Isolation of pheromone synergists of bark beetle,Pityogenes chalcographus,from complex insect-plant odors by fractionation and subtractive-combination bioassay

Capillary gas chromatography with columns of different polarity and two-dimensional fractionation of effluents were used with novel subtrac-tive-combination bioassays to rigorously isolate host- and insect-produced pheromone synergists of the bark beetlePityogenes chalcographus (Coleop-tera: Scolytidae). Methyl (E,Z)-2,4-decadienoate (E,Z-MD) and the previously identified chalcogran were found to be synergistically attractive to both sexes.E,Z-MD was produced sex-specifically in males, and only when they had fed on host-plant tissue. A Norway spruce monoterpene fraction (including -pinene, -pinene, and camphene) increased the attractive response to the pheromone components. Dose-response curves forE,Z-MD and chalcogran in the laboratory bioassay indicated the two components are highly synergistic. The isolation methods are important for further progress in identifying certain semiochemical synergists found in trace amounts in complex chemical mixtures, such as when insects must feed in host plants in order to produce pheromone.     

The aggregation pheromone of <Emphasis Type="Italic">Diorhabda elongata</Emphasis>, a biological control agent of saltcedar(<Emphasis Type="Italic">Tamarix</Emphasis> spp.): identification of two behaviorally active components

The leaf beetle Diorhabda elongata Brullé (Coleoptera: Chrysomelidae)has been introduced as a biological control agent for saltcedars, Tamarix spp., an exotic, invasive weedy tree in the western United State. Gas chromatographic (GC) analysis of volatiles collected from feeding male or female beetles, or saltcedar foliage alone, showed two components produced almost exclusively by males. These compounds elicited responses from antennae of male and female beetles in GC-electroantennographic detection (EAD) analyses. The compounds were identified as (2E,4Z)-2,4-heptadienal (1) and (2E,4Z)-2,4- heptadien-1-ol (2) by GC-mass spectrometry (MS), and confirmed with authentic standards. The two compounds were also detected at trace levels from feeding females and foliage controls, but the amounts from feeding males were 8-40 times higher, typically 55-125 ng per day per male. The amounts of 1 and 2 in collections from females did not differ significantly from amounts collected from control foliage. In field trials, 2 as a single component was as attractive as a 1:1 blend of 1 and 2. Compound 1 as a single component was more attractive than controls, but much less attractive than 2 or the blend. Males and femaleswere attracted in about equal numbers, indicating that this is an aggregation pheromone.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Sensitivity of antennae of male and femaleIps paraconfusus (Coleoptera: Scolytidae) to their natural aggregation pheromone and its enantiomeric components

Antennae of male and femaleIps paraconfusus were equally and highly sensitive to their male-produced, multicomponent aggregation pheromone. Female and male antennae were highly sensitive to the pheromonal component, (S)-(–)-ipsenol, but essentially insensitive to its antipode, (R)-(+)-ipsenol. Further, female and male antennae were more sensitive to the pheromonal component, (S)-cis-verbenol, than to its antipode, (R)-cis-verbenol. Dramatic sexual dimorphism in chiral sensitivity to the ipsdienol enantiortiers was found, with female antennae being more sensitive to the conspecific pheromonal enantiomer, (S)-(+)-ipsdienol, and male antennae being more sensitive to the antipode, (R)-(–)-ipsdienol. Since (R)-(–)-ipsdienol is the principal pheromone of CaliforniaIps pini and interruptsI. paraconfusus aggregation, male antennae appear to be more sensitive to an interspecific allomone than a conspecific pheromone. Of the conspecific pheromonal enantiomers, both male and female antennae were most sensitive to (S)-(+)-ipsdienol, intermediately sensitive to (S)-(–)-ipsenol, and least sensitive to (S)-cis-verbenol. However, when enantiomeric sensitivities were compared to the estimated concentrations of these components in the natural pheromone, (S)-(~)-ipsenol tended to equal or approach the potency of (S)-(+)-ipsdienol as an antennal stimulant, while antennal responsiveness to (S)-cis-verbenol was dramatically less than for the other two pheromonal components. The behavioral implications of such physiological sensitivities are discussed in regard to perception of multicomponent synergistic pheromones and the relative efficacy of each component as an orientation cue.This paper is based in part upon a dissertation submitted to the University of California, Davis, in partial fulfillment of the requirements for the PhD in entomology.     

Lanierone: A new pheromone component fromIps pini (Coleoptera: Scolytidae) in New York

A new pheromone component, lanierone, (2-hydroxy-4,4,6-trimethyl-2,5-cyclohexadien-1-one) was isolated and identified from a Porapak Q collection of volatiles from maleIps pini from New York through GC fractionation, bioassay, and spectrometry. In both the laboratory and the field, synthetic lanierone, in a 1:100 ratio with synthetic ipsdienol, is as attractive as natural pheromone sources. Synthetic ipsdienol alone is not attractive in the laboratory and only weakly attractive in the field. Varying the ratio of lanierone to ipsdienol in the field from 10^–41 to 11 in 10-fold increments resulted in an increased number of beetles trapped at the three lower ratios, but also in an increase in the proportion of males trapped. In the field, all combinations of lanierone to ipsdienol attracted proportionately fewer males than did pheromone-producing male beetles. GC and GC-MS analyses of Porapak Q-trapped volatiles revealed that lanierone is produced in an amount equal to about 0.2% of that of ipsdienol and is produced exclusively by males. The small amount of lanierone produced, together with a GC retention time similar to that of ipsdienol on a nonpolar column, probably confounded its detection in earlier studies.Deceased.     

Enantiomer-based specificity in pheromone communication by two sympatricGnathoirichus species (Coleoptera: Scolytidae)

The aggregation pheromone ofGnathotrichus retusus was isolated and identified as (S)-(+)-sulcatol (6-methyl-5-hepten-2-ol). In laboratory and field experiments,G. retusus responded to (S)-(+)-sulcatol, but not to (±)-sulcatol, which was attractive to the sympatric species,G. sulcatus. G. sulcatus did not respond to optically pure (S)-(+)-sulcatol, but began to respond when 1% (R)-(–)-sulcatol was present in an enantiomeric mixture.Research supported by the National Science Foundation, U.S.A. (Grant BMS-74-13643), and the Natural Sciences and Engineering Research Council of Canada (Co-op grant A0243 and Operating Grant A3881 and A3785).     

Ipsenol: an aggregation pheromone forIps latidens (Leconte) (Coleoptera: Scolytidae)

Ipsenol was identified from the frass of male, but not female,Ips latidens from British Columbia, feeding in phloem tissue of lodgepole pine,Pinus contorta var.latifolia. The responses ofJ. latidens to sources of ips-enol andcis-verbenol were determined with multiple-funnel traps in stands of lodgepole pine in British Columbia. Ipsenol attracted both male and femaleI. latidens, verifying that it is a pheromone for this species. MaleI. latidens showed a slight preference for (S)-(–)-ipsenol.cis-Verbenol was not produced by beetles of either sex and, in contrast to an earlier report, both enantiomers inhibited attraction to ipsenol-baited traps. The predators,Enoclerus sphegeus andThanasimus undatulus (Cleridae), were attracted to traps baited withcis-verbenol and ipsenol.     

3-methyl-3-buten-1-ol: An aggregation pheromone of the four-eyed spruce bark beetle,Polygraphus rufipennis (Kirby) (Coleoptera: Scolytidae)

Porapak Q-captured volatiles of malePolygraphus rufipennis (Kirby) boring in black spruce and white spruce from Newfoundland and British Columbia, respectively, contained 3-methyl-3-buten-1-ol. Volatiles from logs in which the males had been joined by females contained the compound in reduced quantities. Hindgut extracts from male and femaleP. rufipennis disclosed no male-specific volatiles, but 3-methyl-3-buten-1-ol was detected in extracts of male-produced frass. The compound was not present in extracts from fresh phloem tissue.P. rufipennis of both sexes responded strongly in the field to traps baited with 3-methyl-3-buten-1-ol released at 4390 g/day. There was little response to unbaited traps, fresh uninfested black spruce logs, or to 3-methyl-3-buten-1-ol released at lower rates. Combination of 3-methyl-3-buten-1-ol with either one of five terpenes prevalent in black spruce did not enhance beetle catch. Approximately half of 20 black spruce trees baited with 3-methyl-3-buten-1-ol were attacked, compared to 20.0% of 10 unbaited control trees. This new aggregation pheromone could be used to monitor or manageP. rufipennis populations.     

Methylbutynol effectively replaces methylbutenol,a pheromone component ofIps typographus (L.) (Coleoptera: Scolytidae)

In field experiments in Sweden, the constituent 2-methyl-3-buten-2-ol of the aggregation pheromone of the spruce bark bettleIps typographus (L.) was effectively replaced by 2-methyl-3-butyn-2-ol.     

Attraction of bark beetles (Coleoptera: Scolytidae) to a pheromone trap

The movement of bark beetles near an attractive pheromone source is described in terms of mathematical models of the diffusion type. To test the models, two release experiments involving 47,000 marked spruce bark beetles [Ips typographus (L.)] were performed. The attractive source was a pheromone trap, surrounded by eight concentric rings with eight passive trap stations on each ring. Captures were recorded every 2–10 minutes for the pheromone trap and once for the passive traps. The models were fitted to the distribution in time of the central pheromone trap catch and to the spatial distribution of catch among the passive traps. The first model that gives a reasonable fit consists of two phases: Phase one—After release the beetles move according to a diffusion process with drift towards the pheromone trap. The strength of the drift is inversely proportional to the distance from the traps. Phase two—those beetles attracted to, but not caught by, the pheromone trap are no longer influenced by the pheromone, and their movement is described by a diffusion process without drift. In phase two we work with a loss of beetles, whereas the experiment seems to indicate that the loss of beetles in phase one is negligible. As a second model, the following modification of phase one is considered: After release the beetles move according to a diffusion process without drift, until they start responding to the pheromone (with constant probability per unit time), whereafter they start moving according to a diffusion process with drift. This study, like other release experiments, shows that the efficiency of the pheromone trap is rather low. What is specific for the present investigation is that we try to explain this low efficiency in terms of dynamic models for insect movement. Two factors seem to contribute: Some beetles do not respond to pheromone at all, and some beetles disappear again after having been close to the pheromone trap. It also seems that the motility of the beetles decreased after they ceased responding to the pheromone. Furthermore, the data lend some support to the hypothesis that flight exercise increases the response of the beetles to pheromone.     

Influence of aggregation inhibitors (verbenone and ipsidenol) on landing and attack behavior ofDendroctonus brevicomis (Coleoptera: Scolytidae)

The influence of the aggregation inhibitors verbenone and ipsdienol on the response of western pine beetle,Dendroctonus brevicomis, to attractive host trees was investigated. Paired ponderosa pine trees (Pinus ponderosa) were baited with aggregation semiochemicals to stimulate mass attack. One tree in each pair received an inhibitor treatment consisting of five sets of two verbenone and two ipsdienol dispensers spaced 1 m apart vertically along the tree bole. Beetle landing was monitored with sticky traps on the tree bole, and attack density was assessed from bark samples removed four or seven days after baiting. The inhibitor treatment resulted in a significant reduction of both the numbers of beetles landing on trees and the density of attacking beetles compared to control trees (without inhibitors). The ratios of beetle landing density to attacking density were not different between inhibitor-treated and control trees, nor were the vertical distributions of beetles landing or attacking, suggesting that beetle behavior was primarily influenced at a longer range, prior to landing on the tree. Although the application of verbenone and ipsdienol did not preventD. brevicomis from attacking baited trees, our results suggest that when applied to unattacked (and unbaited) trees, their effectiveness at reducing the attack pressure might allow trees having a certain amount of resistance to survive attack by pioneer beetles.     

Synthesis and field testing of 4,6,6-lineatin,the aggregation pheromone ofTrypodendron lineatum (Coleoptera: Scolytidae)

Authentic 4,6,6-lineatin (3,3,7-trimethyl-2,9-dioxatricyclo-[3.3.1.0^4,7]nonane) (I) was produced in low yield via three synthetic pathways. In field tests, microgram amounts of the product from all three syntheses attracted large numbers ofTrypodendron lineatum of both sexes. These results confirm that 4,6,6-lineatin (I) is a population aggregation pheromone forT. lineatum.Research supported by National Science Foundation (U.S.A), Grant PCM4-13643, National Research Council (Canada), Co-op Grant A0243 and Operating Grants A3881 and A3785, and a Canadian Forestry Service Science Subvention Grant.     

Male-produced aggregation pheromone ofCarpophilus obsoletus (Coleoptera: Nitidulidae)

Males ofCarpophilus obsoletus Erichson produce an aggregation pheromone to which both sexes respond. The pheromone was identified by GC-MS as (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-undecatetraene (1), which is also a minor constituent of the pheromone blends ofC. hemipterus (L.),C. freemani Dobson, andC. lugubris Murray. The pheromone was synergized in wind-tunnel bioassays by propyl acetate, a host-type coattractant. In a dose-response study, 50 pg of1, plus propyl acetate, was significantly more attractive than just propyl acetate. Pheromone emission from groups of 65 males, feeding on artificial diet, averaged 2.2 ng/male/day. Emissions from individual males were larger, averaging 72 ng/day and ranging as high as 388 ng/day. Synthetic1 was tested in a date garden in southern California (500 µg/rubber septum), using fermenting whole-wheat bread dough as the coattractant. The pheromone plus dough attracted significantly more beetles than dough alone (means were 4.2 and 0.0 beetles per week per trap). Captured beetles were 54% females. Field trap catches were highest during the months of July and August.All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. Mention of firm names or trade products does not imply endorsement by the U.S. Department of Agriculture over similar firms or products not mentioned.     

Influence of pheromone chirality on response byOryzaephilus surinamensis andOryzaephilus mercator (Coleoptera: Cucujidae)

The response of the sawtoothed grain beetle,Oryzaephilus surinamensis (L.), and the merchant grain beetle,O. mercator (Fauvel), to synthetic racemic and chiral macrolide aggregation pheromones was assessed in pitfall olfactometers.O.mercator utilizes theR enantiomers of (Z)-3-dodecen-11-olide and (Z,Z)-3,6-dodecadien-11-olide.O. surinamensis utilizes theR enantiomers of (Z,Z)-3,6-dodecadien-11-olide and the Synergist (Z,Z)-5,8-tetradecadien-13-olide in combination with achiral (Z,Z)-3,6-dodecadienolide. For both species, the racemates of the respective chiral pheromones were effective attractants. The respectiveS enantiomers were inactive for both species and had no effect on the biological activity of the active antipodes. No diel periodicity in responsiveness to pheromones was detected inOryzaephilus spp. reared either on a 1212 light-dark photoperiod or in darkness. Nonpheromone macrolides, naturally released in trace amounts byOryzaephilus spp., did not affect the aggregation response of either species to its pheromones when these additional macrolides were combined with the pheromone mixtures.Research supported by the Natural Sciences and Engineering Research Council of Canada, Operating Grant A3881, A0851, and A3785, and Strategic Grant G0958.     

(E)- and (Z)-6-nonen-2-one: Biosynthetic precursors ofEndo- andexo-brevicomin in two bark beetles (Coleoptera: Scolytidae)

The ability of (E)- and (Z)-6-nonen-2-one to serve as precursors of the common scolytid pheromonesEndo- andexo-brevicomin was examined in vivo. When mountain pine beetles (MPB),Dendroctonus ponderosae Hopkins, or western balsam bark beetles (WBBB),Dryocoetes confusus Swaine, were exposed to [6,7-D₂](E)-6-nonen-2-one, theEndo-brevicomin produced was enriched with two deuterium atoms per molecule (as determined by GC-MS), indicating that (E)-6-nonen-2-one served as a precursor of this pheromone. Similarly, when the beetles were exposed to [4,4-D₂](Z)-6-nonen-2-one, theexo-brevicomin produced was enriched with two deuterium atoms per molecule. Evidence in support of biological relevance of the latter observation include: (1) (Z)-6-nonen-2-one was found in the volatiles of male MPBs and WBBBs, indicating that this is a natural metabolite; (2) theexo-brevicomin produced by MPB was shown to be of natural (+) chirality by complexation chromatography; and (3) female WBBBs and MPBs (which are not known to produceexo-brevicomin) produced significantly lessexo-brevicomin when exposed to the precursor than did the males.     

Inter- and intrapopulation variation of the pheromone,ipsdienol produced by male pine engravers,Ips pini (Say) (Coleoptera: Scolytidae)

We determined the chirality of ipsdienol in individual male pine engravers,Ips pini (Say), from New York, California, and two localities in British Columbia (BC). Both quantity and chirality of ipsdienol varied significantly between and within populations ofI. pini. Beetles from California and southeastern BC produced primarily (R)-(–)-ipsdienol with mean ratios of (S)-(+) : (R)-(–) of 9 : 91 and 11 : 89, respectively, while beetles from New York produced primarily (S)-(+)-ipsdienol with a mean (S)-(+) : (R)-(–) ratio of 57 : 43. A population from southwestern BC was unlike any other known western population, producing primarily (S)-(+)-ipsdienol with a mean (S)-(+) : (R)-(–) ratio of 66 : 34. In contrast to the unimodal chirality profiles for ipsdienol production in populations from California and southeastern BC, the profiles of the populations from southwestern BC and New York were bimodal, with a common mode at approximately 44 : 56 (S)-(+) : (R)-(–). Bimodality in the profiles of ipsdienol chirality in two populations ofI. pini and remarkably high levels of intrapopulation variation in pheromone chirality in all four populations suggest that evolutionary change in pheromone channels of communication could occur, possibly in response to artificial selection pressures such as mass trapping.     

Seasonal variability in response ofIps pini (Coleoptera: Scolytidae) to ipsdienol in New York

In May,Ips pini in New York did not respond in the field to 50–98.5% (R)-(–)-ipsdienol (synthetic). In September, beetles responded strongly to 50–60% (R)-(–)-ipsdienol (synthetic). In May and June, New York beetles showed marked preference for their own males over Arizona males, which produce an average of 94.1% (R)-(–)-ipsdienol. This suggested that ipsdienol stereochemistry alone does not ensure activity and that an additional compound is necessary for attraction in May. In the second year of field tests, attraction to synthetic ipsdienol and male beetles was tested in the spring, summer, and fall. There was response only to males in the spring and mid-summer and to both males and synthetic ipsdienol in the late summer and fall, causing a significant treatment x sampling period (date) interaction. Laboratory-reared beetles were not significantly more attracted to ipsdienol than to a blank airstream in laboratory assays, while male volatiles were significantly more attractive than ipsdienol and the blank. These data demonstrate that there is one or more unknown semiochemicals necessary for pheromonal response and that the behavioral activity of synthetic ipsdienol varies seasonally.Deceased.     

Green leaf volatiles as antiaggregants for the mountain pine beetle,Dendroctonus ponderosae Hopkins (Coleoptera: Scolytidae)

We tested the hypothesis that green leaf volatiles act as antiaggregants for the mountain pine beetle (MPB),Dendroctonus ponderosac Hopkins. In coupled gas chromatographic-electroantennographic detection (GC-EAD) analysis MPB antennae responded to 30 ng doses of all six-carbon green leaf alcohols tested [1-hexanol, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, (E)-3-hexen-1-ol, and (Z)-3-hexen-1-ol], but not to the aldehydes, hexanal or (E)-2-hexenal, or to alcohol or aldehyde homologues with more or fewer than six carbon atoms. In field trapping experiments a blend of green leaf alcohols [1-hexanol, (Z)-2-hexen-1-ol, (E)-3-hexen-1-ol and (Z)-3-hexen-1-ol] effectively disrupted the response to attractive semiochemicals; a blend of the aldehydes hexanal and (E)-2-hexenal was inactive. The two best disruptants. (E)-2-hexen-1-ol and (Z)-3-hexen-1-ol, reduced catches of both sexes to levels not significantly different from catches in unbaited control traps. They also reduced the attack on trees baited with attractive MBP pheromones to a level not significantly different from that on unbaited control trees. Neither of the clerid predators captured,Enoclerus sphegeus (F.) norThanasimus undatulus (Say), was repelled by green leaf volatiles. Our results suggest that green leaf alcohols are promising disruptants which may be used to supplement the antiaggregation pheromone, verbenone, in protecting single high-value trees as well as carefully selected stands with low-level populations of MPBs.     

Ester components of aggregation pheromone ofDrosophila virilis (Diptera: Drosophilidae)

The male-produced aggregation pheromone ofDrosophila virilis was found to contain five ester components, in addition to a previously identified hydrocarbon, (Z)-10-heneicosene (Z10–21). The five esters were: the methyl, ethyl, and 1-methylethyl (isopropyl) esters of 2-methyl-(E)-2-butenoic (tiglic) acid and the methyl and ethyl esters of hexanoic acid. The esters were not detected in females. Each ester was active by itself in laboratory bioassay tests, and each increased the number of flies responding toZ10–21 ca. 4–5 times. In comparisons among the five esters at 10 ng per compound, ethyl tiglate was the most active, and methyl tiglate, the least. No mixture of esters was found to be significantly more active than ethyl tiglate alone. In a doseresponse study, bioassay activity increased with dose for both ethyl tiglate andZ10–21. Newly emerged males did not have detectable levels of the esters. All five esters increased as sexual maturity was approached. Ethyl tiglate and ethyl hexanoate were the most abundant in mature males, usually over 15 ng per individual. Ratios among the esters were variable. Male flies also contained an as yet unidentified attractant(s) still more polar than the esters, which was synergistic with the esters and hydrocarbon. Food odors also synergized the synthetic compounds.     

Isolation and tentative identification of lineatin,a pheromone from the frass ofTrypodendron lineatum (Coleoptera: Scolytidae)

An attractant compound was isolated from frass produced byTrypodendron lineatum female beetles boring in Douglas fir. The proposed structure is one of two isomeric tricyclic acetals, to which the trivial name lineatin is assigned.Research supported by: National Science Foundation (U.S.A.) Grant No. PCM 74-13643, National Research Council (Canada) Operating Grant No. A3881, Canada Department of Fisheries and Forestry Extramural Research Grant No. F-58, Canadian Forestry Service Science Subvention Grant, and the Council of Forest Industries of British Columbia.     

Male-produced aggregation pheromone ofCarpophilus mutilatus (Coleoptera: Nitidulidae)

Males ofCarpophilus mutilatus Erichson produce an aggregation pheromone to which both sexes respond. The pheromone includes two hydrocarbon components, (3E,5E,7E)-5-ethyl-7-methyl-3,5,7-undecatriene (1) and (3E,5E,7E)-6-ethyl-4-methyl-3,5,7-decatriene (2). These were emitted in a 101 ratio and in a total amount of ca. 5 ng per feeding male per day. All tested doses of1 and2, from 0.03 to 30 ng, were more attractive than controls in wind-tunnel tests, but there was no evidence of synergism between these trienes. Dramatic synergism between the pheromone and a food-type coattractant occurred in the field, however. In a date garden in southern California, traps with a combination of synthetic1 and fermenting whole-wheat bread dough attracted 22 times more beetles than dough by itself and 295 times more than1 by itself. Volatile collections from males also contained three oxygenated compounds that were absent from females. One of these was tetradecanal (ca. 5 ng per male per day), but the structures of the other two are presently undetermined (0.8 and 1.1 ng per male per day). No function for these was demonstrated. One compound originating in the artificial diet, 2-phenylethanol, was particularly attractive in the wind-tunnel bioassay, as was the chromatographic solvent, methanol.