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1.  Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti  
F1000Research  2015;3:305.
Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules.
doi:10.12688/f1000research.5879.2
PMCID: PMC4309172
2.  Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti  
F1000Research  2014;3:305.
Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules.
doi:10.12688/f1000research.5879.1
PMCID: PMC4309172
3.  RNAi-based Demonstration of Direct Link between Specific Odorant Receptors and Mosquito Oviposition Behavior 
The Southern house mosquito, Culex quinquefasciatus - a vector of West Nile virus - is equipped with 130 odorant receptors (ORs), which enable young females to locate plants and blood-meal sources and older females to find suitable sites for oviposition. In our attempts to de-orphanize ORs expressed in female antennae, we identified CquiOR37 and CquiOR99, which were narrowly tuned to two phenolic compounds, 4-methylphenol and 4-ethylphenol. When tested in the Xenopus oocyte recording system the observed EC50s for 4-methylphenol and 4-ethylphenol were 6.4 and 18.2 µM for CquiOR37 and 14.4 and 0.74 µM for CquiOR99 (goodness of fit, R2 =0.88–0.99), respectively. Indoor behavioral assays demonstrated that gravid female mosquitoes laid significantly more eggs in water trays spiked with these compounds than in control water trays. Field studies with gravid traps corroborated that 4-ethylphenol is active in a wide range of doses from 0.1 to 10 µg/l, as required for practical applications. A dsRNA construct based on the two genes, CquiOR37/99-dsRNA was stable in pupa hemolymph for up to 3 h. Pupae injected with CquiOR37/99-dsRNA, β-galactosidasedsRNA or water had more than 40% survival rate at the peak of oviposition (day-9). qPCR analysis showed individual variation, but significant mean reduction in CquiOR37 and CquiOR99 transcript levels in CquiOR37/99-dsRNA-treated mosquitoes. Water-injected females and those treated with the control gene laid significantly more eggs in trays containing 4-ethylphenol than in water trays, whereas CquiOR37/99-dsRNA-treated mosquitoes laid normal number of eggs, but could not discriminate treatment from control. This study linked for the first time specific receptors for 4-ethylphenol with increased oviposition in the important vector Cx. quinquefasciatus.
doi:10.1016/j.ibmb.2013.07.008
PMCID: PMC3800558  PMID: 23911547
Culex quinquefasciatus; CquiOR37; CquiOR99; 4-methylphenol; 4-ethylphenol; RNAi
4.  Silent, Generic and Plant Kairomone Sensitive Odorant Receptors from the Southern House Mosquito 
Journal of insect physiology  2013;59(9):961-966.
The Southern house mosquito Culex quinquefasciatus has the largest repertoire of odorant receptors (ORs) of all mosquitoes and dipteran species whose genomes have been sequenced to date. Previously, we have identified and de-orphanized two ORs expressed in female antennae, CquiOR2 and CquiOR10, which are sensitive to oviposition attractants. In view of a new nomenclature for the Culex genome (VectorBase) we renamed these ORs as CquiOR21 (formerly CquiOR10) and CquiOR121 (CquiOR2). In addition, we selected ORs from six different phylogenetic groups for deorphanization. We cloned four of them by using cDNA from female antennae as a template. Attempts to clone CquiOR87 and CquiOR110 were unsuccessful either because they are pseudogenes or are not expressed in adult female antennae, the main olfactory tissue. By contrast, CquiOR1, CquiOR44, CquiOR73, and CquiOR161 were highly expressed in female antennae. To de-orphanize these ORs, we employed the Xenopus oocyte recording system. CquiORx-CquiOrco-expressed oocytes were challenged with a panel of 90 compounds, including known oviposition attractants, human and vertebrate host odorants, plant kairomones, and naturally occuring repellents. While CquiOR161 did not respond to any test compound in two different laboratories, CquiOR1 showed the features of a generic OR, with strong responses to 1-octen-3-ol and other ligands. CquiOR44 and CquiOR73 showed preference to plant-derived terpenoids and phenolic compounds, respectively. While fenchone was the best ligand for the former, 3,5-dimethylphenol elicited the strongest responses in the latter. The newly de-orphanized ORs may be involved in reception of plant kairomones and/or natural repellents.
doi:10.1016/j.jinsphys.2013.07.004
PMCID: PMC3800014  PMID: 23876610
Culex quinquefasciatus; CquiOR21; CquiOR121; CquiOR1; CquiOR44; CquiOR73; CquiOR161
5.  Probing Insect Odorant Receptors with their Cognate Ligands: Insights into Structural Features 
Biochemical and biophysical research communications  2013;435(3):10.1016/j.bbrc.2013.05.015.
Oodorant receptors (ORs) are essential for insect survival in the environment and thus are ideal molecular targets for the design of insect-inspired modern green chemicals to control populations of agricultural pests and insects of medical importance. Although insect ORs are known for more than a decade, their structural biology is still in its infancy. Here, we unravel the first structural features of ORs from the malaria mosquito, the Southern house mosquito and the silkworm moth. The second extracellular loops (ECL-2s) of their predicted structures are much longer than ECL-1s and ECL-3s. The 27 amino-acid-residue-long of the ECL-2s in mosquito and the 43 amino-acid-residue-long ECL2s in moth ORs are well-conserved. About one-third of the residues are identical, including 3-4 Pro residues. Thorough examination of well-conserved residues in these structures, by point mutation and functional assay with the Xenopus oocyte recording system, strongly suggest that these “loops” include three β-turns and some degree of folding. In the Southern house mosquito three Pro residues in ECL-2 are essential for full activation of the receptor, which is finely tuned to the oviposition attractant 3-methylindole. Additionally, the “corner residues” of prolines, including Gly, Tyr, and Leu are functionally important thus suggesting that turns are stabilized not only by backbone hydrogen bonds, but also by side-chain interactions. Examination of ECL-2s from a distant taxonomical group suggests these ECL-2 loops might be functionally important in all insect ORs. Two of the four Pro residues in the predicted ECL-2 of the bombykol receptor in the silkworm moth, BmorOR1, are essential for function. Experimental evidence indicates that these loops may not be specificity determinants, but they may form a cover to the yet-to-be-identified membrane embedded binding cavities of insect ORs.
doi:10.1016/j.bbrc.2013.05.015
PMCID: PMC3836372  PMID: 23673297
Extracellular loop; AgamOR10; CquiOR10; BmorOR1; 3-methylindole; bombykol
6.  Quasi-Double-Blind Screening of Semiochemicals for Reducing Navel Orangeworm Oviposition on Almonds 
PLoS ONE  2013;8(11):e80182.
A three-step, quasi-double-bind approach was used as a proof-of-concept study to screen twenty compounds for their ability to reduce oviposition of gravid female navel orangeworm(NOW), Ameylois transitella (Lepidoptera: Pyralidae). First, the panel of compounds, whose identity was unknown to the experimenters, was tested by electroantennogram (EAG) using antennae of two-day old gravid females as the sensing element. Of the twenty compounds tested three showed significant EAG responses. These three EAG-active compounds and a negative control were then analyzed for their ability to reduce oviposition via small-cage, two-choice laboratory assays. Two of the three compounds significantly reduced oviposition under laboratory conditions. Lastly, these two compounds were deployed in a field setting in an organic almond orchard in Arbuckle, CA using black egg traps to monitor NOW oviposition. One of these two compounds significantly reduced oviposition on black egg traps under these field conditions. Compound 9 (later identified as isophorone) showed a significant reduction in oviposition in field assays and thus has a potential as a tool to control the navel orangeworm as a pest of almonds.
doi:10.1371/journal.pone.0080182
PMCID: PMC3828197  PMID: 24244643
7.  Identification and Characterization of an Antennae-Specific Aldehyde Oxidase from the Navel Orangeworm 
PLoS ONE  2013;8(6):e67794.
Antennae-specific odorant-degrading enzymes (ODEs) are postulated to inactivate odorant molecules after they convey their signal. Different classes of insect ODEs are specific to esters, alcohols, and aldehydes – the major functional groups of female-produced, hydrophobic sex pheromones from moth species. Esterases that rapidly inactive acetate and other esters have been well-studied, but less is known about aldehyde oxidases (AOXs). Here we report cloning of an aldehyde oxidase, AtraAOX2, from the antennae of the navel orangeworm (NOW), Amyelois transitella, and the first activity characterization of a recombinant insect AOX. AtraAOX2 gene spans 3,813 bp and encodes a protein with 1,270 amino acid residues. AtraAOX2 cDNA was expressed in baculovirus-infected insect Sf21 cells as a ≈280 kDa homodimer with 140 kDa subunits. Recombinant AtraAOX2 degraded Z11Z13–16Ald and plant volatile aldehydes as substrates. However, as expected for aldehyde oxidases, recombinant AtraAOX2 did not show specificity for Z11Z13–16Ald, the main constituent of the sex pheromone, but showed high activity for plant volatile aldehydes. Our data suggest AtraAOX2 might be involved in degradation of a diversity of aldehydes including sex pheromones, plant-derived semiochemicals, and chemical cues for oviposition sites. Additionally, AtraAOX2 could protect the insect's olfactory system from xenobiotics, including pesticides that might reach the sensillar lymph surrounding the olfactory receptor neurons.
doi:10.1371/journal.pone.0067794
PMCID: PMC3691121  PMID: 23826341
8.  Crystallographic Observation of pH-Induced Conformational Changes in the Amyelois transitella Pheromone-Binding Protein AtraPBP1 
PLoS ONE  2013;8(2):e53840.
The navel orangeworm, Amyelois transitella is a major agricultural pest causing large losses in a variety of tree crops. Control of this insect pest may be achieved by interfering with olfactory pathways to block detection of female-produced sex pheromones and consequently, disrupt mating. The first component of this pathway is the pheromone-binding protein AtraPBP1, which recognizes the pheromone and presents it to the odorant receptor housed in a sensory neuron of the male antennae. Release of the ligand depends on a pH-induced conformational change associated with the acidity of the membrane surface. To characterize this conformational change and to understand how pheromones bind, we have determined the high resolution crystal structures of AtraPBP1 in complex with two main constituents of the sex pheromone, i.e., (11Z,13Z)-hexadecadienal and (11Z,13Z)-hexadecadienol. Comparison with the structure of the unliganded form demonstrates a large ∼90° movement of the C-terminal helix which is observed in other pheromone- or odorant-binding proteins accompanied by an unpredicted 37° displacement of the N-terminal helix. Molecular dynamic trajectories suggest that the conformational change of the α1 helix facilitates the movement of the C-terminal helix.
doi:10.1371/journal.pone.0053840
PMCID: PMC3572114  PMID: 23418423
9.  Fatty Acid Solubilizer from the Oral Disk of the Blowfly 
PLoS ONE  2013;8(1):e51779.
Background
Blowflies are economic pests of the wool industry and potential vectors for epidemics. The establishment of a pesticide-free, environmentally friendly blowfly control strategy is necessary. Blowflies must feed on meat in order to initiate the cascade of events that are involved in reproduction including juvenile hormone synthesis, vitellogenesis, and mating. During feeding blowflies regurgitate salivary lipase, which may play a role in releasing fatty acids from triglycerides that are found in food. However, long-chain fatty acids show low solubility in aqueous solutions. In order to solubilize and ingest the released hydrophobic fatty acids, the blowflies must use a solubilizer.
Methodology
We applied native PAGE, Edman degradation, cDNA cloning, and RT-PCR to characterize a protein that accumulated in the oral disk of the black blowfly, Phormia regina. In situ hybridization was carried out to localize the expression at the cellular level. A fluorescence competitive binding assay was used to identify potential ligands of this protein.
Conclusion
A protein newly identified from P. regina (PregOBP56a) belonged to the classic odorant-binding protein (OBP) family. This gene was expressed in a cluster of cells that was localized between pseudotracheae on the oral disk, which are not accessory cells of the taste peg chemosensory sensilla that normally synthesize OBPs. At pH 7 and pH 6, PregOBP56a bound palmitic, stearic, oleic, and linoleic acids, that are mainly found in chicken meat. The binding affinity of PregOBP56a decreased at pH 5. We propose that PregOBP56a is a protein that solubilizes fatty acids during feeding and subsequently helps to deliver the fatty acids to the midgut where it may help in the process of reproduction. As such, PregOBP56a is a potential molecular target for controlling the blowfly.
doi:10.1371/journal.pone.0051779
PMCID: PMC3543412  PMID: 23326317
10.  Specificity of the Receptor for the Major Sex Pheromone Component in Heliothis virescens 
In a previous study, the Drosophila melanogaster OR67dGAL4;UAS system was used to functionally characterize the receptor for the major component of the sex pheromone in the tobacco budworm, Heliothis virescens Fabricius (Lepidoptera: Noctuidae), HvOR13. Electrophysiological and behavioral assays showed that transgenic flies expressing HvOR13 responded to (Z)-11-hexadecenal (Z11-16:Ald). However, tests were not performed to determine whether these flies would also respond to secondary components of the H. virescens sex pheromone. Thus, in this study the response spectrum of HvOR13 expressed in this system was examined by performing single cell recordings from odor receptor neuron in trichoid T1 sensilla on antennae of two Or67dGAL4 [1]; UAS-HvOR13 lines stimulated with Z11-16:Ald and six H. virescens secondary pheromone components. Fly courtship assays were also performed to examine the behavioral response of the Or67dGAL4[1]; UAS-HvOR13 flies to Z11-16:Ald and the secondary component Z9-14:Ald. Our combined electrophysiological and behavioral studies indicated high specificity and sensitivity of HvOR13 to Z11-16:Ald. Interestingly, a mutation leading to truncation in the HvOR13 C-terminal region affected but did not abolish pheromone receptor response to Z11-16:Ald. The findings are assessed in relationship to other HvOR13 heterologous expression studies, and the role of the C-terminal domain in receptor function is discussed. A third line expressing HvOR15 was also tested but did not respond to any of the seven pheromone components.
doi:10.1673/031.013.16001
PMCID: PMC4015405  PMID: 24773407
Drosophila melanogaster; HvORI3; T1 sensilla
11.  Correction: Conserved Odorant-Binding Proteins from Aphids and Eavesdropping Predators 
PLoS ONE  2012;7(12):10.1371/annotation/6fb8a803-8203-429c-b0d1-4d0d995c39e9.
doi:10.1371/annotation/6fb8a803-8203-429c-b0d1-4d0d995c39e9
PMCID: PMC3553189
12.  Specificity Determinants of the Silkworm Moth Sex Pheromone 
PLoS ONE  2012;7(9):e44190.
The insect olfactory system, particularly the peripheral sensory system for sex pheromone reception in male moths, is highly selective, but specificity determinants at the receptor level are hitherto unknown. Using the Xenopus oocyte recording system, we conducted a thorough structure-activity relationship study with the sex pheromone receptor of the silkworm moth, Bombyx mori, BmorOR1. When co-expressed with the obligatory odorant receptor co-receptor (BmorOrco), BmorOR1 responded in a dose-dependent fashion to both bombykol and its related aldehyde, bombykal, but the threshold of the latter was about one order of magnitude higher. Solubilizing these ligands with a pheromone-binding protein (BmorPBP1) did not enhance selectivity. By contrast, both ligands were trapped by BmorPBP1 leading to dramatically reduced responses. The silkworm moth pheromone receptor was highly selective towards the stereochemistry of the conjugated diene, with robust response to the natural (10E,12Z)-isomer and very little or no response to the other three isomers. Shifting the conjugated diene towards the functional group or elongating the carbon chain rendered these molecules completely inactive. In contrast, an analogue shortened by two omega carbons elicited the same or slightly higher responses than bombykol. Flexibility of the saturated C1–C9 moiety is important for function as addition of a double or triple bond in position 4 led to reduced responses. The ligand is hypothesized to be accommodated by a large hydrophobic cavity within the helical bundle of transmembrane domains.
doi:10.1371/journal.pone.0044190
PMCID: PMC3434217  PMID: 22957053
13.  Moth Sex Pheromone Receptors and Deceitful Parapheromones 
PLoS ONE  2012;7(7):e41653.
The insect's olfactory system is so selective that male moths, for example, can discriminate female-produced sex pheromones from compounds with minimal structural modifications. Yet, there is an exception for this “lock-and-key” tight selectivity. Formate analogs can be used as replacement for less chemically stable, long-chain aldehyde pheromones, because male moths respond physiologically and behaviorally to these parapheromones. However, it remained hitherto unknown how formate analogs interact with aldehyde-sensitive odorant receptors (ORs). Neuronal responses to semiochemicals were investigated with single sensillum recordings. Odorant receptors (ORs) were cloned using degenerate primers, and tested with the Xenopus oocyte expression system. Quality, relative quantity, and purity of samples were evaluated by gas chromatography and gas chromatography-mass spectrometry. We identified olfactory receptor neurons (ORNs) housed in trichoid sensilla on the antennae of male navel orangeworm that responded equally to the main constituent of the sex pheromone, (11Z,13Z)-hexadecadienal (Z11Z13-16Ald), and its formate analog, (9Z,11Z)-tetradecen-1-yl formate (Z9Z11-14OFor). We cloned an odorant receptor co-receptor (Orco) and aldehyde-sensitive ORs from the navel orangeworm, one of which (AtraOR1) was expressed specifically in male antennae. AtraOR1•AtraOrco-expressing oocytes responded mainly to Z11Z13-16Ald, with moderate sensitivity to another component of the sex pheromone, (11Z,13Z)-hexadecadien-1-ol. Surprisingly, this receptor was more sensitive to the related formate than to the natural sex pheromone. A pheromone receptor from Heliothis virescens, HR13 ( = HvirOR13) showed a similar profile, with stronger responses elicited by a formate analog than to the natural sex pheromone, (11Z)-hexadecenal thus suggesting this might be a common feature of moth pheromone receptors.
doi:10.1371/journal.pone.0041653
PMCID: PMC3401280  PMID: 22911835
14.  Extrusion of the C-terminal Helix in Navel Orangeworm Moth Pheromone-Binding Protein (AtraPBP1) Controls Pheromone Binding† 
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 Amyelois 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.
doi:10.1016/j.bbrc.2010.11.119
PMCID: PMC3019287  PMID: 21130734
AtraPBP1; NMR; pheromone-binding protein; Amyelois transitella; pheromone; navel orangeworm moth; histidine protonation switch; disulfide bridge
15.  Conserved Odorant-Binding Proteins from Aphids and Eavesdropping Predators 
PLoS ONE  2011;6(8):e23608.
Background
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.
Methodology
We cloned OBP genes from the English grain aphid and two major predators of this aphid species. We then expressed these proteins and compare their binding affinities to the alarm pheromone/kairomone. By using a fluorescence reporter, we tested binding of (E)-ß-farnesene and other electrophysiologically and behaviorally active compounds, including a green leaf volatile attractant.
Conclusion
We found that OBPs from disparate taxa of aphids and their predators are highly conserved proteins, with apparently no orthologue genes in other insect species. Properly folded, recombinant proteins from the English grain aphid, SaveOBP3, and the marmalade hoverfly, EbalOBP3, specifically bind (E)-ß-farnesene with apparent high affinity. For the first time we have demonstrated that insect species belonging to distinct Orders have conserved OBPs, which specifically bind a common semiochemical and has no binding affinity for related compounds.
doi:10.1371/journal.pone.0023608
PMCID: PMC3160308  PMID: 21912599
16.  Generic Insect Repellent Detector from the Fruit Fly Drosophila melanogaster 
PLoS ONE  2011;6(3):e17705.
Background
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.
Methodology
Using olfactory-based choice assay we show here that the fruit fly is repelled by not only DEET, but also IR3535 and picaridin thus suggesting they might have “generic repellent detector(s),” which may be of practical applications in new repellent screenings. We performed single unit recordings from all olfactory sensilla in the antennae and maxillary palps. Although the ab3A neuron in the wild type flies responded to picaridin, it was unresponsive to DEET and IR3535. By contrast, a neuron housed in the palp basiconic sensilla pb1 responded to DEET, IR3535, and picaridin, with apparent sensitivity higher than that of the DEET detectors in the mosquitoes Culex quinquefasciatus and Aedes aegypti. DmOr42a was transplanted from pb1 to the “empty neuron” and showed to be sensitive to the three insect repellents.
Conclusions
For the first time we have demonstrated that the fruit fly avoids not only DEET but also IR3535 and picaridin, and identified an olfactory receptor neuron (ORN), which is sensitive to these three major insect repellents. We have also identified the insect repellent-sensitive receptor, DmOr42a. This generic detector fulfils the requirements for a simplified bioassay for early screening of test insect repellents.
doi:10.1371/journal.pone.0017705
PMCID: PMC3059203  PMID: 21436880
17.  NMR Structure of Navel Orangeworm Moth Pheromone-Binding Protein (AtraPBP1): Implications for pH-Sensitive Pheromone Detection† 
Biochemistry  2010;49(7):1469.
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 the three-dimensional structure of a PBP from Amyelois transitella (AtraPBP1) in solution at pH 4.5 determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments, multi-angle light scattering, and 15N 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 α-helices (α1: L8-L23, α2: D27-F36, α3: R46-V62, α4: A73-M78; α5: D84-S100; α6: R107-L125; α7: 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 (α7) contains hydrophobic residues (M131, V133, V134, V135, V138, L139 and A140) that contact conserved residues (W37, L59, A73, F76, A77, I94, V111, 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.
doi:10.1021/bi9020132
PMCID: PMC2822879  PMID: 20088570
AtraPBP1; NMR; pheromone-binding protein; Amyelois transitella; pheromone; navel orangeworm moth; multi-angle light scattering; histidine protonation switch; disulfide bridge
18.  Odorant-Binding Proteins of the Malaria Mosquito Anopheles funestus sensu stricto 
PLoS ONE  2010;5(10):e15403.
Background
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.
Methodology
In this study, a large screening of over 50 ecologically significant odorant compounds led to the identification of 12 ligands that elicit significant electroantennographic (EAG) responses from An. funestus female antennae. To compare the absolute efficiency/potency of these chemicals, corrections were made for differences in volatility by determining the exact amount in a stimulus puff. Fourteen AfunOBP genes were cloned and their expression patterns were analyzed. AfunOBP1, 3, 7, 20 and 66 showed olfactory tissue specificity by reverse transcriptase PCR (RT-PCR). Quantitative real-time PCR (qRT-PCR) analysis showed that among olfactory-specific OBPs, AfunOBP1 and 3 are the most enriched OBPs in female antennae. Binding assay experiments showed that at pH 7, AfunOBP1 significantly binds to 2-undecanone, nonyl acetate, octyl acetate and 1-octen-3-ol but AfunOBP3, which shares 68% identify with AfunOBP1 at amino acid level, showed nearly no binding activity to the selected 12 EAG-active odorant compounds.
Conclusion
This work presents for the first time a study on the odorants and OBPs of the malaria vector mosquito An. funestus, which may provide insight into the An. funestus olfactory research, assist in a comparative study between major malaria mosquitoes An. gambiae and An. funestus olfactory system, and help developing new mosquito control strategies to reduce malaria transmission.
doi:10.1371/journal.pone.0015403
PMCID: PMC2962654  PMID: 21042539
19.  Odorant Receptor from the Southern House Mosquito Narrowly Tuned to the Oviposition Attractant Skatole 
Journal of Chemical Ecology  2010;36(8):797-800.
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.
doi:10.1007/s10886-010-9828-9
PMCID: PMC2908433  PMID: 20623327
Odorant receptor; CquiOR10; Culex quinquefasciatus; Xenopus oocyte expression system; 3-Methylindole; 2-Methylphenol
20.  Pheromone Binding to General Odorant-binding Proteins from the Navel Orangeworm 
Journal of Chemical Ecology  2010;36(7):787-794.
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.
doi:10.1007/s10886-010-9811-5
PMCID: PMC2895864  PMID: 20535533
Amyelois transitella; AtraGOBP1; AtraGOBP2; AtraPBP1; Circular dichroism; Electroantennogram recording; Female attractants; General odorant-binding proteins; Protein expression
21.  Culex Mosquitoes (Diptera: Culicidae) Egg Laying in Traps Loaded With Bacillus thuringiensis Variety israelensis and Baited With Skatole 
Journal of medical entomology  2010;47(3):345-348.
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.
PMCID: PMC2882852  PMID: 20496581
Bacillus thuringiensis variety israelensis; skatole; grass infusion; West Nile virus; ovitrap
22.  An Odorant Receptor from the Southern House Mosquito Culex pipiens quinquefasciatus Sensitive to Oviposition Attractants 
PLoS ONE  2010;5(4):e10090.
Background
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.
Methodology
We have employed a combination of bioinformatics, molecular cloning and electrophysiology approaches to identify and characterize the response profile of an OR in Cx. quinquefasciatus. First, we have unveiled a large multigenic family of one-hundred-fifty-eight putative ORs in this species, including a subgroup of conserved ORs in three mosquito species. Using the Xenopus oocytes expression system, we have determined the response profile of CquiOR2, an antennae-specific OR, which shares high identity with putative orthologs in Anopheles gambiae (AgamOR2) and Aedes aegypti (AaegOR2).
Conclusion
We show that CquiOR2 is highly sensitive to indole, an oviposition attractant for Cx. quinquefasciatus. The response profile of CquiOR2 expressed in Xenopus oocytes resembles that of an olfactory receptor neuron housed in the antennal short blunt-tipped sensilla (A2) of Cx. quinquefasciatus, which are natural detectors for oviposition attractants. This first Culex OR de-orphanized is, therefore, a potential molecular target for screening oviposition attractants.
doi:10.1371/journal.pone.0010090
PMCID: PMC2851645  PMID: 20386699
23.  Knockdown of a Mosquito Odorant-binding Protein Involved in the Sensitive Detection of Oviposition Attractants 
Journal of Chemical Ecology  2010;36(3):245-248.
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.
doi:10.1007/s10886-010-9762-x
PMCID: PMC2837830  PMID: 20191395
RNA interference; Culex quinquefasciatus antennae; CquiOBP1; EAG; Oviposition attractants; MOP; Skatole; Indole
24.  Structure of an Odorant-Binding Protein from the Mosquito Aedes aegypti Suggests a Binding Pocket Covered by a pH-Sensitive “Lid” 
PLoS ONE  2009;4(11):e8006.
Background
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.
Methodology
Previously, we isolated and cloned a major, female-enriched odorant-binding protein (OBP) from the yellow fever mosquito, AaegOBP1, which was later inadvertently renamed AaegOBP39. We prepared recombinant samples of AaegOBP1 by using an expression system that allows proper formation of disulfide bridges and generates functional OBPs, which are indistinguishable from native OBPs. We crystallized AaegOBP1 and determined its three-dimensional structure at 1.85 Å resolution by molecular replacement based on the structure of the malaria mosquito OBP, AgamOBP1, the only mosquito OBP structure known to date.
Conclusion
The structure of AaegOBP1 ( = AaegOBP39) shares the common fold of insect OBPs with six α-helices knitted by three disulfide bonds. A long molecule of polyethylene glycol (PEG) was built into the electron-density maps identified in a long tunnel formed by a crystallographic dimer of AaegOBP1. Circular dichroism analysis indicated that delipidated AaegOBP1 undergoes a pH-dependent conformational change, which may lead to release of odorant at low pH (as in the environment in the vicinity of odorant receptors). A C-terminal loop covers the binding cavity and this “lid” may be opened by disruption of an array of acid-labile hydrogen bonds thus explaining reduced or no binding affinity at low pH.
doi:10.1371/journal.pone.0008006
PMCID: PMC2778553  PMID: 19956631
25.  1H, 15N, and 13C chemical shift assignments of the mosquito odorant binding protein-1 (CquiOBP1) bound to the mosquito oviposition pheromone 
Biomolecular Nmr Assignments  2009;3(2):195-197.
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°C (BMRB no. 16175).
Electronic supplementary material
The online version of this article (doi:10.1007/s12104-009-9173-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s12104-009-9173-5
PMCID: PMC2772962  PMID: 19888689
Odorant-binding protein; Pheromone signaling; Olfaction; NMR

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