Identification and Cloning of a Female Antenna-specific Odorant-binding Protein in the Mosquito Culex Quinquefasciatus

Journal of Chemical Ecology. Apr, 2002  |  Pubmed ID: 12035932

We have identified and cloned a cDNA encoding the first odorant-binding protein isolated from mosquitoes. The protein isolated from female antennae of Culex quinquefasciatus (CquiOBP) was not detected in legs (control tissue) or in antennal extracts from males, and showed mobility in native polyacrylamide gels similar to that of the pheromone-binding protein from Bombyx mori. The open reading frame of the cloned cDNA encoded a hydrophobic signal peptide (24 residues) and an acid mature protein (pI 5.5) of 125 amino acid residues (calculated molecular mass 14,504 Da). The transcript was detected by RT-PCR with antennal, but not with leg tissues. CquiOPB shared the highest amino acid identity with a product deduced from Drosophila melanogaster PBPRP-3 cDNA (58.6%), OBPs from scarab beetles (35%), and moths (28%). In addition, CquiOBP showed the hallmark of insect odorant-binding proteins, the six Cys residues.

Peripheral Coding of Sex Pheromone and a Behavioral Antagonist in the Japanese Beetle, Popillia Japonica

Journal of Chemical Ecology. May, 2002  |  Pubmed ID: 12049228

Male antennae of the Japanese beetle, Popillia japonica, possess olfactory receptor neurons (ORNs) cocompartmentalized in the same sensilla placodea, one tuned to the sex pheromone, (R)-japonilure, and the other to the detection of a behavioral antagonist, (S)-japonilure. In-depth electrophysiological experiments revealed mutual inhibitory and synergistic effects in ORNs stimulated simultaneously with the two semiochemicals. The olfactory system of P japonica exhibited a remarkable ability to discriminate completely coincident strands of pheromone and behavioral antagonist from strands of the two semiochemicals temporally isolated (by 1.5-3 msec). The mutual inhibition was reflected mainly by the delay of onset or total lack of spikes and by the significant increase in the rise time of potentials generated by blends of (R)- and (S)-japonilure. In contrast, synergist ORNs showed no neural activity (spikes) when stimulated with either the sex pheromone or the behavioral antagonist, but showed clear responses to blends of the two semiochemicals. Evidence for mixture-suppressed responses was observed not only in the Japanese beetle, but also in the Osaka beetle, Anomala osakana, and the Oriental beetle, Exomala orientalis, thus suggesting that it is a common feature in the sensory physiology of scarab beetles.

NMR Structure of the Unliganded Bombyx Mori Pheromone-binding Protein at Physiological PH

FEBS Letters. Nov, 2002  |  Pubmed ID: 12417333

The nuclear magnetic resonance structure of the unliganded pheromone-binding protein (PBP) from Bombyx mori at pH above 6.5, BmPBP(B), consists of seven helices with residues 3-8, 16-22, 29-32, 46-59, 70-79, 84-100, and 107-124, and contains the three disulfide bridges 19-54, 50-108, and 97-117. This polypeptide fold encloses a large hydrophobic cavity, with a sufficient volume to accommodate the natural ligand bombykol. The polypeptide folds in free BmPBP(B) and in crystals of a BmPBP-bombykol complex are nearly identical, indicating that the B-form of BmPBP in solution represents the active conformation for ligand binding.

Cloning of Putative Odorant-degrading Enzyme and Integumental Esterase CDNAs from the Wild Silkmoth, Antheraea Polyphemus

Insect Biochemistry and Molecular Biology. Dec, 2002  |  Pubmed ID: 12429129

Odorant-degrading enzymes have been postulated to participate in the fast deactivation of insect pheromones. These proteins are expressed specifically in the sensillar lymph of insect antennae in such low amounts that, hitherto, isolation and protein-based cDNA cloning has not been possible. Using degenerate primers based on conserved amino acid sequences of insect carboxylesterases and juvenile hormone esterases, we were able to amplify partial cDNA fragments, which were then used for the design of gene-specific primers for RACE. This bioinformatics approach led us to the cloning of cDNAs, encoding a putative odorant-degrading enzyme (Apol-ODE) and a putative integumental esterase (Apol-IE) from the wild silkmoth, Antheraea polyphemus. Apol-ODE had a predicted molecular mass of 59,994 Da, pI of 6.63, three potential N-glycosylation sites, and a putative catalytic site Ser characterized by the sequence Gly(195)-Glu-Ser-Ala-Gly-Ala. Apol-IE gave calculated molecular mass of 61,694 Da, pI of 7.49, two potential N-glycosylation sites, and a putative active site with the sequence Gly(214)-Tyr-Ser-Ala-Gly. The transcript of Apol-ODE was detected by RT-PCR in male antennae and branches (sensillar tissues), but not in female antennae and other control tissues. Apol-IE was detected in male and female antennae as well as legs.

Odorant-binding Proteins from a Primitive Termite

Journal of Chemical Ecology. Sep, 2002  |  Pubmed ID: 12449514

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.

Protein That Makes Sense in the Argentine Ant

Die Naturwissenschaften. Nov, 2002  |  Pubmed ID: 12451452

With a protein-based approach, we have identified and cloned the cDNA encoding a chemosensory protein (LhumCSP) in the Argentine ant, Linepithema humile. The open reading frame of the cloned cDNA encoded a signal peptide (20 residues), and a mature protein (pI 4.62) of 106 amino acid residues. The calculated molecular mass (12,453 Da) was in agreement with the molecular mass measured by on-line chromatography-electrospray ionization mass spectrometry (12,448 Da), given the formation of two disulfide bridges. LhumCSP shared sequence similarity with various CSPs, particularly those identified and/or cloned from moth species. Also, LhumCSP showed the hallmark of the chemosensory proteins, i.e., four well conserved cysteine residues. The antennal protein was not detected in non-olfactory tissues (leg and thorax) contrary to a putative pheromone-binding protein isolated from the thorax of the red imported fire ant, Solenopsis invicta. In addition, these findings suggest that, as in Orthopterans and Phasmids, the protein that makes sense in the Argentine ant is not an odorant-binding protein, but rather a chemosensory protein.

Sex Pheromone of the Scarab Beetle Phyllophaga Elenans and Some Intriguing Minor Components

Journal of Chemical Ecology. Jan, 2003  |  Pubmed ID: 12647850

Three amino acid-derived compounds were identified in extracts from the pheromone glands of the scarab beetle Phyllophaga elenans, i.e., L-isoleucine methyl ester (LIME), N-formyl L-isoleucine methyl ester (For-LIME), and N-acetyl L-isoleucine methyl ester (Ac-LIME). The compounds were characterized from their spectral data (MS and IR), confirmed by synthesis, and their absolute configurations were assigned by gas chromatography with a chiral phase column. The amount of LIME in calling females was ca. 2 microg/gland, whereas only traces of For-LIME and Ac-LIME (0.005% of LIME) were detected in fresh extracts. Regardless of the storage temperature, the amounts of the minor constituents in the extracts increased overtime. Field tests showed that traps baited with For-LIME captured more beetles than control traps. Ac-LIME per se was not attractive, and it did not increase trap catches when combined with For-LIME. Traps baited with LIME caught ca. 150 beetles/trap/day, but catches did not increase with the addition of For-LIME and/or Ac-LIME in binary or tertiary blends.

Trapping of Phyllophaga Elenans with a Female-produced Pheromone

Journal of Chemical Ecology. Jan, 2003  |  Pubmed ID: 12647851

Attraction of Phyllophaga elenas to vaned bucket traps baited with the recently identified female-produced pheromone, L-isoleucine methyl ester (LIME), is efficient. Pheromone-baited vaned buckets with water to retain insects were more effective than buckets without vanes or plastic containers with the sides cut out. Pheromone-baited vaned bucket traps from which water was omitted required the addition of a funnel below the vanes to retain insects. Normally used light traps were about 10 times more effective than pheromone-baited vane bucket traps in capturing P. elenans. Over 95% of P. elenans were captured between 6:00 and 9:00 PM. The male-female ratio was approximately 3-4:1 in both light and pheromone traps, and the ratio was relatively unchanged throughout the capture period. Most P. elenans were captured in the treed areas surrounding sugarcane fields. More P. elenans were captured in treed borders than in grassy borders of sugarcane fields. The effective radius of the pheromone-baited vaned bucket trap is between 5 and 15 m.

Behavioral and Electrophysiological Responses of the Brownbanded Cockroach, Supella Longipalpa, to Stereoisomers of Its Sex Pheromone, Supellapyrone

Journal of Chemical Ecology. Aug, 2003  |  Pubmed ID: 12956508

Females of the brownbanded cockroach, Supella longipalpa, release a sex pheromone (supellapyrone) during a calling behavior and attract males from a distance. Supellapyrone has four possible configurations resulting from two asymmetric carbons at positions 2 and 4 (i.e., 2R,4R; 2R,4S; 2S,4R; and 2S,4S), but only the RR isomer is produced by females. Using pure synthetic stereoisomers in field tests, we showed that males are attracted to RR but also to high concentrations of the isomer SR. To study the activity of the stereoisomers in more detail we developed behavioral and electroantennogram (EAG) dose-response curves for each. Behaviorally, RR was the most active isomer with just 0.3 pg delivered on a filter paper being sufficient to elicit 50% male response in the olfactometer. Males were also attracted to SR and SS in the olfactometer, but at much higher dosages (100x ) than the natural compound; RS did not elicit behavioral responses at any of the doses tested. In EAG assays, the antenna of male S. longipalpa showed high and similar sensitivity to RR and SR, but a much lower (10%) sensitivity to SS and practically no response to RS. The lack of agreement between behavioral and electrophysiological data suggested either that RR and SR stimulate different antennal sensory neuron types, or that some aspect of the interaction between the pheromone and the sensillum environment or the receptor neuron itself is different. To test the first hypothesis we examined the response of the antenna before and after adaptation with each of the four stereoisomers. Positive cross-adaptation between RR and SR suggests that these two compounds stimulate the same receptor cells. Therefore, the lack of agreement between behavioral and EAG dose-response curves could be explained by isomer-specific molecular interactions between the pheromone and the receptor neuron. Although RR and SR produced the same EAG amplitude, stimulation with SR resulted in a slower recovery rate (i.e., wider peaks) than stimulation with RR. To gain further understanding of the response specificity of the antennae to the different stereoisomers we compared EAG responses (amplitude and recovery time) in response to individual stereoisomers and binary mixtures of isomers. These tests showed additive responses of the EAG amplitude to mixtures of compounds, but nonadditive responses of EAG recovery time. Therefore, peak height and width are independent parameters of the EAG, probably representing different intrasensillar events, and likely resulting in the expression of different behavioral responses.

Pheromone Anosmia in a Scarab Beetle Induced by in Vivo Inhibition of a Pheromone-degrading Enzyme

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2004  |  Pubmed ID: 15277687

Previous biochemical evidence suggests that a cytochrome P450 specific to male antennae of the pale-brown chafer, Phyllopertha diversa, has evolved as a pheromone-degrading enzyme. By using a bioinformatics approach, we have now cloned three P450 cDNAs: CYP4AW1, CYP4AW2, and CYP6AT1. RT-PCR indicated that CYP4AW2 is expressed in all tissues examined, that CYP6AT1 is antennae-rich, and that CYP4AW1 is antennae-specific. Both tissue specificity and electrophysiological studies strongly support that CYP4AW1 in P. diversa is a pheromone-degrading enzyme involved in pheromone inactivation. Highly sensitive, pheromone-specific olfactory receptor neurons in male antennae were completely desensitized by direct application of metyrapone into the sensillar lymph. When tested in the same or different individuals, the metyrapone treatment had no effect on olfactory receptor neurons tuned to the plant volatile (Z)-3-hexenyl acetate, which might be inactivated by an esterase. Metyrapone treatment did not affect pheromone reception in the Japanese beetle, Popillia japonica, in the scarab beetle, Anomala octiescostata, or in the Oriental beetle, Exomala orientalis. Metyrapone-induced anosmia was restricted to the pheromone detectors in P. diversa, which became insensitive to physiological concentrations of pheromones for a few minutes. As opposed to previous trials, the specificity of the inhibitor and pheromone system led to unambiguous evidence for the role of pheromone-degrading enzymes in the fast inactivation of pheromones.

Pheromone Unwrapping by PH Flip-flopping

Chemistry & Biology. Aug, 2004  |  Pubmed ID: 15324801

The Asian elephant utilizes the same sex pheromone as a number of moth species, (Z)-7-dodecen-1-yl acetate encapsulated in a serum-derived albumin. The chemical signal is emitted in the urine and received in the mucus of the trunk. The unwrapping of the package is pH mediated.

Intriguing Olfactory Proteins from the Yellow Fever Mosquito, Aedes Aegypti

Die Naturwissenschaften. Sep, 2004  |  Pubmed ID: 15338030

Four antennae-specific proteins (AaegOBP1, AaegOBP2, AaegOBP3, and AaegASP1) were isolated from the yellow fever mosquito, Aedes aegypti and their full-length cDNAs were cloned. RT-PCR indicated that they are expressed in female and, to a lesser extent, in male antennae, but not in control tissues (legs). AaegOBP1 and AaegOBP3 showed significant similarity to previously identified mosquito odorant-binding proteins (OBPs) in cysteine spacing pattern and sequence. Two of the isolated proteins have a total of eight cysteine residues. The similarity of the spacing pattern of the cysteine residues and amino acid sequence to those of previously identified olfactory proteins suggests that one of the cysteine-rich proteins (AaegOBP2) is an OBP. The other (AaegASP1) did not belong to any group of known OBPs. Structural analyses indicate that six of the cysteine residues in AaegOBP2 are linked in a similar pattern to the previously known cysteine pairing in OBPs, i.e., Cys-24-Cys-55, Cys-51-Cys-104, Cys-95-Cys-113. The additional disulfide bridge, Cys-38-Cys-125, knits the extended C-terminal segment of the protein to a predicted alpha2-helix. As indicated by circular dichroism (CD) spectra, the extra rigidity seems to prevent the predicted formation of a C-terminal alpha-helix at low pH.

Behavioral Activity of Stereoisomers and a New Component of the Contact Sex Pheromone of Female German Cockroach, Blattella Germanica

Journal of Chemical Ecology. Sep, 2004  |  Pubmed ID: 15586678

(3S,11S)-3,11-Dimethylnonacosan-2-one is a major component of the courtship stimulating, contact sex pheromone of the female German cockroach. Although the four synthetic stereoisomers of this compound have been tested in behavioral assays, their relative activity remains unresolved. Using isolated male antennae dosed with synthetic test compounds to assay male behavior, we found that at high doses all four stereoisomers elicited responses from 100% of the males. However, at physiologically relevant doses similar to those found on the female antenna, the (3S,11S)-isomer was the least effective of the four stereoisomers at eliciting courtship responses in males. This is the first example of a natural stereoisomer having less bioactivity than related stereoisomers that do not occur naturally. Another component of the sex pheromone blend, 3,11-dimethylheptacosan-2-one, was previously purified from the female's epicuticule and behaviorally assayed, but its activity was not confirmed through synthesis. We now confirm that synthetic (3S,11S)-3,11-dimethylheptacosan-2-one elicits behavioral responses, but less so than its C29 homolog.

Assignments for the Bombyx Mori Pheromone-binding Protein Fragment BmPBP(1-128) at PH 6.5

Journal of Biomolecular NMR. Jan, 2005  |  Pubmed ID: 15692741

Kinetics and Molecular Properties of Pheromone Binding and Release

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2005  |  Pubmed ID: 15784736

Transient kinetic studies have shown that the uptake of the pheromone (bombykol) of the silkworm moth (Bombyx mori), by its pheromone-binding protein (PBP) BmorPBP, proceeds with an "on" rate of 0.068 +/- 0.01 microM(-1).s(-1). With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is approximately equal to 1 ms. A pH-dependent conformational change (BmorPBP(B) --> BmorPBP(A)), associated with the release of pheromone, is a first-order reaction (k = 74.1 +/- 0.32 s(-1); t(1/2), 9.3 ms). Under physiological conditions, both reactions proceed with half-life times on the order of milliseconds, as is required for odorant-oriented navigation in insects. Molecular interactions of bombykol with both native and mutated PBPs were analyzed by a novel binding assay. A recombinant protein with the native conformation (BmorPBP) showed high binding affinity (K(D) = 105 nM) at pH 7 but low affinity (K(D) = 1,600 nM) at pH 5, when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBPDeltaP129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7, indicating that the C-terminal segment is essential for preventing binding at low pH. Binding studies with three mutated proteins (BmorPBPW37F, BmorPBPW127F, and BmorPBPW37A) showed that replacing Trp-37 (with Phe or Ala) or Trp-127 (with Phe) did not affect the binding affinity to bombykol. Fluorescence studies shed light on the contributions of Trp-37 and Trp-127 emissions to the overall fluorescence.

Coil-to-helix Transition and Ligand Release of Bombyx Mori Pheromone-binding Protein

Biochemical and Biophysical Research Communications. Oct, 2005  |  Pubmed ID: 16111659

The transport of hydrophobic insect pheromones through the aqueous medium surrounding their receptors is assisted by pheromone-binding proteins (PBPs). The protein from the silkworm moth Bombyx mori, BmorPBP, exhibits a pH-dependent conformational change postulated to trigger the release of the pheromone bombykol to its receptor. At low pH, an alpha-helix occupies the same binding pocket that houses the pheromone in the BmorPBP-bombykol complex at high pH. We have determined the crystal structure of apo BmorPBP at a resolution of 2.3 angstroms and pH 7.5, which has surprisingly a structure similar to the A-form. These data suggest that BmorPBP undergoes a ligand-dependent conformational change in addition to the previously described pH-dependent conformational change. Analysis of the alpha-helix occupying the binding pocket reveals an amphipathic helix with three acidic residues along one face that are conserved among lepidopteran PBPs and may be involved in a conformational transition of BmorPBP at the receptor membrane.

Selective and PH-dependent Binding of a Moth Pheromone to a Pheromone-binding Protein

Journal of Chemical Ecology. Oct, 2005  |  Pubmed ID: 16132337

Fluorescence and circular dichroism (CD) data suggest that the major pheromone-binding protein (PBP) from the wild silkmoth, Antheraea polyphemus, ApolPBP1, undergoes a pH-dependent conformational change similar to that previously observed for the PBP from the silkworm moth, Bombyx mori, BmorPBP. All three constituents of the sex pheromone, E6,Z11-16Ac, E6,Z11-16Ald, and E4,Z9-14Ac, bound to ApolPBP1 with apparent high affinity at high pH, but reduced binding at low pH when tested individually in a "cold binding assay." In competitive assays, however, ApolPBP1 showed considerable preference for the major constituent of the sex pheromone, E6,Z11-16Ac. These data suggest that specificity of PBPs contributes at least in part to the remarkable selectivity of moth's olfactory system.

Rapid Inactivation of a Moth Pheromone

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2005  |  Pubmed ID: 16172410

We have isolated, cloned, and expressed a male antennae-specific pheromone-degrading enzyme (PDE) [Antheraea polyphemus PDE (ApolPDE), formerly known as Sensillar Esterase] from the wild silkmoth, A. polyphemus, which seems essential for the rapid inactivation of pheromone during flight. The onset of enzymatic activity was detected at day 13 of the pupal stage with a peak at day 2 adult stage. De novo sequencing of ApolPDE, isolated from day 2 male antennae by multiple chromatographic steps, led to cDNA cloning. Purified recombinant ApolPDE, expressed by baculovirus, migrated with the same mobility as the native protein on both native polyacrylamide and isoelectric focusing gel electrophoresis. Concentration of ApolPDE (0.5 microM) in the sensillar lymph is approximately 20,000 lower than that of a pheromone-binding protein. Native and recombinant ApolPDE showed comparable kinetic parameters, with turnover number similar to that of carboxypeptidase and substrate specificity slightly lower than that of acetylcholinesterase. The rapid inactivation of pheromone, even faster than previously estimated, is kinetically compatible with the temporal resolution required for sustained odorant-mediated flight in moths.

The Crystal Structure of an Odorant Binding Protein from Anopheles Gambiae: Evidence for a Common Ligand Release Mechanism

Biochemical and Biophysical Research Communications. Jan, 2006  |  Pubmed ID: 16300742

The Anopheles gambiae mosquito is the main vector of malaria transmission in sub-Saharan Africa. We present here a 1.5A crystal structure of AgamOBP1, an odorant binding protein (OBP) from the A. gambiae mosquito. The protein crystallized as a dimer with a unique binding pocket consisting of a continuous tunnel running through both subunits of the dimer and occupied by a PEG molecule. We demonstrate that AgamOBP1 undergoes a pH dependent conformational change that is associated with reduced ligand binding. A predominance of acid-labile hydrogen bonds involving the C-terminal loop suggests a mechanism in which a drop in pH causes C-terminal loop to open, leaving the binding tunnel solvent exposed, thereby lowering binding affinity for ligand. Because proteins from two distantly related insects also undergo a pH dependent conformational change involving the C-terminus that is associated with reduced ligand affinity, our results suggest a common mechanism for OBP activity.

Identification, Synthesis, and Field Evaluation of the Sex Pheromone from the Citrus Leafminer, Phyllocnistis Citrella

Journal of Chemical Ecology. Jan, 2006  |  Pubmed ID: 16525876

Using male antenna as the sensing element, three electroantennographic detection (EAD)-active peaks were detected from pheromone gland extracts of the citrus leafminer, Phyllocnistis citrella. Based on gas chromatography (GC)-mass spectrometry and GC-infrared data, the semiochemicals were tentatively identified as a novel pheromone, (Z,Z,E)-7,11,13-hexadecatrienal, a previously identified attractant, (Z,Z)-7,11-hexadecadienal, and (Z)-7-hexadecenal in a ratio of 30:10:1, respectively. Identification was confirmed with synthetic compounds, which gave retention times identical to those of the natural products on three capillary columns with polar and nonpolar phases. While traps baited only with the previously identified attractant alone did not catch any males in Brazil, binary and tertiary mixtures with the major constituents caught significantly more male moths than traps baited with five virgin females.

Pheromone Reception in Fruit Flies Expressing a Moth's Odorant Receptor

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2006  |  Pubmed ID: 17060610

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 Deltahalo 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.

Sexual Behavior of the Navel Orangeworm, Amyelois Transitella (Walker) (Lepidoptera: Pyralidae)

Neotropical Entomology. Nov-Dec, 2006  |  Pubmed ID: 17273707

To get a better understanding of the mating behavior of the navel orangeworm, Amyelois transitella (Walker), we developed a robust laboratory colony derived from larvae collected in Bakersfield, California and fed on dried, roasted pistachio. In the lab at 25 degrees C, most of the mating activity was observed during the last hour of the scotophase and for the first 30 min of the photophase. Female calling was characterized by the abdomen being protruded between the wings with the distal segments perpendicular to the body and exposing a pheromone gland, as well as by continuous antennation. Males approached calling females from a short distance by displaying wing fanning and antennation. When a male antennated on a calling female's abdomen, she either accepted the male and lowered the abdomen, or walked away. The accepted male made a final approach parallel to the female's body, but after coupling he rotated 180 masculine with male and female remaining in a linear, abdomen-to-abdomen position for over 3 h in average. In a possible strategy to maximize the chances of mating, the sex ratio was significantly skewed towards males in the first two days of emergence. Almost 80% of mating took place in the first two days after adult emergence, with females mating only once. About 55% of males mated only once and approximately 40% of the observed males mated twice and 5% tree times.

Towards the Identification and Synthesis of the Sex Pheromone of the Citrus Leafminer, Phyllocnistis Citrella Stainton (Lepidoptera: Gracillariidae)

Neotropical Entomology. Jan-Feb, 2006  |  Pubmed ID: 17352064

The objective of this work was to characterize the sexual behavior of the citrus leafminer, Phyllocnistis citrella Stainton, as the foundation for the isolation, identification, and synthesis of the complete sex pheromone of this species. Mating occurred in a time window of 2h, starting 1h before the onset of photophase. The large majority of tested insects mated in the first two days after emergence, with no significant difference between mating at day 1 and day 2. A stereotypical courtship and copulation behavior were described for this species. When mating was successful, the copulation was recorded in average for 49.6 min. In Y-olfactometer tests conducted at the time of mating activity, males were strongly attracted to caged virgin females as well as to extracts from putative pheromone glands.

Synthesis of an Immobilized Bombyx Mori Pheromone-binding Protein Liquid Chromatography Stationary Phase

Talanta. Nov, 2006  |  Pubmed ID: 18970835

The pheromone-binding protein from the silkworm moth, Bombyx mori (BmorPBP) has been covalently bonded to a liquid chromatographic stationary phase. The resulting column was evaluated using radiolabeled bombykol and the immobilized protein retained its ability to bind this ligand. The data also demonstrate that the BmorPBP column was able to distinguish between four compounds, and rank them in their relative order of affinity for the protein from highest to lowest: bombykol>bombykal>1-hexadecanol>(Z,E)-5,7-dodecadien-1-ol, and that the immobilized BmorPBP retained its pH-dependent conformational mobility. The results of this study demonstrate that pheromone-binding protein from the silkworm moth, Bombyx mori and an odorant binding protein (OBP) obtained from the female mosquito Culex quinquefasciatoes have been immobilized on a silica support with retention of ligand-binding activity. The data indicate that proteins from non-mammalian organisms can be used to create liquid chromatography affinity columns.

Maxillary Palps Are Broad Spectrum Odorant Detectors in Culex Quinquefasciatus

Chemical Senses. Oct, 2007  |  Pubmed ID: 17569743

A single type of olfactory sensilla on maxillary palps in many species of mosquitoes houses a very sensitive olfactory receptor neuron (ORN) for carbon dioxide reception. We performed extensive single sensillum recordings from this peg sensillum in Culex quinquefasciatus and have characterized the response threshold and kinetics for CO(2) reception, with a detection threshold less than the CO(2) concentration in the atmosphere. This ORN responded in a tonic mode to lower concentrations of CO(2), whereas higher concentrations generated a phasic-tonic mode of action potential firing. Sensillum potentials accurately represented the response magnitude and kinetics of carbon dioxide-elicited excitatory responses. Stimulation of these ORNs with human breath, a complex mixture of mosquito kairomones and up to 4.5% CO(2), elicited excitatory responses that were reliably detected by CO(2)-sensitive ORNs. Another ORN housed in these sensilla responded to 1-octen-3-ol and to various plant-derived compounds, particularly floral and green leaf volatiles. This ORN showed remarkable sensitivity to the natural enantiomer, (R)-(-)-1-octen-3-ol, rivaling pheromone-detecting ORNs in moths. Maximum neuronal response was elicited with a 10 ng dose. A biological, ecological role of maxillary palps in detection of plant- and nectar-related sources is proposed.

Structural Basis of Ligand Binding and Release in Insect Pheromone-binding Proteins: NMR Structure of Antheraea Polyphemus PBP1 at PH 4.5

Journal of Molecular Biology. Nov, 2007  |  Pubmed ID: 17884092

The NMR structure of the Antheraea polyphemus pheromone-binding protein 1 at pH 4.5, ApolPBP1A, was determined at 20 degrees C. The structure consists of six alpha-helices, which are arranged in a globular fold that encapsulates a central helix alpha7 formed by the C-terminal polypeptide segment 131-142. The 3D arrangement of these helices is anchored by the three disulfide bonds 19-54, 50-108 and 97-117, which were identified by NMR. Superposition of the ApolPBP1A structure with the structure of the homologous pheromone-binding protein of Bombyx mori at pH 4.5, BmorPBPA, yielded an rmsd of 1.7 A calculated for the backbone heavy-atoms N, Calpha and C' of residues 10-142. In contrast, the present ApolPBP1A structure is different from a recently proposed molecular model for a low-pH form of ApolPBP1 that does not contain the central helix alpha7. ApolPBP1 exhibits a pH-dependent transition between two different globular conformations in slow exchange on the NMR chemical shift timescale similar to BmorPBP, suggesting that the two proteins use the same mechanism of ligand binding and ejection. The extensive sequence homology observed for pheromone-binding proteins from moth species further implies that the previously proposed mechanism of ligand ejection involving the insertion of a C-terminal helix into the pheromone-binding site is a general feature of pheromone signaling in moths.

Oviposition Responses of Gravid Female Culex Quinquefasciatus to Egg Rafts and Low Doses of Oviposition Pheromone Under Semifield Conditions

Journal of Chemical Ecology. Mar, 2007  |  Pubmed ID: 17252215

Semifield experiments were conducted to study the oviposition of gravid Culex quinquefasciatus females in response to one or 10 egg rafts, or 3.0 microg of synthetic oviposition pheromone, (-)-(5R, 6S)-6-acetoxy-5-hexadecanolide, a dose equivalent to 10 egg rafts. These treatments were added to the small bowls filled with either hay infusion or water in a small (0.3-m spacing) or a large-square design (3.4-m spacing). Oviposition choice was more pronounced in the "small square" assays. Mean number of egg rafts laid in response to a single egg raft in an oviposition jar filled with hay infusion was significantly greater than with all other treatments. When the oviposition pheromone dose was increased from one to 10 rafts or when 3.0 microg synthetic oviposition pheromone were dispensed on a floating receptacle, synergistic effects were observed between the oviposition pheromone and the hay infusion at both distances. This study is the first demonstration that the amount in a single raft induces oviposition of gravid Cx. quinquefasciatus females under semi-natural conditions.

Bombyx Mori Pheromone-binding Protein Binding Nonpheromone Ligands: Implications for Pheromone Recognition

Structure (London, England : 1993). Sep, 2007  |  Pubmed ID: 17850754

Insect pheromone-binding proteins (PBPs) transport sex pheromones through the aqueous layer surrounding G protein-coupled receptors that initiate signaling events leading to mating. This PBP-receptor system strongly discriminates between ligands with subtle structural differences, but it has proved difficult to distinguish the degree of discrimination of the PBP from that of the G protein-coupled receptor. The three-dimensional structures of the PBP of Bombyx mori, the silkworm moth, both with and without its cognate ligand bombykol ([E,Z]-10,12-hexadecadienol), have been determined by X-ray crystallography and NMR. In this paper, the structures of the same binding protein with bound iodohexadecane and bell pepper odorant were determined at 1.9 and 2.0 A, respectively. These structures illustrate the remarkable plasticity in the ligand binding site of the PBP, but suggest the protein might still act as a filter during pheromone signal processing.

Molecular Switches for Pheromone Release from a Moth Pheromone-binding Protein

Biochemical and Biophysical Research Communications. Aug, 2008  |  Pubmed ID: 18503757

Pheromone-binding proteins (PBPs) are involved in the uptake of pheromones from pores on the antennae, transport through an aqueous environment surrounding the olfactory receptor neurons, and fast delivery to pheromone receptors. We tested the hypothesis that a C-terminal segment and a flexible loop are involved in the release of pheromones to membrane-bound receptors. We expressed in Escherichia coli 11 mutants of the PBP from the silkworm moth, BmorPBP, taking into consideration structural differences between the forms with high and low binding affinity. The N-terminus was truncated and His-69, His-70 and His-95 at the base of a flexible loop, and a cluster of acidic residues at the C-terminus were mutated. Binding assays and circular dichroism analyses support a mechanism involving protonation of acidic residues Asp-132 and Glu-141 at the C-terminus and histidines, His-70 and His-95, in the base of a loop covering the binding pocket. The former leads to the formation of a new alpha-helix, which competes with pheromone for the binding pocket, whereas positive charge repulsion of the histidines opens the opposite side of the binding pocket.

Chiral Discrimination of the Japanese Beetle Sex Pheromone and a Behavioral Antagonist by a Pheromone-degrading Enzyme

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2008  |  Pubmed ID: 18579770

The sophistication of the insect olfactory system is elegantly demonstrated by the reception of sex pheromone by the Japanese beetle. In this insect, two olfactory receptor neurons housed in antennal sensilla placodea are highly sensitive. One neuron specifically detects the sex pheromone produced by conspecific females (R,Z)-5-(-)-(1-decenyl)oxacyclopentan-2-one [(R)-japonilure]. The other neuron is tuned to (S)-japonilure, a sex pheromone from a closely related species and a behavioral antagonist for the Japanese beetle. These chemical signals are enzymatically terminated by antennal esterases that open the lactone rings to form physiologically inactive hydroxyacids. We have isolated a pheromone-degrading enzyme, PjapPDE, from >100,000 antennae of the Japanese beetle. PjapPDE was demonstrated to be expressed only in the antennal tissues housing the pheromone-detecting sensilla placodea. Baculovirus expression generated recombinant PjapPDE with likely the same posttranslational modifications as the native enzyme. Kinetic studies with pure native and recombinant PjapPDE showed a clear substrate preference, with an estimated half-life in vivo for the sex pheromone and a behavioral antagonist of approximately 30 and approximately 90 ms, respectively.

Mosquitoes Smell and Avoid the Insect Repellent DEET

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2008  |  Pubmed ID: 18711137

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.

Reverse and Conventional Chemical Ecology Approaches for the Development of Oviposition Attractants for Culex Mosquitoes

PloS One. 2008  |  Pubmed ID: 18725946

Synthetic mosquito oviposition attractants are sorely needed for surveillance and control programs for Culex species, which are major vectors of pathogens causing various human diseases, including filariasis, encephalitis, and West Nile encephalomyelitis. We employed novel and conventional chemical ecology approaches to identify potential attractants, which were demonstrated in field tests to be effective for monitoring populations of Cx. p. quinquefasciatus in human dwellings. Immunohistochemistry studies showed that an odorant-binding protein from this species, CquiOBP1, is expressed in trichoid sensilla on the antennae, including short, sharp-tipped trichoid sensilla type, which house an olfactory receptor neuron sensitive to a previously identified mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide. CquiOBP1 exists in monomeric and dimeric forms. Monomeric CquiOBP1 bound MOP in a pH-dependent manner, with a change in secondary structure apparently related to the loss of binding at low pH. The pheromone antipode showed higher affinity than the natural stereoisomer. By using both CquiOBP1 as a molecular target in binding assays and gas chromatography-electroantennographic detection (GC-EAD), we identified nonanal, trimethylamine (TMA), and skatole as test compounds. Extensive field evaluations in Recife, Brazil, a region with high populations of Cx. p. quinquefasciatus, showed that a combination of TMA (0.9 microg/l) and nonanal (0.15 ng/microl) is equivalent in attraction to the currently used infusion-based lure, and superior in that the offensive smell of infusions was eliminated in the newly developed synthetic mixture.

GP-9s Are Ubiquitous Proteins Unlikely Involved in Olfactory Mediation of Social Organization in the Red Imported Fire Ant, Solenopsis Invicta

PloS One. 2008  |  Pubmed ID: 19018280

The red imported fire ant (RIFA), Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening) stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9), identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like) and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla.

Potentially Hygroreceptive Sensilla on the Anal Stylus of the Glassy-winged Sharpshooter, Homalodisca Vitripennis

Journal of Insect Science (Online). 2008  |  Pubmed ID: 20302543

Abstract This study begins to elucidate the cues and mechanisms by which the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), select host plants for feeding and oviposition. The electrophysiological response of the anal styli of male and female H. vitripennis to water vapor was examined using a modified electroantennography (EAG) device (stylogram). A strong electrophysiological response of the anal stylus to water vapor was found. Scanning electron microscopic examination of the anal stylus revealed the presence of long mechanosensory hairs, many small coeloconic-type sensilla, and masses of secretory granules termed brochosomes. Each coeloconic sensillum is located in a pit from which protrude finger-like projections. The pit is often blocked by masses of brochosomes and an unidentified dense material. Based on the electrophysiological response of the anal stylus to water vapor, we hypothesize that the coeloconic sensilla on the stylus may be hygroreceptors. H. vitripennis are xylem feeders and may use the sensilla to assist in host selection for the purpose of feeding or oviposition based on detected plant water status. Furthermore, H. vitripennis oviposit into the leaf epidermis, and may use these sensilla to evaluate moisture content to determine host suitability for both oviposition and subsequent feeding of emerged progeny. Understanding the cues and underlying mechanisms of host selection is an important consideration for predicting the movement of H. vitripennis between crops and disease epidemiology.

Genome Analysis and Expression Patterns of Odorant-binding Proteins from the Southern House Mosquito Culex Pipiens Quinquefasciatus

PloS One. 2009  |  Pubmed ID: 19606229

Olfactory-based behaviors in mosquitoes are mediated by odorant-binding proteins (OBPs). They form a multigenic family involved in the peripheral events in insect olfaction, specifically the transport of odorants to membrane-bound odorant receptors. OBPs contribute to the remarkable sensitivity of the insect's olfactory system and may be involved in the selective transport of odorants.We have employed a combination of bioinformatics and molecular approaches to identify and characterize members of the "classic" OBP family in the Southern House mosquito Culex pipiens quinquefasciatus ( = Cx. quinquefasciatus), a vector of pathogens causing several human diseases. By taking advantage of the recently released genome sequences, we have identified fifty-three putative Cx. quinquefasciatus OBP genes by Blast searches. As a first step towards their molecular characterization, expression patterns by RT-PCR revealed thirteen genes that were detected exclusively and abundantly in chemosensory tissues. No clear differences were observed in the transcripts levels of olfactory-specific OBPs between antennae of both sexes using semi-quantitative RT-PCR. Phylogenetic and comparative analysis revealed orthologous of Cx. quinquefasciatus OBPs in Anopheles gambiae and Aedes aegypti. The identification of fifty-three putative OBP genes in Cx. quinquefasciatus highlights the diversity of this family. Tissue-specificity study suggests the existence of different functional classes within the mosquito OBP family. Most genes were detected in chemosensory as well as non chemosensory tissues indicating that they might be encapsulins, but not necessarily olfactory proteins. On the other hand, thirteen "true" OBP genes were detected exclusively in olfactory tissues and might be involved specifically in the detection of "key" semiochemicals. Interestingly, in Cx. quinquefasciatus olfactory-specific OBPs belong exclusively to four distinct phylogenetic groups which are particularly well conserved among three mosquito species.

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth, Amyelois Transitella

PloS One. 2009  |  Pubmed ID: 19789654

The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control--like pheromone-based approaches--are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Acute Olfactory Response of Culex Mosquitoes to a Human- and Bird-derived Attractant

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2009  |  Pubmed ID: 19858490

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 CO(2) synergize, thus, leading to significantly higher catches of Culex mosquitoes in traps baited with binary than in those with individual lures.

(1)H, (15)N, and (13)C Chemical Shift Assignments of the Mosquito Odorant Binding Protein-1 (CquiOBP1) Bound to the Mosquito Oviposition Pheromone

Biomolecular NMR Assignments. Dec, 2009  |  Pubmed ID: 19888689

An odorant-binding protein from the Southern house mosquito, Culex pipiens quinquefasciatus (Cqui-OBP1) binds to the mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide to facilitate the transport of MOP to membrane-bound odorant receptors. We report complete NMR chemical shift assignments of Cqui-OBP1 bound to the MOP pheromone obtained at pH 7.0 and 25 degrees C (BMRB no. 16175).

Structure of an Odorant-binding Protein from the Mosquito Aedes Aegypti Suggests a Binding Pocket Covered by a PH-sensitive "Lid"

PloS One. 2009  |  Pubmed ID: 19956631

The yellow fever mosquito, Aedes aegypti, is the primary vector for the viruses that cause yellow fever, mostly in tropical regions of Africa and in parts of South America, and human dengue, which infects 100 million people yearly in the tropics and subtropics. A better understanding of the structural biology of olfactory proteins may pave the way for the development of environmentally-friendly mosquito attractants and repellents, which may ultimately contribute to reduction of mosquito biting and disease transmission.

NMR Structure of Navel Orangeworm Moth Pheromone-binding Protein (AtraPBP1): Implications for PH-sensitive Pheromone Detection

Biochemistry. Feb, 2010  |  Pubmed ID: 20088570

The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through the aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present the three-dimensional structure of a PBP from A. transitella (AtraPBP1) in solution at pH 4.5 determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments, multiangle light scattering, and (15)N NMR relaxation analysis indicate that AtraPBP1 forms a stable monomer in solution at pH 4.5 in contrast to forming mostly dimers at pH 7. The NMR structure of AtraPBP1 at pH 4.5 contains seven alpha-helices (alpha1, L8-L23; alpha2, D27-F36; alpha3, R46-V62; alpha4, A73-M78; alpha5, D84-S100; alpha6, R107-L125; alpha7, M131-E141) that adopt an overall main-chain fold similar to that of PBPs found in Antheraea polyphemus and Bombyx mori. The AtraPBP1 structure is stabilized by three disulfide bonds formed by C19/C54, C50/C108, and C97/C117 and salt bridges formed by H69/E60, H70/E57, H80/E132, H95/E141, and H123/D40. All five His residues are cationic at pH 4.5, whereas H80 and H95 become neutral at pH 7.0. The C-terminal helix (alpha7) contains hydrophobic residues (M131, V133, V134, V135, V138, L139, and A140) that contact conserved residues (W37, L59, A73, F76, A77, I94, V111, and V115) suggested to interact with bound pheromone. Our NMR studies reveal that acid-induced formation of the C-terminal helix at pH 4.5 is triggered by a histidine protonation switch that promotes rapid release of bound pheromone under acidic conditions.

Chemical Ecology of Animal and Human Pathogen Vectors in a Changing Global Climate

Journal of Chemical Ecology. Jan, 2010  |  Pubmed ID: 20119869

Infectious diseases affecting livestock and human health that involve vector-borne pathogens are a global problem, unrestricted by borders or boundaries, which may be exacerbated by changing global climate. Thus, the availability of effective tools for control of pathogen vectors is of the utmost importance. The aim of this article is to review, selectively, current knowledge of the chemical ecology of pathogen vectors that affect livestock and human health in the developed and developing world, based on key note lectures presented in a symposium on "The Chemical Ecology of Disease Vectors" at the 25th Annual ISCE meeting in Neuchatel, Switzerland. The focus is on the deployment of semiochemicals for monitoring and control strategies, and discusses briefly future directions that such research should proceed along, bearing in mind the environmental challenges associated with climate change that we will face during the 21st century.

Knockdown of a Mosquito Odorant-binding Protein Involved in the Sensitive Detection of Oviposition Attractants

Journal of Chemical Ecology. Mar, 2010  |  Pubmed ID: 20191395

Odorant-binding proteins (OBPs) were discovered almost three decades ago, but there is still considerable debate regarding their role(s) in insect olfaction, particularly due to our inability to knockdown OBPs and demonstrate their direct phenotypic effects. By using RNA interference (RNAi), we reduced transcription of a major OBP gene, CquiOBP1, in the antennae of the Southern house mosquito, Culex quinquefasciatus. Previously, we had demonstrated that the mosquito oviposition pheromone (MOP) binds to CquiOBP1, which is expressed in MOP-sensitive sensilla. Antennae of RNAi-treated mosquitoes showed significantly lower electrophysiological responses to known mosquito oviposition attractants than the antennae of water-injected, control mosquitoes. While electroantennogram (EAG) responses to MOP, skatole, and indole were reduced in the knockdowns, there was no significant difference in the EAG responses from RNAi-treated and water-injected mosquito antennae to nonanal at all doses tested. These data suggest that CquiOBP1 is involved in the reception of some oviposition attractants, and that high levels of OBPs expression are essential for the sensitivity of the insect's olfactory system.

Behavioural Neurobiology: The Treacherous Scent of a Human

Nature. Mar, 2010  |  Pubmed ID: 20203594

An Odorant Receptor from the Southern House Mosquito Culex Pipiens Quinquefasciatus Sensitive to Oviposition Attractants

PloS One. 2010  |  Pubmed ID: 20386699

Insect odorant receptors (ORs) are heteromers comprised of highly variable odorant-binding subunits associated with one conserved co-receptor. They are potential molecular targets for the development of novel mosquito attractants and repellents. ORs have been identified in the malaria mosquito, Anopheles gambiae, and in the yellow fever mosquito, Aedes aegypti. However, they are still unknown in the Southern house mosquito, Culex quinquefasciatus, which transmits pathogens that cause human diseases throughout the world, including West Nile Virus in the United States.

Bombykol Receptors in the Silkworm Moth and the Fruit Fly

Proceedings of the National Academy of Sciences of the United States of America. May, 2010  |  Pubmed ID: 20439725

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the "empty neuron" system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor.

Culex Mosquitoes (Diptera: Culicidae) Egg Laying in Traps Loaded with Bacillus Thuringiensis Variety Israelensis and Baited with Skatole

Journal of Medical Entomology. May, 2010  |  Pubmed ID: 20496581

The Southern house mosquito, Culex quinquefasciatus, is an important human health pest as a vector of several pathogens, including agents of lymphatic filariasis and arboviruses like West Nile virus. We conducted preliminary experiments in Recife, Brazil, to explore applications of Culex oviposition attractants in combination with Bacillus thuringiensis variety israelensis (Bti) in an attract-and-kill approach. Simple, cost-effective oviposition traps, BR-OVT, loaded with Bti and baited with or without attractant, were deployed in 10 homes for 30 d in 2 consecutive yr. Significantly higher numbers of egg rafts were deposited in traps baited with skatole or infusion than the control water traps. In the first year, 2006, significantly higher numbers of eggs were deposited in infusion-baited traps, particularly in the first 15 d of the experiment, than in skatole traps, but in the following year no significant difference was observed between synthetic and natural attractants. The tests strongly demonstrate that skatole or infusion can be used to enhance the number of egg rafts deposited on Bti-treated oviposition traps.

Pheromone Binding to General Odorant-binding Proteins from the Navel Orangeworm

Journal of Chemical Ecology. Jul, 2010  |  Pubmed ID: 20535533

General odorant-binding proteins (GOBPs) of moths are postulated to be involved in the reception of semiochemicals other than sex pheromones, the so-called "general odorants." We have expressed two GOBPs, AtraGOBP1 and AtraGOBP2, which were previously isolated from the antennae of the navel orangeworm, Amyelois transitella. Surprisingly, these two proteins did not bind compounds that are known to attract adult moths, particularly females. The proper folding and functionality of the recombinant proteins was inferred from circular dichroism analysis and demonstration that both GOBPs bound nonanal in a pH-dependent manner. EAG experiments demonstrated that female attractants (1-phenylethanol, propionic acid phenyl ester, and isobutyric acid phenyl ester) are detected with high sensitivity by the antennae of day-0 to day-4 adult females, with response declining in older moths. The same age-dependence was shown for male antennae responding to constituents of the sex pheromone. Interestingly, AtraGOBP2 bound the major constituent of the sex pheromone, Z11Z13-16Ald, with affinity comparable to that shown by a pheromone-binding protein, AtraPBP1. The related alcohol bound to AtraPBP1 with higher affinity than to AtraGOBP2. AtraGOBP1 bound both ligands with low but nearly the same affinity.

Evaluation of an Oviposition-stimulating Kairomone for the Yellow Fever Mosquito, Aedes Aegypti, in Recife, Brazil

Journal of Vector Ecology : Journal of the Society for Vector Ecology. Jun, 2010  |  Pubmed ID: 20618668

A synthetic mixture of an oviposition-stimulating kairomone for the yellow fever mosquito, Aedes aegypti, comprising of 83% tetradecanoic acid, 16% nonanoic acid and 1% tetradecanoic acid methyl ester (NTT, in short) was tested in a dengue endemic area in Recife, Brazil. Gravid female mosquitoes confined to a cage under semi-field conditions deposited significantly higher numbers of eggs in traps baited with NTT at doses ranging from 0.6 to 600 ng/microl than in control (water) traps. When tested in homes, egg-laying in traps baited with 60 ng NTT/microl (final concentration in trap, approximately 3.33 ng/ml) and in control traps was not significantly different, but egg deposited in traps with lower dosage (6 ng NTT/microl; final concentration in trap, approximately 0.33 ng/ml) was significantly higher than in control traps. In subsequent trials, the numbers of eggs laid in traps baited with 0.6 ng NTT/microl (final concentration in trap, approximately 0.033 ng/ml) were not significantly different from the numbers deposited in trap loaded with 6 ng NTT/microl. Egg-laying was significantly higher in these treatments than in control traps.

Odorant Receptor from the Southern House Mosquito Narrowly Tuned to the Oviposition Attractant Skatole

Journal of Chemical Ecology. Aug, 2010  |  Pubmed ID: 20623327

Oviposition attractants are environmental cues that allow Culex gravid female mosquitoes to locate suitable sites for egg-laying and, therefore, may be exploited for environmentally friendly strategies for controlling mosquito populations. Naturally occurring skatole has been identified as an oviposition attractant for the Southern House mosquito, Culex quinquefasciatus. Previously, we identified in Cx. quinquefasciatus female antennae an olfactory receptor neuron (ORN) highly sensitive to skatole and an odorant-binding protein involved in the detection of this semiochemical. Here, we describe the characterization of an odorant receptor (OR), CquiOR10, which is narrowly tuned to skatole when expressed in the Xenopus oocyte system. Odorant-induced response profiles generated by heterologously expressed CquiOR10 suggest that this OR is expressed in the mosquito ORN sensitive to skatole. However, geranylacetone, which stimulates the antennal ORN, was not detected by CquiOR10-expressing oocytes, thus raising interesting questions about reception of oviposition attractants in mosquitoes.

Crystal and Solution Structures of an Odorant-binding Protein from the Southern House Mosquito Complexed with an Oviposition Pheromone

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2010  |  Pubmed ID: 20956299

Culex mosquitoes introduce the pathogens responsible for filariasis, West Nile virus, St. Louis encephalitis, and other diseases into humans. Currently, traps baited with oviposition semiochemicals play an important role in detection efforts and could provide an environmentally friendly approach to controlling their populations. The odorant binding proteins (OBPs) in the female's antenna play a crucial, if yet imperfectly understood, role in sensing oviposition cues. Here, we report the X-ray crystallography and NMR 3D structures of OBP1 for Culex quinquefasciatus (CquiOBP1) bound to an oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide (MOP). In both studies, CquiOBP1 had the same overall six-helix structure seen in other insect OBPs, but a detailed analysis revealed an important previously undescribed feature. There are two models for OBP-mediated signal transduction: (i) direct release of the pheromone from an internal binding pocket in a pH-dependent fashion and (ii) detection of a pheromone-induced conformational change in the OBP·pheromone complex. Although CquiOBP1 binds MOP in a pH-dependent fashion, it lacks the C terminus required for the pH-dependent release model. This study shows that CquiOBP binds MOP in an unprecedented fashion using both a small central cavity for the lactone head group and a long hydrophobic channel for its tail.

Odorant-binding Proteins of the Malaria Mosquito Anopheles Funestus Sensu Stricto

PloS One. 2010  |  Pubmed ID: 21042539

The mosquito Anopheles funestus is one of the major malaria vector species in sub-Saharan Africa. Olfaction is essential in guiding mosquito behaviors. Odorant-binding proteins (OBPs) are highly expressed in insect olfactory tissues and involved in the first step of odorant reception. An improved understanding of the function of malaria mosquito OBPs may contribute to identifying new attractants/repellents and assist in the development of more efficient and environmentally friendly mosquito controlling strategies.

Extrusion of the C-terminal Helix in Navel Orangeworm Moth Pheromone-binding Protein (AtraPBP1) Controls Pheromone Binding

Biochemical and Biophysical Research Communications. Jan, 2011  |  Pubmed ID: 21130734

The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from A. transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129-142) causes more than 100-fold increase in pheromone-binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ∼2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding.

Dynamic Conformational Equilibria in the Physiological Function of the Bombyx Mori Pheromone-binding Protein

Journal of Molecular Biology. May, 2011  |  Pubmed ID: 21396939

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP(B)), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP(A)). This helix α7 is formed by the C-terminal dodecapeptide 131-142, which is flexibly disordered on the protein surface in BmorPBP(B) and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1-128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP(B). At pH 4.5, BmorPBP(1-128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of (15)N-(1)H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.

Generic Insect Repellent Detector from the Fruit Fly Drosophila Melanogaster

PloS One. 2011  |  Pubmed ID: 21436880

Insect repellents are prophylactic tools against a number of vector-borne diseases. There is growing demand for repellents outperforming DEET in cost and safety, but with the current technologies R&D of a new product takes almost 10 years, with a prohibitive cost of $30 million dollar in part due to the demand for large-scale synthesis of thousands of test compounds of which only 1 may reach the market. R&D could be expedited and cost dramatically reduced with a molecular/physiological target to streamline putative repellents for final efficacy and toxicological tests.

Characterization of Olfactory Genes in the Antennae of the Southern House Mosquito, Culex Quinquefasciatus

Journal of Insect Physiology. Jul, 2011  |  Pubmed ID: 21504749

Odorant reception in insects is mediated by different families of olfactory proteins. Here we focus on the characterization of odorant-binding proteins (OBPs), "plus-C" odorant-binding proteins ("plus-C" OBPs), chemosensory proteins (CSPs) and sensory neuron membrane proteins (SNMPs) families from the Southern house mosquito, Culex quinquefasciatus, a vector of pathogens implicated in multiple human diseases. Using bioinformatics and molecular approaches, we have identified a diversity of genes in the genome of Culex quinquefasciatus and examined their expression profiles by RT-PCR and real-time quantitative PCR. Based on their high transcript enrichment in female antennae compared to non-olfactory tissues, we have identified twelve OBPs, two "plus-C" OBPs and two SNMPs that likely play important roles in odorant reception. Transcripts of two genes were clearly enriched in female antennae compared to male antennae, whereas other genes displayed relatively equivalent transcript levels in antennae of both sexes. Additionally, eight genes were found to be transcribed at very high levels in female antennae compared to CquiOR7, suggesting they might encode highly abundant olfactory proteins. Comparative analysis across different mosquito species revealed that olfactory genes of Culex quinquefasciatus are related to putative orthologs in other species, indicating that they might perform similar functions. Understanding how mosquitoes are able to detect ecologically relevant odorant cues might help designing better control strategies. We have identified olfactory genes from different families which are likely important in Culex quinquefasciatus behaviors, thus paving the way towards a better understanding of the diversity of proteins involved in the reception of semiochemicals in this species.

Conserved Odorant-binding Proteins from Aphids and Eavesdropping Predators

PloS One. 2011  |  Pubmed ID: 21912599

The sesquiterpene (E)-ß-farnesene is the main component of the alarm pheromone system of various aphid species studied to date, including the English grain aphid, Sitobion avenae. Aphid natural enemies, such as the marmalade hoverfly Episyrphus balteatus and the multicolored Asian lady beetle Harmonia axyridis, eavesdrop on aphid chemical communication and utilize (E)-ß-farnesene as a kairomone to localize their immediate or offspring preys. These aphid-predator systems are important models to study how the olfactory systems of distant insect taxa process the same chemical signal. We postulated that odorant-binding proteins (OBPs), which are highly expressed in insect olfactory tissues and involved in the first step of odorant reception, have conserved regions involved in binding (E)-ß-farnesene.

Unusual Macrocyclic Lactone Sex Pheromone of Parcoblatta Lata, a Primary Food Source of the Endangered Red-cockaded Woodpecker

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2011  |  Pubmed ID: 22184232

Wood cockroaches in the genus Parcoblatta, comprising 12 species endemic to North America, are highly abundant in southeastern pine forests and represent an important prey of the endangered red-cockaded woodpecker, Picoides borealis. The broad wood cockroach, Parcoblatta lata, is among the largest and most abundant of the wood cockroaches, constituting >50% of the biomass of the woodpecker's diet. Because reproduction in red-cockaded woodpeckers is affected dramatically by seasonal and spatial changes in arthropod availability, monitoring P. lata populations could serve as a useful index of habitat suitability for woodpecker conservation and forest management efforts. Female P. lata emit a volatile, long-distance sex pheromone, which, once identified and synthesized, could be deployed for monitoring cockroach populations. We describe here the identification, synthesis, and confirmation of the chemical structure of this pheromone as (4Z,11Z)-oxacyclotrideca-4,11-dien-2-one [= (3Z,10Z)-dodecadienolide; herein referred to as "parcoblattalactone"]. This macrocyclic lactone is a previously unidentified natural product and a previously unknown pheromonal structure for cockroaches, highlighting the great chemical diversity that characterizes olfactory communication in cockroaches: Each long-range sex pheromone identified to date from different genera belongs to a different chemical class. Parcoblattalactone was biologically active in electrophysiological assays and attracted not only P. lata but also several other Parcoblatta species in pine forests, underscoring its utility in monitoring several endemic wood cockroach species in red-cockaded woodpecker habitats.