We know very little about pollination in the Platanthera Rich (Orchidaceae: Orchidoideae).

The Platanthera Rich. (Orchidoideae) comprise a speciose genus of orchids primarily in the northern hemisphere, with up to 200 known species worldwide. Individual species are known to self-pollinate, but many rely on insect pollinators with characteristics such as floral color, timing of floral odor emissions, nectar rewards, and spur length associated with particular pollination syndromes. As with many orchids, some orchid-pollinator associations are likely highly co-evolved, but we also know that some Platanthera spp. are the result of hybridization events, which implies a lack of pollinator fidelity in some cases. Some Platanthera spp. occur in large numbers which, coupled with the numerous Platanthera-pollinator systems, make them accessible as study species and useful for co-evolutionary studies. Due to the likely effects of climate change and ongoing development on Platanthera spp. habitats, these orchids and their associated pollinators should be a focus of conservation attention and management. However, while there is a fairly substantial literature coverage of Platanthera-pollinator occurrence and interactions, there are still wide gaps in our understanding of the species involved in these systems. In this systematic review, we outline what is current knowledge and provide guidance on further research that will increase our understanding of orchid-insect co-evolutionary relationships. Our review covers 157 orchid species and about 233 pollinator species interacting with 30 Platanthera spp. We provide analyses on aspects of these interactions such as flower morphology, known insect partners of Platanthera species, insect-Platanthera specificity, pollination visitor timing (diurnal vs. nocturnal), floral rewards, and insect behavior affecting pollination outcomes (e.g., pollinia placement). A substantial number of Platanthera spp. and at least a few of their known pollinators are of official (IUCN) conservation concern - and many of their pollinators remain unassessed or even currently unknown - which adds to the urgency of further research on these co-evolved relationships.

Chromosome-level genome assembly reveals genomic architecture of northern range expansion in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae).

Genome sequencing methods and assembly tools have improved dramatically since the 2013 publication of draft genome assemblies for the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). We conducted proximity ligation library sequencing and scaffolding to improve contiguity, and then used linkage mapping and recent bioinformatic tools for correction and further improvement. The new assemblies have dramatically improved contiguity and gaps compared to the originals: N50 values increased 26- to 36-fold, and the number of gaps were reduced by half. Ninety per cent of the content of the assemblies is now contained in 12 and 11 scaffolds for the female and male assemblies, respectively. Based on linkage mapping information, the 12 largest scaffolds in both assemblies represent all 11 autosomal chromosomes and the neo-X chromosome. These assemblies now have nearly chromosome-sized scaffolds and will be instrumental for studying genomic architecture, chromosome evolution, population genomics, functional genomics, and adaptation in this and other pest insects. We also identified regions in two chromosomes, including the ancestral-X portion of the neo-X chromosome, with elevated differentiation between northern and southern Canadian populations.

Identification of genes and gene expression associated with dispersal capacity in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae).

Dispersal flights by the mountain pine beetle have allowed range expansion and major damage to pine stands in western Canada. We asked what the genetic and transcriptional basis of mountain pine beetle dispersal capacity is. Using flight mills, RNA-seq and a targeted association study, we compared strong-flying, weak-flying, and non-flying female beetles from the recently colonized northern end of their range. Nearly 3,000 genes were differentially expressed between strong and weak flying beetles, while weak fliers and nonfliers did not significantly differ. The differentially expressed genes were mainly associated with lipid metabolism, muscle maintenance, oxidative stress response, detoxification, endocrine function, and flight behavior. Three variant loci, two in the coding region of genes, were significantly associated with flight capacity but these genes had no known functional link to flight. Several differentially expressed gene systems may be important for sustained flight, while other systems are downregulated during dispersal and likely to conserve energy before host colonization. The candidate genes and SNPs identified here will inform further studies and management of mountain pine beetle, as well as contribute to understanding the mechanisms of insect dispersal flights.

Random and Directed Movement by Warren Root Collar Weevils (Coleoptera: Curculionidae), Relative to Size and Distance of Host Lodgepole Pine Trees.

Hylobius warreni Wood (Coleoptera: Curculionidae) is a pest of conifers, especially lodgepole pine (Pinus contorta var. latifolia Douglas ex Loudon) (Pinales: Pinaceae) in the Interior of British Columbia. The larvae feed on the roots and root collars and cause girdling damage, resulting in mortality or growth reductions. Previous research has suggested the adult weevils locate potential host trees by using random movements and vision, but likely not chemosensory cues. The purpose of this study is to determine if adult H. warreni respond to particular tree characteristics versus encounter potential hosts at random. Study A was a capture-mark-recapture experiment where weevils were captured on mature pine trees, while Study B was a tracking experiment within a young pine plantation. Weevils showed a preference for larger trees, and for trees that were closer to the weevil's last known location. In Study A, weevils also avoided climbing trees in poor health, while in Study B, the weevils' preference for taller trees increased as their distance from the weevil increased, as well as when taller trees were closer to other trees. Movement rates were similar to those observed in previous studies, were positively correlated with the average spacing of trees, and declined with time after release. This confirms previous findings that H. warreni may locate host trees by both vision and random movements, and that their movements are determined primarily by the size and distribution of potential host trees within their habitat.

Autumn shifts in cold tolerance metabolites in overwintering adult mountain pine beetles.

The mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae) is a major forest pest of pines in western North America. Beetles typically undergo a one-year life cycle with larval cold hardening in preparation for overwintering. Two-year life cycle beetles have been observed but not closely studied. This study tracks cold-hardening and preparation for overwintering by adult mountain pine beetles in their natal galleries. Adults were collected in situ between September and December 2016 for a total of nine time points during 91 days. Concentrations of 41 metabolites in these pooled samples were assessed using quantitative nuclear magnetic resonance (NMR). Levels of glycerol and proline increased significantly with lowering temperature during the autumn. Newly eclosed mountain pine beetles appear to prepare for winter by generating the same cold-tolerance compounds found in other insect larvae including mountain pine beetle, but high on-site mortality suggested that two-year life cycle adults have a less efficacious acclimation process. This is the first documentation of cold acclimation metabolite production in overwintering new adult beetles and is evidence of physiological plasticity that would allow evolution by natural selection of alternate life cycles (shortened or lengthened) under a changing climate or during expansion into new geoclimatic areas.

Single-generation effects on terpenoid defenses in lodgepole pine populations following mountain pine beetle infestation.

The recent mountain pine beetle outbreak in western Canada provides an opportunity to study the selection and heritability of tree defenses. We examined terpenoid-based defenses of seedling lodgepole pines which were offspring of mature trees subjected to high levels of mountain pine beetle selective pressure. Seedlings were grown from one of three types of cones: old cones on live trees; young cones on live trees; and cones on trees killed by beetles. Offspring thus represented crosses of non-surviving (NS) x surviving (S), S x S, and NS x NS parents, respectively. Methyl jasmonate was used to induce a defensive reaction in the seedlings. Seed source had a significant effect on levels of ten different terpenes, but not on total terpene concentrations. When the seedlings were grouped by location and treatment type, the seedlings of different cone types could be almost entirely distinguished by terpene profiles.

DNA barcode-based survey of Trichoptera in the Crooked River reveals three new species records for British Columbia.

Anthropogenic pressures on aquatic systems have placed a renewed focus on biodiversity of aquatic macroinvertebrates. By combining classical taxonomy and DNA barcoding we identified 39 species of caddisflies from the Crooked River, a unique and sensitive system in the southernmost arctic watershed in British Columbia. Our records include three species never before recorded in British Columbia: Lepidostoma togatum (Lepidostomatidae), Ceraclea annulicornis (Leptoceridae), and possibly Cheumatopsyche harwoodi (Hydropsychidae). Three other specimens may represent new occurrence records and a number of other records seem to be substantial observed geographic range expansions within British Columbia.

Seasonal shifts in accumulation of glycerol biosynthetic gene transcripts in mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), larvae.

Winter mortality is a major factor regulating population size of the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Glycerol is the major cryoprotectant in this freeze intolerant insect. We report findings from a gene expression study on an overwintering mountain pine beetle population over the course of 35 weeks. mRNA transcript levels suggest glycerol production in the mountain pine beetle occurs through glycogenolytic, gluconeogenic and potentially glyceroneogenic pathways, but not from metabolism of lipids. A two-week lag period between fall glycogen phosphorylase transcript and phosphoenolpyruvate carboxykinase transcript up-regulation suggests that gluconeogenesis serves as a secondary glycerol-production process, subsequent to exhaustion of the primary glycogenolytic source. These results provide a first look at the details of seasonal gene expression related to the production of glycerol in the mountain pine beetle.

Gene expression analysis of overwintering mountain pine beetle larvae suggests multiple systems involved in overwintering stress, cold hardiness, and preparation for spring development.

Cold-induced mortality has historically been a key aspect of mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), population control, but little is known about the molecular basis for cold tolerance in this insect. We used RNA-seq analysis to monitor gene expression patterns of mountain pine beetle larvae at four time points during their overwintering period-early-autumn, late-autumn, early-spring, and late-spring. Changing transcript profiles over the winter indicates a multipronged physiological response from larvae that is broadly characterized by gene transcripts involved in insect immune responses and detoxification during the autumn. In the spring, although transcripts associated with developmental process are present, there was no particular biological process dominating the transcriptome.

Proteomics indicators of the rapidly shifting physiology from whole mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae), adults during early host colonization.

We developed proteome profiles for host colonizing mountain pine beetle adults, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Adult insects were fed in pairs on fresh host lodgepole pine, Pinus contorta Dougl. ex Loud, phloem tissue. The proteomes of fed individuals were monitored using iTRAQ and compared to those of starved beetles, revealing 757 and 739 expressed proteins in females and males, respectively, for which quantitative information was obtained. Overall functional category distributions were similar for males and females, with the majority of proteins falling under carbohydrate metabolism (glycolysis, gluconeogenesis, citric acid cycle), structure (cuticle, muscle, cytoskeleton), and protein and amino acid metabolism. Females had 23 proteins with levels that changed significantly with feeding (p<0.05, FDR<0.20), including chaperones and enzymes required for vitellogenesis. In males, levels of 29 proteins changed significantly with feeding (p<0.05, FDR<0.20), including chaperones as well as motor proteins. Only two proteins, both chaperones, exhibited a significant change in both females and males with feeding. Proteins with differential accumulation patterns in females exhibited higher fold changes with feeding than did those in males. This difference may be due to major and rapid physiological changes occurring in females upon finding a host tree during the physiological shift from dispersal to reproduction. The significant accumulation of chaperone proteins, a cytochrome P450, and a glutathione S-transferase, indicate secondary metabolite-induced stress physiology related to chemical detoxification during early host colonization. The females' activation of vitellogenin only after encountering a host indicates deliberate partitioning of resources and a balancing of the needs of dispersal and reproduction.

How the mountain pine beetle (Dendroctonus ponderosae) breached the Canadian Rocky Mountains.

The mountain pine beetle (MPB; Dendroctonus ponderosae Hopkins), a major pine forest pest native to western North America, has extended its range north and eastward during an ongoing outbreak. Determining how the MPB has expanded its range to breach putative barriers, whether physical (nonforested prairie and high elevation of the Rocky Mountains) or climatic (extreme continental climate where temperatures can be below -40 °C), may contribute to our general understanding of range changes as well as management of the current epidemic. Here, we use a panel of 1,536 single nucleotide polymorphisms (SNPs) to assess population genetic structure, connectivity, and signals of selection within this MPB range expansion. Biallelic SNPs in MPB from southwestern Canada revealed higher genetic differentiation and lower genetic connectivity than in the northern part of its range. A total of 208 unique SNPs were identified using different outlier detection tests, of which 32 returned annotations for products with putative functions in cholesterol synthesis, actin filament contraction, and membrane transport. We suggest that MPB has been able to spread beyond its previous range by adjusting its cellular and metabolic functions, with genome scale differentiation enabling populations to better withstand cooler climates and facilitate longer dispersal distances. Our study is the first to assess landscape-wide selective adaptation in an insect. We have shown that interrogation of genomic resources can identify shifts in genetic diversity and putative adaptive signals in this forest pest species.

Comparison of lodgepole and jack pine resin chemistry: implications for range expansion by the mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae).

The mountain pine beetle, Dendroctonus ponderosae, is a significant pest of lodgepole pine in British Columbia (BC), where it has recently reached an unprecedented outbreak level. Although it is native to western North America, the beetle can now be viewed as a native invasive because for the first time in recorded history it has begun to reproduce in native jack pine stands within the North American boreal forest. The ability of jack pine trees to defend themselves against mass attack and their suitability for brood success will play a major role in the success of this insect in a putatively new geographic range and host. Lodgepole and jack pine were sampled along a transect extending from the beetle's historic range (central BC) to the newly invaded area east of the Rocky Mountains in north-central Alberta (AB) in Canada for constitutive phloem resin terpene levels. In addition, two populations of lodgepole pine (BC) and one population of jack pine (AB) were sampled for levels of induced phloem terpenes. Phloem resin terpenes were identified and quantified using gas chromatography. Significant differences were found in constitutive levels of terpenes between the two species of pine. Constitutive α-pinene levels - a precursor in the biosynthesis of components of the aggregation and antiaggregation pheromones of mountain pine beetle - were significantly higher in jack pine. However, lower constitutive levels of compounds known to be toxic to bark beetles, e.g., 3-carene, in jack pine suggests that this species could be poorly defended. Differences in wounding-induced responses for phloem accumulation of five major terpenes were found between the two populations of lodgepole pine and between lodgepole and jack pine. The mountain pine beetle will face a different constitutive and induced phloem resin terpene environment when locating and colonizing jack pine in its new geographic range, and this may play a significant role in the ability of the insect to persist in this new host.

An inexpensive feeding bioassay technique for stored-product insects.

ABSTRACT We used the red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), to compare three feeding bioassay techniques using flour disks. The area (scanner or digital photographs) and mass (sensitive balance) of the same flour disks were measured daily for 1 or 2 wk to assess feeding by insects. The loss in mass and area over 4 h was measured, as some variation over time was noticed in the disks with no insects feeding on them. The gravimetric method correlated well with both measurements of the area for the disks held in a growth chamber: scanner (R2 = 0.96), digital photography (R2 = 0.96). There was also a high correlation (R2 = 0.86) between the disk weight and area scanned at normal lab conditions. There were differences in the percentage of the disks remaining over time depending on the temperature and whether they were weighed or scanned. Measuring the mass of the disks resulted in a relatively larger percent of disk remaining compared with the scanned area. Mass measurements required a sensitive balance, handling of the disks and the insects, and appeared slightly more sensitive to humidity and temperature changes over time. Scanning the disks requires flat bed scanner access but less handling of both insects and disks. Digital photographs could be taken quickly, requiring less equipment, although photographs had to be further processed to determine area Scanning or taking digital photographs of flour disk area was an effective technique for measuring insect feeding.

Disentangling detoxification: gene expression analysis of feeding mountain pine beetle illuminates molecular-level host chemical defense detoxification mechanisms.

The mountain pine beetle, Dendroctonus ponderosae, is a native species of bark beetle (Coleoptera: Curculionidae) that caused unprecedented damage to the pine forests of British Columbia and other parts of western North America and is currently expanding its range into the boreal forests of central and eastern Canada and the USA. We conducted a large-scale gene expression analysis (RNA-seq) of mountain pine beetle male and female adults either starved or fed in male-female pairs for 24 hours on lodgepole pine host tree tissues. Our aim was to uncover transcripts involved in coniferophagous mountain pine beetle detoxification systems during early host colonization. Transcripts of members from several gene families significantly increased in insects fed on host tissue including: cytochromes P450, glucosyl transferases and glutathione S-transferases, esterases, and one ABC transporter. Other significantly increasing transcripts with potential roles in detoxification of host defenses included alcohol dehydrogenases and a group of unexpected transcripts whose products may play an, as yet, undiscovered role in host colonization by mountain pine beetle.

Sizing up arthropod genomes: an evaluation of the impact of environmental variation on genome size estimates by flow cytometry and the use of qPCR as a method of estimation.

A study was undertaken to evaluate both a pre-existing method and a newly proposed approach for the estimation of nuclear genome sizes in arthropods. First, concerns regarding the reliability of the well-established method of flow cytometry relating to impacts of rearing conditions on genome size estimates were examined. Contrary to previous reports, a more carefully controlled test found negligible environmental effects on genome size estimates in the fly Drosophila melanogaster. Second, a more recently touted method based on quantitative real-time PCR (qPCR) was examined in terms of ease of use, efficiency, and (most importantly) accuracy using four test species: the flies Drosophila melanogaster and Musca domestica and the beetles Tribolium castaneum and Dendroctonus ponderosa. The results of this analysis demonstrated that qPCR has the tendency to produce substantially different genome size estimates from other established techniques while also being far less efficient than existing methods.

Draft genome of the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major forest pest.

BACKGROUND: The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests. RESULTS: We developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insect's biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle. CONCLUSIONS: Despite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects.

Efficacy of "Verbenone Plus" for protecting ponderosa pine trees and stands from Dendroctonus brevicomis (Coleoptera: Curculionidae) attack in British Columbia and California.

The western pine beetle, Dendroctonus brevicomis LeConte (Coleoptera: Curculionidae, Scolytinae), is a major cause of ponderosa pine, Pinus ponderosa Douglas ex Lawson, mortality in much of western North America. We review several years of research that led to the identification of Verbenone Plus, a novel four-component semiochemcial blend [acetophenone, (E)-2-hexen-1-ol + (Z)-2-hexen-1-ol, and (-)-verbenone] that inhibits the response of D. brevicomis to attractant-baited traps, and examine the efficacy of Verbenone Plus for protecting individual trees and forest stands from D. brevicomis infestations in British Columbia and California. In all experiments, semiochemicals were stapled around the bole of treated trees at approximately equal to 2 m in height. (-)-Verbenone alone had no effect on the density of total attacks and successful attacks by D. brevicomis on attractant-baited P. ponderosa, but significantly increased the percentage of pitchouts (unsuccessful D. brevicomis attacks). Verbenone Plus significantly reduced the density of D. brevicomis total attacks and D. brevicomis successful attacks on individual trees. A significantly higher percentage of pitchouts occurred on Verbenone Plus-treated trees. The application of Verbenone Plus to attractant-baited P. ponderosa significantly reduced levels of tree mortality. In stand protection studies, Verbenone Plus significantly reduced the percentage of trees mass attacked by D. brevicomis in one study, but in a second study no significant treatment effect was observed. Future research should concentrate on determining optimal release rates and spacings of release devices in stand protection studies, and expansion of Verbenone Plus into other systems where verbenone alone has not provided adequate levels of tree protection.

Global and comparative proteomic profiling of overwintering and developing mountain pine beetle, Dendroctonus ponderosae (Coleoptera: Curculionidae), larvae.

BACKGROUND: Mountain pine beetles, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), are native to western North America, but have recently begun to expand their range across the Canadian Rocky Mountains. The requirement for larvae to withstand extremely cold winter temperatures and potentially toxic host secondary metabolites in the midst of their ongoing development makes this a critical period of their lives. RESULTS: We have uncovered global protein profiles for overwintering mountain pine beetle larvae. We have also quantitatively compared the proteomes for overwintering larvae sampled during autumn cooling and spring warming using iTRAQ methods. We identified 1507 unique proteins across all samples. In total, 33 proteins exhibited differential expression (FDR < 0.05) when compared between larvae before and after a cold snap in the autumn; and 473 proteins exhibited differential expression in the spring when measured before and after a steady incline in mean daily temperature. Eighteen proteins showed significant changes in both autumn and spring samples. CONCLUSIONS: These first proteomic data for mountain pine beetle larvae show evidence of the involvement of trehalose, 2-deoxyglucose, and antioxidant enzymes in overwintering physiology; confirm and expand upon previous work implicating glycerol in cold tolerance in this insect; and provide new, detailed information on developmental processes in beetles. These results and associated data will be an invaluable resource for future targeted research on cold tolerance mechanisms in the mountain pine beetle and developmental biology in coleopterans.

Transcriptome and full-length cDNA resources for the mountain pine beetle, Dendroctonus ponderosae Hopkins, a major insect pest of pine forests.

Bark beetles (Coleoptera: Curculionidae: Scolytinae) are major insect pests of many woody plants around the world. The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is a significant historical pest of western North American pine forests. It is currently devastating pine forests in western North America--particularly in British Columbia, Canada--and is beginning to expand its host range eastward into the Canadian boreal forest, which extends to the Atlantic coast of North America. Limited genomic resources are available for this and other bark beetle pests, restricting the use of genomics-based information to help monitor, predict, and manage the spread of these insects. To overcome these limitations, we generated comprehensive transcriptome resources from fourteen full-length enriched cDNA libraries through paired-end Sanger sequencing of 100,000 cDNA clones, and single-end Roche 454 pyrosequencing of three of these cDNA libraries. Hybrid de novo assembly of the 3.4 million sequences resulted in 20,571 isotigs in 14,410 isogroups and 246,848 singletons. In addition, over 2300 non-redundant full-length cDNA clones putatively containing complete open reading frames, including 47 cytochrome P450s, were sequenced fully to high quality. This first large-scale genomics resource for bark beetles provides the relevant sequence information for gene discovery; functional and population genomics; comparative analyses; and for future efforts to annotate the MPB genome. These resources permit the study of this beetle at the molecular level and will inform research in other Dendroctonus spp. and more generally in the Curculionidae and other Coleoptera.

Responses of Dendroctonus brevicomis (Coleoptera: Curculionidae) in behavioral assays: implications to development of a semiochemical-based tool for tree protection.

Currently, techniques for managing western pine beetle, Dendroctonus brevicomis LeConte (Coleoptera: Curculionidae, Scolytinae), infestations are limited to tree removals (thinning) that reduce stand density and presumably host susceptibility, and/or the use of insecticides to protect individual trees. There continues to be significant interest in developing an effective semiochemical-based tool for protecting trees from D. brevicomis attack, largely as an alternative to conventional insecticides. The responses of D. brevicomis to tree volatiles and verbenone were documented in eight experiments (trapping assays) conducted over a 4-yr period in which 88,942 individuals were collected. Geraniol, a tree volatile unique to Pinus ponderosa that elicits female-specific antennal responses in D. brevicomis, did not affect D. brevicomis behavior. Blends of two green leaf alcohols [hexanol + (Z)-3-hexen-1-ol] tested at two release rates (5.0 and 100.0 mg/d) had no effect on the response of D. brevicomis to attractant-baited traps. A nine-component blend [benzaldehyde, benzyl alcohol, guaiacol, nonanal, salicylaldehyde, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, and (-) -verbenone; NAVV] and subsequent revisions of this blend disrupted the response of D. brevicomis to attractant-baited traps in all experiments. The inhibitory effect of a revised five-component blend [nonanal, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, and (-)-verbenone; NAVV5] on the response of mountain pine beetle, D. ponderosae Hopkins, to attractant-baited traps was also documented. Acetophenone significantly reduced D. brevicomis attraction, but was not as effective as verbenone alone. Acetophenone increased the effectiveness of NAVV5 in one of two experiments. Furthermore, by adding acetophenone to NAVV5 we were able to remove the aldehydes from NAVV5 without compromising effectiveness, resulting in a novel four-component blend [acetophenone, (E)-2-hexen-1-ol + (Z)-2-hexen-1-ol, and (-)-verbenone; Verbenone Plus]. We discuss the implications of these and other results to development of Verbenone Plus as a semiochemical-based tool for management of D. brevicomis and D. ponderosae infestations.