The efficacy of 1-allyloxy-4-propoxybenzene (3c{3,6}) against Varroa destructor mites in honey bee colonies from Maryland, USA.

Varroa destructor Oud (Acari: Varroidae) is a harmful ectoparasite of Apis mellifera L. honey bees causing widespread colony losses in Europe and North America. To control populations of these mites, beekeepers have an arsenal of different treatments, including both chemical and nonchemical options. However, nonchemical treatments can be labor intensive, and Varroa has gained resistance to some conventional pesticides, and the use of other chemical treatments is restricted temporally (e.g., cannot be applied during periods of honey production). Thus, beekeepers require additional treatment options for controlling mite populations. The compound 1-allyloxy-4-propoxybenzene (3c{3,6}) is a diether previously shown to be a strong feeding deterrent against Lepidopteran larvae and a repellent against mosquitoes and showed promise as a novel acaricide from laboratory and early field trials. Here we test the effect of the compound, applied at 8 g/brood box on wooden release devices, on honey bees and Varroa in field honey bee colonies located in Maryland, USA, and using a thymol-based commercial product as a positive control. 3c{3,6} had minimal effect on honey bee colonies, but more tests are needed to determine whether it affected egg production by queens. Against Varroa3c{3,6} had an estimated efficacy of 78.5%, while the positive control thymol product showed an efficacy of 91.3%. 3c{3,6} is still in the development stage, and the dose or application method needs to be revisited.

Protein-Ligand Binding and Structural Modelling Studies of Pheromone-Binding Protein-like Sol g 2.1 from Solenopsis geminata Fire Ant Venom.

Sol g 2 is the major protein in Solenopsis geminata fire ant venom. It shares the highest sequence identity with Sol i 2 (S. invicta) and shares high structural homology with LmaPBP (pheromone-binding protein (PBP) from the cockroach Leucophaea maderae). We examined the specific Sol g 2 protein ligands from fire ant venom. The results revealed that the protein naturally formed complexes with hydrocarbons, including decane, undecane, dodecane, and tridecane, in aqueous venom solutions. Decane showed the highest affinity binding (Kd) with the recombinant Sol g 2.1 protein (rSol g 2.1). Surprisingly, the mixture of alkanes exhibited a higher binding affinity with the rSol g 2.1 protein compared to a single one, which is related to molecular docking simulations, revealing allosteric binding sites in the Sol g 2.1 protein model. In the trail-following bioassay, we observed that a mixture of the protein sol g 2.1 and hydrocarbons elicited S. geminata worker ants to follow trails for a longer time and distance compared to a mixture containing only hydrocarbons. This suggests that Sol g 2.1 protein may delay the evaporation of the hydrocarbons. Interestingly, the piperidine alkaloids extracted have the highest attraction to the ants. Therefore, the mixture of hydrocarbons and piperidines had a synergistic effect on the trail-following of ants when both were added to the protein.

Effects of dialkoxybenzenes against Varroa destructor and identification of 1-allyloxy-4-propoxybenzene as a promising acaricide candidate.

The honey bee is responsible for pollination of a large proportion of crop plants, but the health of honey bee populations has been challenged by the parasitic mite Varroa destructor. Mite infestation is the main cause of colony losses during the winter months, which causes significant economic challenges in apiculture. Treatments have been developed to control the spread of varroa. However, many of these treatments are no longer effective due to acaricide resistance. In a search of varroa-active compounds, we tested the effect of dialkoxybenzenes on the mite. A structure-activity relationship revealed that 1-allyloxy-4-propoxybenzene is most active of a series of dialkoxybenzenes tested. We found that three compounds (1-allyloxy-4-propoxybenzene, 1,4-diallyloxybenzene and 1,4-dipropoxybenzene) cause paralysis and death of adult varroa mites, whereas the previously discovered compound, 1,3-diethoxybenzene, which alters host choice of adult mites in certain conditions, did not cause paralysis. Since paralysis can be caused by inhibition of acetylcholinesterase (AChE), a ubiquitous enzyme in the nervous system of animals, we tested dialkoxybenzenes on human, honey bee and varroa AChE. These tests revealed that 1-allyloxy-4-propoxybenzene had no effects on AChE, which leads us to conclude that 1-allyloxy-4-propoxybenzene does not exert its paralytic effect on mites through AChE. In addition to paralysis, the most active compounds affected the ability of the mites to find and remain at the abdomen of host bees provided during assays. A test of 1-allyloxy-4-propoxybenzene in the field, during the autumn of 2019 in two locations, showed that this compound has promise in the treatment of varroa infestations.

Design and Synthesis of Fluorophore-Tagged Disparlure Enantiomers to Study Pheromone Enantiomer Discrimination in the Pheromone-Binding Proteins from the Gypsy Moth, Lymantria dispar.

Fluorescent analogues of the gypsy moth sex pheromone (+)-disparlure (1) and its enantiomer (-)-disparlure (ent-1) were designed, synthesized, and characterized. The fluorescently labelled analogues 6-FAM (+)-disparlure and 1a 6-FAM (-)-disparlure ent-1a were prepared by copper-catalyzed azide-alkyne cycloaddition of disparlure alkyne and 6-FAM azide. These fluorescent disparlure analogues 1a and ent-1a were used to measure disparlure binding to two pheromone-binding proteins from the gypsy moth, LdisPBP1 and LdisPBP2. The fluorescence binding assay showed that LdisPBP1 has a stronger affinity for 6-FAM (-)-disparlure ent-1a, whereas LdisPBP2 has a stronger affinity for 6-FAM (+)-disparlure 1a, consistent with findings from previous studies with disparlure enantiomers. The 6-FAM disparlure enantiomers appeared to be much stronger ligands for LdisPBPs, with binding constants (Kd) in the nanomolar range, compared to the fluorescent reporter 1-NPN (which had Kd values in the micromolar range). Fluorescence competitive binding assays were used to determine the displacement constant (Ki) for the disparlure enantiomers in competition with fluorescent disparlure analogues binding to LdisPBP1 and LdisPBP2. The Ki data show that disparlure enantiomers can effectively displace the fluorescent disparlure from the binding pocket of LdisPBPs and, therefore, occupy the same binding site.

Ligand- and pH-Induced Structural Transition of Gypsy Moth Lymantria dispar Pheromone-Binding Protein 1 (LdisPBP1).

Pheromone-binding proteins (PBPs) are small, water-soluble proteins found in the lymph of pheromone-sensing hairs. PBPs are essential in modulating pheromone partitioning in the lymph and at pheromone receptors of olfactory sensory neurons. The function of a PBP is associated with its ability to structurally convert between two conformations. Although mechanistic details remain unclear, it has been proposed that the structural transition between these forms is affected by two factors: pH and the presence or absence of ligand. To better understand the PBP conformational transition, the structure of the gypsy moth (Lymantria dispar) LdisPBP1 was elucidated at pH 4.5 and 35 °C using nuclear magnetic resonance spectroscopy. In addition, the effects of sample pH and binding of the species' pheromone, (+)-disparlure, (7R,8S)-epoxy-2-methyloctadecane, and its enantiomer were monitored via 15N HSQC spectroscopy. LdisPBP1 in acidic conditions has seven helices, with its C-terminal residues forming the seventh helix within the pheromone-binding pocket and its N-terminal residues disordered. Under conditions where this conformation is made favorable, free LdisPBP1 would have limited ligand binding capacity due to the seventh helix occupying the internal binding pocket. Our findings suggest that even in the presence of 4-fold ligand at acidic pH, LdisPBP1 is only ∼60% in its pheromone-bound form. Furthermore, evidence of a different LdisPBP1 form is seen at higher pH, with the transition pH between 5.6 and 6.0. This suggests that LdisPBP1 at neutral pH exists as a mixture of at least two conformations. These findings have implications concerning the PBP ligand binding and release mechanism.

Chemotaxis by Pseudomonas putida (ATCC 17453) towards camphor involves cytochrome P450cam (CYP101A1).

The camphor-degrading microorganism, Pseudomonas putida strain ATCC 17453, is an aerobic, gram-negative soil bacterium that uses camphor as its sole carbon and energy source. The genes responsible for the catabolic degradation of camphor are encoded on the extra-chromosomal CAM plasmid. A monooxygenase, cytochrome P450cam, mediates hydroxylation of camphor to 5-exo-hydroxycamphor as the first and committed step in the camphor degradation pathway, requiring a dioxygen molecule (O2) from air. Under low O2 levels, P450cam catalyzes the production of borneol via an unusual reduction reaction. We have previously shown that borneol downregulates the expression of P450cam. To understand the function of P450cam and the consequences of down-regulation by borneol under low O2 conditions, we have studied chemotaxis of camphor induced and non-induced P. putida strain ATCC 17453. We have tested camphor, borneol, oxidized camphor metabolites and known bacterial attractants (d)-glucose, (d) - and (l)-glutamic acid for their elicitation chemotactic behavior. In addition, we have used 1-phenylimidazole, a P450cam inhibitor, to investigate if P450cam plays a role in the chemotactic ability of P. putida in the presence of camphor. We found that camphor, a chemoattractant, became toxic and chemorepellent when P450cam was inhibited. We have also evaluated the effect of borneol on chemotaxis and found that the bacteria chemotaxed away from camphor in the presence of borneol. This is the first report of the chemotactic behaviour of P. putida ATCC 17453 and the essential role of P450cam in this process.

Selecting of a cytochrome P450cam SeSaM library with 3-chloroindole and endosulfan - Identification of mutants that dehalogenate 3-chloroindole.

Cytochrome P450cam (a camphor hydroxylase) from the soil bacterium Pseudomonas putida shows potential importance in environmental applications such as the degradation of chlorinated organic pollutants. Seven P450cam mutants generated from Sequence Saturation Mutagenesis (SeSaM) and isolated by selection on minimal media with either 3-chloroindole or the insecticide endosulfan were studied for their ability to oxidize of 3-chloroindole to isatin. The wild-type enzyme did not accept 3-chloroindole as a substrate. Mutant (E156G/V247F/V253G/F256S) had the highest maximal velocity in the conversion of 3-chloroindole to isatin, whereas mutants (T56A/N116H/D297N) and (G60S/Y75H) had highest kcat/KM values. Six of the mutants had more than one mutation, and within this set, mutation of residues 297 and 179 was observed twice. Docking simulations were performed on models of the mutant enzymes; the wild-type did not accommodate 3-chloroindole in the active site, whereas all the mutants did. We propose two potential reaction pathways for dechlorination of 3-chloroindole. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Synthesis of Enantiopure Alicyclic Ethers and Their Activity on the Chemosensory Organ of the Ectoparasite of Honey Bees, Varroa destructor.

The preparation of enantiopure conformationally restricted alicyclic ethers and their inhibitory activities on the chemosensory organ of the Varroa destructor, a parasite of honey bees, are reported in this article. We tested the effect of enantiopure ethers of cis-5-(2'-hydroxyethyl)cyclopent-2-en-1-ol on the Varroa chemosensory organ by electrophysiology, for their ability to inhibit the responses to two honey bee-produced odors that are important for the mite to locate its host: nurse bee head space odor and (E)-ß-ocimene, a honey bee brood pheromone. Previous work with the racemic compounds showed that they suppress the mite's olfactory response to its bee host, which led to incorrect host choice. Based on a structure-activity relationship, we predicted that the two most active compounds-cis-1-butoxy-5-(2'-methoxyethyl)cyclopent-2-ene, cy{4,1}, and (cis-1-ethoxy-5-(2'ethoxyethyl)cyclopent-2-ene, cy{2,2}-could have opposite active enantiomers. Here we studied the enantiomers of both ethers, whose preparation involved enzymatic resolution of racemic diol cis-5-(2'-hydroxyethyl)cyclopent-2-en-1-ol using Lipase AK with vinyl acetate. The racemic diol was prepared from commercially available 2,5-norbornadiene. We observed that the responses of the chemosensory organ to honey bee head space volatiles were significantly decreased by both enantiomers of cy{4,1} and cy{2,2}, but that responses to (E)-ß-ocimene were decreased significantly only by (+)-cy{4,1} (1R,5S) and (-)-cy{2,2} (1S,5R) and not by their respective enantiomers. The importance of this result is that the racemates could be used to inhibit olfactory detection of bee odors by mites, without a loss in activity relative to the more expensive enantiopure compounds.

NADPH oxidase-derived H2O2 subverts pathogen signaling by oxidative phosphotyrosine conversion to PB-DOPA.

Strengthening the host immune system to fully exploit its potential as antimicrobial defense is vital in countering antibiotic resistance. Chemical compounds released during bidirectional host-pathogen cross-talk, which follows a sensing-response paradigm, can serve as protective mediators. A potent, diffusible messenger is hydrogen peroxide (H2O2), but its consequences on extracellular pathogens are unknown. Here we show that H2O2, released by the host on pathogen contact, subverts the tyrosine signaling network of a number of bacteria accustomed to low-oxygen environments. This defense mechanism uses heme-containing bacterial enzymes with peroxidase-like activity to facilitate phosphotyrosine (p-Tyr) oxidation. An intrabacterial reaction converts p-Tyr to protein-bound dopa (PB-DOPA) via a tyrosinyl radical intermediate, thereby altering antioxidant defense and inactivating enzymes involved in polysaccharide biosynthesis and metabolism. Disruption of bacterial signaling by DOPA modification reveals an infection containment strategy that weakens bacterial fitness and could be a blueprint for antivirulence approaches.

Gypsy moth pheromone-binding protein-ligand interactions: pH profiles and simulations as tools for detecting polar interactions.

Pheromone-binding proteins (PBPs) are believed to control diffusion of pheromones in sensory hairs of insects. The interactions of gypsy moth (Lymantria dispar) PBPs with the sex attractant pheromone, (+)-Disparlure ((7R,8S)-epoxy-2-methyloctadecane), and the enantioselectivity of recognition are not completely understood. Enantioselectivity is important for L. dispar, because (-)-disparlure cancels the attraction of (+)-disparlure, so these moths use enantiopure (+)-disparlure for communication. We performed docking simulations of the protonated homology PBP models with the enantiomers of disparlure, 5-oxadisparlure, 10-oxadisparlure, 5-thiadisparlure and 10-thiadisparlure, together with a binding assay experiment, in which the pH profiles for the PBP-ligand combinations were surveyed. The molecular simulations revealed different amino acid residues in the binding sites, movement of specific amino acid residues at certain pH values, distinct amino acid-ligand interactions (side chain donors/acceptors, H-arene bonding, backbone donors/acceptors) and differences in the conformations of each protein-ligand complex. The pKa values obtained from the binding experiment and the results from the molecular simulations served as tools for detecting polar interactions between the PBPs and ligands. The differences found between structures docked with ligand enantiomers reveal the enantioselectivity of the gypsy moth PBPs towards the pheromone and its antipode, as well as towards enantiomers of pheromone analogs with heteroatom substitutions.

Endogenous fatty acids in olfactory hairs influence pheromone binding protein structure and function in Lymantria dispar.

The gypsy moth utilizes a pheromone, (7R,8S)-2-methyl-7,8-epoxyoctadecane, for mate location. The pheromone is detected by sensory hairs (sensilla) on the antennae of adult males. Sensilla contain the dendrites of olfactory neurons bathed in lymph, which contains pheromone binding proteins (PBPs). We have extracted and identified free fatty acids from lymph of sensory hairs, and we demonstrate that these function as endogenous ligands for gypsy moth PBP1 and PBP2. Homology modeling of both PBPs, and docking of fatty acids reveal multiple binding sites: one internal, the others external. Pheromone binding assays suggest that these fatty acids increase PBP-pheromone binding affinity. We show that fatty acid binding causes an increase in α-helix content in the N-terminal domain, but not in the C-terminal peptide of both proteins. The C-terminal peptide was shown to form a α-helix in a hydrophobic, homogeneous environment, but not in the presence of fatty acid micelles. Through partition assays we show that the fatty acids prevent adsorption of the pheromone on hydrophobic surfaces and facilitate pheromone partition into an aqueous phase. We propose that lymph is an emulsion of fatty acids and PBP that influence each other and thereby control the partition equilibria of hydrophobic odorants.

Neuronal plasticity in the mushroom body calyx during adult maturation in the honeybee and possible pheromonal influences.

Honeybee workers express a pronounced age-dependent polyethism switching from various indoor duties to foraging outside the hive. This transition is accompanied by tremendous changes in the sensory environment that sensory systems and higher brain centers have to cope with. Foraging and age have earlier been shown to be associated with volume changes in the mushroom bodies (MBs). Using age- and task-controlled bees this study provides a detailed framework of neuronal maturation processes in the MB calyx during the course of natural behavioral maturation. We show that the MB calyx volume already increases during the first week of adult life. This process is mainly driven by broadening of the Kenyon cell dendritic branching pattern and then followed by pruning of projection neuron axonal boutons during the actual transition from indoor to outdoor duties. To further investigate the flexible regulation of division of labor and its neuronal correlates in a honeybee colony, we studied the modulation of the nurse-forager transition via a chemical communication system, the primer pheromone ethyl oleate (EO). EO is found at high concentrations on foragers in contrast to nurse bees and was shown to delay the onset of foraging. In this study, EO effects on colony behavior were not as robust as expected, and we found no direct correlation between EO treatment and synaptic maturation in the MB calyx. In general, we assume that the primer pheromone EO rather acts in concert with other factors influencing the onset of foraging with its effect being highly adaptive.

Can we disrupt the sensing of honey bees by the bee parasite Varroa destructor?

BACKGROUND: The ectoparasitic mite, Varroa destructor, is considered to be one of the most significant threats to apiculture around the world. Chemical cues are known to play a significant role in the host-finding behavior of Varroa. The mites distinguish between bees from different task groups, and prefer nurses over foragers. We examined the possibility of disrupting the Varroa--honey bee interaction by targeting the mite's olfactory system. In particular, we examined the effect of volatile compounds, ethers of cis 5-(2'-hydroxyethyl) cyclopent-2-en-1-ol or of dihydroquinone, resorcinol or catechol. We tested the effect of these compounds on the Varroa chemosensory organ by electrophysiology and on behavior in a choice bioassay. The electrophysiological studies were conducted on the isolated foreleg. In the behavioral bioassay, the mite's preference between a nurse and a forager bee was evaluated. PRINCIPAL FINDINGS: We found that in the presence of some compounds, the response of the Varroa chemosensory organ to honey bee headspace volatiles significantly decreased. This effect was dose dependent and, for some of the compounds, long lasting (>1 min). Furthermore, disruption of the Varroa volatile detection was accompanied by a reversal of the mite's preference from a nurse to a forager bee. Long-term inhibition of the electrophysiological responses of mites to the tested compounds was a good predictor for an alteration in the mite's host preference. CONCLUSIONS: These data indicate the potential of the selected compounds to disrupt the Varroa--honey bee associations, thus opening new avenues for Varroa control.

An inexpensive feeding bioassay technique for stored-product insects.

ABSTRACT We used the red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae), to compare three feeding bioassay techniques using flour disks. The area (scanner or digital photographs) and mass (sensitive balance) of the same flour disks were measured daily for 1 or 2 wk to assess feeding by insects. The loss in mass and area over 4 h was measured, as some variation over time was noticed in the disks with no insects feeding on them. The gravimetric method correlated well with both measurements of the area for the disks held in a growth chamber: scanner (R2 = 0.96), digital photography (R2 = 0.96). There was also a high correlation (R2 = 0.86) between the disk weight and area scanned at normal lab conditions. There were differences in the percentage of the disks remaining over time depending on the temperature and whether they were weighed or scanned. Measuring the mass of the disks resulted in a relatively larger percent of disk remaining compared with the scanned area. Mass measurements required a sensitive balance, handling of the disks and the insects, and appeared slightly more sensitive to humidity and temperature changes over time. Scanning the disks requires flat bed scanner access but less handling of both insects and disks. Digital photographs could be taken quickly, requiring less equipment, although photographs had to be further processed to determine area Scanning or taking digital photographs of flour disk area was an effective technique for measuring insect feeding.

Biodegradation of 1-allyloxy-4-propoxybenzene by selected strains of Pseudomonas putida.

Dialkoxybenzenes constitute a class of organic compounds with anti feeding and oviposition effects on the cabbage looper, Trichoplusia ni. Among them, 1-allyloxy-4-propoxybenzene has the highest feeding deterrence activity and potential for development as commercial insect control agent. To develop this compound, its fate in the environment needs to be studied. The fate of organic compounds in the environment depends on their biodegradability in the soil. We present results of laboratory biodegradation experiments of 1-allyloxy-4-propoxybenzene with three strains of Pseudomonas putida. Two of the three strains of P. putida tested were able to metabolize 1-allyloxy-4-propoxybenzene. Both strains required induction of the catabolic pathway. Specifically, strain ATCC 17453 (which contains the CAM plasmid) metabolized 1-allyloxy-4-propoxybenzene by first dealkylating. This gave both possible monoalkoxy phenols after five days, followed by dihydroquinone after 8 days. In vitro tests with CYP101A1 (cytochrome P450cam, a camphor hydroxylase), revealed that the dealkylation is catalyzed by this enzyme.

Feeding Deterrence of Cabbage Looper (Lepidoptera: Noctuidae) by 1-Allyloxy-4-Propoxybenzene, Alone and Blended With Neem Extract.

The cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), is one of the most damaging insect pests of cabbage (Brassica oleracea variety capitata) and broccoli (B. oleracea variety italica) in North America. Leaf-feeding larvae attack crucifer and vegetable crops in greenhouses and fields. Here, we have studied a synthetic feeding deterrent, 1-allyloxy-4-propoxybenzene, and a botanical deterrent, neem (an extract from seeds of Azadirachta indica A. de Jussieu (Meliaceae)), in leaf disc choice bioassays with T. ni. We tested the two deterrents and the combination, and we found that the blend exhibits synergy between the two deterrents. We also tested the deterrents in assays with whole cabbage plants in ventilated enclosures and found that 1-allyloxy-4-propoxybenzene evaporated and, therefore, in that context addition of 1-allyloxy-4-propoxybenzene to neem did not enhance deterrence against T. ni.

Partition, sorption and structure activity relation study of dialkoxybenzenes that modulate insect behavior.

Some dialkoxybenzenes are promising new insect control agents. These compounds mimic naturally occurring odorants that modulate insect behavior. Before applying these compounds, however, their persistence and biodegradability at the application site and in the environment should be understood. The fate of organic compounds in the environment is a complex phenomenon which is influenced by many processes such as sorption to soil components, sedimentation, volatilization, and uptake by plants, as well as biotic and abiotic chemical degradation. In this study, the octanol-water partition coefficient, volatility and sorption on soil components (sand, clay and organic matter) of selected dialkoxybenzenes as well as structure activity relationships with regard to partition, volatility and sorption were investigated. Additionally, calculations of partition, molar volume and molecular surface areas were done, to understand structure-activity relationships of the physical properties.

Water oxidation by a cytochrome p450: mechanism and function of the reaction.

P450(cam) (CYP101A1) is a bacterial monooxygenase that is known to catalyze the oxidation of camphor, the first committed step in camphor degradation, with simultaneous reduction of oxygen (O2). We report that P450(cam) catalysis is controlled by oxygen levels: at high O2 concentration, P450(cam) catalyzes the known oxidation reaction, whereas at low O2 concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using (17)O and (2)H NMR, that the hydrogen atom added to camphor comes from water, which is oxidized to hydrogen peroxide (H2O2). This is the first time a cytochrome P450 has been observed to catalyze oxidation of water to H2O2, a difficult reaction to catalyze due to its high barrier. The reduction of camphor and simultaneous oxidation of water are likely catalyzed by the iron-oxo intermediate of P450(cam) , and we present a plausible mechanism that accounts for the 1:1 borneol:H2O2 stoichiometry we observed. This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O2, for two reasons: 1) the borneol and H2O2 mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450(cam) and its electron transfer partners. Since the reaction described here only occurs under low O2 conditions, the down-regulation only occurs when O2 is scarce.

Enantiomer and conformer recognition of (+) and (-)-disparlure and their analogs by the pheromone binding proteins of the gypsy moth, Lymantria dispar.

Adult female gypsy moths produce a sex pheromone (+)-(7R,8S)-2-methyl-7,8-epoxyoctadecane, (+)-disparlure, to attract male gypsy moths. To better understand the recognition of (+)-disparlure by the male's olfactory system, we synthesized racemic and enantiopure oxa and thia analogs of (+)-disparlure (ee>98%). Ab initio calculations of the conformeric landscapes around the dihedral angles C5-6-7-8 and C7-8-9-10 of (+)-disparlure and corresponding dihedral angles of analogs revealed that introduction of the heteroatom changes the conformeric landscape around these important epitopes. The energy difference between HOMO and LUMO decreased after oxygen or sulfur was introduced into the backbone. Consistent with this, an enhancement of binding affinity between sulfur analogs and the pheromone-binding proteins (PBPs) was observed in vitro. Docking of the pheromone and analogs onto models of the two known PBPs of the gypsy moth revealed that the internal binding pocket of PBP1 showed higher selectivity than that of PBP2, consistent with in vitro binding assays. Further energy analysis revealed that enantiomers adopted different conformations with different energies when docked in the internal binding pocket of PBPs, resulting in enantiomer discrimination of PBPs towards disparlure and its analogs.