Comparison of volatiles from beetle-transmittedCeratocystis fagacearum and four non-insect-dependent fungi

Ceratocystis fagacearum (Bretz) Hunt is the causative agent of oak wilt disease, which is transmitted primarily by nitidulid beetles. This fungus was compared with four non-insect-dependent fungi for their volatile profiles using gas chromatography and mass spectrometry (GC-MS) and for their attractiveness to nitidulids using a wind-tunnel bioassay. The four additional fungi includedXerula radicata Sing,Pluteus atricapillus Kumm,Tyromyces chioneus Karst, andBotrytis cinerea. Nitidulids have been reported in association with each of these fungi, but unlikeC. fagacearum, they are dispersed primarily by wind or rain. Significant attraction of three nitidulid species,Carpophilus hemipterus (Linne),C. lugubris Murray, andStelidota geminata (Say) was elicited byC. fagacearum and to a lesser extent byX. radicata, but not by the others. A comparison of headspace volatile profiles showed that the odor ofC. fagacearum was the strongest, both with regard to the number of components and in their rates of production. Chemical characterization of the headspace profile ofC. fagacearum revealed 16 components: one aldehyde, one ketone, five alcohols, and nine esters. These components were all common fruit-odor constituents and many of them were previously shown to be attractive to nitidulid beetles. The results of this study suggest that, by mimicking food odors,C. fagacearum odor is an adaptation for attracting nitidulid and possibly other insect vectors.     

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 source's 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.     

Development of Synthetic Food-Related Attractant for Carpophilus davidsoni and its Effectiveness in the Stone Fruit Orchards in Southern Australia

An effective synthetic attractant, based on host-related volatile compounds, was developed for the nitidulid beetle, Carpophilus davidsoni, as part of a program for managing this pest in Australian peach orchards. Fermenting peach juice and whole peaches and nectarines served as chemical models for the attractant, and synthetic formulations were sought that matched the natural sources, both with respect to emission rates of key chemicals (in ng/min) and attractiveness to flying beetles. All volatile sources were kept in airstreams during laboratory chemical measurements, and these airstreams were subsequently directed to the wind-tunnel behavioral assay, allowing the determined emission rates of compounds to be associated with particular levels of attractiveness. Chemical sampling of airstreams was by solid-phase microextraction, and analysis was by gas chromatography and mass spectrometry. A volatile delivery system was coupled to the wind tunnel and allowed facile manipulation of natural and synthetic scents. The final blend, modeled after fermenting peach juice, contained ethanol as the main constituent and 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, acetaldehyde, and ethyl acetate as minor constituents, all in water solution. Blend components were synergistic, and use of a mixture was essential for optimal attractiveness. The synthetic blend was highly effective in the field and is intended to replace fermenting peach juice and overripe peaches, which were previously used as synergists of the Carpophilus spp. aggregation pheromones in attract-and-kill stations.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.     

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.     

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.     

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.     

Enhanced maize (Zea mays L.) pericarp browning: Associations with insect resistance and involvement of oxidizing enzymes

The kernel pericarp of a maize (Zea mays L.) inbred, Mp313E, that browns rapidly at milk stage when damaged and that is resistant toAspergillus flavus Link and the dusky sap beetleCarpophilus lugubris Murray compared to a susceptible inbred, SC212M, was examined for differing oxidizing enzymes (peroxidases) and their substrate specificity. Additional pericarp enzymes were constitutively produced by the Mp313E inbred compared to the SC212M inbred, as indicated by gel electrophoresis and isoelectric focusing. These enzymes oxidized relevent pericarp substrates such as ferulic acid. Similar results were seen with two varieties of maize containing theCh mutant gene, which brown upon senescence in that enhanced oxidation of ferulic acid was seen in homogenates of browned pericarp compared to that which was cold-shocked and did not brown. Corn powder that was browned by mixing oxidizing enzymes with corn pericarp components ferulic acid and coumaric acid were typically less preferred/more toxic to caterpillars such asHelicoverpa zea (Boddie) and sap beetles such asC. lugubris. Thus, enhanced browning of maize pericarp can promote resistance to insects and is at least partly influenced by the presence of oxidizing enzymes. This mechanism may also promote resistance to maize pathogens, including those that produce mycotoxins.     

Attraction ofCarpophilus Spp. (Coleoptera: Nitidulidae) to volatile compounds present in figs

Various volatile compounds present in ripening figs were attractive toCarpophilus hemipterus, C. mutilatus, C.freemani, andC. lugubris. Field tests demonstrated that traps baited with a mixture of acetaldehyde, ethyl alcohol, and ethyl acetate caught more adultC. hemipterus than those baited with (1) other mixtures involving various combinations of 19 compounds, (2) with 16 single compounds, or (3) with fig paste.This investigation was supported in part by a grant from the California Dried Fig Advisory Board.     

Aggregation pheromone of driedfruit beetle,Carpophilus hemipterus Wind-tunnel bioassay and identification of two novel tetraene hydrocarbons

A male-produced aggregation pheromone was demonstrated inCarpophilus hemipterus (L.) (Coleoptera: Nitidulidae) using a wind-tunnel bioassay. Both sexes responded to the pheromone, but the beetles flew in the wind tunnel only after they had been starved for at least several hours. The attractiveness of the pheromone was greatly enhanced by volatiles from a food source, and combinations of pheromone and food volatiles typically attracted 3–10 times more beetles than either source by itself. A variety of food-related sources of volatiles were effective. These included apple juice; a mixture of baker's yeast plus banana; the pinto bean diet used for rearing this beetle; the chemicals propyl acetate, ethanol; and a mixture of acetaldehyde, ethyl acetate, and ethanol. The pheromonal activity resided with a series of 10 male-specific, unsaturated hydrocarbons of 13, 14, and 15 carbon atoms. These were partially separated by HPLC. No single compound was absolutely required for pheromonal activity to be observed, and various subsets of these compounds were active. The most abundant component was (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-decatetraene. One minor component was (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-undecatetraene. These structures were proven by synthesis. Together, the synthetic compounds were as active in the wind tunnel as the beetle-derived pheromone.     

Host-derived volatiles as attractants and pheromone synergists for driedfruit beetleCarpophilus hemipterus

The attractiveness of representative host materials, host extracts, and individual host volatiles (primarily carboxylic acids, alcohols, and esters) toCarpophilus hemipterus (L.) (Coleoptera: Nitidulidae) adults in wind-tunnel bioassays was examined. Attractiveness of the materials was examined alone and in combination with the aggregation pheromone. Host materials and extracts were often attractive on their own, and the attractancy was synergized when they were combined with the pheromone. Propanoic and butanoic acids, methanol, 2-propanol, 1-heptanol, methyl butanoate, and propanal were among the most effective attractants relative to the pheromone, but many other compounds significantly synergized the pheromone (typically three- to four fold). Attractiveness and synergism were influenced by the carbon chain length and branching of the substitutents. Straight-chain compounds that had at least three carbon atoms were generally effective as synergists. Many branched-chain compounds were also effective synergists. In general, the degree of attractiveness and synergism could be predicted fairly well with the physicochemical steric (Es) parameter, although the lipophilicity (Pi) parameter also appeared to be useful in explaining the lower activity of short-chain substituents. Thus, many compounds that had only limited attractiveness on their own may nevertheless play and important role in synergizing the pheromone. Structure-activity studies appear to be appropriate not only for determining optimal attractants for these insects, but also for determining effective synergists for the pheromone.The mention of firms or trade names does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.