Identification of a Male-Produced Volatile Pheromone for Phymatodes dimidiatus (Coleoptera: Cerambycidae) and Seasonal Flight Phenology of Four Phymatodes Species Endemic to the North American Intermountain West.

Research over the last 15 yr has shown widespread pheromone parsimony within the coleopteran family Cerambycidae, with a number of highly conserved pheromone motifs, often shared within and across subfamilies, tribes, and genera. Our goals were to increase our understanding of the evolution of volatile pheromones within the Cerambycidae, their role in reproductive isolation and to identify pheromones for use in the development of lures for monitoring cerambycids. Over 3 yr, we tested 12 compounds known to be cerambycid pheromones as possible attractants at sites across Idaho. This study focused on species within the cerambycine genus Phymatodes (Tribe: Callidiini). We also collected and analyzed headspace volatiles of captured Phymatodes dimidiatus (Kirby). Our results demonstrate that (R)-2-methylbutan-1-ol is a male-produced volatile pheromone for P. dimidiatus. These results are consistent with prior research suggesting that (R)-2-methylbutan-1-ol and (R)-3-hydroxyhexan-2-one, individually or in a blend of both compounds, commonly serve as pheromones for Phymatodes spp. We captured Phymatodes starting in mid-May, continuing through mid-August. Our data indicate that flight periods of Phymatodes spp. in Idaho overlap. These species may be utilizing various mechanisms to ensure reproductive isolation, such as the production of different volatile pheromones, minor components, and/or proportions of components, utilizing different host species and/or host volatiles, differing daily activity periods, and/or occupying different heights in the tree canopy. Our results contribute to the basic understanding of the chemical and behavioral ecology of the Cerambycidae and can be applied to the development of pheromone lures for monitoring of economically important or endangered species.

Mating Disruption for Managing Prionus californicus (Coleoptera: Cerambycidae) in Hop and Sweet Cherry.

Larvae of Prionus californicus Motschulsky feed on the roots of many woody perennial plants and are economically important pests of hop Humulus lupulus L. (Urticales: Cannabaceae) and sweet cherry Prunus avium (L.) (Magnoliopsida: Rosaceae) in the United States Pacific Northwest and Intermountain West. Adult males are strongly attracted to a volatile sex pheromone, (3R,5S)-3,5-dimethyldodecanoic acid, produced by females. Here, we summarize the results of field experiments evaluating the synthetic pheromone in a blend of all four possible stereoisomers as a means for managing P. californicus in hop yards and sweet cherry orchards by mating disruption (MD). Mean capture of male beetles was lower, in all 3 yr of the study, from plots in commercial hop yards and sweet cherry orchards treated with synthetic P. californicus pheromone than from similar, untreated plots. Although trap catch was lower in sweet cherry, relative differences between trap catches from MD and nonmating disruption plots were similar to that seen in hop yards. The number of P. californicus larvae recovered from plots in hop yards treated for three consecutive growing seasons with synthetic pheromone was lower than in similar plots that were not treated with the pheromone or treated with the soil fumigant ethoprop. Our research demonstrates that deployment of synthetic P. californicus pheromone effectively reduces mate-finding by males, can effectively reduce larvae populations in pheromone-treated hop yards, and thus, has excellent potential for managing P. californicus in hop, sweet cherry, and perhaps in other crops where it or Prionus species are pests.

Effects of residual novaluron on reproduction in alfalfa leafcutting bees, Megachile rotundata F. (Megachilidae).

BACKGROUND: The chitin synthesis inhibitor novaluron can suppress pests that affect alfalfa seed production, but can negatively affect reproductive success in the alfalfa pollinator Megachile rotundata. Novaluron is considered to be a reduced-risk insecticide because it disrupts ecdysis and is non-lethal to adult insects, but some exposed adults have fewer eggs and suppressed egg hatch. For this experiment, bees nested in field cages where they were exposed to alfalfa that had never been treated with novaluron, alfalfa that had recently been sprayed or alfalfa that had been sprayed 1 and 2 weeks earlier. RESULTS: Compared with the control, greater proportions of dead eggs and larvae and lower proportions of live prepupae occurred when bees were exposed to recent novaluron sprays as well as one- or two-week old spray residues. Two possible routes of residual pesticide exposure were revealed. Mother bees become contaminated through ingestion or direct contact, or pollen-nectar provisions become contaminated with novaluron (1) on or within leaf pieces that surround provisions or (2) transferred from mother bees' bodies to provisions. CONCLUSION: We found strong immature mortality effects of novaluron and its residues on M. rotundata. Understanding all possible pesticide exposure routes for pollinating bees enhances decision-making for maintaining bee populations while protecting crops. © 2016 Society of Chemical Industry.

North American Species of Cerambycid Beetles in the Genus Neoclytus Share a Common Hydroxyhexanone-Hexanediol Pheromone Structural Motif.

Many species of cerambycid beetles in the subfamily Cerambycinae are known to use male-produced pheromones composed of one or a few components such as 3-hydroxyalkan-2-ones and the related 2,3-alkanediols. Here, we show that this pheromone structure is characteristic of the cerambycine genus Neoclytus Thomson, based on laboratory and field studies of 10 species and subspecies. Males of seven taxa produced pheromones composed of (R)-3-hydroxyhexan-2-one as a single component, and the synthetic pheromone attracted adults of both sexes in field bioassays, including the eastern North American taxa Neoclytus caprea (Say), Neoclytus mucronatus mucronatus (F.), and Neoclytus scutellaris (Olivier), and the western taxa Neoclytus conjunctus (LeConte), Neoclytus irroratus (LeConte), and Neoclytus modestus modestus Fall. Males of the eastern Neoclytus acuminatus acuminatus (F.) and the western Neoclytus tenuiscriptus Fall produced (2S,3S)-2,3-hexanediol as their dominant or sole pheromone component. Preliminary data also revealed that males of the western Neoclytus balteatus LeConte produced a blend of (R)-3-hydroxyhexan-2-one and (2S,3S)-2,3-hexanediol but also (2S,3S)-2,3-octanediol as a minor component. The fact that the hydroxyketone-hexanediol structural motif is consistent among these North American species provides further evidence of the high degree of conservation of pheromone structures among species in the subfamily Cerambycinae.

2,3-Hexanediols as sex attractants and a female-produced sex pheromone for cerambycid beetles in the prionine genus Tragosoma.

Recent work suggests that closely related cerambycid species often share pheromone components, or even produce pheromone blends of identical composition. However, little is known of the pheromones of species in the subfamily Prioninae. During field bioassays in California, males of three species in the prionine genus Tragosoma were attracted to 2,3-hexanediols, common components of male-produced aggregation pheromones of beetles in the subfamily Cerambycinae. We report here that the female-produced sex pheromone of Tragosoma depsarium "sp. nov. Laplante" is (2R,3R)-2,3-hexanediol, and provide evidence from field bioassays and electroantennography that the female-produced pheromone of both Tragosoma pilosicorne Casey and T. depsarium "harrisi" LeConte may be (2S,3R)-2,3-hexanediol. Sexual dimorphism in the sculpting of the prothorax suggests that the pheromone glands are located in the prothorax of females. This is the second sex attractant pheromone structure identified from the subfamily Prioninae, and our results provide further evidence of pheromonal parsimony within the Cerambycidae, in this case extending across both subfamily and gender lines.

Evaluation of mass trapping and mating disruption for managing Prionus californicus (Coleoptera: Cerambycidae) in hop production yards.

Larvae of Prionus californicus Motschulsky (Coleoptera: Cerambycidae) feed on the roots of many types of woody perennial crops and are serious pests of hop in the northwestern United States. The adult males are strongly attracted to a volatile sex pheromone, (3R,5S)-3,5-dimethyldodecanoic acid, that is produced by females. Here, we summarize the results of field experiments that evaluated the potential for using the synthetic pheromone (in a blend of all four possible stereoisomers) to manage infestations of P. californicus in commercial hop yards by mass trapping or mating disruption. Our research provides evidence that mass trapping may be effective in reducing mating success of the females: positioning surrogate females (sentinel traps baited with a low dose of pheromone) within a square of eight pheromone-baited traps resulted in an 88% reduction in the number of wild males that reached the sentinel traps compared with sentinel traps that were surrounded by traps baited with blank lures. Similarly, surrogate females that were surrounded by pheromone lures (without traps) were reached by 84% fewer wild males than surrogate females surrounded by blank lures, suggesting that mating disruption also may be effective. A mark-recapture experiment indicated that male P. californicus were attracted to traps baited with 1 mg of pheromone from as far away as 585 m. These studies indicate that 3,5-dimethyldodecanoic acid has very good potential for managing P. californicus in hop yards, and perhaps in other crops where it is a pest.

Synthesis and field tests of possible minor components of the sex pheromone of Prionus californicus.

Earlier work has shown that adult male Prionus californicus Motschulsky (Coleoptera: Cerambycidae) are attracted to the female-produced compound (3R,5S)-3,5-dimethyldodecanoic acid, and to a synthetic mixture of the four stereoisomers of 3,5-dimethyldodecanoic acid. Here, we report the results of field trials that tested whether or not three structurally related compounds (methyl 3,5-dimethyldodecanoate, 3,5-dimethyltridecanoic acid, and 3,5-dimethylpentadecanoic acid), present in extracts of virgin females, are attractive, and whether or not they influence attraction to 3,5-dimethyldodecanoic acid. In a trial with single components, only traps baited with the acid or its methyl ester captured more beetles than did control traps; catches to the acid were five times higher than to the methyl ester. Another trial, excluding 3,5-dimethyldodecanoic acid, confirmed the activity of the methyl ester. Finally, addition of the three compounds to 3,5-dimethyldodecanoic acid, in the ratio found in extracts from female beetles, gave a catch similar to that of traps baited with 3,5-dimethyldodecanoic acid alone. Consequently, the function of these minor compounds remains undetermined.

Effects of the insect growth regulator, novaluron on immature alfalfa leafcutting bees, Megachile rotundata.

Alfalfa leafcutting bees, Megachile rotundata F. (Hymenoptera: Megachilidae), are the most common pollinators of alfalfa in the Pacific Northwest. Reports from users of M. rotundata in Idaho, Utah and Colorado have indicated exceptionally poor bee return from fields treated with novaluron to control Lygus spp. Our goal was to evaluate novaluron toxicity to immature M. rotundata using two different possible mechanisms of exposure. One goal was to assess immature mortality via treating nectar-pollen provisions and adults with novaluron. Immature M. rotundata mortality in all novaluron provision dosing treatments was significantly higher than the water or blank controls, providing evidence that novaluron is toxic to progeny in nest cells. The mean cumulative frequency showed that more eggs and 1st-2nd instars died compared to older instars. Female M. rotundata nested similarly in field cages during the field cage experiment; however, there was greater immature mortality in cages where females were fed sugar-water + novaluron compared to sugar-water only. Although females provided adequate provisions, there was a low percentage of egg hatch and larval development when females ingested novaluron before mating and nesting. Novaluron was also present in egg provision of bees collecting resources from novaluron-sprayed plants. At least 84% of progeny died when the females were allowed to mate and nest 24 hours after a novaluron application. Novaluron could be contributing to poor bee return in alfalfa grown for seed. Timely insecticide applications to suppress Lygus spp. is an important consideration to improve ongoing bee health.

Determination of the relative and absolute configurations of the female-produced sex pheromone of the cerambycid beetle Prionus californicus.

We previously identified the basic structure of the female-produced sex attractant pheromone of the cerambycid beetle, Prionus californicus Motschulsky (Cerambycidae: Prioninae), as 3,5-dimethyldodecanoic acid. A synthesized mixture of the four stereoisomers of 3,5-dimethyldodecanoic acid was highly attractive to male beetles. Here, we describe stereoselective syntheses of three of the four possible stereoisomers, and the results of laboratory and field bioassays showing that male beetles are attracted specifically to (3R,5S)-3,5-dimethyldodecanoic acid, but not to its enantiomer, (3S,5R)-3,5-dimethyldodecanoic acid, indicating that the (3R,5S)-enantiomer is the active pheromone component. The diastereomeric (3R,5R)- and (3S,5S)-enantiomers were excluded from consideration because their gas chromatographic retention times were different from that of the insect-produced compound. The mixture of the four stereoisomers of 3,5-dimethyldodecanoic acid was as attractive to male P. californicus as the (3R,5S)-enantiomer, indicating that none of the other three stereoisomers inhibited responses to the active enantiomer. Beetles responded to as little as 10 ng and 10 µg of synthetic 3,5-dimethyldodecanoic acid in laboratory and field studies, respectively. Field studies indicated that capture rate did not increase with dosages of 3,5-dimethyldodecanoic acid greater than 100 µg. In field bioassays, males of a congeneric species, P. lecontei Lameere, were captured in southern California but not in Idaho.

Treating panel traps with a fluoropolymer enhances their efficiency in capturing cerambycid beetles.

The most effective traps for capturing cerambycids and other saproxylic beetles are intercept designs such as funnel traps and cross-vane panel traps. We have observed that adult cerambycids of many species often alight and walk upon panel traps, and few are actually captured. In an effort to improve trap capture and retention, researchers have treated intercept traps with Rain-X, a polysiloxane formulation that renders surfaces more slippery. Here, we summarize experiments that compared the efficacies of Rain-X and Fluon, a PTFE fluoropolymer dispersion, assurface treatments for panel traps that are deployed to capture cerambycid beetles, using untreated traps as controls. Fluon-treated traps captured on average > 14x the total number of beetles, and many more cerambycid species, than were captured by Rain-X-treated or control traps. Beetles captured by Fluon-treated traps ranged in body length by 350%. They could not walk on vertical panels treated with Fluon but easily walked on those treated with Rain-X and on untreated traps. Moreover, a single Fluon treatment remained effective for the entire field season, even in inclement weather. We conclude that treating panel traps with Fluon greatly improves their efficiency in capturing cerambycid beetles. This increased efficacy will be particularly important when traps are deployed to detect very low-density populations, such as incursions of exotic species, or remnant communities of rare and endangered species. The influence of Fluon on trap efficiency may vary with product formulation and its source and also with climatic conditions.

Identification and synthesis of a female-produced sex pheromone for the cerambycid beetle Prionus californicus.

Females of the large cerambycid beetle Prionus californicus produce a powerful sex pheromone that attracts males. The pheromone was adsorbed on solid phase microextraction (SPME) fibers inserted into the ovipositor sheath and analyzed by coupled gas chromatography-electroantennogram detection and GC-mass spectrometry. The pheromone was identified as an isomer of 3,5-dimethyldodecanoic acid by a combination of retention index comparisons and mass spectral interpretation. The mass spectrum was misleading because it exhibited enlarged fragment ions that were not representative of branch points or other obvious stabilizing structural elements. The structure was verified by synthesis of 3,5-dimethyldodecanoic acid as a mixture of all four possible isomers, and this mixture was highly attractive to male beetles in field bioassays. The SPME extracts also contained several other compounds that were tentatively identified as chain-extended homologs of the main pheromone component. This pheromone should prove useful for sampling and management of the beetle, which is an important pest of hops, and an occasional pest in a variety of orchard crops. Although this is the first female-produced pheromone to be identified from the Cerambycidae, there is considerable evidence for pheromone production by females of other species in the subfamily Prioninae. Thus, this pheromone and the associated methodology used in its identification should be useful in the identification of female-produced attractant pheromones from other members of the subfamily.

Male-produced aggregation pheromone of the cerambycid beetle Rosalia funebris.

We report the identification, synthesis, and field bioassays of a volatile, male-produced aggregation pheromone of a long-horned beetle, the banded alder borer, Rosalia funebris Mots. Headspace collections from males contained a major male-specific compound, (Z)-3-decenyl (E)-2-hexenoate, and several minor components, identified as (Z)-3-decenol, (Z)-3-nonenyl (E)-2-hexenoate, and (Z)-3-decenyl (E)-3-hexenoate. The antennae of both males and females responded strongly to (Z)-3-decenyl (E)-2-hexenoate. We collected significant numbers of adult R. funebris in field bioassays using traps baited with this compound. This pheromone structure is unprecedented in the literature of cerambycid pheromones and distinct from the more common diol/hydroxyketone pheromone motif of many other species of the diverse subfamily Cerambycinae. This is the first pheromone identified for a species in the tribe Rosaliini.

Using generic pheromone lures to expedite identification of aggregation pheromones for the cerambycid beetles Xylotrechus nauticus, Phymatodes lecontei, and Neoclytus modestus modestus.

Males of several species of longhorned beetles in the subfamily Cerambycinae produce sex or aggregation pheromones consisting of 2,3-hexanediols and/or hydroxyhexanones. We tested the hypothesis that this diol/hydroxyketone pheromone motif is highly conserved within the subfamily, and the resulting prediction that multiple cerambycine species will be attracted to compounds of this type. We also tested the concept that live traps baited with generic blends of these compounds could be used as a source of live insects from which pheromones could be collected and identified. Traps placed in a mature oak woodland and baited with generic blends of racemic 2-hydroxyhexan-3-one and 3-hydroxyhexan-2-one captured adults of both sexes of three cerambycine species: Xylotrechus nauticus (Mannerheim), Phymatodes lecontei Linsley, and Phymatodes decussatus decussatus (LeConte). Odors collected from male X. nauticus contained a 9:1 ratio of two male-specific compounds, (R)- and (S)-3-hydroxyhexan-2-one. Field trials with synthetic compounds determined that traps baited with (R)-3-hydroxyhexan-2-one (94% ee), alone or in blends with other isomers, attracted similar numbers of X. nauticus of both sexes, whereas (S)-3-hydroxyhexan-2-one (94% ee) attracted significantly fewer beetles. Phymatodes lecontei and P. d. decussatus also were caught in traps baited with hydroxyhexanones, as well as a few specimens of two other cerambycine species, Neoclytus modestus modestus Fall (both sexes) and Brothylus gemmulatus LeConte (only females). Male N. m. modestus produced (R)-3-hydroxyhexan-2-one, which was not present in extracts from females. Neoclytus m. modestus of both sexes also responded to lures that included (R)-3-hydroxyhexan-2-one as one of the components. The only male-specific compound found in extracts from P. lecontei was (R)-2-methylbutan-1-ol, and adults of both sexes were attracted to racemic 2-methylbutan-1-ol in field bioassays. Surprisingly, P. lecontei of both sexes also were attracted to (R)- and (S)-3-hydroxyhexan-2-ones, although neither compound was detected in extracts from this species. Males of all five beetle species had gland pores on their prothoraces that were similar in structure to those that have been associated with volatile pheromone production in other cerambycine species. The attraction of multiple cerambycine species of two tribes to (R)-3-hydroxyhexan-2-one in this study, and in earlier studies with other cerambycine species, suggests that this compound is a widespread aggregation pheromone component in this large and diverse subfamily. Overall, the attraction of multiple species from different cerambycine tribes to this compound at a single field site supports the hypothesis that the hydroxyketone pheromone structural motif is highly conserved within this subfamily.

Parasitism of Lygus spp. nymphs by the parasitoid wasp, Peristenus howardi, in the alfalfa seed-growing region of the Pacific Northwest.

The parasitoid, Peristenus howardi Shaw (Hymenoptera: Braconidae) has been found to parasitize a large proportion of Lygus species in some Washington and Idaho alfalfa seed fields. During 2002-2003 a survey was conducted to estimate the proportion of Lygus spp. (Hemiptera: Miridae) parasitized and the amountof that parasitism attributable to P. howardi in crop and non-crop plants attacked by Lygus in the alfalfa seed growing region of southwestern Idaho and eastern Oregon. Percentage parasitism was estimated from dissection of field-collected Lygus nymphs. Polymerase chain reaction (PCR) was used to amplify DNA extracted from parasitoid larvae followed by restriction endonuclease digestion of PCR products to distinguish P. howardi from other potentially co-occurring Peristenus species. Peak parasitism of Lygus nymphs occurred between the first and third weeks of July for both years for all host plants sampled. Of the parasitoid larvae recovered from Lygus nymphs in our study, 75% to 80% tested positive as Peristenus spp. and 76% of these matched the endonuclease digestion banding pattern for P. howardi. The identity of the remaining 20% to 25% of the parasitoids is not known.

Distinguishing the parasitic wasp, Peristenus howardi, from some of its congeners using polymerase chain reaction and restriction endonuclease digestion.

A molecular procedure incorporating polymerase chain reaction (PCR) of the COI gene and restriction endonuclease digestion of PCR products was used to distinguish Peristenus howardi (Hymenoptera: Braconidae) from four other Peristenus species. Non-solvent extraction of parasite DNA using a commercially available kit proved to be very effective in producing amplifiable template. Use of SfcI endonuclease produced restriction fragments with banding patterns in agarose gel electrophoresis that readily separated P. howardi, P. digoneutis, P. conradi, P. pallipes, and P. pseudopallipes. However, while the restriction fragment banding patterns of both P. pallipes and P. pseudopallipes were easily distinguishable from the other Peristenus species, they could not be reliably separated from one another. This molecular procedure can be used in applied and ecological research to better understand the role of P. howardi in the Peristenus-Lygus parasite-host system within the Pacific Northwest. Consensus sequences of our amplimers for all five Peristenus spp. are deposited in GenBank.