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.

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.

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.

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.

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.

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.

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.

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.

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.

Sensory reception of the primer pheromone ethyl oleate.

Social work force distribution in honeybee colonies critically depends on subtle adjustments of an age-related polyethism. Pheromones play a crucial role in adjusting physiological and behavioral maturation of nurse bees to foragers. In addition to primer effects of brood pheromone and queen mandibular pheromone--both were shown to influence onset of foraging--direct worker-worker interactions influence adult behavioral maturation. These interactions were narrowed down to the primer pheromone ethyl oleate, which is present at high concentrations in foragers, almost absent in young bees and was shown to delay the onset of foraging. Based on chemical analyses, physiological recordings from the antenna (electroantennograms) and the antennal lobe (calcium imaging), and behavioral assays (associative conditioning of the proboscis extension response), we present evidence that ethyl oleate is most abundant on the cuticle, received by olfactory receptors on the antenna, processed in glomeruli of the antennal lobe, and learned in olfactory centers of the brain. The results are highly suggestive that the primer pheromone ethyl oleate is transmitted and perceived between individuals via olfaction at close range.

Enrichment of H(2)(17)O from tap water, characterization of the enriched water, and properties of several (17)O-labeled compounds.

A low-abundance form of water, H(2)(17)O, was enriched from 0.04% to ∼90% by slow evaporation and fractional distillation of tap water. The density and refractive index for H(2)(17)O are reported. Gas chromatography-mass spectrometry (GC-MS) of (16)O- and (17)O-1-hexanols and their trimethyl silyl ethers and of (16)O- and (17)O-hexamethyl disiloxanes was used to determine the percentage of (17)O enrichment in the H(2)(17)O. Furthermore, the chemical shifts of labeled and nonlabeled water dissolved in CDCl(3) differed sufficiently that we could verify the enrichment of H(2)(17)O. (17)O hexanol was synthesized by the reaction of iodohexane with Na(17)OH. (17)O-Labeled trimethylsilanol and (17)O-labeled hexamethyldisiloxane were prepared by the reaction of H(2)(17)O with bis(trimethylsilyl)trifluoroacetamide (BSTFA). To generate standards for (17)O NMR, H(2)(17)O(2), and (17)O camphor were prepared. H(2)(17)O was electrolyzed to form (17)O-labeled hydrogen peroxide which was quantified using two colorimetric assays. (17)O-Labeled camphor was prepared by exchanging the ketone oxygen of camphor using H(2)(17)O. The (17)O-labeled compounds were characterized using (17)O, (1)H, and (13)C NMR and GC-MS. While we were characterizing the labeled camphor, we also detected an unexpected oxygen exchange reaction of primary alcohols, catalyzed by electrophilic ketones such as camphor. The reaction is a displacement of the alcohol OH group by water. This is an example of the usefulness of (17)O NMR in the study of a reaction mechanism that has not been noticed previously.

Effects of aromatic compounds on antennal responses and on the pheromone-binding proteins of the gypsy moth (Lymantria dispar).

Female gypsy moths emit a pheromone, (+)-disparlure, which the males follow until they locate the emitter. The male moths' antennae are covered with innervated sensory hairs, specialized in detection of the pheromone. The neurons in these sensory hairs are bathed by a solution rich in pheromone-binding protein (PBP). PBPs are soluble proteins that bind the pheromone and other odorants reversibly with variable thermodynamic and kinetic selectivity and are essential for olfactory responses. Here, we have studied the interaction between 2 gypsy moth PBPs with aromatic compounds that modulate the responses of male moth antennae to (+)-disparlure. The aromatic compounds do not elicit responses by themselves, but when administered together with pheromone, they inhibit, enhance, or prolong the electrophysiological response to the pheromone. Three interactions between the compounds and PBPs were studied: 1) the equilibrium binding of the compounds by themselves to the PBPs, 2) the equilibrium binding of the compounds in the presence of pheromone or a fluorescent reporter ligand, and 3) the effect of the compounds on the conformation of the pheromone-PBP complex. A subset of compounds causes a prolongation of the electroantennogram response, and from this study, we conclude that these compounds follow a structure-activity pattern and stabilize a particular conformer of the PBPs that appears to activate the olfactory response.

Identification of camphor oxidation and reduction products in Pseudomonas putida: new activity of the cytochrome P450cam system.

P450 enzymes are known for catalyzing hydroxylation reactions of non-activated C-H bonds. For example, P450(cam) from Pseudomonas putida oxidizes (1R)-(+)-camphor to 5-exo-hydroxy camphor and further to 5-ketocamphor. This hydroxylation reaction proceeds via a catalytic cycle in which the reduction of dioxygen (O(2)) is coupled to the oxidation of the substrate. We have observed that under conditions of low oxygen, P. putida and isolated P450(cam) reduce camphor to borneol. We characterized the formation of borneol under conditions of low oxygen or when the catalytic cycle is shunted by artificial oxidants like m-chloro perbenzoic acid, cumene hydroperoxide, etc. We also tested the toxicity of camphor and borneol with P. putida and Escherichia coli. We have found that in P. putida borneol is less toxic than camphor, whereas in E. coli borneol is more toxic than camphor. We discuss a potental ecological advantage of the camphor reduction reaction for P. putida.

Binding conformation and kinetics of two pheromone-binding proteins from the Gypsy moth Lymantria dispar with biological and nonbiological ligands.

Pheromone-binding proteins (PBPs) in insects can bind various substances and selectively deliver the message of a signal molecule to the downstream components of the olfactory system. This can be achieved either through a ligand-specific conformational change of the C-terminal peptide of the PBP or by selectively binding/releasing the ligand. PBP may also act as a scavenger to protect the sensory neurons from saturating at high ligand doses. We have compared two PBPs from the gypsy moth (PBP1 and PBP2) and their truncated forms (TPBPs), which lack the C-terminal peptide, in this study. Stopped-flow kinetics with N-phenyl-1-naphthylamine (NPN) have revealed a diffusion-controlled collisional step, between PBP and NPN, after which the NPN relocates into a hydrophobic environment. This work supports the hypothesis that binding between PBPs and ligands occurs stepwise. With the method of tryptophan fluorescence quenching, we have shown different local conformational changes around Trp 37, induced by different ligands, manifested in changes of both the steric and electronic environment around the residue. Importantly, we have noticed a significant difference in the changes induced by the biological ligand (the pheromone) and nonbiological ligands. Therefore, we hypothesize that PBP may serve a different function in each kinetic step, displaying a unique P.L conformation.

New insights into honey bee (Apis mellifera) pheromone communication. Is the queen mandibular pheromone alone in colony regulation?

BACKGROUND: In social insects, the queen is essential to the functioning and homeostasis of the colony. This influence has been demonstrated to be mediated through pheromone communication. However, the only social insect for which any queen pheromone has been identified is the honey bee (Apis mellifera) with its well-known queen mandibular pheromone (QMP). Although pleiotropic effects on colony regulation are accredited to the QMP, this pheromone does not trigger the full behavioral and physiological response observed in the presence of the queen, suggesting the presence of additional compounds. We tested the hypothesis of a pheromone redundancy in honey bee queens by comparing the influence of queens with and without mandibular glands on worker behavior and physiology. RESULTS: Demandibulated queens had no detectable (E)-9-oxodec-2-enoic acid (9-ODA), the major compound in QMP, yet they controlled worker behavior (cell construction and queen retinue) and physiology (ovary inhibition) as efficiently as intact queens. CONCLUSIONS: We demonstrated that the queen uses other pheromones as powerful as QMP to control the colony. It follows that queens appear to have multiple active compounds with similar functions in the colony (pheromone redundancy). Our findings support two hypotheses in the biology of social insects: (1) that multiple semiochemicals with synonymous meaning exist in the honey bee, (2) that this extensive semiochemical vocabulary exists because it confers an evolutionary advantage to the colony.