A blood test to monitor bee health across a European network of agricultural sites of different land-use by MALDI BeeTyping mass spectrometry.

There are substantial concerns about impaired honey bee health and colony losses due to several poorly understood factors. We used MALDI profiling (MALDI BeeTyping®) analysis to investigate how some environmental and management factors under field conditions across Europe affected the honey bee haemolymph peptidome (all peptides in the circulatory fluid), as a profile of molecular markers representing the immune status of Apis mellifera. Honey bees were exposed to a range of environmental stressors in 128 agricultural sites across eight European countries in four biogeographic zones, with each country contributing eight sites each for two different cropping systems: oilseed rape (OSR) and apple (APP). The full haemolymph peptide profiles, including the presence and levels of three key immunity markers, namely the antimicrobial peptides (AMPs) Apidaecin, Abaecin and Defensin-1, allowed the honey bee responses to environmental variables to be discriminated by country, crop type and site. When considering just the AMPs, it was not possible to distinguish between countries by the prevalence of each AMP in the samples. However, it was possible to discriminate between countries on the amounts of the AMPs, with the Swedish samples in particular expressing high amounts of all AMPs. A machine learning model was developed to discriminate the haemolymphs of bees from APP and OSR sites. The model was 90.6 % accurate in identifying the crop type from the samples used to build the model. Overall, MALDI BeeTyping® of bee haemolymph represents a promising and cost-effective "blood test" for simultaneously monitoring dozens of peptide markers affected by environmental stressors at the landscape scale, thus providing policymakers with new diagnostic and regulatory tools for monitoring bee health.

Sex pheromone biosynthesis in the sugarcane borer Diatraea saccharalis: paving the way for biotechnological production.

BACKGROUND: The sugarcane borer Diatraea saccharalis (Lepidoptera) is a key pest on sugarcane and other grasses in the Americas. Biological control as well as insecticide treatments are used for pest management, but economic losses are still significant. The use of female sex pheromones for mating disruption or mass trapping in pest management could be established for this species, provided that economical production of pheromone is available. RESULTS: Combining in vivo labelling studies, differential expression analysis of transcriptome data and functional characterisation of insect genes in a yeast expression system, we reveal the biosynthetic pathway and identify the desaturase and reductase enzymes involved in the biosynthesis of the main pheromone component (9Z,11E)-hexadecadienal, and minor components hexadecanal, (9Z)-hexadecenal and (11Z)-hexadecenal. We next demonstrate heterologous production of the corresponding alcohols of the pheromone components, by expressing multiple steps of the biosynthetic pathway in yeast. CONCLUSION: Elucidation of the genetic basis of sex pheromone biosynthesis in D. saccharalis, and heterologous expression in yeast, paves the way for biotechnological production of the pheromone compounds needed for pheromone-based pest management of this species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Seed treatment with clothianidin induces changes in plant metabolism and alters pollinator foraging preferences.

Neonicotinoids, systemic insecticides that are distributed into all plant tissues and protect against pests, have become a common part of crop production, but can unintentionally also affect non-target organisms, including pollinators. Such effects can be direct effects from insecticide exposure, but neonicotinoids can affect plant physiology, and effects could therefore also be indirectly mediated by changes in plant phenology, attractiveness and nutritional value. Under controlled greenhouse conditions, we tested if seed treatment with the neonicotinoid clothianidin affected oilseed rape's production of flower resources for bees and the content of the secondary plant products glucosinolates that provide defense against herbivores. Additionally, we tested if seed treatment affected the attractiveness of oilseed rape to flower visiting bumblebees, using outdoor mesocosms. Flowers and leaves of clothianidin-treated plants had different profiles of glucosinolates compared with untreated plants. Bumblebees in mesocosms foraged slightly more on untreated plants. Neither flower timing, flower size nor the production of pollen and nectar differed between treatments, and therefore cannot explain any preference for untreated oilseed rape. We instead propose that this small but significant preference for untreated plants was related to the altered glucosinolate profile caused by clothianidin. Thereby, this study contributes to the understanding of the complex relationships between neonicotinoid-treated crops and pollinator foraging choices, by suggesting a potential mechanistic link by which insecticide treatment can affect insect behavior.

Green Chemistry Production of Codlemone, the Sex Pheromone of the Codling Moth (Cydia pomonella), by Metabolic Engineering of the Oilseed Crop Camelina (Camelina sativa).

Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C12 chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional ∆9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T2 generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T4 generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone.

Tetranorsesquiterpenoids as Attractants of Yucca Moths to Yucca Flowers.

The obligate pollination mutualism between Yucca and yucca moths is a classical example of coevolution. Oviposition and active pollination by female yucca moths occur at night when Yucca flowers are open and strongly scented. Thus, floral volatiles have been suggested as key sensory signals attracting yucca moths to their host plants, but no bioactive compounds have yet been identified. In this study, we showed that both sexes of the pollinator moth Tegeticula yuccasella are attracted to the floral scent of the host Yucca filamentosa. Chemical analysis of the floral headspace from six Yucca species in sections Chaenocarpa and Sarcocarpa revealed a set of novel tetranorsesquiterpenoids putatively derived from (E)-4,8-dimethyl-1,3,7-nonatriene. Their structure elucidation was accomplished by NMR analysis of the crude floral scent sample of Yucca treculeana along with GC/MS analysis and confirmed by total synthesis. Since all these volatiles are included in the floral scent of Y. filamentosa, which has been an important model species for understanding the pollination mutualism, we name these compounds filamentolide, filamentol, filamental, and filamentone. Several of these compounds elicited antennal responses in pollinating (Tegeticula) and non-pollinating (Prodoxus) moth species upon stimulation in electrophysiological recordings. In addition, synthetic (Z)-filamentolide attracted significant numbers of both sexes of two associated Prodoxus species in a field trapping experiment. Highly specialized insect-plant interactions, such as obligate pollination mutualisms, are predicted to be maintained through "private channels" dictated by specific compounds. The identification of novel bioactive tetranorsesquiterpenoids is a first step in testing such a hypothesis in the Yucca-yucca moth interaction.

Metapopulation dynamics over 25 years of a beetle, Osmoderma eremita, inhabiting hollow oaks.

Osmoderma eremita is a species of beetle that inhabits hollows in ancient trees, which is a habitat that has decreased significantly during the last century. In southeastern Sweden, we studied the metapopulation dynamics of this beetle over a 25 year period, using capture-mark-recapture. The metapopulation size had been rather stable over time, but in most of the individual trees there had been a positive or negative trend in population development. The probability of colonisation was higher in well-connected trees with characteristics reflecting earlier successional stages, and the probability of extinction higher in trees with larger diameter (i.e. in later successional stages), which is expected from a habitat-tracking metapopulation. The annual tree mortality and fall rates (1.1% and 0.4%, respectively) are lower than the colonisation and extinction rates (5-7%), indicating that some of the metapopulation dynamics are due to the habitat dynamics, but many colonisations and extinctions take place for other reasons, such as stochastic events in small populations. The studied metapopulation occurs in an area with a high density of hollow oaks and where the oak pastures are still managed by grazing. In stands with fewer than ten suitable trees, the long-term extinction risk may be considerable, since only a small proportion of all hollow trees harbours large populations, and the population size in trees may change considerably during a decade.

Identification and Synthesis of Putative Pheromone Components of the Threatened Salt Marsh Bagworm Moth, Whittleia retiella (Lepidoptera: Psychidae).

Whittleia retiella (Newman, 1847) is a threatened salt marsh species of the bagworm moth family Psychidae. For its preservation it is necessary to develop efficient tools to survey its distribution and habitat requirements in order to use appropriate conservation methods. Such tools may be pheromone-based monitoring systems, which have documented efficacy in establishing the occurrence of cryptic insect species in nature. By using gas chromatography combined with electroantennographic detection (GC-EAD), we found two compounds in female W. retiella headspace samples and whole-body extracts that elicited electrophysiological activity in male antennae. Gas chromatograpy coupled with mass spectrometry (GC-MS) operating in electron impact (EI) mode and comparison of the analytical data with those of synthetic reference compounds showed the chemical structures of these putative pheromone components to be (1S)-1-methylpropyl (5Z)-dec-5-enoate and 1-methylethyl (5Z)-dec-5-enoate. Field assays using baits loaded with synthetic compounds revealed that conspecific males were attracted to (1S)-1-methylpropyl (5Z)-dec-5-enoate alone or in combination with 1-methylethyl (5Z)-dec-5-enoate, whereas 1-methylethyl (5Z)-dec-5-enoate neither attracted nor repelled males in the field assays when tested alone. This study shows the potential of using (1S)-1-methylpropyl (5Z)-dec-5-enoate for monitoring W. retiella to gather more detailed information about the geographic distribution and habitat needs of this rare moth.

Characterization of olfactory sensory neurons in the red clover seed weevil, Protapion trifolii (Coleoptera: Brentidae) and comparison to the closely related species P. fulvipes.

Protapion trifolii and P. fulvipes (Coleoptera: Brentidae) are major pests in European clover seed production. Previous studies have reported a high host plant fidelity of these weevils for red and white clover species, respectively, driven by host plant olfactory cues. Given the specific host preferences observed in these weevils, we aimed to elucidate to which extent such selectivity is reflected in their peripheral olfactory systems. Using an electrophysiological approach, we performed the first functional characterisation of olfactory sensory neurons (OSNs) in P. trifolii to a panel of volatile compounds emitted by red clover plants, and compared the results with the reported OSN types of P. fulvipes. Nineteen OSN classes were characterized in P. trifolii, with the majority of these neurons responding strongly to common volatiles released by the host plant. Based on response profiles, eight of these OSN classes have clear matches to OSN classes in P. fulvipes. The OSN colocalisation patterns and antennal frequency of these classes are similar in the two species. Additionally, the responses of these OSNs are generally highly conserved in the two species, with clear response shifts only revealed for two of the OSN classes. These response shifts in combination with additional response dissimilarities for compounds that vary in abundance between red and white clover plants may underlie the species-specific host preferences. Further behavioural and field experiments should focus on these differentially detected compounds to elucidate their potential role in host selection and use in semiochemical-based control of these pests.

Pheromones and Barcoding Delimit Boundaries between Cryptic Species in the Primitive Moth Genus Eriocrania (Lepidoptera: Eriocraniidae).

Animal classification is primarily based on morphological characters, even though these may not be the first to diverge during speciation. In many cases, closely related taxa are actually difficult to distinguish based on morphological characters alone, especially when there is no substantial niche separation. As a consequence, the diversity of certain groups is likely to be underestimated. Lepidoptera -moths and butterflies- represent the largest group of herbivorous insects. The extensive diversification in the group is generally assumed to have its origin in the spectacular radiation of flowering plants and the resulting abundance of ecological niches. However, speciation can also occur without strong ecological divergence. For example, reproductive isolation can evolve as the result of divergence in mate preference and the associated pheromone communication system. We combined pheromone trapping and genetic analysis to elucidate the evolutionary relationships within a complex of primitive moth species (Lepidoptera: Eriocraniidae). Mitochondrial and nuclear DNA markers provided evidence that Eriocrania semipurpurella, as currently defined by morphological characters, includes three cryptic species in Northern and Western Europe. Male moths of these cryptic species, as well as of the closely related E. sangii, exhibited relative specificity in terms of their attraction to specific ratios of two major pheromone components, (2S,6Z)-nonen-2-ol and (2R,6Z)-nonen-2-ol. Our data suggest strong assortative mating in these species in the absence of apparent niche separation, indicating that Eriocrania moths may represent an example of non-ecological speciation. Finally, our study argues in favour of combining pheromone investigations and DNA barcoding as powerful tools for identifying and delimitating species boundaries.

The Pattern of Straight Chain Hydrocarbons Released by Yucca Flowers (Asparagaceae).

The hydrocarbon pattern in the floral scent of Yucca species was found to comprise a group of unbranched, mid-chain alkanes, alkenes, and an alkadiene. In Y. reverchonii, highly dominant (Z)-8-heptadecene is accompanied by (6Z,9Z)-6,9-heptadecadiene and heptadecane as minor components and by traces of other saturated and unsaturated hydrocarbons with similar chain length. Some of these volatiles proved to be perceived by the antennae of Tegeticula cassandra (pollinating seed-eater of Yucca) and Prodoxus decipiens (herbivore on Yucca). The possible biosynthesis of the compounds is discussed.

Challenges of pheromone-based mating disruption of Cydia strobilella and Dioryctria abietella in spruce seed orchards.

Seed orchards function as the primary source of high-quality seeds for reforestation in many European countries, but their seed yields can be severely reduced due to seed- and cone-feeding insects. We evaluated various parameters of pheromone-based mating disruption for control of the moths Cydia strobilella and Dioryctria abietella, which are major pests in European Picea abies seed orchards. We applied different types of pheromone dispensers (rubber septa or wax droplets) at different densities and heights, and with different amounts of active components, covering whole orchards or part of an orchard. The efficacy of the treatment was evaluated by analysing male captures in pheromone-baited assessment traps, and presence of larvae in cones. A dramatic decrease (94-100%) in capture of males in traps occurred in all pheromone-treated plots compared to control plots for both moth species. In contrast, a subsequent reduction in larval numbers in cones was only achieved when wax droplets were used as the dispensing formulation at high density and at the highest pheromone dose tested, and only in half of the trials for each pest species. Electrophysiological recordings using antennae of male C. strobilella indicated elevated pheromone concentrations in a treated plot versus a control plot. Our results show that mating disruption has potential to reduce cone damage in spruce seed orchards caused by C. strobilella and D. abietella, but optimisation of the technique is required to achieve consistent and efficient population suppression of these pests.

Visual cues of oviposition sites and spectral sensitivity of Cydia strobilella L.

We investigated whether the spruce seed moth (Cydia strobilella L., Tortricidae: Grapholitini), an important pest in seed orchards of Norway spruce (Picea abies (L.) Karst.), can make use of the spectral properties of its host when searching for flowers to oviposit on. Spectral measurements showed that the flowers, and the cones they develop into, differ from a background of P. abies needles by a higher reflectance of long wavelengths. These differences increase as the flowers develop into mature cones. Electroretinograms (ERGs) in combination with spectral adaptation suggest that C. strobilella has at least three spectral types of photoreceptor; an abundant green-sensitive receptor with maximal sensitivity at wavelength λmax=526nm, a blue-sensitive receptor with λmax=436nm, and an ultraviolet-sensitive receptor with λmax=352nm. Based on our spectral measurements and the receptor properties inferred from the ERGs, we calculated that open flowers, which are suitable oviposition sites, provide detectable achromatic, but almost no chromatic contrasts to the background of needles. In field trials using traps of different spectral properties with or without a female sex pheromone lure, only pheromone-baited traps caught moths. Catches in baited traps were not correlated with the visual contrast of the traps against the background. Thus, visual contrast is probably not the primary cue for finding open host flowers, but it could potentially complement olfaction as a secondary cue, since traps with certain spectral properties caught significantly more moths than others.

Floral scent of Joshua trees (Yucca brevifolia sensu lato): Divergence in scent profiles between species but breakdown of signal integrity in a narrow hybrid zone.

PREMISE OF THE STUDY: The role of floral scent in facilitating reproductive isolation between closely related plants remains poorly understood. Yucca brevifolia and Yucca jaegeriana are pollinated by different moth species in allopatry, but in a narrow contact zone, pollinator-host specificity breaks down, resulting in hybridization between species. We explored the chemical basis for reproductive isolation and hybridization in these Joshua trees by characterizing the floral scent of each species in allopatry, analyzing scent profiles from trees in the contact zone, and matching these data with genotypic and phenotypic data. METHODS: We analyzed floral volatiles using gas chromatography-mass spectrometry, tested for species divergence of scent profiles and classified trees in the contact zone as hybrid or either parental species. We used floral and vegetative morphological data and genotypic data to classify trees and analyzed whether certain trait combinations were more correlated than others with respect to assignment of trees and whether frequencies of classified tree types differed depending on which data set was used. KEY RESULTS: The Joshua tree floral scent included oxygenated 8-carbon compounds not reported for other yuccas. The two species differed (P < 0.001) in scent profiles. In the contact zone, many hybrids were found, and phenotypic traits were generally weakly correlated, which may be explained by extensive gene flow between species or by exposure to different selection pressures. CONCLUSIONS: Although the two Joshua tree species produce distinct floral scent profiles, it is insufficient to prevent attraction of associated pollinators to both hosts. Instead, floral morphology may be the key trait mediating gene flow between species.

Antennal fine morphology of the threatened beetle Osmoderma eremita (Coleoptera: Scarabaeidae), revealed by scanning electron microscopy.

The aim of this study was to characterize the antennal morphology of Osmoderma eremita, a threatened scarab beetle inhabiting tree hollows. O. eremita males produce a sex pheromone, (R)-(+)-γ-decalactone, responsible mainly for the attraction of females but also other males. Gross and fine morphology of microstructures including sensilla, microsculpture and pores were analyzed using Scanning Electron Microscopy. The antenna of O. eremita showed the typical lamellicorn shape of scarab beetles, with a basal scape, a pedicel, a funicle composed of five antennomeres and a club composed of three lamellae. Six different subtypes of sensilla chaetica (Ch.1 - 6), Böhm sensilla (Bo), one subtype of sensilla basiconica (Ba.1), two subtypes of sensilla coeloconica (Co.1 - 2), two subtypes of sensilla placodea (Pl.1 - 2), pores and peculiar folds were described. The two sexes did not show any significant differences in the occurrence and number of the sensilla placodea, known to be responsible for the pheromone reception. Instead, some sexual differences were found on the occurrence and topology of three different microstructures: (1) one subtype of sensillum chaeticum (Ch.2) occurring on the pedicel only in males; (2) a characteristic pore occurring on the funicle only in males; (3) a peculiar fold occurring on different antennomeres of the funicle in the two sexes, on the fourth in males and on the fifth in females. A comparison between sensilla of O. eremita and those of other Scarabaeoidea is provided.

Field Abundance Patterns and Odor-Mediated Host Choice by Clover Seed Weevils, Apion fulvipes and Apion trifolii (Coleoptera: Apionidae).

The clover seed weevils Apion fulvipes Geoffroy, 1785 and Apion trifolii L., 1768 (Coleoptera: Apionidae) cause major losses to seed production of white clover (Trifolium repens L.) and red clover (Trifolium pratense L.), respectively. Clover is important as animal forage and an alternative to inorganic fertilizers. Because clover is mainly pollinated by bees, the use of insecticides in management of these weevils is discouraged. To gain basic knowledge for development of alternative management strategies, we investigated weevil field abundance over two growing seasons, as well as feeding and olfactory host preferences by A. fulvipes and A. trifolii. Field trap catches in southern Sweden revealed that white clover was dominated by A. fulvipes and red clover by A. trifolii. For both weevil species, female catches were positively correlated to the number of clover buds and flowers in the field. In feeding and olfactory bioassays, females of A. fulvipes and A. trifolii showed a preference for T. repens and T. pratense, respectively. However, the feeding preference was lost when the antennae were removed, indicating a significant role of olfaction in host choice. Male weevils of both species did not show clear olfactory or feeding preferences for host plant species. The field study and laboratory bioassays demonstrate that, at least for female weevils, olfaction is important for selection of host plants. We discuss these novel results in the context of managing these important pests of clover by exploiting olfaction and behavioral attraction to host plant volatiles.

Enhanced science-stakeholder communication to improve ecosystem model performances for climate change impact assessments.

In recent years, climate impact assessments of relevance to the agricultural and forestry sectors have received considerable attention. Current ecosystem models commonly capture the effect of a warmer climate on biomass production, but they rarely sufficiently capture potential losses caused by pests, pathogens and extreme weather events. In addition, alternative management regimes may not be integrated in the models. A way to improve the quality of climate impact assessments is to increase the science-stakeholder collaboration, and in a two-way dialog link empirical experience and impact modelling with policy and strategies for sustainable management. In this paper we give a brief overview of different ecosystem modelling methods, discuss how to include ecological and management aspects, and highlight the importance of science-stakeholder communication. By this, we hope to stimulate a discussion among the science-stakeholder communities on how to quantify the potential for climate change adaptation by improving the realism in the models.

Identification, synthesis, and behavioral activity of 5,11-dimethylpentacosane, a novel sex pheromone component of the greater wax moth, Galleria mellonella (L.).

The greater wax moth, Galleria mellonella (L.), is a serious and widespread pest of the honeybee, Apis mellifera L. In contrast to most moths, for which long-range mate finding is mediated by female-produced sex pheromones, G. mellonella males attract conspecific females over long distances by emitting large amounts of a characteristic scent in combination with bursts of ultrasonic calls. The male scent for this species was previously identified as a blend of nonanal and undecanal. When these compounds were bioassayed, characteristic short-range sexual behavior, including wing fanning, was triggered in conspecific females, but the aldehyde blend failed to elicit attraction over longer distances. We identified, via analysis and synthesis, a third male-specific compound, 5,11-dimethylpentacosane. We show that it acts as a behavioral synergist to the aldehydes. In wind tunnel experiments, very few female moths responded to the aldehyde blend or to 5,11-dimethylpentacosane tested separately, but consistently showed orientation and source contact when a combination of all three compounds was applied. The level of attraction to the three-component mixture was still lower than that to male extract, indicating that the composition of compounds in the synthetic blend is suboptimal, or that additional pheromone components of G. mellonella are yet to be identified. The identification of 5,11-dimethylpentacosane is an important step for the development of an efficient long-range attractant that will be integrated with other environmentally safe strategies to reduce damage to beehives caused by wax moths.

Active pollination favours sexual dimorphism in floral scent.

Zoophilous flowers often transmit olfactory signals to attract pollinators. In plants with unisexual flowers, such signals are usually similar between the sexes because attraction of the same animal to both male and female flowers is essential for conspecific pollen transfer. Here, we present a remarkable example of sexual dimorphism in floral signal observed in reproductively highly specialized clades of the tribe Phyllantheae (Phyllanthaceae). These plants are pollinated by species-specific, seed-parasitic Epicephala moths (Gracillariidae) that actively collect pollen from male flowers and pollinate the female flowers in which they oviposit; by doing so, they ensure seeds for their offspring. We found that Epicephala-pollinated Phyllanthaceae plants consistently exhibit major qualitative differences in scent between male and female flowers, often involving compounds derived from different biosynthetic pathways. In a choice test, mated female Epicephala moths preferred the scent of male flowers over that of female flowers, suggesting that male floral scent elicits pollen-collecting behaviour. Epicephala pollination evolved multiple times in Phyllantheae, at least thrice accompanied by transition from sexual monomorphism to dimorphism in floral scent. This is the first example in which sexually dimorphic floral scent has evolved to signal an alternative reward provided by each sex, provoking the pollinator's legitimate altruistic behaviour.

Characterization of olfactory sensory neurons in the white clover seed weevil, Apion fulvipes (Coleoptera: Apionidae).

Seed-eating Apion weevils (Coleoptera: Apionidae) cause large economic losses in white and red clover seed production across Europe. Monitoring and control of clover weevils would be facilitated by semiochemical-based methods. Until now, however, nothing was known about physiological or behavioral responses to semiochemicals in this insect group. Here we analyzed the antenna of the white clover (Trifolium repens L.) specialist Apion fulvipes Geoffroy with scanning electron microscopy, and used single sensillum recordings with a set of 28 host compounds to characterize 18 classes of olfactory sensory neurons (OSNs). Nine of the OSN classes responded strongly to synthetic compounds with high abundance in clover leaves, flowers, or buds. Eight classes responded only weakly to the synthetic stimuli, whereas one collective class responded exclusively to volatiles released from a crushed clover leaf. The OSNs showed a remarkable degree of specificity, responding to only one or a few chemically related compounds. In addition, we recorded a marked difference in the temporal dynamics of responses between different neurons, compounds, and doses. The identified physiologically active compounds will be screened for behavioral activity, with the ultimate goal to develop an odor-based control strategy for this pest.

Chemical ecology of obligate pollination mutualisms: testing the 'private channel' hypothesis in the Breynia-Epicephala association.

*Obligate mutualisms involving actively pollinating seed predators are among the most remarkable insect-plant relationships known, yet almost nothing is known about the chemistry of pollinator attraction in these systems. The extreme species specificity observed in these mutualisms may be maintained by specific chemical compounds through 'private channels'. Here, we tested this hypothesis using the monoecious Breynia vitis-idaea and its host-specific Epicephala pollinator as a model. *Headspace samples were collected from both male and female flowers of the host. Gas chromatography with electroantennographic detection (GC-EAD), coupled gas chromatography-mass spectrometry, and olfactometer bioassays were used to identify the floral compounds acting as the pollinator attractant. *Male and female flowers of B. vitis-idaea produced similar sets of general floral compounds, but in different ratios, and male flowers emitted significantly more scent than female flowers. A mixture of 2-phenylethyl alcohol and 2-phenylacetonitrile, the two most abundant compounds in male flowers, was as attractive to female moths as the male flower sample, although the individual compounds were slightly less attractive when tested separately. *Data on the floral scent signals mediating obligate mutualisms involving active pollination are still very limited. We show that system-specific chemistry is not necessary for efficient host location by exclusive pollinators in these tightly coevolved mutualisms.