Walter S. Leal

Odorant Reception in Insects: Roles of Receptors, Binding Proteins, and Degrading Enzymes

Our knowledge of the molecular basis of odorant reception in insects has grown exponentially over the past decade. Odorant receptors (ORs) from moths, fruit flies, mosquitoes, and the honey bees have been deorphanized, odorant-degrading enzymes (ODEs) have been isolated, and the functions of odorant-binding proteins (OBPs) have been unveiled. OBPs contribute to the sensitivity of the olfactory system by transporting odorants through the sensillar lymph, but there are competing hypotheses on how they act at the end of the journey. A few ODEs that have been demonstrated to degrade odorants rapidly may act in signal inactivation alone or in combination with other molecular traps. Although ORs in Drosophila melanogaster respond to multiple odorants and seem to work in combinatorial code involving both periphery and antennal lobes, reception of sex pheromones by moth ORs suggests that their labeled lines rely heavily on selectivity at the periphery.

Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein–bombykol complex

BACKGROUND Insects use volatile organic molecules to communicate messages with remarkable sensitivity and specificity. In one of the most studied systems, female silkworm moths (Bombyx mori) attract male mates with the pheromone bombykol, a volatile 16-carbon alcohol. In the male moths antennae, a pheromone-binding protein conveys bombykol to a membrane-bound receptor on a nerve cell. The structure of the pheromone-binding protein, its binding and recognition of bombykol, and its full role in signal transduction are not known. RESULTS The three-dimensional structure of the B. mori pheromone-binding protein with bound bombykol has been determined by X-ray diffraction at 1.8 A resolution. CONCLUSIONS The pheromone binding protein of B. mori has six helices, and bombykol binds in a completely enclosed hydrophobic cavity formed by four antiparallel helices. Bombykol is bound in this cavity through numerous hydrophobic interactions, and sequence alignments suggest critical residues for specific pheromone binding.

NMR structure reveals intramolecular regulation mechanism for pheromone binding and release

Odorants are transmitted by small hydrophobic molecules that cross the aqueous sensillar lymph surrounding the dendrites of the olfactory neurons to stimulate the olfactory receptors. In insects, the transport of pheromones, which are a special class of odorants, is mediated by pheromone-binding proteins (PBPs), which occur at high concentrations in the sensillar lymph. The PBP from the silk moth Bombyx mori (BmPBP) undergoes a pH-dependent conformational transition between the forms BmPBPA present at pH 4.5 and BmPBPB present at pH 6.5. Here, we describe the NMR structure of BmPBPA, which consists of a tightly packed arrangement of seven α-helices linked by well defined peptide segments and knitted together by three disulfide bridges. A scaffold of four α-helices that forms the ligand binding site in the crystal structure of a BmPBP–pheromone complex is preserved in BmPBPA. The C-terminal dodecapeptide segment, which is in an extended conformation and located on the protein surface in the pheromone complex, forms a regular helix, α7, which is located in the pheromone-binding site in the core of the unliganded BmPBPA. Because investigations by others indicate that the pH value near the membrane surface is reduced with respect to the bulk sensillar lymph, the pH-dependent conformational transition of BmPBP suggests a novel physiological mechanism of intramolecular regulation of protein function, with the formation of α7 triggering the release of the pheromone from BmPBP to the membrane-standing receptor.

Conformational change in the pheromone-binding protein from Bombyx mori induced by pH and by interaction with membranes.

The pheromone-binding protein (PBP) fromBombyx mori was expressed in Escherichia coliperiplasm. It specifically bound radiolabeled bombykol, the natural pheromone for this species. It appeared as a single band both in native and SDS-polyacrylamide gel electrophoresis and was also homogeneous in most chromatographic systems. However, in ion-exchange chromatography, multiple forms sometimes appeared. Attempts to separate them revealed that they could be converted into one another. Analysis of the protein by circular dichroism and fluorescence spectroscopy demonstrated that its tertiary structure was sensitive to pH changes and that a dramatic conformational transition occurred between pH 6.0 and 5.0. This high sensitivity to pH contrasted markedly with its thermal stability and resistance to denaturation by urea. There was also no significant change in CD spectra in the presence of the pheromone. The native protein isolated from male antennae displayed the same changes in its spectroscopic properties as the recombinant material, demonstrating that this phenomenon is not an artifact arising from the expression system. This conformational transition was reproduced by interaction of the protein with anionic (but not neutral) phospholipid vesicles. Unfolding of the PBP structure triggered by membranes suggests a plausible mechanism for ligand release upon interaction of the PBP-pheromone complex with the surface of olfactory neurons. This pH-linked structural flexibility also explains the heterogeneity reported previously for B. mori PBP and other members of this class of proteins.

Mosquitoes smell and avoid the insect repellent DEET

The insect repellent DEET is effective against a variety of medically important pests, but its mode of action still draws considerable debate. The widely accepted hypothesis that DEET interferes with the detection of lactic acid has been challenged by demonstrated DEET-induced repellency in the absence of lactic acid. The most recent hypothesis suggests that DEET masks or jams the olfactory system by attenuating electrophysiological responses to 1-octen-3-ol. Our research shows that mosquitoes smell DEET directly and avoid it. We performed single-unit recordings from all functional ORNs on the antenna and maxillary palps of Culex quinquefasciatus and found an ORN in a short trichoid sensillum responding to DEET in a dose-dependent manner. The same ORN responded with higher sensitivity to terpenoid compounds. SPME and GC analysis showed that odorants were trapped in conventional stimulus cartridges upon addition of a DEET-impregnated filter paper strip thus leading to the observed reduced electrophysiological responses, as reported elsewhere. With a new stimulus delivery method releasing equal amounts of 1-octen-3-ol alone or in combination with DEET we found no difference in neuronal responses. When applied to human skin, DEET altered the chemical profile of emanations by a “fixative” effect that may also contribute to repellency. However, the main mode of action is the direct detection of DEET as indicated by the evidence that mosquitoes are endowed with DEET-detecting ORNs and corroborated by behavioral bioassays. In a sugar-feeding assay, both female and male mosquitoes avoided DEET. In addition, mosquitoes responding only to physical stimuli avoided DEET.

Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori

Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris‐(2‐carboxyethyl)phosphine, followed by digestion with endoproteinase Lys‐C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on‐line liquid chromatography‐electrospray ionization mass spectrometry (LC/ESI‐MS) and protein sequencing supported the assignment of disulfide bridges at Cys‐19‐Cys‐54, Cys‐50‐Cys‐108 and Cys‐97‐Cys‐117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.

Acute olfactory response of Culex mosquitoes to a human- and bird-derived attractant

West Nile virus, which is transmitted by Culex mosquitoes while feeding on birds and humans, has emerged as the dominant vector borne disease in North America. We have identified natural compounds from humans and birds, which are detected with extreme sensitivity by olfactory receptor neurons (ORNs) on the antennae of Culex pipiens quinquefasciatus (Cx. quinquefasciatus). One of these semiochemicals, nonanal, dominates the odorant spectrum of pigeons, chickens, and humans from various ethnic backgrounds. We determined the specificity and sensitivity of all ORN types housed in different sensilla types on Cx. quinquefasciatus antennae. Here, we present a comprehensive map of all antennal ORNs coding natural ligands and their dose-response functions. Nonanal is detected by a large array of sensilla and is by far the most potent stimulus; thus, supporting the assumption that Cx. quinquefasciatus can smell humans and birds. Nonanal and CO2 synergize, thus, leading to significantly higher catches of Culex mosquitoes in traps baited with binary than in those with individual lures.

Green leaf volatile-detecting olfactory receptor neurones display very high sensitivity and specificity in a scarab beetle

In the Japanese scarab beetle, Phyllopertha diversa, olfactory receptor neurones specific for the detection of so‐called general green leaf volatiles (GLV) display a high specificity and sensitivity. Three main types of green‐leaf‐volatile‐detecting receptor neurones specific to (Z)‐3‐hexenyl acetate, (E)‐2‐hexenal and (Z)‐3‐hexenol, respectively, were identified. Each type responded at a very low stimulus concentration to the key stimulus, and required at least a thousand‐fold increase in concentration to respond to any of the other GLVs tested. Flower‐odour‐and pheromone‐detecting receptor neurones were also identified. Olfactory sensilla housing plant‐odour‐ or pheromone‐detecting receptor neurones displayed clear morphological differences, and were also separated into different antennal regions.

Multifunctional communication inRiptortus clavatus (Heteroptera: Alydidae): Conspecific nymphs and egg parasitoidOoencyrtus nezarae use the same adult attractant pheromone as chemical cue

The bean bug,Riptortus clavatus lays scattered eggs (as opposed to the egg masses of pentatomids) on host as well as nonhost plants. Therefore, the first feeding stage (second-instar) nymphs emerging from eggs laid on nonhost plants need a signal that enables them to locate a food source at the lowest energy cost. Male-released (E)-2-hexenyl (E)-2-hexenoate, (E)-2-hexenyl (Z)-3-hexenoate, and myristyl isobutyrate play the double role of attractant pheromone for adults as well as aggregation pheromone, which enables the second-instar nymphs to find the host food plant. These male-specific semiochemicals are released only when foodstuff is available. On the other hand, females ofOoencyrtus nezarae, the most effective parasitoid of the host in Kumamoto, Japan (where the field experiments were conducted), utilize these semiochemicals as kairomones in order to locate the potential host community. Field experiments revealed that the synthetic pheromone rivaled 10 live males in the attraction of adults and second-instar nymphs. Captures of the egg parasitoidO. nezarae females in cylindrical sticky traps were significantly higher in traps baited with the synthetic semiochemicals than in control traps. The number of females captured was significantly higher than the number of males, although the captures in the sticky suction trap system revealed that the populations of male and female were not significantly different.

Pheromone reception in fruit flies expressing a moth's odorant receptor

We have expressed a male-specific, pheromone-sensitive odorant receptor (OR), BmorOR1, from the silkworm moth Bombyx mori in an “empty neuron” housed in the ab3 sensilla of a Drosophila Δhalo mutant. Single-sensillum recordings showed that the BmorOR1-expressing neurons in the transgenic flies responded to the B. mori pheromone bombykol, albeit with low sensitivity. These transgenic flies responded to lower doses of bombykol in an altered stimulation method with direct delivery of pheromone into the sensillum milieu. We also expressed a B. mori pheromone-binding protein, BmorPBP, in the BmorOR1-expressing ab3 sensilla. Despite the low levels of BmorPBP expression, flies carrying both BmorOR1 and BmorPBP showed significantly higher electrophysiological responses than BmorOR1 flies. Both types of BmorOR1-expressing flies responded to bombykol, and to a lesser extent to a second compound, bombykal, even without the addition of organic solvents to the recording electrode buffer. When the semiochemicals were delivered by the conventional puffing of stimulus on the antennae, the receptor responded to bombykol but not to bombykal. The onset of response was remarkably slow, and neural activity extended for an unusually long time (>1 min) after the end of stimulus delivery. We hypothesize that BmorOR1-expressing ab3 sensilla lack a pheromone-degrading enzyme to rapidly inactivate bombykol and terminate the signal. We also found an endogenous receptor in one of the sensillum types on Drosophila antenna that responds to bombykol and bombykal with sensitivity comparable to the pheromone-detecting sensilla on B. mori male antennae.