Inter- and intra-specific variation in stem phloem phenolics of paper birch (Betula papyrifera) and European white birch (Betula pendula).

Outbreaks of bronze birch borer (BBB) (Agrilus anxius), a wood-boring beetle endemic to North America, have been associated with widespread mortality of birch (Betula spp.). There is substantial inter- and intra-specific variation in birch resistance to BBB. Species endemic to North America, such as paper birch (B. papyrifera), have coevolved with BBB and are more resistant than European and Asian birch species, such as European white birch (B. pendula), which lack an evolutionary history with BBB. Borer larvae feed on stem phloem tissue. Therefore, in search of potential resistance mechanisms against BBB, we compared the constitutive phenolic profile of stem phloem tissue of paper birch with that of European white birch. We also analyzed intraspecific variation in phenolic composition among clones and/or half-siblings of both species. Three phenolics (coumaroylquinic acid, betuloside pentoside A, and a diarylheptanoid hexoside) were detected only in paper birch, and concentrations of six other phenolics were significantly higher in paper birch. These differences may contribute to the high resistance of paper birch to BBB relative to European white birch. There was significant intraspecific variation in four of 17 phenolics found in paper birch and in five of 14 found in European white birch, but clones and half-siblings within each species could not be distinguished by phenolic composition using multivariate analysis.

High light intensity: a critical factor in the wind-tunnel flight of two scarabs, the rose chafer and Japanese beetle.

We analyzed the flight of the rose chafer Macrodactylus subspinosus (F.) (Melolonthinae: Scarabaeidae) and the Japanese beetle Popillia japonica Newman (Rutelinae: Scarabaeidae) in a wind tunnel with controlled humidity, temperature, light, and airflow. The data indicate that an optimum combination of light and temperature dramatically improves their response to lures. Both species took off upwind, oriented to, and contacted the odor source well (40-60%) when light intensity was > 50% of a clear day, temperature was 26-27 degrees C, and relative humidity was 65-75%.

Evaluation of individual components of plum odor as potential attractants for adult plum curculios.

We evaluated olfactory attraction of overwintered plum curculio (PC) adults, Conotrachelus nenuphar, to 16 individual volatile components of unripe plum odor in the laboratory using a still-air dual-choice bioassay system and in the field using baited cotton dental wicks attached to boll-weevil traps placed on the ground beneath the canopy of unsprayed apple trees. Two compounds, ethyl isovalerate and limonene, were significantly attractive in both laboratory bioassays and field experiments. In laboratory bioassays, as concentration was decreased across five orders of magnitude, a greater number of compounds elicited responses suggestive of attractancy (except at the lowest concentration). Even so, linalool, 2-hexanone, and 3-hydroxy-2-butanone were the only other compounds showing significant attractiveness in laboratory bioassays, but none of these (nor any other compounds) were significantly attractive in field assays. We suggest that the use of ethyl isovalerate and/or limonene as odor attractants offers potential to increase the efficacy of current traps for monitoring PCs immigrating into fruit orchards during spring.

Bioassay approaches to assessing behavioral responses of plum curculio adults (Coleoptera: Curculionidae) to host fruit odor.

We evaluated several approaches to developing a simple, sensitive, and reliable laboratory bioassay of responses of overwintered adult plum curculios (PCs),Conotrachelus nenuphar (Herbst), to host fruit odor or its attractive components. A high proportion of assayed PCs responded positively to odor of wild plums under no-choice, moving-air conditions in a wind tunnel and under dual-choice, still-air conditions in enclosed Petri dishes. Positive response to controls lacking host odor, however, was much greater in the wind tunnel, arguing in favor of bioassays under dual-choice conditions in still air to provide greater PC discrimination. Response to host odor (from wild plums or hexane extract of wild plums or Liberty apples) in Petri dish bioassay chambers proved greatest: (1) during the scotophase of PCs under total dark or dim red light conditions, (2) when Petri dishes were completely enclosed, (3) when PCs were starved for 24 or 48 hr, and (4) when PCs were tested within seven weeks after apple tree petal fall. Neither the sex of a PC nor the direction in which a PC was obliged to move to find the source of host odor (upward through a port in the Petri dish lid or downward through a port in the base) had a substantial effect on level of response to host odor or discrimination of host odor from a nonodorous control. We conclude that an enclosed Petri dish bioassay chamber of the type described here should be a valuable asset in the process of chemically identifying components of host fruit odor attractive to PCs.

Quantitative and temporal analysis of effects of twospotted spider mite (acari: Tetranychidae) female sex pheromone on male guarding behavior.

Responses of male twospotted spider mite,Tetranychus urticae (Koch), to female sex pheromone were described by a glass slide bioassay and computerized pathway digitizer. Pheromone was extracted from quiescent deutonymphs and fractionated by HPLC, and responses of guarding males to each fraction, all fractions combined, unfractionated extract, and a hexane control were bioassayed for 6 min. Mean angular velocities, linear velocities, percent time stationary, and distance from the 3-mm-diameter treatment circle were calculated for each mite at 20-sec intervals and these behavioral parameters regressed on time. Analysis of variance of regression-equation intercepts showed that no differences in initial male angular velocities were observed among treatments, but initial linear velocities were greater in response to all fractions combined and to extract than to individual fractions, and greater in response to individual fractions than to the control. Angular velocities decreased and linear velocities increased more rapidly in response to individual fractions than to all fractions combined and extract, while males turned preferentially in one direction and were stationary (no displacement) more often in response to all-fraction combinations than to individual fractions and the control. Significant differences were observed among the parameter values elicited by individual fractions, suggesting that active fractions differed qualitatively. The described changes in movement parameters show that maleT. urticae response to presence of pheromone occurs almost immediately, and that multiple pheromonal components are necessary to elicit maximum male response. The overall effect of pheromonal components was to decrease the rate at which males moved away from the treatment cycle, thereby causing male arrestment.

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.

Arrestment of male twospotted spider mite caused by female sex pheromone.

A glass slide bioassay was used to evaluate male twospotted spider mite,Tetranychus urticae Koch, arrestment caused by quiescent deutonymph extract. Males that were guarding quiescent deutonymphs prior to being tested were arrested by a 3-mm-diameter circle of quiescent deutonymph extract; nonguarding males and adult females were not arrested. Extracts of allT. urticae instars tested caused male arrestment, but mean arrestment duration was longest with quiescent deutonymph extract. Arrestment by volatile perception of pheromone and upwind orientation to point sources of extract were not observed. The mono- and sesquiterpene alcohols previously identified as components of the pheromone did not arrest males. HPLC separation of extract resulted in four active fractions; a subtractive bioassay showed that three were essential to elicit maximum male response.

Identification and bioassay of sex pheromone components of carob moth,Ectomyelois ceratoniae (Zeller).

Three sex pheromone components of the carob moth were isolated and identified from the extract of female pheromone glands, using a variety of techniques including coupled gas chromatographic-electroantennographic recordings, coupled gas chromatographic-mass spectrometric analysis, microozonolysis, electroantennographic assays of monounsaturated standards, wind-tunnel bioassays, and field trials. The major component was identified as (Z,E)-9,11,13-tetradecatrienal, a novel lepidopterous pheromone component structure. Two minor components, either one of which improves the upwind flight response of males when blended with the major component, were identified as (Z,E)-9,11-tetradecadienal, and (Z)-9-tetra-decenal.

Identification of food volatiles attractive toGlischrochilus quadrisignatus andGlischrochilus fasciatus (Coleoptera: Nitidulidae).

Seven volatile compounds identified from the headspace of whole wheat bread dough were investigated for their role in attractingGlischrochilus quadrisignatus andG. fasciatus in the field. Traps baited with either whole wheat bread dough or a synthetic seven-component bread dough odor caught similar numbers of these beetles, suggesting that the seven-compound combination could simulate the behavioral effect of bread dough. A series of trials using traps baited with various combinations of these chemicals showed that five compounds were significantly active in attractingG. quadrisignatus andG. fasciatus, but not all were essential for maximum response. The simplest blend eliciting a level of response comparable to the seven-component combination included ethyl acetate, acetaldehyde, ethanol, and racemic 2-methylbutanol, of which ethyl acetate, ethanol, and acetaldehyde were essential and 2-methylbutanol was replaceable with 2-methylpropanol forG. quadrisignatus attraction. Ethyl acetate and ethanol were essential for comparable attraction ofG. fasciatus. The chemical mediation of food finding in G.quadrisignatus andG. fasciatus is discussed in the context of volatile blends characterized for other nitidulid species.

Mediation of host selection by cuticular hydrocarbons in the honeybee tracheal MiteAcarapis woodi (Rennie).

Using a simple two-choice bioassay and video analysis of individual locomotory tracks, it was determined that a preference for young-bee hosts over old-bee hosts in female honeybee tracheal mites,Acarapis woodi (Rennie), is chemically mediated. When presented with a choice of cuticular extracts from 5-day-old and <1-day-old adult bees, mites showed a significant preference for the young-bee extract in three of four bee colony sources. This discrimination was due apparently to a greater positive response elicited by the young-bee extract rather than a deterrent effect of old-bee extract, as the extract of old bees evoked a significantly higher response than a hexane control. A bioassay of silica-gel fractions of young-bee extract indicated that cuticular hydrocarbons alone were responsible for tracheal mite response. Further fractionation of a hydrocarbon fraction by argentation chromatography demonstrated that both saturated and unsaturated hydrocarbons were involved in the mite response, but when presented in opposition, mites showed a stronger response to the saturated than to the unsaturated components. Mites placed in zones treated with cuticular extract of young bees exhibited higher angular velocities than those placed on hexane, causing them to remain in the extract-treated zones for extended periods. These results point to a possible control strategy whereby migration of tracheal mites to young-bee hosts could be disrupted by artificially altering the chemical profile of the honeybee cuticle.

Characterization of chemicals mediating ovipositional host-plant finding byAmyelois transitella females.

Ovipositional host-finding in the navel orangeworm,Amyelois transitella (Walker), is brought about by an in-flight response to host odors. Wind-tunnel studies of the response of gravid females to almonds showed that this response is mediated primarily by long-chain fatty acids, particularly oleic acid and linoleic acid. Evidence for the behavioral activity of fatty acids is based on the fact that: (1) behavioral activity of almond oil was concentrated in a single liquid chromatographic fraction whose composition was predominantly long-chain fatty acids, (2) behavioral activity was lost when either almond oil or the active fraction of that oil was treated with diazomethane, (3) full activity was elicited by a selective extraction of free fatty acids from crude almond oil, and (4) upwind response by females was elicited by a blend of synthetic oleic and linoleic acids, albeit at a level less than that elicited by almond oil. Five fatty acids identified from the almond oil were: myristic acid (1%), palmitic acid (16%), stearic acid (3%), oleic acid (58%), and linoleic (22%). Attraction to various combinations of synthetic acids was observed only when oleic acid was present, and oleic acid elicited upwind flights to the source when presented alone; however, short-range responses were enhanced by the addition of linoleic acid, which elicited no long-range orientation by itself. Despite significant levels of attraction to synthetic blends, the percentage of females flying to the source was lower than that flying to acidulated almond oil, the best natural attractant tested. Thus, although longrange response may be mediated primarily by a blend of oleic and linoleic acids, additional and as yet unidentified components must also play an important role. Long-range chemically modulated host finding in this and other generalist plant feeders is discussed with respect to current models of the evolution of host finding, and it is argued that suggestions that long-range host finding should be correlated with narrowness of host utilization are logically flawed and are not supported by our current understanding of specific examples of host finding.

Chemical characterization of fruit and fungal volatiles attractive to dried-fruit beetle,Carpophilus hemipterus (L.) (Coleoptera: Nitidulidae).

The chemical basis underlying orientation to fruit and fungal odors was investigated for the dried-fruit beetle,Carpophilus hemipterus (L.). In wind-tunnel bioassays of walking and flight response from 1.8 m, beetles were attracted to odors of the yeastSaccharomyces cerevisiae on agar, aseptic banana, or banana inoculated withS. cerevisiae, although both banana substrates elicited greater response than the yeast alone. When presented in a two-choice bioassay, the yeast-inoculated banana attracted approximately twice as many beetles as did the aseptic banana. GC-MS analysis of the headspace volatiles above these odor sources revealed a somewhat more complex and concentrated volatile profile for yeast-inoculated banana than for aseptic banana. The odor from yeast on agar had fewer components, and these were present at lower concentrations than the odors of either banana substrate. By blending mineral-oil or aqueous solutions of the 18 components of inoculated-banana odor in varying concentrations, it was possible to mimic closely the headspace profile of the natural odor. This synthetic odor also elicited beetle attraction in the wind tunnel at levels comparable to the inoculated banana. Through a series of bioassays in which individual components were subtracted from or added to a synthetic odor blend, it was determined that ethyl acetate, acetaldehyde, 2-pentanol, and 3-methylbutanol comprised the simplest blend of compounds evoking full behavioral response. However, 2-methylpropanol or butanol were apparently interchangeable with 3-methylbutanol in this blend, and comparable response could also be elicited by replacing acetaldehyde with a combination of both 2-pentanone and 3-hydroxy-2-butanone. Thus, our results suggest that this generalist insect herbivore locates its hosts by a long-range response to a variety of blends of common fruit volatiles, whose concentrations are enhanced by fungi.

Identification of food volatiles attractive to dusky sap beetle,Carpophilus lugubris (Coleoptera: Nitidulidae).

The chemical mediation of host-finding was investigated for the dusky sap beetle,Carpophilus lugubris Murray. GC-MS analysis of the headspace volatiles above whole-wheat bread dough inoculated with baker's yeast, a substrate previously determined to be an effective attractant, revealed seven major components in the following order of decreasing concentration: ethanol, acetaldehyde, 2-methylpropanol, 3-methylbutanol, propanol, 2-methylbutanol, and ethyl acetate. Solutions of these seven compounds blended so as to mimic the odor of whole-wheat bread dough elicited upwind orientation from 1.8 m in a wind tunnel byC. lugubris at a level comparable to that elicited by the bread dough. A series of bioassays investigating the role of individual components from the synthetic blend determined that all seven compounds contributed to behavioral activity; however, the simplest blend evoking attraction comparable to bread dough included acetaldehyde, ethyl acetate, 2-methylpropanol, and 3-methylbutanol. Of these compounds, acetaldehyde was essential, ethyl acetate was interchangeable with ethanol or partially replaceable with propanol, and 2-methylpropanol and 3-methylbutanol were partially replaceable with a combination of the other alcohols. Headspace volatiles above aseptic or fungus-inoculated tomato, banana, sweet corn, and strawberry were also qualitatively and quantitatively analyzed. This study suggests thatC. lugubris locates its food sources by response to variable blends of common volatile constituents of plants and fungi.

Interaction between visual and olfactory stimuli during host-finding by leafhopper,Dalbulus maidis (Homoptera: Cicadellidae).

Virtually nothing is known about the role plant volatiles play in host-finding by Homoptera in the Suborder Auchenorrhyncha. In laboratory bioassays, we examined the influence of plant volatiles on orientation and postcontact behaviors of the leafhopper,Dalbulus maidis, and determined the relationship between visual and olfactory stimuli during host-finding. When compared to the number of contacts made with reflected green light in the presence of a hexane control,D. maidis made more contacts when exposed to volatile extracts from its preferred host, maize; a similar number of contacts when exposed to volatiles from a marginal host, gamagrass; and fewer contacts when exposed to volatiles from a nonhost, sorghum. There was no difference between males and females in the number of contacts made with green light when exposed to maize volatiles compared to hexane alone. More contacts were made with green light than with white light of similar intensity, both in the presence and in the absence of olfactory stimuli; however, maize volatiles acted as a Synergist by increasing the number of contacts leafhoppers made with green light. After contacting the green light, exposure of maize volatiles significantly increased, relative to hexane, the amount of stationary time, but did not influence the amount of time spent moving, the distance traveled, or the speed while moving when within the boundaries of the green light. This study provides the first evidence for an interaction between visual and olfactory stimuli during host-finding for a leafhopper and also for olfactory mediation of postcontact behaviors not associated with feeding.

Regulation of pheromone biosynthesis by a brain hormone in two moth species.

Experiments were performed to characterize the action of a brain hormone on pheromone biosynthesis in female redbanded leafroller and cabbage looper moths. Results showed that the two species differed in their respective control mechanisms. In the cabbage looper, pheromone titer from decapitated females that received either saline or brain extract injections was not significantly different from control females, suggesting that pheromone biosynthesis was not dependent on the presence of the brain hormone. In contrast, with redbanded leafroller females, studies using radiolabeled acetate incorporation as well as incorporation of deuterium-labeled hexadecanoic acid showed that (i) the brain hormone was required for pheromone biosynthesis, (ii) the brain hormone regulated pheromone biosynthesis by activating synthesis of octadecanoyl and hexadecanoyl intermediates, and (iii) the brain hormone did not control other enzymes in the pathway. Regulation of fatty acid synthetase was unlikely since assays of the enzyme from decapitated and normal females showed no differences in the amount or distribution of the 18- and 16-carbon acyl end products. These results in conjunction with those from organ cultures of the pheromone gland suggest that the brain hormone acts by increasing the substrate supply for fatty acid synthesis.

Evolution of male pheromones in moths: reproductive isolation through sexual selection?

Central to our understanding of the species concept is knowledge of the nature and evolution of reproductive isolating mechanisms. The once widely accepted model of Dobzhansky, which holds that isolation evolves through selection against hybrids of differentially adapted populations, is now largely rejected. This rejection is due to both theoretical difficulties and a paucity of examples of the predicted pattern of reproductive character displacement. From a survey of five families of Lepidoptera, entailing more than 800 species, evidence is given that male courtship pheromones have evolved within the context of sexual isolation as an adaptive response to mating mistakes between differentially adapted populations; however, distinct from the natural selection model of Dobzhansky, this report suggests the mechanism for change to be sexual selection.

Chemical identification and behavioral characterization of male wing pheromone ofEphestia elutella (Pyralidae).

Behavioral and chemical evidence is presented for the identity of the male wing pheromone ofEphestia elutella (Hübner) and the role of this pheromone in courtship success is evaluated. Males with the forewing removed experienced a mating success rate less than half of that of either intact males or males that had only the wing gland area remaining of their forewings. GC-MS analysis and microchemical reactions indicated the presence of (E)-phytol and a series of saturated γ-lactones in a methylene chloride extract of the wings. Using an assay of female courtship behavioral response, (E)-phytol was found to evoke an intermediate level of response in females when presented alone, while the complete array of insect-derived γ-lactones produced no significant response. The combination of either γ-decalactone or γ-undecalactone with (E)-phytol in a 1∶2 ratio, however, elicited a female response equivalent to that produced by the unfractionated wing extract.