Geosmin, a Food- and Water-Deteriorating Sesquiterpenoid and Ambivalent Semiochemical, Activates Evolutionary Conserved Receptor OR11A1.

Geosmin, a ubiquitous volatile sesquiterpenoid of microbiological origin, is causative for deteriorating the quality of many foods, beverages, and drinking water, by eliciting an undesirable "earthy/musty" off-flavor. Moreover, and across species from worm to human, geosmin is a volatile, chemosensory trigger of both avoidance and attraction behaviors, suggesting its role as semiochemical. Volatiles typically are detected by chemosensory receptors of the nose, which have evolved to best detect ecologically relevant food-related odorants and semiochemicals. An insect receptor for geosmin was recently identified in flies. A human geosmin-selective receptor, however, has been elusive. Here, we report on the identification and characterization of a human odorant receptor for geosmin, with its function being conserved in orthologs across six mammalian species. Notably, the receptor from the desert-dwelling kangaroo rat showed a more than 100-fold higher sensitivity compared to its human ortholog and detected geosmin at low nmol/L concentrations in extracts from geosmin-producing actinomycetes.

Trail pheromone identification in the ant Crematogaster scutellaris.

In this work, we identified the trail pheromone of the ant Crematogaster scutellaris. We combined gas chromatography-mass spectrometry analysis of extracts from the hind tibia, the location of the respective glands, with automated trail following assays. The study found tridecan-2-ol to be the strongest discriminator between hind tibia and other body part extracts. Tridecan-2-ol elicited trail-following behaviour at concentrations of 1 ng/µL. A separation of the enantiomers showed responses to (R)-tridecan-2-ol already at 0.001 ng/µL and only at a 1000-fold higher concentration for (S)-tridecan-2-ol, suggesting that only the R enantiomer is used by C. scutellaris in its natural environment. We also found strong behavioural responses to 2-dodecanol, a substance that was not detectable in the hind tibia extract of C. scutellaris, but which has been reported to be the trail pheromone of the related species C. castanea. We discuss the contribution of these results to the 'dissection and reconstruction' of strategies and mechanisms underlying the social organization of ants.

Allelochemical root-growth inhibitors in low-molecular-weight cress-seed exudate.

BACKGROUND AND AIMS: Cress seeds release allelochemicals that over-stimulate the elongation of hypocotyls of neighbouring (potentially competing) seedlings and inhibit their root growth. The hypocotyl promoter is potassium, but the root inhibitor was unidentified; its nature is investigated here. METHODS: Low-molecular-weight cress-seed exudate (LCSE) from imbibed Lepidium sativum seeds was fractionated by phase partitioning, paper chromatography, high-voltage electrophoresis and gel-permeation chromatography (on Bio-Gel P-2). Fractions, compared with pure potassium salts, were bioassayed for effects on Amaranthus caudatus seedling growth in the dark for 4 days. KEY RESULTS: The LCSE robustly promoted amaranth hypocotyl elongation and inhibited root growth. The hypocotyl inhibitor was non-volatile, hot acid stable, hydrophilic and resistant to incineration, as expected for K+. The root inhibitor(s) had similar properties but were organic (activity lost on incineration). The root inhibitor(s) remained in the aqueous phase (at pH 2.0, 6.5 and 9.0) when partitioned against butan-1-ol or toluene, and were thus hydrophilic. Activity was diminished after electrophoresis, but the remaining root inhibitors were neutral. They became undetectable after paper chromatography; therefore, they probably comprised multiple compounds, which separated from each other, in part, during fractionation. On gel-permeation chromatography, the root inhibitor co-eluted with hexoses. CONCLUSIONS: Cress-seed allelochemicals inhibiting root growth are different from the agent (K+) that over-stimulates hypocotyl elongation and the former probably comprise a mixture of small, non-volatile, hydrophilic, organic substances. Abundant components identified chromatographically and by electrophoresis in cress-seed exudate fitting this description include glucose, fructose, sucrose and galacturonic acid. However, none of these sugars co-chromatographed and co-electrophoresed with the root-inhibitory principle of LCSE, and none of them (in pure form at naturally occurring concentrations) inhibited root growth. We conclude that the root-inhibiting allelochemicals of cress-seed exudate remain unidentified.

Passive permeability controls synthesis for the allelochemical sorgoleone in sorghum root exudate.

Competition for soil nutrients and water with other plants foster competition within the biosphere for access to these limited resources. The roots for the common grain sorghum produce multiple small molecules that are released via root exudates into the soil to compete with other plants. Sorgoleone is one such compound, which suppresses weed growth near sorghum by acting as a quinone analog and interferes with photosynthesis. Since sorghum also grows photosynthetically, and may be susceptible to sorgoleone action if present in tissues above ground, it is essential to exude sorgoleone efficiently. However, since the P450 enzymes that synthesize sorgoleone are intracellular, the release mechanism for sorgoleone remain unclear. In this study, we conducted an in silico assessment for sorgoleone and its precursors to passively permeate biological membranes. To facilitate accurate simulation, CHARMM parameters were newly optimized for sorgoleone and its precursors. These parameters were used to conduct 1 µs of unbiased molecular dynamics simulations to compare the permeability of sorgoleone with its precursors molecules. We find that interleaflet transfer is maximized for sorgoleone, suggesting that the precursor molecules may remain in the same leaflet for access by biosynthetic P450 enzymes. Since no sorgoleone was extracted during unbiased simulations, we compute a permeability coefficient using the inhomogeneous solubility diffusion model. The requisite free energy and diffusivity profiles for sorgoleone through a sorghum membrane model were determined through Replica Exchange Umbrella Sampling (REUS) simulations. The REUS calculations highlight that any soluble sorgoleone would quickly insert into a lipid bilayer, and would readily transit. When sorgoleone forms aggregates in root exudate as indicated by our equilibrium simulations, aggregate formation would lower the effective concentration in aqueous solution, creating a concentration gradient that would facilitate passive transport. This suggests that sorgoleone synthesis occurs within sorghum root cells and that sorgoleone is exuded by permeating through the cell membrane without the need for a transport protein once the extracellular sorgoleone aggregate is formed.

Transcriptional response of a target plant to benzoxazinoid and diterpene allelochemicals highlights commonalities in detoxification.

BACKGROUND: Plants growing in proximity to other plants are exposed to a variety of metabolites that these neighbors release into the environment. Some species produce allelochemicals to inhibit growth of neighboring plants, which in turn have evolved ways to detoxify these compounds. RESULTS: In order to understand how the allelochemical-receiving target plants respond to chemically diverse compounds, we performed whole-genome transcriptome analysis of Arabidopsis thaliana exposed to either the benzoxazinoid derivative 2-amino- 3H-phenoxazin-3-one (APO) or momilactone B. These two allelochemicals belong to two very different compound classes, benzoxazinoids and diterpenes, respectively, produced by different Poaceae crop species. CONCLUSIONS: Despite their distinct chemical nature, we observed similar molecular responses of A. thaliana to these allelochemicals. In particular, many of the same or closely related genes belonging to the three-phase detoxification pathway were upregulated in both treatments. Further, we observed an overlap between genes upregulated by allelochemicals and those involved in herbicide detoxification. Our findings highlight the overlap in the transcriptional response of a target plant to natural and synthetic phytotoxic compounds and illustrate how herbicide resistance could arise via pathways involved in plant-plant interaction.

Feather chemicals contain information about the major histocompatibility complex in a highly scented seabird.

Mate choice informed by the immune genes of the major histocompatibility complex (MHC) may provide fitness benefits including offspring with increased immunocompetence. Olfactory cues are considered the primary mechanism organisms use to evaluate the MHC of potential mates, yet this idea has received limited attention in birds. Motivated by a finding of MHC-dependent mate choice in the Leach's storm-petrel (Oceanodroma leucorhoa), we examined whether the chemical profiles of this highly scented seabird contain information about MHC genes. Whereas previous studies in birds examined non-volatile compounds, we used gas chromatography-mass spectrometry to measure the volatile compounds emitted from feathers that potentially serve as olfactory infochemicals about MHC and coupled this with locus-specific genotyping of MHC IIB genes. We found that feather chemicals reflected individual MHC diversity through interactions with sex and breeding status. Furthermore, similarity in MHC genotype was correlated with similarity in chemical profiles within female-female and male-female dyads. We provide the first evidence that volatile chemicals from bird feathers can encode information about the MHC. Our findings suggest that olfaction likely aids MHC-based mate choice in this species and highlight a role for chemicals in mediating genetic mate choice in birds where this mode of communication has been largely overlooked.

Why does an obligate autogamous orchid produce insect attractants in nectar? - a case study on Epipactis albensis (Orchidaceae).

BACKGROUND: The flowers of some species of orchids produce nectar as a reward for pollination, the process of transferring pollen from flower to flower. Epipactis albensis is an obligatory autogamous species, does not require the presence of insects for pollination, nevertheless, it has not lost the ability to produce nectar, the chemical composition of which we examined by gas chromatography-mass spectrometry (GC-MS) method for identification of potential insect attractants. RESULTS: During five years of field research, we did not observe any true pollinating insects visiting the flowers of this species, only accidental insects as ants and aphids. As a result of our studies, we find that this self-pollinating orchid produces in nectar inter alia aliphatic saturated and unsaturated aldehydes such as nonanal (pelargonal) and 2-pentenal as well as aromatic ones (i.e., syringaldehyde, hyacinthin). The nectar is low in alkenes, which may explain the absence of pollinating insects. Moreover, vanillin and eugenol derivatives, well-known as important scent compounds were also identified, but the list of chemical compounds is much poorer compared with a closely related species, insect-pollinating E. helleborine. CONCLUSION: Autogamy is a reproductive mechanism employed by many flowering plants, including the orchid genus Epipactis, as an adaptation to growing in habitats where pollinating insects are rarely observed due to the lack of nectar-producing plants they feed on. The production of numerous chemical attractants by self-pollinated E. albensis confirms the evolutionary secondary process, i.e., transition from ancestral insect-pollinating species to obligatory autogamous.

Antennae of psychodid and sphaerocerid flies respond to a high variety of floral scent compounds of deceptive Arum maculatum L.

Insect-pollinated plants often release complex mixtures of floral scents to attract their pollinators. Yet scent compounds eliciting physiological or behavioural responses in pollinators have only been identified in few plant species. The sapromyiophilous aroid Arum maculatum releases a highly diverse dung-like scent with overall more than 300 different compounds recorded so far to attract its psychodid and other fly pollinators. The volatiles' role in pollinator attraction is mostly unknown. To identify potential behaviourally active compounds, we recorded electroantennographic responses of four Psychodidae and one Sphaeroceridae species to (1) inflorescence scents of A. maculatum and (2) the scents released by cow dung, likely imitated by the plant species. Here we show that these flies are sensitive to 78 floral volatiles of various chemical classes, 18 of which were also found in cow dung. Our study, which for the first time determined physiologically active compounds in the antennae of Psychoda spp. and Sphaeroceridae, identified various volatiles not known to be biologically active in any floral visitors so far. The obtained results help deciphering the chemical basis that enables A. maculatum and other plants, pollinated by psychodids and sphaerocerids, to attract and deceive their pollinators.

Anti-attractant activity of (+)-trans-4-thujanol for Eurasian spruce bark beetle Ips typographus: Novel potency for females.

BACKGROUND: Eurasian spruce bark beetles (Ips typographus) use both attractant and anti-attractant semiochemicals to find suitable mature host trees. Trans-4-thujanol is abundant in young, unsuitable spruce trees. Electrophysiological studies have demonstrated its high activity levels, but field data are lacking. RESULTS: Enantioselective GC-MS analysis showed that only (1R,4S)-(+)-trans-4-thujanol was present in Norway spruce bark volatiles. In a factorial design field-trapping experiment, trans-4-thujanol alone was not attractive to Ips typographus. Traps baited with I. typographus' aggregation pheromone and trans-4-thujanol or the known anti-attractant 1,8-cineole caught fewer beetles than those baited with the aggregation pheromone alone. Catches for trans-4-thujanol and 1,8-cineole were dose-dependent. Intermediate doses of trans-4-thujanol and 1,8-cineole had a similar effect. Surprisingly, in contrast to 1,8-cineole and other known Ips anti-attractants, which all inhibit males more strongly than females, the addition of trans-4-thujanol to the aggregation pheromone reduced the attraction of females more. CONCLUSION: The Norway spruce volatile (+)-trans-4-thujanol is a novel I. typographus anti-attractant with potency comparable to the known anti-attractants 1,8-cineole and verbenone, and is more effective for females than for males. Incorporating (+)-trans-4-thujanol into anti-attractant lures could improve protection of trees from mass attack by I. typographus.

Assessment of the In Vivo and In Vitro Release of Chemical Compounds from Vespa velutina.

Vespa velutina has been rapidly expanding throughout Galicia since 2012. It is causing human health risks and well-known losses in the beekeeping sector. Control methods are scarce, unspecific, and ineffective. Semiochemicals are insect-derived chemicals that play a role in communication and they could be used an integrated pest management tool alternative to conventional pesticides. A previous determination of the organic chemical profile should be the first step in the study of these semiochemicals. HS-SPME in living individuals and the sting apparatus extraction followed by GC-MS spectrometry were combined to extract a possible profile of these compounds in 43 hornets from Galicia. The identified compounds were hydrocarbons, ketones, terpenes, and fatty acid, and fatty acid esters. Nonanal aldehyde appeared in important concentrations in living individuals. While pentadecane, 8-hexyl- and ethyl oleate were mainly extracted from the venom apparatus. Ketones 2-nonanone, 2-undecanone and 7-nonen-2-one, 4,8-dimethyl- were identified by both procedures, as was 1,7-Nonadiene, 4,8-dimethyl-. Some compounds were detected for the first time in V. velutina such as naphthalene, 1,6-dimethyl-4-(1-methylethyl). The chemical profile by caste was also characterized.

Highly sensitive scent-detection of COVID-19 patients in vivo by trained dogs.

Timely and accurate diagnostics are essential to fight the COVID-19 pandemic, but no test satisfies both conditions. Dogs can scent-identify the unique odors of volatile organic compounds generated during infection by interrogating specimens or, ideally, the body of a patient. After training 6 dogs to detect SARS-CoV-2 by scent in human respiratory secretions (in vitro diagnosis), we retrained 5 of them to search and find the infection by scenting the patient directly (in vivo screening). Then, efficacy trials were designed to compare the diagnostic performance of the dogs against that of the rRT-PCR in 848 human subjects: 269 hospitalized patients (COVID-19 prevalence 30.1%), 259 hospital staff (prevalence 2.7%), and 320 government employees (prevalence 1.25%). The limit of detection in vitro was lower than 10-12 copies ssRNA/mL. During in vivo efficacy experiments, our 5 dogs detected 92 COVID-19 positive patients among the 848 study subjects. The alert (lying down) was immediate, with 95.2% accuracy and high sensitivity (95.9%; 95% C.I. 93.6-97.4), specificity (95.1%; 94.4-95.8), positive predictive value (69.7%; 65.9-73.2), and negative predictive value (99.5%; 99.2-99.7) in relation to rRT-PCR. Seventy-five days after finishing in vivo efficacy experiments, a real-life study (in vivo effectiveness) was executed among the riders of the Metro System of Medellin, deploying the human-canine teams without previous training or announcement. Three dogs were used to examine the scent of 550 volunteers who agreed to participate, both in test with canines and in rRT-PCR testing. Negative predictive value remained at 99.0% (95% C.I. 98.3-99.4), but positive predictive value dropped to 28.2% (95% C.I. 21.1-36.7). Canine scent-detection in vivo is a highly accurate screening test for COVID-19, and it detects more than 99% of infected individuals independent of key variables, such as disease prevalence, time post-exposure, or presence of symptoms. Additional training is required to teach the dogs to ignore odoriferous contamination under real-life conditions.

Identification and quantification of 11 airborne biochemicals emitted by the brown recluse and another primitive hunting spider using headspace solid-phase microextraction-GC/MS.

Loxosceles reclusa, or brown recluse spider, is a harmful household spider whose habitat extends throughout the Midwest in the USA and other regions in the world. The pheromones and other biomolecules that facilitate signaling for brown recluses and other spider species are poorly understood. A rapid and sensitive method is needed to analyze airborne spider signaling biomolecules to better understand the structure and function of these biochemicals in order to control the population of the spiders. In this study, we developed a novel headspace solid-phase microextraction (HS-SPME)-GC/MS method to analyze potential pheromones and biomolecules emitted by the brown recluse spider. The method is highly selective and sensitive for biomolecule identification and quantification from a single live spider. Using this novel non-destructive HS-SPME-GC/MS technique, we identified 11 airborne biomolecules, including 4-methylquinazoline, dimethyl sulfone, 2-methylpropanoic acid, butanoic acid, hexanal, 3-methylbutanoic acid, 2-methylbutanoic acid, 2,4-dimethylbenzaldehyde, 2-phenoxyethanol, and citral (contains both isomers of neral and geranial). Some of these airborne biomolecules were also reported as semiochemicals associated with biological functions of other spiders and insects. The method was also applied to study the airborne biochemicals of Plectreurys tristis, another primitive hunting spider with a poor web, enabling quantitation of the same compounds and demonstrating a difference in signaling molecule concentrations between the two species. This method has potential application in the study of pheromones and biological signaling in other species, which allows for the possibility of utilizing attractant or deterrent functions to limit household populations of harmful species.

Multimodal learning of pheromone locations.

Memorizing pheromonal locations is critical for many mammalian species as it involves finding mates and avoiding competitors. In rodents, pheromonal information is perceived by the main and accessory olfactory systems. However, the role of somatosensation in context-dependent learning and memorizing of pheromone locations remains unexplored. We addressed this problem by training female mice on a multimodal task to locate pheromones by sampling volatiles emanating from male urine through the orifices of varying dimensions or shapes that are sensed by their vibrissae. In this novel pheromone location assay, female mice' preference toward male urine scent decayed over time when they were permitted to explore pheromones vs neutral stimuli, water. On training them for the associations involving olfactory and whisker systems, it was established that they were able to memorize the location of opposite sex pheromones, when tested 15 days later. This memory was not formed either when the somatosensory inputs through whisker pad were blocked or when the pheromonal cues were replaced with that of same sex. The association between olfactory and somatosensory systems was further confirmed by the enhanced expression of the activity-regulated cytoskeleton protein. Furthermore, the activation of main olfactory bulb circuitry by pheromone volatiles did not cause any modulation in learning and memorizing non-pheromonal volatiles. Our study thus provides the evidence for associations formed between different sensory modalities facilitating the long-term memory formation relevant to social and reproductive behaviors.

Identification and Behavioral Assays of Alarm Pheromone in the Vetch Aphid Megoura viciae.

Aphids are destructive pests, and alarm pheromones play a key role in their chemical ecology. Here, we conducted a detailed analysis of terpenoids in the vetch aphid, Megoura viciae, and its host plant Pisum sativum using gas chromatography/mass spectrometry. Four major components, (-)-ß-pinene (49.74%), (E)-ß-farnesene (32.64%), (-)-α-pinene (9.42%) and ( +)-limonene (5.24%), along with trace amounts of ( +)-sabinene, camphene and α-terpineol) (3.14%) were found in the aphid. In contrast, few terpenoids were found in the host plant, consisting mainly of squalene (66.13%) and its analog 2,3-epoxysqualene (31.59%). Quantitative analysis of the four major terpenes in different developmental stages of the aphid showed that amounts of the monoterpenes increased with increasing stage, while the sesquiterpene amount peaked in the 3rd instar. (-)-ß-Pinene was the most abundant terpene at all developmental stages. Behavioral assays using a three-compartment olfactometer revealed that the repellency of single compounds varied in a concentration-dependent manner, but two mixtures [(-)-α-pinene: (-)-ß-pinene: (E)-ß-farnesene: ( +)-limonene = 1:44.4:6.5:2.2 or 1:18.4:1.3:0.8], were repellent at all concentrations tested. Our results suggest that (-)-α-pinene and (-)-ß-pinene are the major active components of the alarm pheromone of M. viciae, but that mixtures play a key role in the alarm response. Our study contributes to the understanding of the chemical ecology of aphids and may help design new control strategies against this aphid pest.

Attraction of Brown Marmorated Stink Bugs, Halyomorpha halys, to Blooming Sunflower Semiochemicals.

The polyphagous invasive brown marmorated stink bug, Halyomorpha halys, reportedly discriminates among phenological stages of host plants. To determine whether olfaction is involved in host plant stage discrimination, we selected (dwarf) sunflower, Helianthus annuus, as a model host plant species. When adult females of a still-air laboratory experiment were offered a choice of four potted sunflowers at distinct phenological stages (vegetative, pre-bloom, bloom, seeding), most females settled onto blooming plants but oviposited evenly on plants of all four stages. In moving-air two-choice olfactometer experiments, we then tested each plant stage versus filtered air and versus one another, for attraction of H. halys females. Blooming sunflowers performed best overall, but no one plant stage was most attractive in all experiments. Capturing and analyzing (by GC-MS) the headspace odorants of each plant stage revealed a marked increase of odorant abundance (e.g., monoterpenes) as plants transitioned from pre-bloom to bloom. Analyzing the headspace odorant blend of blooming sunflower by gas chromatographic-electroantennographic detection (GC-EAD) revealed 13 odorants that consistently elicited responses from female H. halys antennae. An 11-component synthetic blend of these odorants attracted H. halys females in laboratory olfactometer experiments. Furthermore, in field settings, the synthetic blend enhanced the attractiveness of synthetic H. halys pheromone as a trap lure, particularly in spring (April to mid-June). A simpler yet fully effective sunflower semiochemical blend could be developed and coupled with synthetic H. halys aggregation pheromones to improve monitoring efforts or could improve the efficacy of modified attract-and-kill control tactics for H. halys.

α-Terpineol: An Aggregation Pheromone in Optatus palmaris (Coleoptera: Curculionidae) (Pascoe, 1889) Enhanced by Its Host-Plant Volatiles.

The Annonaceae fruits weevil (Optatus palmaris) causes high losses to the soursop production in Mexico. Damage occurs when larvae and adults feed on the fruits; however, there is limited research about control strategies against this pest. However, pheromones provide a high potential management scheme for this curculio. Thus, this research characterized the behavior and volatile production of O. palmaris in response to their feeding habits. Olfactometry assays established preference by weevils to volatiles produced by feeding males and soursop. The behavior observed suggests the presence of an aggregation pheromone and a kairomone. Subsequently, insect volatiles sampled by solid-phase microextraction and dynamic headspace detected a unique compound on feeding males increased especially when feeding. Feeding-starvation experiments showed an averaged fifteen-fold increase in the concentration of a monoterpenoid on males feeding on soursop, and a decrease of the release of this compound males stop feeding. GC-MS analysis of volatiles identified this compound as α-terpineol. Further olfactometry assays using α-terpineol and soursop, demonstrated that this combination is double attractive to Annonaceae weevils than only soursop volatiles. The results showed a complementation effect between α-terpineol and soursop volatiles. Thus, α-terpineol is the aggregation pheromone of O. palmaris, and its concentration is enhanced by host-plant volatiles.

An evolutionary conserved olfactory receptor for foodborne and semiochemical alkylpyrazines.

Molecular recognition is a fundamental principle in biological systems. The olfactory detection of both food and predators via ecological relevant odorant cues are abilities of eminent evolutionary significance for many species. Pyrazines are such volatile cues, some of which act as both human-centered key food odorants (KFOs) and semiochemicals. A pyrazine-selective odorant receptor has been elusive. Here we screened 2,3,5-trimethylpyrazine, a KFO and semiochemical, and 2,5-dihydro-2,4,5-trimethylthiazoline, an innate fear-associated non-KFO, against 616 human odorant receptor variants, in a cell-based luminescence assay. OR5K1 emerged as sole responding receptor. Tested against a comprehensive collection of 178 KFOs, we newly identified 18 pyrazines and (2R/2S)-4-methoxy-2,5-dimethylfuran-3(2H)-one as agonists. Notably, OR5K1 orthologs in mouse and domesticated species displayed a human-like, potency-ranked activation pattern of pyrazines, suggesting a domestication-led co-evolution of OR5K1 and its orthologs. In summary, OR5K1 is a specialized olfactory receptor across mammals for the detection of pyrazine-based key food odors and semiochemicals.

Dufour's gland analysis reveals caste and physiology specific signals in Bombus impatiens.

Reproductive division of labor in insect societies is regulated through multiple concurrent mechanisms, primarily chemical and behavioral. Here, we examined if the Dufour's gland secretion in the primitively eusocial bumble bee Bombus impatiens signals information about caste, social condition, and reproductive status. We chemically analyzed Dufour's gland contents across castes, age groups, social and reproductive conditions, and examined worker behavioral and antennal responses to gland extracts. We found that workers and queens each possess caste-specific compounds in their Dufour's glands. Queens and gynes differed from workers based on the presence of diterpene compounds which were absent in workers, whereas four esters were exclusive to workers. These esters, as well as the total amounts of hydrocarbons in the gland, provided a separation between castes and also between fertile and sterile workers. Olfactometer bioassays demonstrated attraction of workers to Dufour's gland extracts that did not represent a reproductive conflict, while electroantennogram recordings showed higher overall antennal sensitivity in queenless workers. Our results demonstrate that compounds in the Dufour's gland act as caste- and physiology-specific signals and are used by workers to discriminate between workers of different social and reproductive status.

Responses of the Pheromone-Binding Protein of the Silk Moth Bombyx mori on a Graphene Biosensor Match Binding Constants in Solution.

An electronic biosensor for odors was assembled by immobilizing the silk moth Bombyx mori pheromone binding protein (BmorPBP1) on a reduced graphene oxide surface of a field-effect transistor. At physiological pH, the sensor detects the B. mori pheromones, bombykol and bombykal, with good affinity and specificity. Among the other odorants tested, only eugenol elicited a strong signal, while terpenoids and other odorants (linalool, geraniol, isoamyl acetate, and 2-isobutyl-3-methoxypyrazine) produced only very weak responses. Parallel binding assays were performed with the same protein and the same ligands, using the common fluorescence approach adopted for similar proteins. The results are in good agreement with the sensor's responses: bombykol and bombykal, together with eugenol, proved to be strong ligands, while the other compounds showed only poor affinity. When tested at pH 4, the protein failed to bind bombykol both in solution and when immobilized on the sensor. This result further indicates that the BmorPBP1 retains its full activity when immobilized on a surface, including the conformational change observed in acidic conditions. The good agreement between fluorescence assays and sensor responses suggests that ligand-binding assays in solution can be used to screen mutants of a binding protein when selecting the best form to be immobilized on a biosensor.

Identification of a non-host semiochemical from miniature pinscher, Canis lupus familiaris, that repels Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

It is already known that the beagle breed of domestic dogs produces semiochemicals capable of repelling the brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.). With a view to discovering new non-host semiochemicals as tick repellents, we compared the semiochemicals produced by a putative tick-resistant breed of dog, miniature pinscher, with known tick-resistant (beagle) and tick-susceptible (English cocker spaniel) breeds. Two non-host compounds produced by beagles, i.e. 2-hexanone and benzaldehyde, were shown to be present in samples collected from all three breeds. Furthermore, two compounds, 6-methyl-5-hepten-2-one and 1,2,4-trimethylbenzene, were found in higher amounts in samples collected from miniature pinscher dogs. The mean amounts of benzaldehyde, 2-hexanone and 1,2,4-trimethylbenzene were similar for beagles and miniature pinschers (P > 0.05) and higher than the means observed for cocker spaniels (P < 0.05), whereas the mean amount of 6-methyl-5-hepten-2-one produced by miniature pinschers was significantly higher (P < 0.05) than for the other breeds of dogs. In Petri-dish assays with adult R. sanguineus s.l., 6-methyl-5-hepten-2-one was repellent for all observation periods evaluated for the two highest concentrations (0.100 and 0.200 mg.cm-2, P < 0.01). The obtained results support our hypothesis that miniature pinschers are a tick-resistant dog breed and agree with previous observations of miniature pinschers being the breed least parasitized by ticks. Furthermore, the non-host semiochemical 6-methyl-5-hepten-2-one has potential to be developed for use as a repellent for the protection of susceptible dogs from R. sanguineus s.l. ticks.