Secure Food-Allergen Determination by Combining Smartphone-Based Raw Image Analyses and Liquid Chromatography-Mass Spectrometry for the Quantification of Proteins Contained in Lateral Flow Assays.

The current food safety testing system, based on laboratory-based quantification, is difficult to scale up in line with the growth in the export market and does not enable traceability through the nodes of the food supply system. Screening assays, for example, lateral flow assays (LFAs), can improve traceability but often lack the required reliability to guarantee compliance. Here, we present an alternative pipeline for secure on-site compliance testing, using allergens as a case study. The pipeline features smartphone-driven LFA quantification and an liquid chromatography-mass spectrometry (LC-MS) method enabling direct quantification of the allergens contained in the LFA. The system enables swift and objective screening and provides a control measure to verify LFA assay reliability. For the smartphone assay, 8-bit RGB and grayscale colorimetric channels were compared with 16-bit raw intensity values. The latter outperformed RGB and grayscale channels in sensitivity, repeatability, and precision, while ratiometric ambient light correction resulted in excellent robustness for light-intensity variation. Calibration curves for peanut determination using two commercial LFAs featured excellent analytical parameters (R2 = 0.97-0.99; RSD 7-1%; LOD 3-7 ppm). Gluten determination with a third commercial LFA was equally established. A prediction error of 13 ± 11% was achieved for the best performing assay. Good performance-calibration curves (R2 = 0.93-0.99) and CVs (<15%)- were observed for the analyte quantification from the LFA by LC-MS. The LOD for the LC-MS assay was 0.5 ppm, well below the LODs reported for the LFAs. This method creates a digital, fast, and secure food safety compliance testing paradigm that can benefit the industry and consumer alike.

Biomarkers and biosensors for the diagnosis of noncompliant pH, dark cutting beef predisposition, and welfare in cattle.

Meat quality can be affected by stress, exhaustion, feed composition, and other physical and environmental conditions. These stressors can alter the pH in postmortem muscle, leading to high pH and low-quality dark cutting (DC) beef, resulting in considerable economic loss. Moreover, the dark cutting prediction may equally provide a measure for animal welfare since it is directly related to animal stress. There are two needs to advance on-site detection of dark cutters: (1) a clear indication that biomarker (signature compounds) levels in cattle correlate with stress and DC outcome; and (2) measuring these biomarkers rapidly and accurately on-farm or the abattoir, depending on the objectives. This critical review assesses which small molecules and proteins have been identified as potential biomarkers of stress and dark cutting in cattle. We discuss the potential of promising small molecule biomarkers, including catecholamine/cortisol metabolites, lactate, succinate, inosine, glucose, and ß-hydroxybutyrate, and we identify a clear research gap for proteomic biomarker discovery in live cattle. We also explore the potential of chemical-sensing and biosensing technologies, including direct electrochemical detection improved through nanotechnology (e.g., carbon and gold nanostructures), surface-enhanced Raman spectroscopy in combination with chemometrics, and commercial hand-held devices for small molecule detection. No current strategy exists to rapidly detect predictive meat quality biomarkers due to the need to further validate biomarkers and the fact that different biosensor types are needed to optimally detect different molecules. Nonetheless, several biomarker/biosensor combinations reported herein show excellent potential to enable the measurement of DC potential in live cattle.

Resonance Energy Transfer-Based Biosensors for Point-of-Need Diagnosis-Progress and Perspectives.

The demand for point-of-need (PON) diagnostics for clinical and other applications is continuing to grow. Much of this demand is currently serviced by biosensors, which combine a bioanalytical sensing element with a transducing device that reports results to the user. Ideally, such devices are easy to use and do not require special skills of the end user. Application-dependent, PON devices may need to be capable of measuring low levels of analytes very rapidly, and it is often helpful if they are also portable. To date, only two transduction modalities, colorimetric lateral flow immunoassays (LFIs) and electrochemical assays, fully meet these requirements and have been widely adopted at the point-of-need. These modalities are either non-quantitative (LFIs) or highly analyte-specific (electrochemical glucose meters), therefore requiring considerable modification if they are to be co-opted for measuring other biomarkers. Förster Resonance Energy Transfer (RET)-based biosensors incorporate a quantitative and highly versatile transduction modality that has been extensively used in biomedical research laboratories. RET-biosensors have not yet been applied at the point-of-need despite its advantages over other established techniques. In this review, we explore and discuss recent developments in the translation of RET-biosensors for PON diagnoses, including their potential benefits and drawbacks.

Molecular and Functional Evolution at the Odorant Receptor Or22 Locus in Drosophila melanogaster.

Insect odorant receptor (Or) genes determine the responses of sensory neurons that mediate critical behaviors. The Drosophila melanogaster Or22 locus represents an interesting example of molecular evolution, with high levels of sequence divergence and copy number variation between D. melanogaster and other Drosophila species, and a corresponding high level of variability in the responses of the neuron it controls, ab3A. However, the link between Or22 molecular and functional diversity has not been established. Here, we show that several naturally occurring Or22 variants generate major shifts in neuronal response properties. We determine the molecular changes that underpin these response shifts, one of which represents a chimeric gene variant previously suggested to be under natural selection. In addition, we show that several alternative molecular genetic mechanisms have evolved for ensuring that where there is more than one gene copy at this locus, only one functional receptor is generated. Our data thus provide a causal link between the striking levels of phenotypic neuronal response variation found in natural populations of D. melanogaster and genetic variation at the Or22 locus. Since neuronal responses govern animal behavior, we predict that Or22 may be a key player in underlying one or more olfactory-driven behaviors of significant adaptive importance.

Drosophila melanogaster odorant receptors as volatile compound detectors in forensic science: a proof-of-concept study.

The ability to detect and identify substances based on the volatile compounds (odors) they emit is relied upon heavily for numerous investigative purposes. Animals have an innate olfactory sensitivity and selectivity that out-performs current instrumentation. This has led to immense interest in their employment as chemical sensors for a range of applications, including forensic science, both as whole organisms and as sensing elements in biosensors. Using electrophysiological and calcium imaging assays, this research examined the response of Drosophila melanogaster olfactory receptors (ORs) to odor compounds significant in forensic science and assessed their potential utility as volatile compound sensors. This investigation illustrated the different sensitivities, selectivities, and sensing features of individual ORs and demonstrated that their employment for detection purposes is feasible. While further research expanding on this study will be required to demonstrate the performance characteristics that an OR-based detection system will ultimately possess, this research provides an encouraging first step towards the goal of utilizing isolated biological ORs as volatile compound sensors in forensic science.

Molecular characterization of sugar taste receptors in the cotton bollworm Helicoverpa armigera.

Insects utilize sugars as their essential energy and nutrient sources; therefore, the sense of sugar detection plays a critical role in insect behaviours. Previously, using genomic and transcriptomic approaches, we identified eight putative sugar gustatory receptor (GR) genes from the cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Here, we further validated these annotated sugar receptor genes (HarmGr4-HarmGr8 and HarmGr10-HarmGr12) and found HarmGr10 may be a pseudogene carrying a stop codon in the open reading frame. Sequence alignment revealed H. armigera sugar GR sequences are conserved at C-terminus and phylogenetic analysis showed that insect sugar GRs have evolved in a family-specific manner. Interestingly, all eight H. armigera sugar GRs are localized in a tandem array on the same scaffold of the genome. In silico gene expression and reverse transcription (RT)-PCR analysis showed that HarmGr10 is specifically expressed in male adult testes while HarmGr11 is specifically expressed in female adult ovaries, suggesting H. armigera sugar GRs may be involved in reproduction-related functions. This study improves our knowledge on insect sugar receptors and gustatory systems.

Chemosensory genes identified in the antennal transcriptome of the blowfly Calliphora stygia.

BACKGROUND: Blowflies have relevance in areas of forensic science, agriculture, and medicine, primarily due to the ability of their larvae to develop on flesh. While it is widely accepted that blowflies rely heavily on olfaction for identifying and locating hosts, there is limited research regarding the underlying molecular mechanisms. Using next generation sequencing (Illumina), this research examined the antennal transcriptome of Calliphora stygia (Fabricius) (Diptera: Calliphoridae) to identify members of the major chemosensory gene families necessary for olfaction. RESULTS: Representative proteins from all chemosensory gene families essential in insect olfaction were identified in the antennae of the blowfly C. stygia, including 50 odorant receptors, 22 ionotropic receptors, 21 gustatory receptors, 28 odorant binding proteins, 4 chemosensory proteins, and 3 sensory neuron membrane proteins. A total of 97 candidate cytochrome P450s and 39 esterases, some of which may act as odorant degrading enzymes, were also identified. Importantly, co-receptors necessary for the proper function of ligand-binding receptors were identified. Putative orthologues for the conserved antennal ionotropic receptors and candidate gustatory receptors for carbon dioxide detection were also amongst the identified proteins. CONCLUSIONS: This research provides a comprehensive novel resource that will be fundamental for future studies regarding blowfly olfaction. Such information presents potential benefits to the forensic, pest control, and medical areas, and could assist in the understanding of insecticide resistance and targeted control through cross-species comparisons.

Carbon dioxide receptor genes in cotton bollworm Helicoverpa armigera.

Carbon dioxide (CO2) is important in insect ecology, eliciting a range of behaviours across different species. Interestingly, the numbers of CO2 gustatory receptors (GRs) vary among insect species. In the model organism Drosophila melanogaster, two GRs (DmelGR21a and DmelGR63a) have been shown to detect CO2. In the butterfly, moth, beetle and mosquito species studied so far, three CO2 GR genes have been identified, while in tsetse flies, four CO2 GR genes have been identified. In other species including honeybees, pea aphids, ants, locusts and wasps, no CO2 GR genes have been identified from the genome. These genomic differences may suggest different mechanisms for CO2 detection exist in different insects but, with the exception of Drosophila and mosquitoes, limited attention has been paid to the CO2 GRs in insects. Here, we cloned three putative CO2 GR genes from the cotton bollworm Helicoverpa armigera and performed phylogenetic and expression analysis. All three H. armigera CO2 GRs (HarmGR1, HarmGR2 and HarmGR3) are specifically expressed in labial palps, the CO2-sensing tissue of this moth. HarmGR3 is significantly activated by NaHCO3 when expressed in insect Sf9 cells but HarmGR1 and HarmGR2 are not. This is the first report characterizing the function of lepidopteran CO2 receptors, which contributes to our general understanding of the molecular mechanisms of insect CO2 gustatory receptors.

Two general-odorant binding proteins in Spodoptera litura are differentially tuned to sex pheromones and plant odorants.

Moths have evolved a sensitive and sophisticated olfactory system to sense a variety of semiochemicals from the external environment. In chemosensory processes, the odorant binding protein (OBP) is an essential element for filtering, binding and transporting hydrophobic odorant molecules to the specific receptors. Here focusing on a major sub-class of lepidopteran OBPs, general-odorant binding proteins (GOBPs), we explored the relationship and functional difference between two GOBP members from a noctuid species Spodoptera litura. Using genomic DNA as the template, we demonstrated that SlitGOBP2 and three SlitPBPs are clustered on the same chromosome within a close proximity. qPCR results showed that two SlitGOBPs were primarily expressed in antennae at similar levels between females and males, but GOBP2 displayed much higher expression than GOBP1. Binding studies revealed that both SlitGOBP1 and 2 strongly bound C14-C16 alcohol-pheromone analogs with high affinities (Ki<1.0 µM). However, SlitGOBP2 also strongly bound most acetate- and aldehyde-sex pheromone components and analogs, while SlitGOBP1 could not. For tested plant odorants, SlitGOBP1 showed a relatively broad ligand-binding spectrum with moderate affinities, while SlitGOBP2 was tuned to some compounds with strong binding activities (Ki<5.0 µM). Finally, by molecular docking we explored the differences in protein structures and potential key residues in the binding pockets between the two SlitGOBPs. Taken together, our study strongly suggests that SlitGOBP2 and SlitPBPs evolved by gene duplication events, and two SlitGOBPs have functionally differentiated in odorant recognition.

Identification and characterization of three chemosensory receptor families in the cotton bollworm Helicoverpa armigera.

BACKGROUND: Chemosensory receptors including olfactory receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) play a central role in sensing chemical signals and guiding insect behaviours, and are potential target genes in insect pest control. The cotton bollworm Helicoverpa armigera is one of the most destructive pest species that can feed on over 200 different plant species. This diversity of host plants is likely linked to a complex chemosensory system. Here we built on previous work to characterize crucial chemosensory tissues linked to environmental interactions including larval antennae, larval mouthparts and larval fat bodies, as well as male and female adult heads, male and female adult tarsi, and female abdomens. RESULTS: Using transcriptome sequencing, Trinity RNA-seq assemblies and extensive manual curation, we identified a total of 91 candidate chemosensory receptors (60 candidate ORs, 10 GRs and 21 IRs). Thirty-five of these candidates present full-length transcripts. First, we performed in silico differential expression analysis on different sequenced tissues. Further, we created extensive expression profiles using reverse transcription (RT)-PCR on a variety of adult and larval stages. We found that the expression profile of HarmOR51 was limited to adult male antenna suggesting a role in mating that was further supported by a phylogenetic analysis clustering it into the pheromone receptor clade. HarmOR51 in calcium imaging analysis did not show responses to either of the two H. armigera sex pheromone components (Z9-16:Ald or Z11-16:Ald) inviting a future detailed study. In addition, we found four novel HarmORs (OR1, 53, 54 and 58) that appeared to be larvae-antennal specific. Finally, our expression profiling showed that four "divergent" HarmIRs (IR2, 7d.1, 7d.2 and 7d.3) were expressed in both adult and larval antennae, suggesting a functional divergence from their Drosophila homologues. CONCLUSIONS: This study explored three chemoreceptor superfamily genes using a curated transcriptomic approach coupled with extensive expression profiling and a more limited functional characterization. Our results have now provided an extensive resource for investigating the chemoreceptor complement of this insect pest, and meanwhile allow for targeted experiments to identify potential molecular targets for pest control and to investigate insect-plant interactions.

Comparisons of contact chemoreception and food acceptance by larvae of polyphagous Helicoverpa armigera and oligophagous Bombyx mori.

We compared food choice and the initial response to deterrent treated diet between fifth instars of Helicoverpa armigera, a polyphagous generalist pest, and Bombyx mori, an oligophagous specialist beneficial. Bombyx mori was more behaviorally sensitive to salicin than to caffeine. The relative sensitivities were reversed for H. armigera, which was tolerant to the highest levels of salicin found in natural sources but sensitive to caffeine. A single gustatory receptor neuron (GRN) in the medial styloconic sensillum of B. mori was highly sensitive to salicin and caffeine. The styloconic sensilla of H. armigera did not respond consistently to either of the bitter compounds. Phagostimulants also were tested. Myo-inositol and sucrose were detected specifically by two GRNs located in B. mori lateral styloconic sensillum, whereas, in H. armigera, sucrose was sensed by a GRN in the lateral sensillum, and myo-inositol by a GRN in the medial sensillum. Myo-inositol responsiveness in both species occurred at or below 10(-3) mM, which is far below the naturally occurring concentration of 1 mM in plants. Larval responses to specific plant secondary compounds appear to have complex determinants that may include host range, metabolic capacity, and gustatory repertoire.

Safe food through better labelling; a robust method for the rapid determination of caprine and bovine milk allergens.

Accidental milk cross-contamination is one of the most common causes for costly food recalls. Yet, quantifying trace-levels of allergen is time-consuming and current methods are not adapted for routine analyses making quality control for trace-level allergen content impractical. This perpetuates voluntary "may-contain" statements that are unhelpful for people suffering from food allergies. Here, we developed a rapid LC-MS method enabling milk allergen quantification by comparing all tryptic-peptides of major milk allergens. The bovine-specific αS-2 casein peptide and allergen-epitope NAVPITPTLNR provided excellent performance in sensitivity (LOD 1 mg.kg-1; LOQ 2 mg.kg-1) across various dairy products, good recovery rates in baked croissants (77% with a 10% inter-day RSD) and a linear range of 2-2,000 mg.kg-1. The method can be used for routine determination of trace-contamination with bovine milk allergen and the adulteration of high-value caprine dairy products with lower-value bovine milk products, protecting consumer trust and the growing population suffering from food allergies.

A sugar gustatory receptor identified from the foregut of cotton bollworm Helicoverpa armigera.

Helicoverpa armigera (Hübner) is one of the most polyphagous and cosmopolitan pest species, the larvae of which feed on numerous important crops. The gustatory system is critical in guiding insect feeding behavior. Here, we identified a gustatory receptor from H. armigera, HaGR9, which shows high levels of identity to DmGR43a from Drosophila melanogaster and BmGR9 from Bombyx mori. Reverse transcriptase PCR (RT-PCR) revealed HaGR9 is highly expressed in larval foregut, with little or no expression in other chemosensory tissues. Membrane topology studies indicated that, like two previously studied B. mori GRs, BmGR8 and BmGR53, HaGR9 has an inverted topology relative to G protein-coupled receptors (GPCRs), an intracellular N-terminus and an extracellular C-terminus. Calcium imaging studies confirmed HaGR9 is a sugar receptor showing dose-dependent responses to D-galactose, D-maltose, and D-fructose. This highly-expressed foregut-specific gustatory receptor may contribute to the regulation of larval feeding behavior.

The present and the future of protein biosensor engineering.

Protein biosensors play increasingly important roles in cell and neurobiology and have the potential to revolutionise the way clinical and industrial analytics are performed. The gradual transition from multicomponent biosensors to fully integrated single chain allosteric biosensors has brought the field closer to commercial applications. We evaluate various approaches for converting constitutively active protein reporter domains into analyte operated switches. We discuss the paucity of the natural receptors that undergo conformational changes sufficiently large to control the activity of allosteric reporter domains. This problem can be overcome by constructing artificial versions of such receptors. The design path to such receptors involves the construction of Chemically Induced Dimerisation systems (CIDs) that can be configured to operate single and two-component biosensors.

Targeted proteomics for rapid and robust peanut allergen quantification.

This study improves LC-MS-based trace level peanut allergen quantification in processed food by refining method robustness, total analysis time and method sensitivity. Extraction buffer (six compared) and peptide choice were optimised and found to profoundly affect method robustness. A rapid extraction and in-solution digestion method was developed omitting subsequent reduction, alkylation and sample clean-up steps effectively reducing total analysis time from the previously reported ∼5.5-20 h to ∼2.5 h. For the three best performing peptides, accurate quantification (CVs < 15%) with matrix-matched calibration curves (R2 = 0.99-0.97) was achieved for peanut muffin and ice-cream with excellent linearity (0.25-1000 mg kg-1). The best performing peptide enabled excellent recovery rates in ice-cream (106.0 ± 15.1%) and peanut muffin (72.7 ± 13.4%). Sensitivity (LOD = 0.25-0.5 mg kg-1; LOQ = 0.5-1.0 mg kg-1) was 2- to 20-fold improved compared to previous methods depending on the peptide. These methodological improvements contribute to robust peanut detection in food and can be translated to additional food-borne allergens.

Natural variation at the Drosophila melanogaster Or22 odorant receptor locus is associated with changes in olfactory behaviour.

In insects, many critical olfactory behaviours are mediated by the large odorant receptor (Or) gene family, which determines the response properties of different classes of olfactory receptor neurons (ORNs). While ORN responses are generally conserved within and between Drosophila species, variant alleles of the D. melanogaster Or22 locus have previously been shown to alter the response profile of an ORN class called ab3A. These alleles show potential clinal variation, suggesting that selection is acting at this locus. Here, we investigated if the changes seen in ab3A responses lead to changes in olfactory-related behaviours. We show that variation at the Or22 locus and in the ab3A neurons are not fully compensated for by other ORNs and lead to overall changes in antennal odorant detection. We further show that this correlates with differences in odorant preference behaviour and with differences in oviposition site preference, with flies that have the chimaeric short allele strongly preferring to oviposit on banana. These findings indicate that variation at the Or22 locus leads to changes in olfactory-driven behaviours, and add support to the idea that the ab3A neurons are of especial importance to the ecology of Drosophila flies.

Application of a Microfluidic Gas-to-Liquid Interface for Extraction of Target Amphetamines and Precursors from Air Samples.

The investigation of clandestine laboratories poses serious hazards for first responders, emergency services, investigators and the surrounding public due to the risk of exposure to volatile organic compounds (VOCs) used in the manufacture of illicit substances. A novel gas sampling interface using open microfluidic channels that enables the extraction of VOCs out of the gas phase and into a liquid, where it can be analysed by conventional detection systems, has recently been developed. This paper investigates the efficiency and effectiveness of such a gas-to-liquid (GTL) extraction system for the extraction of amphetamine-type substances (ATS) and their precursors from the vapour phase. The GTL interface was evaluated across a range of different ATS and their precursors (methamphetamine, dimethylamphetamine, N-formylmethamphetamine, benzaldehyde, phenyl-2-propanone, ephedrine and pseudoephedrine) at concentrations ranging between 10 and 32 mg m-3. These gas samples were produced by a gas generation system directly in Tedlar® bags and gas canisters for controlled volume sampling. When using gas sampled from Tedlar® bags, four of the seven compounds were able to be extracted by the GTL interface, with the majority of the VOCs having extraction yields between 0.005% and 4.5%, in line with the results from an initial study. When samples were taken from gas canisters, only benzaldehyde was able to be detected, with extraction efficiencies between 0.2% and 0.4%. A custom-built mount for the GTL interface helped to automate the extraction process, with the aim of increasing extraction efficiency or reducing variability. However, the extraction efficiency did not improve when using this accessory, but the procedure did become more efficient. The results from the study indicated that the GTL interface could be employed for the collection of gaseous ATS and incorporated into mobile detection systems for onsite collection and analysis of volatile compounds related to ATS manufacture.

Development and characterisation of a compact device for rapid real-time-on-chip detection of thrombin activity in human serum using bioluminescence resonance energy transfer (BRET).

Bioluminescence resonance energy transfer (BRET) is a sensitive optical detection method that can monitor changes in the relative orientation and the physical proximity of molecules in real-time. Since the light is generated internally by a bioluminescent protein, BRET does not rely on an external light source. The use of BRET simultaneously simplifies the hardware required for sensing and offers improved detection limits and sensitivity for applications targeting point-of-care bio-sensing. In this paper, we report a compact micro reactor integrating a thermostat with a re-useable glass-chip comprising a chaotic mixer, an incubation channel and optical detection chamber. The device was optimised to detect thrombin activities in serum, achieving a thrombin detection limit of 38 µU/µl in 10% (v/v) human serum in a 5 min assay time. This is a 90% assay time reduction, compared with previous BRET-based work or other technologies. It matches sensitivity levels achieved when the assay is deployed on a commercially available plate-reader. The device can be used continuously with low concentrations (3.4 µM) of luciferase substrate. The low cost associated with this approach, low interference from human serum and other proteases and good reproducibility (CV = 0.2-3.6%), establish new performance standards for point-of-care diagnostics with samples of human serum. Importantly, measuring protease activity levels, rather than concentrations, is the most informative approach for clinical diagnostics. Of the recently reported ultra-sensitive thrombin sensing techniques, this is the only one to measure thrombin activity in serum dilutions, rather than simply quantifying thrombin concentrations.

A phylogenomics approach to characterizing sensory neuron membrane proteins (SNMPs) in Lepidoptera.

Sensory neuron membrane proteins (SNMPs) play a critical role in the insect olfactory system but there is a deficit of functional studies beyond Drosophila. Here, we use a combination of available genome sequences, manual curation, genome and transcriptome data, phylogenetics, expression profiling and gene knockdown to investigate SNMP superfamily in various insect species with a focus on Lepidoptera. We curated 81 genes from 36 insect species and identified a novel lepidopteran SNMP gene family, SNMP3. Phylogenetic analysis shows that lepidopteran SNMP3, but not the previously annotated lepidopteran SNMP2, is the true homologue of the dipteran SNMP2. Digital expression, microarray and qPCR analyses show that the lepidopteran SNMP1 is specifically expressed in adult antennae. SNMP2 is widely expressed in multiple tissues while SNMP3 is specifically expressed in the larval midgut. Microarray analysis suggest SNMP3 may be involved in the silkworm immunity response to virus and bacterial infections. We functionally characterized SNMP1 in the silkworm using RNA interference (RNAi) and behavioral assays. Our results suggested that Bombyx mori SNMP1 is a functional orthologue of the Drosophila melanogaster SNMP1 and plays a critical role in pheromone detection. Split-ubiquitin yeast hybridization study shows that BmorSNMP1 has a protein-protein interaction with the pheromone receptor (BmorOR1), and the co-receptor (BmorOrco). Concluding, we propose a novel molecular model in which BmorOrco, BmorSNMP1 and BmorOR1 form a heteromer in the detection of the silkworm sex pheromone bombykol.

Odorant receptors from the light brown apple moth (Epiphyas postvittana) recognize important volatile compounds produced by plants.

Moths recognize a wide range of volatile compounds, which they use to locate mates, food sources, and oviposition sites. These compounds are recognized by odorant receptors (OR) located within the dendritic membrane of sensory neurons that extend into the lymph of sensilla, covering the surface of insect antennae. We have identified 3 genes encoding ORs from the tortricid moth, Epiphyas postvittana, a pest of horticulture. Like Drosophila melanogaster ORs, they contain 7 transmembrane helices with an intracellular N-terminus, an orientation in the plasma membrane opposite to that of classical GPCRs. EpOR2 is orthologous to the coreceptor Or83b from D. melanogaster. EpOR1 and EpOR3 both recognize a range of terpenoids and benzoates produced by plants. Of the compounds tested, EpOR1 shows the best sensitivity to methyl salicylate [EC(50) = 1.8 x 10(-12) M], a common constituent of floral scents and an important signaling compound produced by plants when under attack from insects and pathogens. EpOR3 best recognizes the monoterpene citral to low concentrations [EC(50) = 1.1 x 10(-13) M]. Citral produces the largest amplitude electrophysiological responses in E. postvittana antennae and elicits repellent activity against ovipositing female moths. Orthologues of EpOR3 were found across 6 families within the Lepidoptera, suggesting that the ability to recognize citral may underpin an important behavior.