Glucosinolates on the leaf surface perceived by insect herbivores: review of ambiguous results and new investigations

Herbivorous insects identify their host plants either by structural features, chemical cues, or a combination. Some insects probe the host leaf prior feeding or oviposition, other species use olfactorial cues or compounds somewhere on the surface. Insects attacking Brassicaceae are no exception, some are attracted and stimulated by volatile isothiocyanates (ITC), many others depend fully on the non-volatile glucosinolates (GS) for host-plant recognition and acceptance. Since most insects have no access to the leaf interior investigators concluded that GS must be present on the leaf surface and ITC in the headspace. However, peelings of mechanically removed surface waxes were devoid of measurable amounts of GS, whereas solvent surface extractions revealed a correlation between stomatal conditions and GS concentrations. Both observations lead to the conclusion that the presence of GS on the top leaf surface is rather unlikely. In the experimental part we show that a chloroform/methanol/water (2:1:1 vol/vol/vol) solvent leaf extract contains GS and, in addition, thia-triaza-fluorenes (TTF), other oviposition stimulants of the cabbage root fly, Delia radicum. Electrophysiological investigations showed that both, GS and TTF stimulated specific receptor neurones of the fly. We suggest that these compounds probably originated from deeper leaf layers and that herbivorous insects may penetrate the wax layer and perceive the stimulating compounds in deeper layers or through the stomata.     

Representation of binary pheromone blends by glomerulus-specific olfactory projection neurons

An outstanding challenge in olfactory neurobiology is to explain how glomerular networks encode information about stimulus mixtures, which are typical of natural olfactory stimuli. In the moth Manduca sexta, a species-specific blend of two sex-pheromone components is required for reproductive signaling. Each component stimulates a different population of olfactory receptor cells that in turn target two identified glomeruli in the macroglomerular complex of the males antennal lobe. Using intracellular recording and staining, we examined how responses of projection neurons innervating these glomeruli are modulated by changes in the level and ratio of the two essential components in stimulus blends. Compared to projection neurons specific for one component, projection neurons that integrated information about the blend (received excitatory input from one component and inhibitory input from the other) showed enhanced ability to track a train of stimulus pulses. The precision of stimulus-pulse tracking was furthermore optimized at a synthetic blend ratio that mimics the physiological response to an extract of the females pheromone gland. Optimal responsiveness of a projection neuron to repetitive stimulus pulses therefore appears to depend not only on stimulus intensity but also on the relative strength of the two opposing synaptic inputs that are integrated by macroglomerular complex projection neurons.     

Responses of olfactory receptor neurons in the spiny lobster to binary mixtures are predictable using a noncompetitive model that incorporates excitatory and inhibitory transduction pathways

Coding of binary mixtures by a population of olfactory receptor neurons in the spiny lobster (Panulirus argus) was examined. Extracellular single-unit responses of 50 neurons to seven compounds and their binary mixtures were recorded. The ability of a noncompetitive model with correction for binding inhibition to predict responses to mixtures based on responses to their components was compared with the predictive abilities of other models. This model assumes that different compounds activate different transduction processes in the same neuron leading to excitation or inhibition, and it includes a term quantifying the degree to which binding of an odorant to its receptor sites is inhibited by other compounds. The model accurately predicted the absolute response magnitude of the population of neurons for 13 of 15 mixtures assessed, which is superior to the predictive power of any of the other models. The model also accurately predicted the across neuron patterns generated by the binary mixtures, as evaluated by multidimensional scaling analysis. The results suggest that there is no emergence of unique qualities for binary mixtures relative to components of these mixtures.Abbreviations AMP or A adenosine-5-monophosphate - ANP across neuron pattern - ARM absolute response magnitude - ASW artificial sea water - Bet or B betaine - Cys or C L-cysteine - Glu or G L-glutamate - MDS multidimensional scaling - MID mixture interaction distance - NC model noncompetitive model - NCBI model noncompetitive model with correction for binding inhibition - C model competitive model - CBI model competitive model with correction for binding inhibition - MEC more effective component - NH ₄ or N ammonium chloride - ORN olfactory receptor neuron - Suc or S DL-succinate - Tau or T taurine     

Boundary-layer effect on chemical signal movement near the antennae of the sphinx moth, Manduca sexta : temporal filters for olfaction

For olfaction to occur, signal molecules must move through the environment from the source to the receptor cells. As molecules approach receptor structures they pass through a boundary layer surrounding those receptor structures. Within boundary layers the interaction between the forces causing chemical dispersion changes. To investigate how the boundary layer changes the dynamics of the chemical signals, we measured chemical dynamics within the boundary layer around the moth antennae using microelectrodes. The results showed that the boundary layer amplified three aspects of the chemical signal: peak height, peak onset, and decay time. Spectral analysis of turbulent signals showed that the temporal aspects of the chemical signal were altered. The boundary layers around the male and female antennae have different effects on the spectrum of chemical temporal fluctuations. Specifically, at a flow speed of 0.12 m s⁻¹, the analysis showed distinct amplification patterns for each sex. Thus, the fluid flow around the antennae functions as a filter, altering the structure of the chemical signal that is arriving at the receptors. The results illustrated in this study show that male and female moths have different physical filters that can alter the information that can be extracted from odor plumes. Accepted: 1 September 1997     

Direct nuclear magnetic resonance observation of odorant binding to mouse odorant receptor MOR244-3

Mammals are able to perceive and differentiate a great number of structurally diverse odorants through the odorant's interaction with odorant receptors (ORs), proteins found within the cell membrane of olfactory sensory neurons. The natural gas industry has used human olfactory sensitivity to sulfur compounds (thiols, sulfides, etc.) to increase the safety of fuel gas transport, storage, and use through the odorization of this product. In the United States, mixtures of sulfur compounds are used, but the major constituent of odorant packages is 2-methylpropane-2-thiol, also known as tert-butyl mercaptan. It has been fundamentally challenging to understand olfaction and odorization due to the low affinity of odorous ligands to the ORs and the difficulty in expressing a sufficient number of OR proteins. Here, we directly observed the binding of tert-butyl mercaptan and another odiferous compound, cis-cyclooctene, to mouse OR MOR244-3 on living cells by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. This effort lays the groundwork for resolving molecular mechanisms responsible for ligand binding and resulting signaling, which in turn will lead to a clearer understanding of odorant recognition and competition.     

Intensity and the ratios of compounds in the scent of snapdragon flowers affect scent discrimination by honeybees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Floral scent is used by pollinators during foraging to identify and discriminate among flowers. The ability to discriminate among scents may depend on both scent intensity and the ratios of the concentrations of the volatile compounds of a complex mixture rather than on the presence of a few compounds. We used four scent-emitting cultivars of snapdragon (Antirrhinum majus) to test this hypothesis by examining the ability of honeybees to differentiate among their scents. Each cultivar produced three monoterpenes (myrcene, E--ocimene, and linalool) and five phenylpropanoids (methylbenzoate, acetophenone, dimethoxytoluene, cis-methylcinnamate, and trans-methylcinnamate). Cultivars were reliably classified by their scents in a canonical discriminant analysis. Honeybees were unable to discriminate among the scents of flowers of the same cultivar in our assay. The ability of honeybees to discriminate among the scents of different cultivars was a function of the intensity of the floral scent. Discrimination was also correlated to the distance among the scents described by the discriminant analysis; the cultivars that had the greatest differences observed in the discriminant analysis were the easiest to discriminate. Our results show that honeybees are capable of using all of the floral volatiles to discriminate subtle differences in scent.     

Geometric considerations of nitric oxide-cyclic GMP signalling in the glomerular neuropil of the locust antennal lobe

Using NADPH-diaphorase staining as a marker for nitric oxide (NO) synthase and an antiserum against cyclic GMP, we recently reported the anatomical distribution of nitric oxide donor and target cells in the antennal lobe, the principal olfactory neuropile of the locust. The most striking NADPH-diaphorase activity in the olfactory pathway is concentrated in a cluster of intensely stained local interneurons innervating the glomeruli. After incubation of tissue in a nitric oxide donor and inhibition of phospodiesterase activity, neurons of this cluster expressed cyclic GMP-immunoreactivity in the cell body and neurites. Here we examine the importance of the arrangement of NO donor and target cells for information processing in the glomeruli. The cellular organization of the NO-cyclic GMP system in olfactory interneurons, and the dendritic branching pattern, suggest that nitric oxide may not only act as intercellular, but also as intracellular messenger molecule in the glomerular neuropile of the antennal lobe. <br>     

ORA1, a Zebrafish Olfactory Receptor Ancestral to All Mammalian V1R Genes,Recognizes 4-Hydroxyphenylacetic Acid,a Putative Reproductive Pheromone

The teleost v1r-related ora genes are a small, highly conserved olfactory receptor gene family of only six genes, whose direct orthologues can be identified in lineages as far as that of cartilaginous fish. However, no ligands for fish olfactory receptor class A related genes (ORA) had been uncovered so far. Here we have deorphanized the ORA1 receptor using heterologous expression and calcium imaging. We report that zebrafish ORA1 recognizes with high specificity and sensitivity 4-hydroxyphenylacetic acid. The carboxyl group of this compound is required in a particular distance from the aromatic ring, whereas the hydroxyl group in the para-position is not essential, but strongly enhances the binding efficacy. Low concentrations of 4-hydroxyphenylacetic acid elicit increases in oviposition frequency in zebrafish mating pairs. This effect is abolished by naris closure. We hypothesize that 4-hydroxyphenylacetic acid might function as a pheromone for reproductive behavior in zebrafish. ORA1 is ancestral to mammalian V1Rs, and its putative function as pheromone receptor is reminiscent of the role of several mammalian V1Rs as pheromone receptors.