Bruce W. Zilkowski

Male-specific sesquiterpenes from Phyllotreta and Aphthona flea beetles

It was previously reported that males of the crucifer flea beetle, Phyllotreta cruciferae, feeding on host foliage are attractive to both males and females in the field. Based on this evidence for an aggregation pheromone, volatiles were collected from male and female P. cruciferae feeding on cabbage (Brassica oleracea) and analyzed. For comparison, volatiles were also collected from males and females of three other flea beetle species, Aphthona flava,A. czwalinae, and A. cyparissiae, all feeding on their host, leafy spurge foliage (Euphorbia esula). Six male-specific compounds were isolated from P. cruciferae, and the same compounds plus two additional ones were isolated from males of Aphthona flava,A. czwalinae, and A. cyparissiae. The blends of compounds were relatively consistent within species, but there were characteristic differences between species. Compound structures were studied by mass spectrometry, NMR spectroscopy, UV spectroscopy, polarimetry, chiral and achiral gas chromatography, molecular modeling, and microchemical tests. Three of the compounds were identified as (+)-ar-himachalene; (+)-trans-α-himachalene; (+)-γ-cadinene. Two others were new enantiomers of himachalene hydrocarbons that were previously identified from the fir trees, Abies alba and Abies nordmanniana. Finally, there were two himachalene alcohols and one norsesquiterpene ketone that is a himachalene analog. Only (+)-ar-himachalene and (+)-γ-cadinene are previously known natural products. Electrophysiological activity was demonstrated for five of the compounds. The chemical and electrophysiological patterns are consistent with, but do not prove, a pheromonal function.

IDENTIFICATION OF HOST-RELATED VOLATILES ATTRACTIVE TO PINEAPPLE BEETLE Carpophilus humeralis

Volatiles collected from oranges fed upon by Carpophilus humeralis of either sex were consistently more attractive than volatiles from beetle-free oranges in wind-tunnel bioassays. Three compounds were identified as attractants from this system: 4-ethyl-2-methoxyphenol (1), 2,5-diisopropylpyrazine (2) (a new natural product), and 2-phenylethanol (3). Identifications were confirmed with synthetic compounds that had matching chromatographic and spectral properties. Compounds 1, 2, and 3 had only slight activity alone, but were highly synergistic with each other and with propyl acetate (PA), a fruity ester that is mildly attractive to Carpophilus beetles. Compound 2 was the most active in the wind tunnel; its threshold dose was 0.5 ng when PA was present. The structural specificity for these compounds was high. Twelve phenol analogs of 1 were tested, but only one of these, 2-methoxyphenol, was more attractive than the control. Similarly, the analogs of 2, 2-isopropylpyrazine and 2,6-diisopropylpyrazine, were completely inactive. In the field, a combination of 1, 2, and 3 was not attractive by itself, but it strongly synergized attraction to fermentation volatiles, Carpophilus pheromones, or both. Compounds 1, 2, and 3 apparently have a microbial origin because all three were detected when the host fruit was pineapples instead of oranges, because they could occur in the absence of beetles, and because autoclaved pineapple began to produce the compounds after inoculation from an attractive piece of fruit. The study demonstrated that host location for this generalist species can be far more complex than responding simply to the bouquet of low-molecular-weight volatiles normally associated with fermentation.

Nonequilbrium quantitation of volatiles in air streams by solid-phase microextraction.

Solid-phase microextraction (SPME) is a valuable technique for analyzing air-borne organic compounds; one important application is measuring concentrations when these are constant over time. Quantitation normally relies on the SPME fiber being fully equilibrated with the sample medium. Unfortunately, relatively heavy compounds do not equilibrate within a reasonable amount of time, and this has limited the scope of SPME. The ability to quantitate during equilibration was needed and was the focus of this investigation. This entailed having an accurate description of SPME kinetics, and the kinetics of extraction by poly(dimethylsiloxane) fibers was studied for alkanes of 9-22 carbons, primary alcohols of 6-13 carbons, and methyl esters of 6-16-carbon acids. Sampling was from air streams in which analyte concentrations were effectively constant, and sampling times ranged from 30 min to 3 days. Other experimental variables included sampling temperature, fiber coating thickness, air flow rate, and tubing diameter in which the SPME sampling took place. Over 1900 data points were acquired. Previous theoretical kinetic models were not applicable to the present experimental conditions, but a simple kinetic equation was formulated that described the data very well; its key property is an explicit relationship between fiber sensitivity and equilibration time. Using nonlinear regression, the equation parameters were linked to known properties of the analyte (the functional group and GC retention index on a nonpolar column) and to certain sampling conditions (temperature, sampling duration, air flow rate, tubing diameter). The regression equation serves as a practical quantitation formula and allows the absolute concentration of the analyte in the air stream to be calculated directly from the amount extracted by the SPME fiber (which is easily measured by GC), regardless of whether equilibrium has been established or not, as long as the above analyte properties and sampling conditions are known. The residual variability for the model (RSD = 9.4%) was only slightly larger than the variability inherent in SPME alone (∼5%). Considerations for SPME sampling from air are discussed, and new fiber calibration information is presented for the larger hydrocarbons, alcohols, and methyl esters.

Pheromone Components of the Wheat Stem Sawfly: Identification, Electrophysiology, and Field Bioassay

Volatiles collections and cuticular extracts of the wheat stem sawfly, Cephus cinctus Norton (Hymenoptera: Cephidae), were analyzed by coupled gas chromatographic–electroantennographic detection (GC-EAD) in order to detect possible pheromone components of this species. Volatiles collections from male and female sawflies contained the same 13 GC-EAD-active compounds that stimulated both male and female antennae. GC-EAD-active compounds were identified by mass spectrometry, and the identities were verified by acquiring or synthesizing authentic standards with identical GC retention times and mass spectra. 9-Acetyloxynonanal gave the strongest EAD response. Additional GC-EAD-active compounds included 13-acetyloxytridecanal, aldehydes with 9–16 carbon chain lengths, acids with 8–10 carbon chain lengths, and phenylacetic acid. The last was instantly detectable by human nose when several males were allowed to interact, but not from isolated insects. On a per-insect basis, grouped males overall released higher amounts of the GC-EAD-active compounds than grouped females. Notable quantitative differences between the sexes were relatively higher amounts of 9-acetyloxynonanal, phenylacetic acid, and tetradecanal from males and hexadecanal from females. In the absence of an absolute, qualitative difference between sexes, these relative differences might be important for sex recognition. Field bioassay demonstrated the attractiveness of 9-acetyloxynonanal for male and female C. cinctus. The major compounds of male and female cuticular extracts showed no GC-EAD activity, but ozonolysis of extracts gave dramatically increased amounts of GC-EAD-active material. Hence, double-bond-containing cuticular compounds are suggested as precursors of the aldehydic pheromone components.

Pheromonal Activity of Compounds Identified from Male Phyllotreta cruciferae: Field Tests of Racemic Mixtures, Pure Enantiomers, and Combinations with Allyl Isothiocyanate

Four himachalene sesquiterpenes and (+)-γ-cadinene, previously identified as possible pheromone components from males of a North American population of Phyllotreta cruciferae Goeze (Coleoptera, Chrysomelidae), were tested for attractiveness in field trapping experiments in Hungary. A mixture of the four synthetic racemic himachalene derivatives and (+)-γ-cadinene from a botanical source was slightly attractive to beetles, but much more attractive when blended with the known host-plant-derived attractant allyl isothiocyanate. This result was consistent with a previous study in North America. In tests with optically pure synthetic compounds, a blend of the same himachalene enantiomers found from male beetles was equivalent to the corresponding blend of racemic compounds, whereas a blend of the opposite enantiomers was not active. Through subtraction tests, it was found that the single compound, (6R,7S)-2,2,6,10-tetramethylbicyclo[5.4.0.]undeca-9,11-diene [compound (+)-A in this study], was as active as the whole mixture, suggesting that this compound is the key pheromone component of the European population of P. cruciferae. During field trials, several congeneric species, including P. vittula, P. nemorum, P. nodicornis, and P. ochripes, also were caught, suggesting that the same compound(s) may be relatively widespread as pheromone components in this genus.

Dimethylfuran-lactone pheromone from males of Galerucella calmariensis and Galerucella pusilla.

Male Galerucella calmariensis and Galerucella pusilla (Coleoptera: Chrysomelidae) emit an aggregation pheromone while feeding on host foliage. Isolation of the compound from collected volatiles was guided by comparisons of gas chromatograms of extracts from males and females and by gas chromatography–electroantennographic detection. The compound was identified by a combination of spectrometric methods and microchemical tests as the novel dimethylfuran lactone, 12,13-dimethyl-5,14-dioxabicyclo[9.2.1]tetradeca-1(13),11-dien-4-one. The structure was confirmed by synthesis, and the synthetic compound attracted males and females of both species in field bioassays. These beetles were previously introduced into North America as biological control agents for the invasive wetland weed, purple loosestrife Lythrum salicaria, and the pheromone could become a tool for monitoring populations. A new method is described for distinguishing the two species based on the tibial spurs of the males.

Diapause in the leaf beetle Diorhabda elongata (Coleoptera: Chrysomelidae), a biological control agent for tamarisk (Tamarix spp.)

Abstract The tamarisk leaf beetle, Diorhabda elongata Brullé deserticola Chen, was collected in northwestern China and has been released in the western United States to control tamarisk (Tamarix spp.). Characteristics of diapause and reproductive development in D. elongata were examined to improve management as a biocontrol agent. Under long days, 16:8 (L:D) h, males began to emit aggregation pheromone within 2–3 d of adult emergence, mating occurred, and females oviposited within 7 d of adult emergence. Under short days, 12:12 (L:D) h, males did not emit pheromone, mating did not occur, and both males and females entered reproductive diapause marked by inconspicuous gonads and hypertrophied fat body. Ovaries of diapausing females lacked vitellogenic oocytes, and the ovarioles were clear and narrow, whereas reproductive females had enlarged ovaries with two to three yellow oocytes per ovariole. Diapausing males had thin, transparent accessory glands and ejaculatory ducts, whereas reproductive males had thick white accessory glands and white opaque ejaculatory ducts. Sensitivity to diapause-inducing photoperiods extended into the adult stage. Reproductive females ceased oviposition, resorbed oocytes, and entered diapause when switched from long to short days. Diapause-destined insects ceased feeding and entered the leaf litter 10–20 d after adult emergence, whereas reproductive insects remained on the plants and fed for at least 30 d. Reproductive insects exhibited dispersal behaviors, such as attempted flights, whereas diapause-destined insects did not show dispersal behaviors. Information gained from these studies will be used to better manage populations in the field and to improve rearing and storage in the laboratory.

Aggregation pheromone of the cereal leaf beetle: field evaluation and emission from males in the laboratory

The previously identified, male-specific compound of the cereal leaf beetle (CLB, Chrysomelidae; Oulema melanopus), (E)-8-hydroxy-6-methyl-6-octen-3-one, was studied further with respect to field activity and emission rate from male beetles. In a 5-week field experiment in Oregon, the compound was shown to function as an aggregation pheromone in attracting male and female CLBs migrating from overwintering sites in spring. Traps baited with the synthetic compound (500 μg per rubber septum) caught 3.3 times more CLBs than control traps. Lower doses of the pheromone (50 and 150 μg) were less attractive than the 500 μg dose. One relatively abundant, volatile compound from the host plant (oats), (Z)-3-hexenyl acetate, that elicited responses from beetle antennae was not attractive, either by itself or as a synergist of the pheromone. Both sexes were captured about equally for all treatments. We also measured daily pheromone emission by male beetles in the laboratory. Individual males feeding on oat seedlings under greenhouse conditions emitted as much as 6 μg per day, which is about 500 times higher than had been previously observed under incubator conditions. The pheromone emission rate was at least five times higher during the day than at night, and in one male, emission spanned a period of 28 d. The release rate of synthetic pheromone from the 500 μg septa was very similar to the maximum from single males; thus, future experiments should evaluate even higher doses. The field results indicate that the pheromone has potential as a monitoring tool for early detection of CLBs as they move from their overwintering sites into newly planted cereal crops in spring.

Chemistry and Defensive Efficacy of Secretion of Burrowing Bug (Sehirus cinctus cinctus)

Adult Sehirus cinctus cinctus emit a volatile secretion from their metathoracic scent glands when tactually stimulated. We identified the volatile components by gas chromatography, high-performance liquid chromatography, and mass spectrometry. The secretion of both sexes contained (1R)-(+)-α-pinene, (1S)-(–)-β-pinene, β-myrcene, (R)-(+)-limonene, and α-terpinolene. Two additional compounds were found in only female secretions: (E)-2-hexenyl acetate and (E)-2-octenal. We also tested the defensive capability of this insect by offering it to various predators. Anoles, starlings, and a killdeer rejected S. c. cinctus after an initial sampling. These findings suggest that the secretion plays a defensive role.

A Versatile and Quantitative Volatile-Delivery System for Laboratory Bioassays

A versatile moving-air system is described for delivering volatiles into a wind tunnel or other bioassay device. The system controls up to four volatile sources at one time. There is a calibrated, adjustable splitter for each source so that any percentage of a sources airstream, or none of it, can be directed to the system outlet at any moment. Thus, the system allows the sample volatiles to be bioassayed in any order and at any level or in mixtures of any desired proportions. Volatile sources of many types can be used, including single chemicals in slow-release formulations, mixtures of chemicals, or volatiles from living organisms. The volatile stream can be sampled by solid-phase microextraction (SPME) just before it enters the wind tunnel. Analysis of the SPME sample by gas chromatography allows absolute delivery rates of volatile components to be calculated. System performance was characterized with physical measurements and with bioassay experiments involving Carpophilus humeralis (F.) (Coleoptera: Nitidulidae). One bioassay experiment demonstrated how volatiles from a microbial culture (fermenting bread dough) and a synthetic counterpart (an aqueous solution of acetaldehyde, ethanol, 1-propanol, isobutanol, 3-methyl-1-butanol, 2-methyl-1-butanol, and ethyl acetate) could be compared at a range of dose levels, with just one sample of each type. These natural and synthetic volatile sources delivered very similar amounts of the above compounds and produced nearly identical dose–response curves. In another experiment, three bread dough volatiles (ethanol, acetaldehyde, and ethyl acetate) were tested in mixtures. Each component was used at four different levels (giving a total of 64 experimental treatments), but just one physical sample was needed for each chemical. The experiment provided clear information about response thresholds and interactions among these host volatiles. The volatile delivery system is versatile, easy to operate, and can be constructed from inexpensive materials.