetles. The 300 + species of disteniids are mostly native to tropical and subtropical regions, with the only described North American species north of Mexico being Elytrimitatrix undata (F.). Here we describe the identification and field testing of (1R,4R)-quercivorol as a male-produced aggregation-sex pheromone component for E. undata. This is the first pheromone identified for any species within the family Disteniidae.

Semiochemical Release and Ontogenetic Changes in a Primary Scent Gland of Podisus maculiventris.

The spined shoulder bug, Podisus maculiventris, is a generalist predator studied for its biocontrol potential. Despite our growing understanding of gland development, the conditions that elicit releases are largely unknown. To determine if male age or gland development affects the chemical composition and release behavior, we dissected adult male bugs and profiled the chemical composition of the male DAG 1, 7, and 14 d post-eclosion. To determine if gland development is related to sexual maturity, we counted the number of sperm present in the seminal vesicles at the same time points. Finally, we measured the diurnal release patterns of different aged males and in various male-female combinations. We observed that newly eclosed adults have under-developed glands and male seminal vesicles contained few sperm. One week post-eclosion the DAG contained previously reported semiochemical compounds and males contained many sperm. Mirroring the trend in reproductive maturation and gland development, the number of semiochemical releases increased with age and the majority of releases followed a scotophase pattern unaffected by sexual composition. These findings link male age to 1) dorsal abdominal gland development 2) release behavior and 3) sexual maturity, which will help our understanding of when these olfactory cues are present for other organisms, like prey, to perceive. Given the results, releasing adults that are at least 1 week post eclosion will maximize the non-consumptive effects of this biocontrol agent.

A meta-analytic investigation of the potential for plant volatiles and sex pheromones to enhance detection and management of Lepidopteran pests.

Effective early detection, monitoring and management methods are critical for reducing the impacts of insect pests in agriculture and forestry. Combining host plant volatiles with sex pheromones could enhance trapping methodologies, whilst the use of non-host volatiles could improve the effectiveness of pest management through repellency effects. In this meta-analysis approach, we analysed 51 studies that used electroantennograms (EAG), wind tunnels and/or field traps to evaluate the antennal and behavioural responses of Lepidoptera to sex pheromones combined with attractant or repellent plant volatiles. Proposed attractant plant volatiles had a positive association with female Lepidoptera responses to sex pheromone, but effects on males were highly variable, with unexpected repellency reported in some studies. Proposed repellent plant volatiles were significantly or near-significantly negatively associated with male attraction to sex pheromones but were scarcely studied. Sub-group analysis identified that male responses to sex pheromone were reduced when the dose of attractant plant volatile relative to sex pheromone was increased. Green-leaf volatiles were associated with the strongest positive effects for males in field traps. Multiple-compound attractant plant volatile blends were less effective than single compounds in field studies. Our analysis demonstrates, (i) the potential value of combining host plant volatiles with sex pheromones to capture females rather than only males, (ii) the importance of identifying appropriate host plant volatiles and optimal relative doses, and (iii) the potential for non-host plant volatile use in pest management strategies.

Molecular identification and functional analysis of Niemann-Pick type C2 proteins，carriers for semiochemicals and other hydrophobic compounds in the brown dog tick, Rhipicephalus linnaei.

Ticks are important vectors of many pathogens with tremendous impact on human and animal health. Studies of semiochemical interactions and mechanisms underlying chemoreception can provide important tools in tick management. Niemann-Pick type C2 (NPC2) proteins have been proposed as one type of chemoreceptor in arthropods. Here, we cloned two NPC2 genes in the brown dog tick, Rhipicephalus linnaei, the tropical lineage previously named R. sanguineus sensu lato and characterized them functionally. R.linNPC2a and R.linNPC2b genes were found to be expressed at each developmental stage with the highest level in adult males. By using quantitative real-time PCR we revealed expression in multiple tissues, including midgut, ovary, salivary glands and legs. Ligand binding analysis revealed that R.linNPC2b bound a wide spectrum of compounds, with ß-ionone, α-amylcinnamaldehyde, 2-nitrophenol and benzaldehyde displaying the strongest binding affinity (Ki < 10 µM), whereas R.linNPC2a showed a more narrow ligand binding range, with intermediate binding affinity to α-amylcinnamaldehyde and 2-nitrophenol (Ki < 20 µM). Molecular docking indicated that the amino acid residue Phe89, Leu77 and Val131 of R.linNPC2a and Phe70, Leu132 and Phe73 of R.linNPC2b could bind multiple ligands. These residues might thus play a key role in the identification of the volatiles. Our results contribute to the understanding of olfactory mechanisms of R. linnaei and can offer new pathways towards new management strategies.

Eight-carbon volatiles: prominent fungal and plant interaction compounds.

Signaling via volatile organic compounds (VOCs) has historically been studied mostly by entomologists; however, botanists and mycologists are increasingly aware of the physiological potential of chemical communication in the gas phase. Most research to date focuses on the observed effects of VOCs on different organisms such as differential growth or metabolite production. However, with the increased interest in volatile signaling, more researchers are investigating the molecular mechanisms for these effects. Eight-carbon VOCs are among the most prevalent and best-studied fungal volatiles. Therefore, this review emphasizes examples of eight-carbon VOCs affecting plants and fungi. These compounds display different effects that include growth suppression in both plants and fungi, induction of defensive behaviors such as accumulation of mycotoxins, phytohormone signaling cascades, and the inhibition of spore and seed germination. Application of '-omics' and other next-generation sequencing techniques is poised to decipher the mechanistic basis of volatiles in plant-fungal communication.

Sea Lamprey Alarm Cue Comprises Water- and Chloroform- Soluble Components.

A diversity of aquatic organisms manage predation risk by avoiding waters activated with conspecific alarm cues, a chemical mixture released from injuries. The sea lamprey (Petromyzon marinus) is a nocturnal migratory species that relies on its alarm cue to navigate around areas of predation risk when moving through river channels. Identification of the cue's chemistry would allow managers to harness this innate behavioral response to guide migrating sea lamprey to traps (invasive population in the Laurentian Great Lakes) or to fish passage devices where dams block migrations in their native range. We pursued isolation of the sea lamprey alarm cue through behaviorally guided fractionation, fractionating the alarm cue into water-soluble and chloroform-soluble fractions, each of which elicited a substantial avoidance response. Recombining the two fractions restored full reactivity, suggesting the alarm cue mixture contains components that exhibit high solubility in water (e.g., nitrogenous compounds), chloroform (e.g., lipids), or perhaps materials that dissolve readily in either solvent. We further screened 13 individual compounds or pure isolates and 6 sub-fractions from the water-soluble fraction and found one of the pure isolates, isoleucine, evoked an avoidance response on its own, but not consistently when found in other mixtures. In a third experiment, we observed no behavioral response after recombining 32 compounds isolated and identified from the water-soluble fraction. These results confirm other suggestions that the process of elucidating alarm cue constituents is challenging. However, we suggest the pursuit is worthwhile given the strong evidence for the utility of alarm cues for use in the conservation and management of fishes and other aquatic organisms.

If All Else Fails: Impact of Silicon Accumulation in Maize Leaves on Volatile Emissions and Oviposition Site Selection of Spodoptera exigua Hübner.

Silicon (Si) fertilization alleviates biotic stresses in plants. Si enhances plant resistance against phytophagous insects through physical and biochemical mechanisms. In particular, Si modifies jasmonic acid levels and the emissions of herbivore-induced plant volatiles (HIPVs). Here, we investigated whether Si accumulation in the tissues of maize leaves modifies the emissions of constitutive and herbivore-induced plant volatiles, with cascade deterrent effects on oviposition site selection by Spodoptera exigua Hübner (Lepidoptera: Noctuidae). Maize plants were cultivated in a hydroponic system under three Si concentrations, resulting in three groups of plants expressing different Si concentrations in their tissues (0.31 ± 0.04, 4.69 ± 0.49, and 9.56 ± 0.30 g Si. Kg- 1 DW). We collected volatiles from undamaged and caterpillar-infested plants, and found that Si concentration in plant tissues had no significant impact. Jasmonic acid content was high in insect-infested plants, but was similar across all Si treatments. Oviposition site selection bioassays using fertilized S. exigua females showed that Si concentration in plant tissues did not affect the number of eggs laid on Si-treated plants. In conclusion, our study shows that the Si content in maize tissues does not impact the semiochemical interactions with S. exigua.

Field Response of Black Turpentine Beetle to Pine Resin Oxidation and Pheromone Displacement.

The black turpentine beetle, Dendroctonus terebrans, is an economically important pest of pines in the Southeastern U.S., with a high potential for invasion to other pine-rich regions. Dendroctonus terebrans attraction to an injured host tree lessens over time as the host material degrades. Likewise, kairomonal volatiles emitted from the host change as constituents of the defensive resin oxidize. Therefore we hypothesized that volatiles associated with a fresh host would be more attractive to D. terebrans than those associated with a dead or dying host. We replicated the natural oxidation process of turpentine, fractionated the distilled products to isolate the oxidized products, and deployed the complex mixtures to measure field attraction based on the amount of oxidation performed. Contrasting with previous studies, our results suggest that D. terebrans attraction is not primarily based on host tree degradation. In a second experiment incorporating Dendroctonus pheromones, we demonstrate D. terebrans has a displacement-dependent response to endo-brevicomin, a pheromone associated with the sympatric southern pine beetle, D. frontalis. This has implications not only for possible interspecific signaling, but also for the role of endo-brevicomin in D. terebrans colonization behavior. The results from this study broaden the understanding of D. terebrans chemical ecology and directly contribute to the development of an effective lure-based monitoring system that will benefit future research and management efforts. This may become important if the species is established outside its native range, as in the closely related red turpentine beetle, Dendroctonus valens, which caused mass pine tree mortality following its introduction to Asia.

Comparison of surveillance trapping methods to monitor Culicoides biting midge activity in Trinidad, West Indies.

Culicoides biting midges (Diptera: Ceratopogonidae) are biting nuisances and arbovirus vectors of both public health and veterinary significance in Trinidad. We compared sampling methods to define the behaviour and bionomics of adult Culicoides populations at a commercial dairy goat farm. Three static trap designs were compared: (a) Centre for Disease Control (CDC) downdraft UV trap; (b) CDC trap with an incandescent bulb and (c) CDC trap with semiochemical lure consisting of R-(-)-1-octen-3-ol and CO2 (no bulb). Sweep netting was used to define diel periodicity. A total of 30,701 biting midges were collected using static traps, dominated by female Culicoides furens (>70% of trap collections across all three designs). There was no significant difference in the Margalef's index between the three traps; however, trap designs A and C collected a significantly greater number of individuals than trap B, and trap C gained highest species richness. The greatest species richness and abundance of Culicoides collected by sweep net was observed between 6:00 and 6:15 pm and notable differences in the crepuscular activity pattern of several species were identified. Comparative data on Culicoides species richness, abundance, sex and reproductive status is discussed and can be used to improve surveillance strategies, research designs and risk management.

Burying Beetle Parents Adaptively Manipulate Information Broadcast from a Microbial Community.

AbstractMicrobial volatiles provide essential information for animals, which compete to detect, respond to, and perhaps control this information. Burying beetle parents have the opportunity to influence microbially derived semiochemicals, because they monopolize a small carcass for their family, repairing feeding holes and applying exudates that alter the microbial community. To study adaptive manipulation of microbial cues, we integrated mechanistic and functional approaches. We contrasted gas chromatography-mass spectrometry (GC-MS) volatile profiles from carcasses that were or were not prepared by a resident pair of Nicrophorus orbicollis. Methyl thiocyanate (MeSCN), the primary attractant for burying beetles seeking a fresh carcass, was reduced 20-fold by carcass preparation, while dimethyl trisulfide (DMTS), which deters breeding beetles, was increased 20-fold. These results suggest that parental care serves to make previously public information more private (crypsis, MeSCN) and to disinform rivals with a deterrent (DMTS). Functional tests in the field demonstrated that carcass preparation reduced discovery and use by congeners (threefold) as well as by dipteran rivals. Because microbes and their chemicals influence nearly every aspect of animal ecology, animal manipulation of microbial cues may be as widespread as manipulation of their own signals.

Identification and quantification of 11 airborne biochemicals emitted by the brown recluse and another primitive hunting spider using headspace solid-phase microextraction-GC/MS.

Loxosceles reclusa, or brown recluse spider, is a harmful household spider whose habitat extends throughout the Midwest in the USA and other regions in the world. The pheromones and other biomolecules that facilitate signaling for brown recluses and other spider species are poorly understood. A rapid and sensitive method is needed to analyze airborne spider signaling biomolecules to better understand the structure and function of these biochemicals in order to control the population of the spiders. In this study, we developed a novel headspace solid-phase microextraction (HS-SPME)-GC/MS method to analyze potential pheromones and biomolecules emitted by the brown recluse spider. The method is highly selective and sensitive for biomolecule identification and quantification from a single live spider. Using this novel non-destructive HS-SPME-GC/MS technique, we identified 11 airborne biomolecules, including 4-methylquinazoline, dimethyl sulfone, 2-methylpropanoic acid, butanoic acid, hexanal, 3-methylbutanoic acid, 2-methylbutanoic acid, 2,4-dimethylbenzaldehyde, 2-phenoxyethanol, and citral (contains both isomers of neral and geranial). Some of these airborne biomolecules were also reported as semiochemicals associated with biological functions of other spiders and insects. The method was also applied to study the airborne biochemicals of Plectreurys tristis, another primitive hunting spider with a poor web, enabling quantitation of the same compounds and demonstrating a difference in signaling molecule concentrations between the two species. This method has potential application in the study of pheromones and biological signaling in other species, which allows for the possibility of utilizing attractant or deterrent functions to limit household populations of harmful species.

Chemical Ecology of the Asian Longhorn Beetle, Anoplophora glabripennis.

The Asian longhorn beetle (ALB), Anoplophora glabripennis (Motschulsky), is a destructive forest pest in its native range, East Asia, or a high-risk invasive species in many other parts of the world. Extensive research has been directed toward the development of ALB management strategies. However, semiochemical-based trap lures, which are one of the effective tools for detecting, monitoring, and potentially assisting in eradicating cerambycids, have not reached operational efficacy for ALB to date, which is probably due to a grossly incomplete understanding of its chemical ecology. Here, we summarize the current progress in ALB chemical ecology including host selection and location, pheromone identification, trapping techniques, olfactory system, and related biology and behavior. We also briefly review the known semiochemicals in the subfamily Lamiinae, particularly the ALB congener, A. chinensis. Based on this knowledge, we highlight a potentially important role of some host-original chemicals, such as sesquiterpenes, in ALB host and mate location, and emphasize the basic studies on the biology and behavior of adult ALB. Last, we formulate suggestions for further research directions that may contribute to a better understanding of ALB chemical ecology and improved lure efficacy.

Individual Chemical Profiles in the Leach's Storm-Petrel.

Avian chemical communication, once largely overlooked, is a growing field that has revealed the important role that olfaction plays in the social lives of some birds. Leach's storm-petrels (Oceanodroma leucorhoa) have a remarkable sense of smell and a strong, musky scent. This long-lived, monogamous seabird relies on olfaction for nest relocation and foraging, but whether they use scent for communication is less well studied. They are nocturnally active at the breeding colony and yet successfully reunite with their mate despite poor night-vision, indicating an important role for non-visual communication. We investigated the chemical profiles of Leach's storm-petrels to determine whether there is socially relevant information encoded in their plumage odor. To capture the compounds comprising their strong scent, we developed a method to study the compounds present in the air surrounding their feathers using headspace stir bar sorptive extraction coupled with gas chromatography-mass spectrometry. We collected feathers from Leach's storm-petrels breeding on Bon Portage Island in Nova Scotia, Canada in both 2015 and 2016. Our method detected 142 commonly occurring compounds. We found interannual differences in chemical profiles between the two sampling years. Males and females had similar chemical profiles, while individuals had distinct chemical signatures across the two years. These findings suggest that the scent of the Leach's storm-petrel provides sociochemical information that could facilitate olfactory recognition of individuals and may inform mate choice decisions.

Trap Assays of the Walnut Twig Beetle, Pityophthorus juglandis Blackman (Coleoptera: Curculionidae: Scolytinae), Reveal an Effective Semiochemical Repellent Combination.

Thousand cankers disease (TCD), is an invasive insect-disease complex caused by the walnut twig beetle, Pityophthorus juglandis, and fungal pathogen, Geosmithia morbida. Semiochemical interruption is a viable option for protecting walnut trees from P. juglandis attack. The goal of this study was to test beetle responses to potential repellent compounds. The results of five, flight-intercept assays are reported. Assays 1-3 tested four compounds at variable release rates: (S)-(-)-verbenone, (R)-(+)-verbenone, racemic chalcogran, and racemic trans-conophthorin. Trapping results indicated that the highest release rate tested for each compound was the most effective in reducing the number of beetles caught. (S)-(-)-Verbenone was the least effective, reducing P. juglandis trap catches by 66%. (R)-(+)-Verbenone reduced the number of P. juglandis by 84%. Neither enantiomer of verbenone performed as well as chalcogran or trans-conophthorin, which both reduced the number of beetles caught by ca. 98%. Following individual assays, the most effective compounds were tested in subtractive-combination assays. Combinations of high release rates for (R)-(+)-verbenone, trans-conophthorin, and two stereoisomers of limonene (tested in a previous study) were tested in two assays. The subtractive-combination assays were inconclusive in that trap catches were similar across all treatments. All combination treatments were highly effective, achieving approximately 99% reduction in the number of beetles caught. Based on the trapping results, commercial availability, and cost of the semiochemicals tested, we conclude that a combination of (R)-(+)-limonene, trans-conophthorin, and (R)-(+)-verbenone constitutes an effective tool for reducing P. juglandis trap catches.