(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.

Chemical Ecology, Biochemistry, and Molecular Biology of Insect Hydrocarbons.

Insect cuticular hydrocarbons (CHCs) consist of complex mixtures of straight-chain alkanes and alkenes, and methyl-branched hydrocarbons. In addition to restricting water loss through the cuticle and preventing desiccation, they have secondarily evolved to serve a variety of functions in chemical communication and play critical roles as signals mediating the life histories of insects. In this review, we describe the physical properties of CHCs that allow for both waterproofing and signaling functions, summarize their roles as inter- and intraspecific chemical signals, and discuss the influences of diet and environment on CHC profiles. We also present advances in our understanding of hydrocarbon biosynthesis. Hydrocarbons are biosynthesized in oenocytes and transported to the cuticle by lipophorin proteins. Recent work on the synthesis of fatty acids and their ultimate reductive decarbonylation to hydrocarbons has taken advantage of powerful new tools of molecular biology, including genomics and RNA interference knockdown of specific genes, to provide new insights into the biosynthesis of hydrocarbons.

Chemically-mediated colonization of black cherry by the peach bark beetle, Phloeotribus liminaris.

The peach bark beetle (Phloeotribus liminaris Harris, PBB) affects the health, quality, and value of black cherry (Prunus serotina Ehrh.) within the Central Hardwoods Forest Region of North America. When colonized by adult beetles, black cherry trees produce a defensive exudate, or 'gum', staining the wood and decreasing its value up to 90%. Current management tactics are inadequate to avoid extensive damage to most veneer-sized black cherry in the region. We test the hypothesis that PBB colonization behavior is chemically-mediated and determine the extent to which PBB is attracted to compounds associated with wounded or PBB-infested cherry wood. Through olfactometer and field bioassays, we determined that adult PBB were attracted to cherry branches infested with female beetles. We then used dynamic headspace sampling to collect volatiles associated with wounded and infested bolts of black cherry. The volatile benzaldehyde dominated these collections and was more abundant in aerations of female-infested bolts than other odor sources. In subsequent field bioassays, we evaluated the bioactivity of benzaldehyde, as well as α-longipinene, in combination with several chemical carriers. Traps baited with benzaldehyde captured more PBB than all other treatments, irrespective of other lure components. Moreover, PBB were not attracted to traps baited solely with ethanol, a common attractant for bark beetles that colonize hardwood trees. This is the first report of benzaldehyde as an attractant for a species of bark beetle and could aid in developing semiochemical-based management tactics for this important pest.

Conventional Gel Electrophoresis and TaqMan Probes Enable Rapid Confirmation of Thousand Cankers Disease from Diagnostic Samples.

Thousand cankers disease (TCD) is caused by the fungal pathogen Geosmithia morbida and vectored by the walnut twig beetle Pityophthorus juglandis. In infected walnut and butternut (Juglans spp.) hosts and wingnut species (Pterocarya spp.) hosts, tree decline and death results in ecological disruption and economic losses. A rapid molecular detection protocol for TCD using microsatellite markers can confirm the presence of insect vector or fungal pathogen DNA, but it requires specialized expensive equipment and technical expertise. Using four different experimental approaches, capillary and conventional gel electrophoresis, and traditional polymerase chain reaction (PCR) and quantitative PCR (qPCR), we describe simplified and inexpensive processes for diagnostic confirmation of TCD. The improved and rapid detection protocols reported in this study reduce time and equipment costs associated with detection of molecular pest and pathogen DNA by (1) using conventional gel electrophoresis or TaqMan molecular probes to elucidate the detection limits for G. morbida and P. juglandis DNA and (2) identifying resources that allow visualization of positive test results for infected host plant tissue samples. Conventional gel electrophoresis and TaqMan molecular probe protocols detected presence of DNA from TCD-associated fungal and insect samples. These procedural improvements can be readily adopted by diagnostic end-users and adapted for use with other complex disease systems to enable rapid pest and pathogen detection.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Bed bugs, Cimex lectularius L., exhibiting metabolic and target site deltamethrin resistance are susceptible to plant essential oils.

Pyrethroid resistance has been a major hurdle limiting the effective control of bed bugs (Cimex lectularius L.). Alternative approaches that include the use of plant essential oils (EOs) have been proposed for effective management of bed bugs. However, EO resistance level comparisons between pyrethroid susceptible and resistant bed bug populations have not been previously conducted. The goal of this study was twofold: (i) determine deltamethrin resistance levels and associated resistance mechanisms in the field-collected Knoxville strain and (ii) quantify resistance levels of the Knoxville strain to five EOs (thyme, oregano, clove, geranium and coriander), their major insecticidal constituents (thymol, carvacrol, eugenol, geraniol and linalool) and an EO-based product (EcoRaider®). First, we found that the Knoxville strain was 72,893 and 291,626 fold more resistant to topically applied deltamethrin in comparison to the susceptible Harlan strain at the LD25 and LD50 lethal dose levels, respectively. Synergist bioassays and detoxification enzyme assays revealed significantly higher activity of cytochrome P450 and esterase enzymes in the resistant Knoxville strain. Further, Sanger sequencing revealed the presence of the L925I mutation in the voltage-sensitive sodium channel α subunit gene. The Knoxville strain that possesses both enzymatic and target site deltamethrin resistance, however, did not show any resistance to EOs, their major insecticidal constituents and EcoRaider® in topical bioassays (resistance ratio of ~1). In conclusion, this study demonstrated that a deltamethrin-resistant strain of bed bugs is susceptible to EOs and their insecticidal constituents.

Volatile and Contact Chemical Cues Associated with Host and Mate Recognition Behavior of Sphenophorus venatus and Sphenophorus parvulus (Coleoptera: Dryophthoridae).

Beetles in the genus Sphenophorus Schönherr, or billbugs, potentially utilize both volatile and non-volatile behavior-modifying chemical signals. These insects are widely distributed across North America, often occurring in multi-species assemblages in grasses. However, details about their host- and mate-finding behavior are poorly understood. This study tested the hypothesis that volatile organic compounds from host-plants and conspecifics direct the dispersal behavior of hunting billbug S. venatus Say. Further, we characterized the cuticular hydrocarbon profiles of two widespread pest species, S. venatus and bluegrass billbug S. parvulus Gyllenhaal, to assess the potential role of contact pheromones in mate-recognition. In Y-tube olfactometer bioassays, S. venatus males were attracted to a combination of conspecifics and Cynodon dactylon host-plant material, as well as C. dactylon plant material alone. S. venatus females were attracted to a combination of male conspecifics and host-plants but were also attracted to male conspecifics alone. Field evaluation of a putative male-produced aggregation pheromone, 2-methyl-4-octanol, identified from two congeners, S. levis Vaurie and S. incurrens Gyllenhaal, did not support the hypothesis that S. venatus and S. parvulus were also attracted to this compound. Gas chromatography-mass spectrometry analysis of S. venatus and S. parvulus whole-body cuticular extracts indicated a series of hydrocarbons with qualitative and quantitative interspecific variation in addition to intraspecific quantitative variation between males and females. This study provides the first evidence that S. venatus orients toward host- and insect-derived volatile organic compounds and substantiates the presence of species-specific cuticular hydrocarbons that could serve as contact pheromones for sympatric Sphenophorus species.

Determination of the Absolute Configuration of Female-Produced Contact Sex Pheromone Components of the Longhorned Beetle, Neoclytus acuminatus acuminatus (F).

Cuticular hydrocarbons play important roles in contact chemical communication in insects. Many of these compounds are methyl-branched hydrocarbons with one or more chiral centers, which can exist in two or more stereoisomeric forms. Although the importance of chirality for the volatile semiochemicals that insects use for long-range communication is well established, almost nothing is known about the role of chirality in insect contact chemoreception. Here, we used reverse phase high performance liquid chromatography (RP-HPLC) and digital polarimetry to isolate and determine the absolute configuration of a component of the female-produced contact sex pheromone of the cerambycid beetle, Neoclytus acuminatus acuminatus (F.). The pheromone consists of 7-methylpentacosane (7-MeC25), 7-methylheptacosane (7-MeC27), and 9-methylheptacosane (9-MeC27). The absolute configuration of the most abundant pheromone component, 7-MeC25, was found to be (R). We then utilized enantiomerically pure synthetic pheromone components to test the hypothesis that males would respond more strongly to (R)- than to (S)-enantiomers of the three pheromone components. We also tested blends of (R)-7-MeC27, the most bioactive component, with the (S)-enantiomers of the minor components and vice versa to determine if unnatural stereoisomers might decrease behavioral responses. Males responded most strongly to solvent-washed females treated with the blend of (R)-pheromone components, and to a lesser extent to (R)-7-MeC27 alone. A blend of (R)-7-MeC27 with the (S)-minor components elicited an intermediate response. Together, these findings suggest that the insects can discriminate the absolute configuration of the major and minor pheromone components, and that the configuration of all three components is likely to be (R).

Toxicity of Turmeric Extracts to the Termite Reticulitermes flavipes (Blattodea: Rhinotermitidae).

Turmeric is an important spice crop with documented human health benefits associated with chemicals called curcuminoids. In this study, the termite Reticulitermes flavipes (Kollar) was exposed to different solvent extracts of turmeric to investigate potential termiticidal properties. Treating termites with hexane extracts of purified lab-grade curcuminoids had no effect on termites. However, in continuous exposure assays, the LC(50) for hexane extracts of crude turmeric powder was 9.6 mg, or 1.0 mg starting material per square centimeter of filter paper substrate. These active components were soluble in a range of polar and apolar solvents, but only hexane could selectively fractionate active components away from the inactive curcuminoids. The active constituents of turmeric separated by thin layer chromatography (TLC) fluoresced in short-wave UV light but were not visible in long-wave UV light. By re-extracting TLC-separated bands in hexane and performing bioassays and gas chromatography-mass spectrometry, we demonstrated that termiticidal components of turmeric are extractable as a blend containing mainly ar-turmerone, turmerone, and curlone. This determination is consistent with findings of preceding work by other researchers that investigated insecticidal properties of turmeric in other pest insects.

Volatiles from nutritional fungal symbiont influence the attraction of Anisandrus maiche (Coleoptera: Curculionidae) to ethanol-baited traps.

Anisandrus maiche Stark (Coleoptera: Curculionidae: Scolytinae) is a non-native ambrosia beetle from central Asia that has been spreading throughout the eastern United States since 2005. Preferred hosts of A. maiche are not well characterized within its currently invaded range, but it is established in managed and natural forests throughout Indiana. Current monitoring and detection efforts for this beetle rely on ethanol-baited traps, but fungal volatiles may alter the attraction of A. maiche to ethanol. In this study, we conducted trapping experiments in Indiana to determine the extent to which a suite of common fungal alcohols influences the response of A. maiche to ethanol-baited traps. We then evaluated isoamyl and isobutyl alcohol as potential attractants for A. maiche and their ability to enhance attraction to ethanol. Lastly, we used SPME-GC-MS to identify volatiles from Ambrosiella cleistominuta (Mayers & Harr.), the fungal symbiont of A. maiche, grown for 7 and 14 days on malt extract agar. Benzyl alcohol, isobutyl alcohol, hexanol, methyl phenylacetate, phenethyl alcohol, and piperitone reduced the attraction of A. maiche to ethanol-baited traps in the field. Moreover, adding methyl benzoate and isoamyl alcohol individually to ethanol-baited traps did not further increase A. maiche capture. When paired with ethanol, isoamyl alcohol repelled beetles in the early flight period but did not significantly increase trap capture during the fall flight. These results represent a first step in understanding the role of fungal volatiles in the colonization behavior of A. maiche and may ultimately inform management strategies for this species.

Lethal and sublethal heat-exposure of bed bugs (Cimex lectularius L.) causes alarm pheromone emission and elicits a movement response in nearby recipients.

Many gregarious insect species use aggregation and alarm pheromones. The bed bug, Cimex lectularius L., emits an alarm pheromone (AP), a 70/30 blend of (E)-2-hexenal and (E)-2-octenal, when threatened. Bed bugs avoid temperatures above 43 °C, which are lethal to bugs and used commercially as spatial heat treatments to manage infestations. However, the interaction of bed bug AP in heat avoidance has not been investigated. The goal of this research was to: 1) determine if bed bugs emit AP as an alarm response to heat exposure, and 2) quantify the behavioral responses of conspecifics to AP emitted by heat-exposed bed bugs. Using a selected ion flow tube mass spectrometer, we found that bed bugs responded to lethal and sublethal heat exposure by emitting AP. The Harlan laboratory population emitted more pheromone than a laboratory adapted field population from Florida (McCall). Harlan females emitted the most AP, followed by Harlan males, McCall females and males. In separate behavioral experiments, we showed that conspecifics (i.e., recipients) reacted to AP released by heat exposed bed bugs (i.e., emitters) by frantically moving within 50 mm and 100 mm test arenas. The Harlan recipients reacted to AP in 100 mm areas, whereas the McCall strain did not, indicating a short area of effectiveness of the AP. Synthetic AP components tested in behavioral experiments caused identical effects as the natural AP blend released by heat-exposed bed bugs.

The Global Forest Health Crisis: A Public-Good Social Dilemma in Need of International Collective Action.

Society is confronted by interconnected threats to ecological sustainability. Among these is the devastation of forests by destructive non-native pathogens and insects introduced through global trade, leading to the loss of critical ecosystem services and a global forest health crisis. We argue that the forest health crisis is a public-good social dilemma and propose a response framework that incorporates principles of collective action. This framework enables scientists to better engage policymakers and empowers the public to advocate for proactive biosecurity and forest health management. Collective action in forest health features broadly inclusive stakeholder engagement to build trust and set goals; accountability for destructive pest introductions; pooled support for weakest-link partners; and inclusion of intrinsic and nonmarket values of forest ecosystems in risk assessment. We provide short-term and longer-term measures that incorporate the above principles to shift the societal and ecological forest health paradigm to a more resilient state.

First contact pheromone identified for a longhorned beetle (Coleoptera: Cerambycidae) in the subfamily Prioninae.

Little is known of the reproductive behavior of longhorned beetles (Coleoptera: Cerambycidae) in the subfamily Prioninae. Mallodon dasystomus (Say), the hardwood stump borer, is a widely distributed prionine that is native to the southern U.S. Here, we explored the chemically-mediated mating behavior of M. dasystomus, and tested the hypothesis that males recognize females by a contact pheromone. In mating bioassays, all males tested attempted to mate with females only after contacting females with their antennae. Moreover, all males attempted to mate with solvent-washed dead females treated with as little as 0.15 ± 0.03 female equivalents of conspecific cuticular extracts, confirming that compounds on the cuticle of females are essential for mate recognition. Cuticular hydrocarbon profiles of females contained 13 compounds that were not present in profiles of males. Among the female-specific compounds, two co-dominant methyl-branched alkanes, 2-methylhexacosane (2Me-C(26)) and 2-methyloctacosane (2Me-C(28)), accounted for 17% of the total hydrocarbons. Our strategy for identifying the contact pheromone was to synthesize and test the bioactivity of female specific compounds, starting with the most abundant. In bioassays, males displayed mating behavior in response to synthetic 2Me-C(26) and 2Me-C(28) when tested individually. Furthermore, when these compounds were tested in combination, they elicited the full progression of mating behaviors, suggesting that 2Me-C(26) and 2Me-C(28) make up the contact pheromone. These findings are further evidence of the critical role of contact pheromones in mating systems of longhorned beetles.

Two regulatory mechanisms of monoterpenoid pheromone production in Ips spp. of bark beetles.

Bark beetles use aggregation pheromones to coordinate host colonization and mating. These monoterpenoid chemical signals are produced de novo in midgut cells via the mevalonate pathway, and pheromone production is induced when an adult beetle feeds on phloem of a host tree. In Ips pini, juvenile hormone (JH) III influences key regulatory enzymes along the mevalonate pathway that leads to pheromone production. In fact, topically applied JH III is sufficient to stimulate pheromone production in unfed males. In this study, we explore the influence of feeding and JH III treatment on pheromone production in male Ips confusus, the pinyon Ips. We also characterize the influence of feeding and JH III treatment on transcript levels and activity of three key enzymes involved in pheromone biosynthesis: 3-hydroxy-3-methylglutaryl-CoA (HMG) synthase (HMGS), HMG-CoA reductase (HMGR) and geranyl diphosphate synthase (GPPS). We also extend the current understanding of the regulation of pheromone biosynthesis in I. pini, by measuring the influence of feeding and JHIII treatment on enzymatic activity of HMGS and GPPS. Feeding on host phloem alone strongly induces pheromone production in male I. confusus, while JH III treatment has no effect. However, feeding and JH III both significantly up-regulate mRNA levels of key mevalonate pathway genes. Feeding up-regulates these genes to a maximum at 32 h, whereas with JH III-treatment, they are up-regulated at 4, 8, and 16 h, but return near to non-treatment levels at 32 h. Feeding, but not JH III treatment, also increases the activity of all three enzymes in I. confusus, while both feeding or treatment with JH III increase HMGS and GPPS activity in I. pini. Our data suggest that pheromone production in Ips is not uniformly controlled by JH III and feeding may stimulate the release of some other regulatory factor, perhaps a brain hormone, required for pheromone production.

Male Megacyllene robiniae (Coleoptera: Cerambycidae) use multiple tactics when aggressively competing for mates.

Adult male Megacyllene robiniae (Förster) (Coleoptera: Cerambycidae) that are paired with a female often are challenged by conspecific males that attempt to displace them. In staged laboratory bouts, challenging males used seven distinct tactics to displace defending males, including wedging their head between the defender and the female (termed wedging), straddling the mated pair and pulling the defender off (prying), pulling it with the mandibles, batting it with the antennae, or pushing, biting, or kicking the defender. Individual challengers attempted as many as six different tactics in a single bout, repeating certain tactics multiple times. They often attempted tactics that were not very effective. For example, prying was one of the most common attempted tactics but was rarely effective. However, few challengers attempted to push defenders off the female, even though that tactic often was effective. Challengers apparently were influenced by context in their choice of particular tactics. For example, males that approached the mated pair from the side were likely to use wedging, whereas those approaching head on were more likely to bat with the antennae. Choice of tactic apparently was not influenced by absolute size of challengers, nor was it strongly influenced by relative size of defenders. However, the effectiveness of tactics varied significantly with relative body size: larger challengers were most successful when prying or pushing, while smaller challengers were most successful when biting and kicking. By using different tactics, relatively small males were as adept as larger males at displacing rivals.

Identifying Possible Pheromones of Cerambycid Beetles by Field Testing Known Pheromone Components in Four Widely Separated Regions of the United States.

The pheromone components of many cerambycid beetles appear to be broadly shared among related species, including species native to different regions of the world. This apparent conservation of pheromone structures within the family suggests that field trials of common pheromone components could be used as a means of attracting multiple species, which then could be targeted for full identification of their pheromones. Here, we describe the results of such field trials that were conducted in nine states in the northeastern, midwestern, southern, and western United States. Traps captured 12,742 cerambycid beetles of 153 species and subspecies. Species attracted in significant numbers to a particular treatment (some in multiple regions) included 19 species in the subfamily Cerambycinae, 15 species in the Lamiinae, one species in the Prioninae, and two species in the Spondylidinae. Pheromones or likely pheromones for many of these species, such as 3-hydroxyhexan-2-one and syn- and anti-2,3-hexanediols for cerambycine species, and fuscumol and/or fuscumol acetate for lamiine species, had already been identified. New information about attractants (in most cases likely pheromone components) was found for five cerambycine species (Ancylocera bicolor [Olivier], Elaphidion mucronatum [Say], Knulliana cincta cincta [Drury], Phymatodes aeneus LeConte, and Rusticoclytus annosus emotus [Brown]), and five lamiine species (Ecyrus dasycerus dasycerus [Say], Lepturges symmetricus [Haldeman], Sternidius misellus [LeConte], Styloleptus biustus biustus [LeConte], and Urgleptes signatus [LeConte]). Consistent attraction of some species to the same compounds in independent bioassays demonstrated the utility and reliability of pheromone-based methods for sampling cerambycid populations across broad spatial scales.

Colonization of Artificially Stressed Black Walnut Trees by Ambrosia Beetle, Bark Beetle, and Other Weevil Species (Coleoptera: Curculionidae) in Indiana and Missouri.

Thousand cankers disease (TCD) is a new disease of black walnut (Juglans nigra L.) in the eastern United States. The disease is caused by the interaction of the aggressive bark beetle Pityophthorus juglandis Blackman and the canker-forming fungus, Geosmithia morbida M. Kolarik, E. Freeland, C. Utley & Tisserat, carried by the beetle. Other insects also colonize TCD-symptomatic trees and may also carry pathogens. A trap tree survey was conducted in Indiana and Missouri to characterize the assemblage of ambrosia beetles, bark beetles, and other weevils attracted to the main stems and crowns of stressed black walnut. More than 100 trees were girdled and treated with glyphosate (Riverdale Razor Pro, Burr Ridge, Illinois) at 27 locations. Nearly 17,000 insects were collected from logs harvested from girdled walnut trees. These insects represented 15 ambrosia beetle, four bark beetle, and seven other weevil species. The most abundant species included Xyleborinus saxeseni Ratzburg, Xylosandrus crassiusculus Motschulsky, Xylosandrus germanus Blandford, Xyleborus affinis Eichhoff, and Stenomimus pallidus Boheman. These species differed in their association with the stems or crowns of stressed trees. Multiple species of insects were collected from individual trees and likely colonized tissues near each other. At least three of the abundant species found (S. pallidus, X. crassiusculus, and X. germanus) are known to carry propagules of canker-causing fungi of black walnut. In summary, a large number of ambrosia beetles, bark beetles, and other weevils are attracted to stressed walnut trees in Indiana and Missouri. Several of these species have the potential to introduce walnut canker pathogens during colonization.

Growth of Larval Agrilus planipennis (Coleoptera: Buprestidae) and Fitness of Tetrastichus planipennisi (Hymenoptera: Eulophidae) in Blue Ash (Fraxinus quadrangulata) and Green Ash (F. pennsylvanica).

Emerald ash borer (Agrilus planipennis Fairmaire) is an invasive primary pest of North American ash (Fraxinus spp.) trees. Blue ash (F. quadrangulata) is less susceptible to emerald ash borer infestations in the forest than other species of North American ash. Whereas other studies have examined adult host preferences, we compared the capacity of emerald ash borer larvae reared from emerald ash borer eggs in the field and in the laboratory to survive and grow in blue ash and the more susceptible green ash (F. pennsylvanica). Emerald ash borer larval survivorship was the same on both ash species. Mortality due to wound periderm formation was only observed in living field grown trees, but was low (<4%) in both green and blue ash. No difference in larval mortality in the absence of natural enemies suggests that both green and blue ash can support the development of emerald ash borer. Larvae reared from eggs on blue ash were smaller than on green ash growing in the field and also in bolts that were infested under laboratory conditions. In a laboratory study, parasitism rates of confined Tetrastichus planipennisi were similar on emerald ash borer larvae reared in blue and green ash bolts, as were fitness measures of the parasitoid including brood size, sex ratio, and adult female size. Thus, we postulate that emerald ash borer larvae infesting blue ash could support populations of T. planipennisi and serve as a potential reservoir for this introduced natural enemy after most of the other native ash trees have been killed.

Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations.

Ips pini bark beetles use myrcene hydroxylases to produce the aggregation pheromone component, ipsdienol, from myrcene. The enantiomeric ratio of pheromonal ipsdienol is an important prezygotic mating isolation mechanism of I. pini and differs among geographically distinct populations. We explored the substrate and product ranges of myrcene hydroxylases (CYP9T2 and CYP9T3) from reproductively-isolated western and eastern I. pini. The two cytochromes P450 share 94% amino acid identity. CYP9T2 mRNA levels were not induced in adults exposed to myrcene-saturated atmosphere. Functional assays of recombinant enzymes showed both hydroxylated myrcene, (+)- and (-)-α-pinene, 3-carene, and R-(+)-limonene, but not α-phellandrene, (-)-ß-pinene, γ-terpinene, or terpinolene, with evidence that CYP9T2 strongly preferred myrcene over other substrates. They differed in the enantiomeric ratios of ipsdienol produced from myrcene, and in the products resulting from different α-pinene enantiomers. These data provide new information regarding bark beetle pheromone evolution and factors affecting cytochrome P450 structure-function relationships.

Myrcene hydroxylases do not determine enantiomeric composition of pheromonal ipsdienol in Ips spp.

Myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation is likely one of the final reactions involved in the production of the Ips spp. (Coleoptera: Scolytidae) aggregation pheromone components, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) and ipsenol (2-methyl-6-methylene-7-octen-4-ol). To gain insight into the evolution of pheromone production, we isolated a full-length cDNA from the pinyon ips, Ips confusus (LeConte), that encodes a pheromone-biosynthetic cytochrome P450, I. confusus CYP9T1 (IcCYP9T1). The recovered cDNA is 1.70 kb, and the open reading frame encodes a 532 amino acid protein. IcCYP9T1 is 94% identical to the pine engraver, Ips pini (Say), CYP9T2 ortholog that hydroxylates myrcene. Quantitative real-time PCR experiments showed that IcCYP9T1, as does CYP9T2, has an expression pattern similar to other pheromone-biosynthetic genes in I. pini. Basal expression levels were higher in males than females, and expression was significantly induced in male, but not in female, anterior midguts by feeding on host phloem. Microsomes, prepared from Sf9 cells co-expressing baculoviral-mediated recombinant IcCYP9T1 and house fly (Musca domestica) NADPH-cytochrome P450 reductase, converted myrcene to ~85%-(R)-(-)-ipsdienol. These results are consistent with IcCYP9T1 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway. Since the I. confusus pheromone blend contains >90%-(S)-(+)-ipsdienol, these results confirm further that Ips spp. myrcene hydroxylases do not control the final ipsdienol enantiomeric blend. Other enzymes are required following myrcene hydroxylation to achieve the critical quantity and enantiomeric composition of pheromonal ipsenol and ipsdienol used by different Ips spp.

Antennally mediated negative feedback regulation of pheromone production in the pine engraver beetle, Ips pini.

Bark beetles use monoterpenoid aggregation pheromones to coordinate host colonization and mating. These chemical signals are produced de novo in midgut cells via the mevalonate pathway, and pheromone production may be regulated by a negative feedback system mediated through the antennae. In this study, we explored the effect of antennectomy on pheromone production and transcript levels of key mevalonate pathway genes in juvenile hormone III-treated male pine engraver beetles, Ips pini (Say). Antennectomized males produced significantly greater amounts of pheromone than podectomized males and those with intact antennae. Likewise, mRNA levels of three mevalonate pathway genes important in pheromone biosynthesis were measured by quantitative real-time PCR and found to be induced to a greater extent with antennectomy, suggesting a transcriptional regulation of pheromone production.