Stephen C. Trowell

Female‐biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori

Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition. Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla. However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide. We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera.

Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain.

The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochrome-P450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species, and the corresponding absence of data on rates of insecticide metabolism by functionally expressed P450s. To facilitate P450 gene isolation from H. armigera we used degenerate primers in the reverse transcriptase-polymerase chain reaction (RT-PCR) to clone P450 gene fragments from the RNA of a pyrethroid-resistant strain. Here we report the isolation of eight new P450 genes: seven from the CYP4 family and one CYP9. One of these genes, CYP4G8, is two-fold over-expressed in the resistant strain, whereas the other CYP4s showed either similar or undetectable levels of expression. CYP9A3 appears to be a homolog of the putatively resistance-associated CYP9A1 of Heliothis virescens. However, no difference in expression between the H. armigera strains was detected. CYP6B2, a gene previously reported to be over-expressed in a different pyrethroid-resistant strain of H. armigera, also revealed non-detectable levels of expression in both strains. These observations suggest that different P450s may be over-expressed in different resistant strains, and emphasize that recombinant expression will be necessary in order to define precisely their individual substrate specificities and ability to metabolize pyrethroids. The gene fragments described here represent an important first step in this direction.

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.

Molecular basis of female-specific odorant responses in Bombyx mori

Males and females of many moth species exhibit important differences in sexual behaviours. Much research in this field has focused on the male-specific behaviour, electrophysiology and molecular biology of sex pheromone reception. Female-specific behaviours have been less well studied although, like male-specific behaviours, they could provide opportunities for intervention and management of lepidopteran pests. Previously, we identified genes encoding putative odorant receptors (ORs) from the genome of the silkworm, Bombyx mori, some of which have higher levels of steady-state transcript in the antennae of adult females compared with males. We have identified the full-length cDNA sequences of some of these ORs and described a novel OR that is part of a female-biased clade. Using expression in Sf9 cells and a calcium-imaging assay, we tested a range of compounds for their ability to activate the most highly female-biased ORs, BmOR19, BmOR30, BmOR45 and BmOR47. BmOR19 responds to linalool, while BmOR45 and BmOR47 respond to benzoic acid > 2-phenylethanol > benzaldehyde. No activating ligands were found for BmOR30. RNA in situ hybridisation experiments reveal that BmOR19 is expressed in female olfactory sensory neurons that are co-located in the same sensilla as a second ORN expressing BmOR45 and/or BmOR47. Taken together the activity and expression of these receptors is likely explanatory of the observed electrophysiology of long sensilla trichoidea of female B. mori, previously shown to each contain one terpene (BmOR19) and one benzoic acid (BmOR45, BmOR47) sensory neuron. Plant volatiles such as linalool, benzoic acid, 2-phenylethanol and benzaldehyde are oviposition cues for females of some moths. These compounds have also been found in male-produced pheromone blends extracted from the hair pencils of many noctuid species. Hair pencil structures have not previously been reported for B. mori, but we have found hair pencil-like structures in adult male B. mori that are absent in female moths. It is proposed that BmOR19, BmOR45 and BmOR47 account for some of the female-specific odorant responses in B. mori, such as oviposition and/or detection of an as yet unidentified male-produced sex pheromone.

Topological and Functional Characterization of an Insect Gustatory Receptor

Insect gustatory receptors are predicted to have a seven-transmembrane structure and are distantly related to insect olfactory receptors, which have an inverted topology compared with G-protein coupled receptors, including mammalian olfactory receptors. In contrast, the topology of insect gustatory receptors remains unknown. Except for a few examples from Drosophila, the specificity of individual insect gustatory receptors is also unknown. In this study, the total number of identified gustatory receptors in Bombyx mori was expanded from 65 to 69. BmGr8, a silkmoth gustatory receptor from the sugar receptor subfamily, was expressed in insect cells. Membrane topology studies on BmGr8 indicate that, like insect olfactory receptors, it has an inverted topology relative to G protein-coupled receptors. An orphan GR from the bitter receptor family, BmGr53, yielded similar results. We infer, from the finding that two distantly related BmGrs have an intracellular N-terminus and an odd number of transmembrane spans, that this is likely to be a general topology for all insect gustatory receptors. We also show that BmGr8 functions independently in Sf9 cells and responds in a concentration-dependent manner to the polyalcohols myo-inositol and epi-inositol but not to a range of mono- and di-saccharides. BmGr8 is the first chemoreceptor shown to respond specifically to inositol, an important or essential nutrient for some Lepidoptera. The selectivity of BmGr8 responses is consistent with the known responses of one of the gustatory receptor neurons in the lateral styloconic sensilla of B. mori, which responds to myo-inositol and epi-inositol but not to allo-inositol.

Geographical origin of Sauvignon Blanc wines predicted by mass spectrometry and metal oxide based electronic nose.

Analysis of 34 Sauvignon Blanc wine samples from three different countries and six regions was performed by gas chromatography-mass spectrometry (GC-MS). Linear discriminant analysis (LDA) showed that there were three distinct clusters or classes of wines with different aroma profiles. Wines from the Loire region in France and Australian wines from Tasmania and Western Australia were found to have similar aroma patterns. New Zealand wines from the Marlborough region as well as the Australian ones from Victoria were grouped together based on the volatile composition. Wines from South Australia region formed one discrete class. Seven analytes, most of them esters, were found to be the relevant chemical compounds that characterized the classes. The grouping information obtained by GC-MS, was used to train metal oxide based electronic (MOS-Enose) and mass spectrometry based electronic (MS-Enose) noses. The combined use of solid phase microextraction (SPME) and ethanol removal prior to MOS-Enose analysis, allowed an average error of prediction of the regional origins of Sauvignon Blanc wines of 6.5% compared to 24% when static headspace (SHS) was employed. For MS-Enose, the misclassification rate was higher probably due to the requirement to delimit the m/z range considered.

Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET)

Our goal is to develop a general transduction system for G-protein coupled receptors (GPCRs). GPCRs are present in most eukaryote cells and transduce diverse extracellular signals. GPCRs comprise not only the largest class of integral membrane receptors but also the largest class of targets for therapeutic drugs. In all cases studied, binding of ligand to a GPCR leads to a sub-nanometer intramolecular rearrangement. Here, we report the creation of a novel chimaeric BRET-based biosensor by insertion of sequences encoding a bioluminescent donor and a fluorescent acceptor protein into the primary sequence of a GPCR. The BRET(2)-ODR-10 biosensor was expressed in membranes of Saccharomyces cerevisiae. Assays conducted on isolated membranes indicated an EC(50) in the femtomolar range for diacetyl. The response was ligand-specific and was abolished by a single point mutation in the receptor sequence. Novel BRET-GPCR biosensors of this type have potential application in many fields including explosive detection, quality control of food and beverage production, clinical diagnosis and drug discovery.

Experimental Determination of the Förster Distance for Two Commonly Used Bioluminescent Resonance Energy Transfer Pairs

Förster resonance energy transfer (RET) is the nonradiative transfer of energy from a donor to an acceptor fluorophore. The Förster distance (R(0)), being the fluorophore separation corresponding to 50% of the maximum RET efficiency (E(RET)), is a critical parameter for optimization of RET biosensors. Sensitive RET-based monitoring of molecular rearrangements requires that the separation of the donor and acceptor RET pair is matched to their Förster distance. Here, for the first time, we experimentally determine the Förster distance for BRET(1), R(0) = 4.4 nm, and for BRET(2), R(0) = 7.5 nm. The latter is the largest reported value for a genetically encoded RET pair.

Bio-Benchmarking of Electronic Nose Sensors

Background Electronic noses, E-Noses, are instruments designed to reproduce the performance of animal noses or antennae but generally they cannot match the discriminating power of the biological original and have, therefore, been of limited utility. The manner in which odorant space is sampled is a critical factor in the performance of all noses but so far it has been described in detail only for the fly antenna. Methodology Here we describe how a set of metal oxide (MOx) E-Nose sensors, which is the most commonly used type, samples odorant space and compare it with what is known about fly odorant receptors (ORs). Principal Findings Compared with a flys odorant receptors, MOx sensors from an electronic nose are on average more narrowly tuned but much more highly correlated with each other. A set of insect ORs can therefore sample broader regions of odorant space independently and redundantly than an equivalent number of MOx sensors. The comparison also highlights some important questions about the molecular nature of fly ORs. Conclusions The comparative approach generates practical learnings that may be taken up by solid-state physicists or engineers in designing new solid-state electronic nose sensors. It also potentially deepens our understanding of the performance of the biological system.

Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage.

In this study, a representative FRET system (CFP donor and YFP acceptor) is compared with the BRET(2) system (Renilla luciferase donor, green fluorescent protein(2) (GFP(2)) acceptor and coelenterazine 400a substrate). Cleavage of a thrombin-protease-sensitive peptide sequence inserted between the donor and acceptor proteins was detected by the RET signal. Complete cleavage by thrombin changed the BRET(2) signal by a factor of 28.9+/-0.2 (R.S.D. (relative standard deviation), n=3) and the FRET signal by a factor of 3.2+/-0.1 (R.S.D., n=3). The BRET(2) technique was 50 times more sensitive than the FRET technique for monitoring thrombin concentrations. Detection limits (blank signal+3sigma(b), where sigma(b)=the standard deviation (S.D.) of the blank signal) were calculated to be 3.05 and 0.22nM thrombin for FRET and BRET(2), respectively. This direct comparison suggests that the BRET(2) technique is more suitable than FRET for use in proximity assays such as protease cleavage assays or protein-protein interaction assays.