Surface Properties and Architectures of Male Moth Trichoid Sensilla Investigated Using Atomic Force Microscopy.

The surfaces of trichoid sensilla on male moth antennae have been sculpted over evolutionary time to capture pheromone odorant molecules emitted by the females of their species and transport the molecules in milliseconds into the binding protein milieu of the sensillum lumen. The capture of pheromone molecules likely has been optimized by the topographies and spacings of the numerous ridges and pores on these sensilla. A monolayer of free lipids in the outer epicuticle covers the sensillar surfaces and must also be involved in optimal pheromone odorant capture and transport. Using electro-conductive atomic force microscopy probes, we found that electrical surface potentials of the pores, ridges and flat planar areas between ridges varied in consistent ways, suggesting that there is a heterogeneity in the distribution of surface lipid mixtures amongst these structures that could help facilitate the capture and transport of pheromone molecules down through the pores. We also performed experiments using peak force atomic force microscopy in which we heated the sensilla to determine whether there is a temperature-related change of state of some of the surface lipid exudates such as the prominent domes covering many of the pores. We found that these exudates were unaffected by heating and did not melt or change shape significantly under high heat. Additionally, we measured and compared the topographies of the trichoid sensilla of five species of moths, including the distributions, spacings, heights and diameters of ridges, pores and pore exudates.

Pheromone Odorant Receptor Responses Reveal the Presence of a Cryptic, Redundant Sex Pheromone Component in the European Corn Borer, Ostrinia nubilalis.

Two odorant receptors (ORs), OnubOR3 and OnubOR6, in the sex pheromone communication systems of E- and Z-strain European corn borers, Ostrinia nubilalis, were broadly receptive to analogs of their pheromone components. In addition to responding to their natural 14-carbon pheromone components, (Z)-11- and (E)-11-tetradecenyl acetates (Z11- and E11-14:OAc), these pheromone ORs responded to the longer-chain compounds, (Z)-11- and (E)-11-hexadecenyl acetate (Z11- and E11-16:OAc). Z11-16:OAc is a pheromone gland constituent of E-strain O. nubilalis females in Europe but has not previously been shown to have behavioral activity to males. Here, we demonstrate that Z11-16:OAc evokes high levels of upwind flight and source location in E-strain males when substituted for Z11-14:OAc (minor component) in the E-strain blend. Since Z11-16:OAc is found in the gland and has behavioral activity when Z11-14:OAc is missing, then it should be classified as a cryptic, redundant minor pheromone component in E-strain O. nubilalis. The opposite geometric isomer, E11-16:OAc, also functions in Z-strain O. nubilalis, substituting behaviorally for the E11-14:OAc minor component, but has not been found in Z-strain female glands. Single-sensillum recordings showed that sensory neurons of E- and Z-strain male antennae expressing OnubOR3 and OnubOR6 produced responses to these hexadecenyl acetates similar to those evoked by the natural (tetradecenyl acetate) pheromone components. We postulate that the wide responsiveness of these two ORs to the 16-carbon acetates could be a preadaptation for O. nubilalis to use these compounds as minor components in lieu of the respective 14-carbon acetates. Alternatively, the responsiveness of OnubOR3 to E11-16:OAc and OnubOR6 to Z11-16:OAc could represent a vestigial state of these receptors, with the 16-carbon acetates having previously acted as functional minor components in an ancestral blend.

Proximate Mechanisms of Host Plant Location by a Specialist Phytophagous Insect, the Grape Berry Moth, Paralobesia Viteana.

There are contrasting hypotheses regarding the role of plant volatiles in host plant location. We used the grape berry moth (GBM; Paralobesia viteana)-grape plant (Vitis spp.) complex as a model for studying the proximate mechanisms of long distance olfactory-mediated, host-plant location and selection by a specialist phytophagous insect. We used flight tunnel assays to observe GBM female in-flight responses to host (V. riparia) and non-host (apple, Malus domestica; and gray dogwood, Cornus racimosa,) odor sources in the form of plant shoots, extracts of shoots, and synthetic blends. Gas chromatography-electroantennographic detection and gas chromatography/mass spectrometry analyses were used to identify antennal-active volatile compounds. All antennal-active compounds found in grape shoots were also present in dogwood and apple shoots. Female GBM flew upwind to host and non-host extracts and synthetic blends at similar levels, suggesting discrimination is not occurring at long distance from the plant. Further, females did not land on sources releasing plant extracts and synthetic blends, suggesting not all landing cues were present. Additionally, mated and unmated moths displayed similar levels of upwind flight responses to all odor sources, supporting the idea that plant volatiles are not functioning solely as ovipositional cues. The results of this study support a hypothesis that GBM females are using volatile blends to locate a favorable habitat rather than a specific host plant, and that discrimination is occurring within the habitat, or even post-landing.

Response to a Synthetic Pheromone Source by OX4319L, a Self-Limiting Diamondback Moth (Lepidoptera: Plutellidae) Strain, and Field Dispersal Characteristics of its Progenitor Strain.

The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), is a global pest that infests vegetable and field crops within the Brassica family. A genetically engineered strain of P. xylostella, OX4319L, carrying a 'self-limiting' gene, has shown potential for managing P. xylostella populations, using sustained releases of OX4319L male moths. In order for such a strain to provide control, the transgenic individuals must exhibit attraction to female P. xylostella sex pheromone and adequate dispersal in the field. In this study, we tested these key traits. First, we compared the responses of the OX4319L male moths to a synthetic female sex pheromone source in wind tunnel trials to those of males from three other strains. We found that OX4319L males responded comparably to strains of non-engineered males, with all males flying upwind towards the pheromone source. Second, we used mark-release-recapture studies of a wildtype P. xylostella strain, from which the OX4319L strain was originally developed, to assess dispersal under field conditions. Released males were recaptured using both pheromone-baited and passive traps within a 2.83 ha circular cabbage field, with a recapture rate of 7.93%. Males were recaptured up to the boundary of the field at 95 m from the central release point. The median dispersal of males was 14 m. These results showed the progenitor strain of OX4319L retained its ability to disperse within a host field. The results of these experiments are discussed in relation to the potential for the effective use of engineered male-selecting P. xylostella strains under field conditions.

Identification of a New Blend of Host Fruit Volatiles from Red Downy Hawthorn, Crataegus mollis, Attractive to Rhagoletis pomonella Flies from the Northeastern United States.

A new blend of volatiles was identified for the fruit of downy red hawthorn, Crataegus mollis, that is attractive to Rhagoletis pomonella flies infesting this host in the northeastern USA. The new blend was as attractive as the previously identified mixture but is more complex in the number of odorants (six in the old versus ten in the new) and differs significantly in the ratio of three volatiles, 3-methylbutan-1-ol, butyl hexanoate, and dihydro-ß-ionone, that are common to both blends and exerted agonist or antagonist effects on behavior in a flight tunnel assay. However, behavioral results with the old and new northern hawthorn blends, as well as modified blends with substituted ratios of 3-methylbutan-1-ol, butyl hexanoate, dihydro-ß-ionone, indicated that the 'agonist' or 'antagonist' effects of these volatiles depended on the ratio, or balance of compounds within the blend. In addition, the new blend contains a number of esters identified from the headspace of domesticated apple, Malus domestica, that are attractive to apple-origin R. pomonella, and present in the five other blends from southern hawthorns, including the southern C. mollis var. texana blend, but are not part of the previously identified blend from northern C. mollis fruit. This finding supports the hypothesis that in addition to providing specificity to the odor blends of the northern and southern hawthorn populations, the presence of the significant amounts of ester compounds in the new northern hawthorn blend might have provided a source of standing variation that could help explain the shift in host preference by C. mollis-infesting flies to introduced apple in the mid-1800's.

A Multiple-Choice Bioassay Approach for Rapid Screening of Key Attractant Volatiles.

Fermentation volatiles attract a wide variety of insects and are used for integrated pest management. However, identification of the key behavior modifying chemicals has often been challenging due to the time consuming nature of thorough behavioral tests and unexpected discrepancies between laboratory and field results. Thus we report on a multiple-choice bioassay approach that may expedite the process of identifying field-worthy attractants in the laboratory. We revisited the four-component key chemical blend (acetic acid, ethanol, acetoin, and methionol) identified from 12 antennally active wine and vinegar chemicals for Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). The identification of this blend took 2 yr of continuous laboratory two-choice assays and then similarly designed field trials. This delay was mainly due to a discrepancy between laboratory and field results that laboratory two-choice assay failed to identify methionol as an attractant component. Using a multiple-choice approach, we compared the co-attractiveness of the 12 potential attractants to an acetic acid plus ethanol mixture, known as the basal attractant for D. suzukii, and found similar results as the previous field trials. Only two compounds, acetoin and, importantly, methionol, increased attraction to a mixture of acetic acid and ethanol, suggesting the identification of the four-component blend could have been expedited. Interestingly, the co-attractiveness of some of the 12 individual compounds, including a key attractant, methionol, appears to change when they were tested under different background odor environments, suggesting that background odor can influence detection of potential attractants. Our findings provide a potentially useful approach to efficiently identify behaviorally bioactive fermentation chemicals.

Robust Manipulations of Pest Insect Behavior Using Repellents and Practical Application for Integrated Pest Management.

In agricultural settings, examples of effective control strategies using repellent chemicals in integrated pest management (IPM) are relatively scarce compared to those using attractants. This may be partly due to a poor understanding of how repellents affect insect behavior once they are deployed. Here we attempt to identify potential hallmarks of repellent stimuli that are robust enough for practical use in the field. We explore the literature for success stories using repellents in IPM and we investigate the mechanisms of repellency for two chemical oviposition deterrents for controlling Drosophila suzukii Matsumura, a serious pest of small fruit crops. Drosophila suzukii causes injury by laying her eggs in ripening fruit and resulting larvae make fruit unmarketable. In caged choice tests, reduced oviposition was observed in red raspberry fruit treated with volatile 1-octen-3-ol and geosmin at two initial concentrations (10% and 1%) compared to untreated controls. We used video monitoring to observe fly behavior in these caged choice tests and investigate the mode of action for deterrence through the entire behavioral repertoire leading to oviposition. We observed fewer visitors and more time elapsed before flies first landed on 1-octen-3-ol-treated fruits than control fruits and concluded that this odor primarily inhibits behaviors that occur before D. suzukii comes in contact with a potential oviposition substrate (precontact). We observed some qualitative differences in precontact behavior of flies around geosmin-treated fruits; however, we concluded that this odor primarily inhibits behaviors that occur after D. suzukii comes in contact with treated fruits (postcontact). Field trials found reduced oviposition in red raspberry treated with 1-octen-3-ol and a combination of 1-octen-3-ol and geosmin, but no effect of geosmin alone. Recommendations for further study of repellents for practical use in the field are discussed.

Comparative Responses of Rhagoletis zephyria and Rhagoletis pomonella (Diptera: Tephritidae) to Commercial and Experimental Sticky Traps and Odors in Washington State.

Rhagoletis zephyria Snow and Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) are morphologically similar flies that attack white-colored snowberry fruit (Symphoricarpos spp.) and yellow/red or dark-colored apple/hawthorn fruit (Malus/Crataegus spp.), respectively. The two flies are caught together on traps in R. pomonella surveys in the western United States, increasing labor needed to process catches. Comparing responses of the two species with different traps could help identify best practices for reducing R. zephyria captures in these surveys and could contribute to understanding population divergence in Rhagoletis flies. In Washington State, United States, we found that R. zephyria responded most to yellow rectangles and more to white than red spheres (RSs) baited with ammonium carbonate (AC), whereas R. pomonella responded most to RSs with AC. Yellow plastic rectangles with AC were more effective in capturing R. zephyria than cardboard rectangles, as has been found for R. pomonella. R. zephyria did not respond to apple fruit volatiles associated with RSs that were attractive to R. pomonella. In contrast, R. zephyria responded more to yellow rectangles with snowberry than apple volatiles. Both species responded to AC. Our results suggest that RSs are better than yellow rectangles for surveying R. pomonella when snowberries are abundant. However, if discrimination from R. zephyria is paramount, RSs with apple volatiles should be used. Differences in the species' responses to traps appear related to odor/color cues of the flies' host fruit, while commonalties appear related to visual/olfactory stimuli associated with protein feeding, for which AC is a general attractant.

Identification of Host Fruit Volatiles from Snowberry (Symphoricarpos albus), Attractive to Rhagoletis zephyria Flies from the Western United States.

A mixture of behaviorally active volatiles was identified from the fruit of snowberry, Symphoricarpos albus laevigatus, for Rhagoletis zephyria flies reared from snowberry fruit. A nine-component blend containing 3-methylbutan-1-ol (3%), dimethyl trisulfide (1%), 1-octen-3-ol (40%), myrcene (8%), nonanal (9%), linalool (13%), (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT, 6%), decanal (15%), and ß-caryophyllene (5%) was identified that gave consistent electroantennogram activity and was behaviorally active in flight tunnel tests. In other flight tunnel assays, snowberry flies from two sites in Washington state, USA, displayed significantly greater levels of upwind oriented flight to sources with the snowberry volatile blend compared with previously identified volatile blends from domestic apple (Malus domestica) and downy hawthorn (Crataegus mollis) fruit from the eastern USA, and domestic apple, black hawthorn (C. douglasii) and ornamental hawthorn (C. monogyna) from Washington state. Selected subtraction assays showed that whereas removal of DMNT or 1-octen-3-ol significantly reduced the level of upwind flight, removal of myrcene and ß-caryophyllene, or dimethyl trisulfide alone did not significantly affect the proportion of upwind flights. Our findings add to previous studies showing that populations of Rhagoletis flies infesting different host fruit are attracted to unique mixtures of volatile compounds specific to their respective host plants. Taken together, the results support the hypothesis that differences among flies in their behavioral responses to host fruit odors represent key adaptations involved in sympatric host plant shifts, contributing to host specific mating and generating prezygotic reproductive isolation among members of the R. pomonella sibling species complex.

Transplant Antennae and Host Brain Interact to Shape Odor Perceptual Space in Male Moths.

Behavioral responses to odors rely first upon their accurate detection by peripheral sensory organs followed by subsequent processing within the brain's olfactory system and higher centers. These processes allow the animal to form a unified impression of the odor environment and recognize combinations of odorants as single entities. To investigate how interactions between peripheral and central olfactory pathways shape odor perception, we transplanted antennal imaginal discs between larval males of two species of moth Heliothis virescens and Heliothis subflexa that utilize distinct pheromone blends. During metamorphic development olfactory receptor neurons originating from transplanted discs formed connections with host brain neurons within olfactory glomeruli of the adult antennal lobe. The normal antennal receptor repertoire exhibited by males of each species reflects the differences in the pheromone blends that these species employ. Behavioral assays of adult transplant males revealed high response levels to two odor blends that were dissimilar from those that attract normal males of either species. Neurophysiological analyses of peripheral receptor neurons and central olfactory neurons revealed that these behavioral responses were a result of: 1. the specificity of H. virescens donor olfactory receptor neurons for odorants unique to the donor pheromone blend and, 2. central odor recognition by the H. subflexa host brain, which typically requires peripheral receptor input across 3 distinct odor channels in order to elicit behavioral responses.

Flight Tunnel Response of Male European Corn Borer Moths to Cross-Specific Mixtures of European and Asian Corn Borer Sex Pheromones: Evidence Supporting a Critical Stage in Evolution of a New Communication System.

Previous flight tunnel studies showed that 3-5 % of male European corn borer (ECB) moths, Ostrinia nubilalis, could fly upwind and make contact with sources releasing the sex pheromone of the closely related Asian corn borer (ACB), Ostrina furnacalis, [2:1 (Z)-12-tetradecenyl acetate (Z12-14:OAc) : (E)-12-teradecenyl acetate (E12-14:OAc)] and that 2-4 % of ACB males could similarly fly upwind to the sex pheromone blends of the ECB Z- [97:3 (Z)-tetradecenyl acetate (Z11-14:OAc) : (E)-tetradecenyl acetate (E11-14:Ac)] and E-strains (1:99 Z/E11-14:OAc) pheromones. The results supported the hypothesis that the evolution of the ACB pheromone system from an ECB-like ancestor included a stage in which males could be attracted to the unusual females emitting Z12- and E12-14:OAc while retaining their responsiveness to the ancestral pheromone blend of Z11- and E11-14:OAc. Here, we showed further that ECB E-strain males exhibited upwind oriented flight and source contacts to sources containing all combinations of ECB and ACB components. Maximal response levels were observed with the E-strain 99:1 E11/Z11-14:OAc blend, and high response levels also were observed with two other blends containing E11-14:OAc as the major component (E11:E12 and E11:Z12). Upwind flight and source contact also occurred at lower levels with the remaining blend combinations in which Z11-, E12-, or Z12-14:OAc was the major component. Our current results support the hypothesis concerning the evolution of ACB from an ECB-like ancester by showing that males were able to respond to females producing either the 12-14:Ac isomers, 11-14:Ac isomers, or even mixtures of all four components.

Ammonium carbonate is more attractive than apple and hawthorn fruit volatile lures to Rhagoletis pomonella (Diptera: Tephritidae) in Washington State.

The apple maggot fly, Rhagoletis pomonella (Walsh), is an introduced, quarantine pest of apple (Malus domestica Borkhausen) in the Pacific Northwest of the United States. In the eastern United States where the fly is native, fruit volatiles have been reported to be more attractive than ammonia compounds to R. pomonella. However, the opposite may be true in the western United States. Here, we determined whether newly identified western apple and western hawthorn fruit volatiles are more attractive than ammonium carbonate (AC) to R. pomonella in apple, black hawthorn, and ornamental hawthorn trees in western Washington State. In all three host trees, sticky red sphere or yellow panel traps baited with AC generally caught more flies than traps baited with lures containing the four newly developed fruit blends (modified eastern apple, western apple, western ornamental hawthorn, and western black hawthorn) or two older blends (eastern apple and eastern downy hawthorn). Fruit volatiles also displayed more variation among trapping studies conducted at different sites, in different host trees, and across years than AC. The results imply that traps baited with AC represent the best approach to monitoring R. pomonella in Washington State.

Chemical and behavioral analysis of the cuticular hydrocarbons from Asian citrus psyllid, Diaphorina citri.

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the vector of the phloem-inhabiting bacterium, Candidatus Liberibacter asiaticus, which is presumed to cause HLB in Florida citrus. Laboratory and field studies were conducted to examine the behavioral responses of male and female D. citri to their cuticular extracts. In olfactometer assays, more male D. citri were attracted to one, five, or 10 female cuticular extract equivalent units than blank controls. The results were confirmed in field studies in which clear or yellow traps baited with 10 female cuticular extract equivalent units attracted proportionately more males than clear traps baited with male cuticular extract or unbaited traps. Analyses of cuticular constituents of male and female D. citri revealed differences between the sexes in chemical composition of their cuticular extracts. Laboratory bioassays with synthetic chemicals identified from cuticular extracts indicated that dodecanoic acid attracted more males than clean air. Traps baited with dodecanoic acid did not increase total catch of D. citri as compared with blank traps at the dosages tested; however, the sex ratio of psyllid catch was male biased on traps baited with the highest lure loading dosage tested (10.0 mg).

Influence of trap design on upwind flight behavior and capture of female grape berry moth (Lepidoptera: Tortricidae) with a kairomone lure.

Oil-coated clear panel traps baited with a host plant-based kairomone lure have successfully been used for monitoring female grape berry moth, Paralobesia viteana (Clemens) (Lepidoptera: Tortricidae), but low capture rates as well as difficulty in servicing these traps makes them unsuitable for commercial use. We compared the performance of different trap designs in a flight tunnel and in a vineyard by using a 7-component synthetic kairomone blend, with a focus on trap visual cues. In flight tunnel experiments, a clear delta trap performed better than other traps. When we tested clear delta, green delta, or clear wing traps baited with a cut grape shoot, >50% of female grape berry moths made complete upwind flights. However, the clear delta trap was the only design that resulted in female moths entering the trap. Similar results were observed when females were tested with different traps (clear delta, green delta, white delta, clear wing, or green wing traps) baited with the kairomone lure. Adding a visual pattern that mimicked grape shoots to the outside surface of the clear delta trap resulted in 66% of the females that made upwind flights entering the trap. However, the positive effect of adding a visual pattern to the trap was not observed in a vineyard setting, where clear delta traps with or without a visual pattern caught similar numbers of females. Still, the number of male and female grape berry moths captured in clear delta traps with or without a visual pattern was not significantly different from the number of male and female grape berry moths captured in panel traps, suggesting that the use of these delta traps could be a less cumbersome alternative to oil-coated panel traps for monitoring female grape berry moth.

The response to selection for broad male response to female sex pheromone and its implications for divergence in close-range mating behavior in the European corn borer moth, Ostrinia nubilalis.

Coordinated sexual communication systems, seen in many species of moths, are hypothesized to be under strong stabilizing natural selection. Stabilized communication systems should be resistant to change, but there are examples of species/populations that show great diversification. A possible solution is that it is directional sexual selection on variation in male response that drives evolution. We tested a component of this model by asking whether 'rare' males (ca. 5 % of all males in a population) of the European corn borer moth (ECB), Ostrinia nubilalis, that respond to the sex pheromones of both ECB and a different Ostrinia species (O. furnacalis, the Asian corn borer, ACB), might play an important role in diversification. We specifically tested, via artificial selection, whether this broad male response has an evolvable genetic component. We increased the frequency of broad male response from 5 to 70 % in 19 generations, showing that broad-responding males could be important for the evolution of novel communication systems in ECB. We did not find a broader range of mating acceptance of broad males by females of the base population, however, suggesting that broad response would be unlikely to increase in frequency without the involvement of other factors. However, we found that ECB selection-line females accepted a broader range of courting males, including those of ACB, than did females of the base population. Thus, a genetic correlation exists between broad, long-range response to female sex pheromone and the breadth of female acceptance of males at close range. These results are discussed in the context of evolution of novel communication systems in Ostrinia.

Behavioral evidence for fruit odor discrimination and sympatric host races of Rhagoletis pomonella flies in the Western United States.

The recent shift of Rhagoletis pomonella (Diptera: Tephritidae) from its native host downy hawthorn, Crataegus mollis, to introduced domesticated apple, Malus domestica, in the eastern United States is a model for sympatric host race formation. However, the fly is also present in the western United States, where it may have been introduced via infested apples within the last 60 years. In addition to apple, R. pomonella also infests two hawthorns in the West, one the native black hawthorn, C. douglasii, and the other the introduced English ornamental hawthorn, C. monogyna. Here, we test for behavioral evidence of host races in the western United States. through flight tunnel assays of western R. pomonella flies to host fruit volatile blends. We report that western apple, black hawthorn, and ornamental hawthorn flies showed significantly increased levels of upwind-directed flight to their respective natal compared to nonnatal fruit volatile blends, consistent with host race status. We discuss the implications of the behavioral results for the origin(s) of western R. pomonella, including the possibility that western apple flies were not introduced, but may represent a recent shift from local hawthorn fly populations.

On the scent of standing variation for speciation: behavioral evidence for native sympatric host races of Rhagoletis pomonella (Diptera: Tephritidae) in the southern United States.

Standing variation can be critical for speciation. Here, we investigate the origins of fruit odor discrimination for Rhagoletis pomonella underlying the fly's sympatric shift in the northeastern United States from downy hawthorn (Crataegus mollis) to apple (Malus domestica). Because R. pomonella mate on host fruit, preferences for natal fruit volatiles generate prezygotic isolation. Apples emit volatiles that appear to be missing from gas chromatography/electroantennographic detection profiles for flies infesting downy hawthorns, raising the question of how R. pomonella evolved a preference for apple. In the southern United States, R. pomonella attacks several native hawthorns. Behaviorally active volatile blends for R. pomonella infesting southern hawthorns contain the missing apple volatiles, potentially explaining why downy hawthorn flies could have evolved to be sensitive to a blend of apple volatiles. Flight tunnel assays imply that southern hawthorn populations were not the antecedent of a preassembled apple race, as southern flies were not attracted to the apple volatile blend. Instead, behavioral evidence was found for southern host races on native hawthorns, complementing the story of the historical sympatric shift to introduced apple in the North and illustrating how R. pomonella may evolve novel combinations of agonist and antagonist responses to volatiles to use new fruit resources.

Single mutation to a sex pheromone receptor provides adaptive specificity between closely related moth species.

Sex pheromone communication, acting as a prezygotic barrier to mating, is believed to have contributed to the speciation of moths and butterflies in the order Lepidoptera. Five decades after the discovery of the first moth sex pheromone, little is known about the molecular mechanisms that underlie the evolution of pheromone communication between closely related species. Although Asian and European corn borers (ACB and ECB) can be interbred in the laboratory, they are behaviorally isolated from mating naturally by their responses to subtly different sex pheromone isomers, (E)-12- and (Z)-12-tetradecenyl acetate and (E)-11- and (Z)-11-tetradecenyl acetate (ACB: E12, Z12; ECB; E11, Z11). Male moth olfactory systems respond specifically to the pheromone blend produced by their conspecific females. In vitro, ECB(Z) odorant receptor 3 (OR3), a sex pheromone receptor expressed in male antennae, responds strongly to E11 but also generally to the Z11, E12, and Z12 pheromones. In contrast, we show that ACB OR3, a gene that has been subjected to positive selection (ω = 2.9), responds preferentially to the ACB E12 and Z12 pheromones. In Ostrinia species the amino acid residue corresponding to position 148 in transmembrane domain 3 of OR3 is alanine (A), except for ACB OR3 that has a threonine (T) in this position. Mutation of this residue from A to T alters the pheromone recognition pattern by selectively reducing the E11 response ∼14-fold. These results suggest that discrete mutations that narrow the specificity of more broadly responsive sex pheromone receptors may provide a mechanism that contributes to speciation.

Identification of host fruit volatiles from domestic apple (Malus domestica), native black hawthorn (Crataegus douglasii) and introduced ornamental hawthorn (C. monogyna) attractive to Rhagoletis pomonella flies from the western United States.

The apple maggot fly, Rhagoletis pomonella, infests apple (Malus domestica) and hawthorn species (most notably the downy hawthorn, Crataegus mollis) in the eastern USA. Evidence suggests that the fly was introduced into the western USA sometime in the last 60 years. In addition to apple, R. pomonella also infests two species of hawthorns in the western USA as major hosts: the native black hawthorn (C. douglasii) and the introduced ornamental English hawthorn, C. monogyna. Apple and downy hawthorn-origin flies in the eastern USA use volatile blends emitted from the surface of their respective ripening fruit to find and discriminate among host trees. To test whether the same is true for western flies, we used coupled gas chromatography and electroantennographic detection (GC-EAD) and developed a 7-component apple fruit blend for western apple-origin flies, an 8-component black hawthorn fruit blend for flies infesting C. douglasii, and a 9-component ornamental hawthorn blend for flies from C. monogyna. Crataegus douglasii and C. monogyna-origin flies showed similar levels of upwind directed flight to their respective natal synthetic fruit blends in flight tunnel assays compared to whole fruit adsorbent extracts, indicating that the blends contain all the behaviorally relevant fruit volatiles to induce maximal response levels. The black and ornamental hawthorn blends shared four compounds in common including 3-methylbutan-1-ol, which appears to be a key volatile for R. pomonella populations in the eastern, southern, and western USA that show a preference for fruit from different Crataegus species. However, the blends also differed from one another and from domesticated apple in several respects that make it possible that western R. pomonella flies behaviorally discriminate among fruit volatiles and form ecologically differentiated host races, as is the case for eastern apple and hawthorn flies.

Abundance of apple maggot, Rhagoletis pomonella, across different areas in central Washington, with special reference to black-fruited hawthorns.

The apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), infests non-commercial apple (Malus domestica (Borkh.) Borkh.) and native black-fruited hawthorns (mostly Crataegus douglasii Lindl.) in central Washington, but little has been published on the abundance of the fly in this region. In this paper, the abundance of R. pomonella across different sites near apple-growing areas in central Washington is documented in order to assess the threat of the fly to commercial apple orchards. The fly was first detected on traps in Klickitat, Yakima, and Kittitas Counties in 1981, 1995, and 1997, respectively. From 1981-2010 in Kittitas and Yakima Counties, only 0 to 4.7% of traps on apple, crabapple, and hawthorn trees were positive for flies, whereas in Klickitat County, located farther from commercial apple orchards, 0 to 41.9% of traps were positive. In 2008, in Yakima County and Goldendale in Klickitat County, 7.8% of black-fruited hawthorn trees were infested, with 0 to 0.00054 larvae per fruit. In 2010, in Kittitas and Yakima Counties and Goldendale in Klickitat County, 25.0% of C. douglasii trees were infested, with 0.00042 to 0.00248 larvae per fruit. In 2010, in a remote forested area of Klickitat County far from commercial apple orchards, 94.7% of C. douglasii trees were infested, with 0.20813 larvae per fruit. Overall results suggest R. pomonella is unlikely to develop high populations rapidly near major commercial apple-growing areas in central Washington, including in black-fruited hawthorns, increasing chances it can be kept out of commercial orchards.