The greening-causing agent alters the behavioral and electrophysiological responses of the Asian citrus psyllid to a putative sex pheromone.

The Asian Citrus Psyllid (ACP), Diaphorina citri, is a vector of the pathological bacterium Candidatus Liberibacter asiaticus (CLas), which causes the most devastating disease to the citrus industry worldwide, known as greening or huanglongbing (HLB). Earlier field tests with an acetic acid-based lure in greening-free, 'Valencia' citrus orange groves in California showed promising results. The same type of lures tested in São Paulo, Brazil, showed unsettling results. During the unsuccessful trials, we noticed a relatively large proportion of females in the field, ultimately leading us to test field-collected males and females for Wolbachia and CLas. The results showed high rates of Wolbachia and CLas infection in field populations. We then compared the olfactory responses of laboratory-raised, CLas-free, and CLas-infected males to acetic acid. As previously reported, CLas-uninfected males responded to acetic acid at 1 µg. Surprisingly, CLas-infected males required 50 × higher doses of the putative sex pheromone, thus explaining the failure to capture CLas-infected males in the field. CLas infection was also manifested in electrophysiological responses. Electroantennogram responses from CLas-infected ACP males were significantly higher than those obtained with uninfected males. To the best of our knowledge, this is the first report of a pathogen infection affecting a vector's response to a sex attractant.

Circabidian rhythm of sex pheromone reception in a scarab beetle.

Animals have endogenous clocks that regulate their behavior and physiology. These clocks rely on environmental cues (time givers) that appear approximately every 24 h due to the Earth's rotation; thus, most insects exhibit a circadian rhythm. One notable exception is the scarab beetle, Holotrichia parallela, a severe agricultural pest in China, Japan, South Korea, and India. Females emerge from the soil every other night, reach the canopy of host plants, evert an abdominal gland, and release a pheromone bouquet comprising l-isoleucine methyl ester (LIME) and l-linalool. To determine whether this circa'bi'dian rhythm affects the olfactory system, we aimed to identify H. parallela sex pheromone receptor(s) and study their expression patterns. We cloned 14 odorant receptors (ORs) and attempted de-orphanizing them in the Xenopus oocyte recording system. HparOR14 gave robust responses to LIME and smaller responses to l-linalool. Structural modeling, tissue expression profile, and RNAi treatment followed by physiological and behavioral studies support that HparOR14 is a sex pheromone receptor-the first of its kind discovered in Coleoptera. Examination of the HparOR14 transcript levels throughout the adult's life showed that on sexually active days, gene expression was significantly higher in the scotophase than in the photophase. Additionally, the HparOR14 expression profile showed a circabidian rhythm synchronized with the previously identified pattern of sex pheromone emission. 48 h of electroantennogram recordings showed that responses to LIME were abolished on non-calling nights. In contrast, responses to the green leaf volatile (Z)-3-henexyl acetate remained almost constant throughout the recording period.

Single amino acid residue mediates reciprocal specificity in two mosquito odorant receptors.

The southern house mosquito, Culex quinquefasciatus, utilizes two odorant receptors, CquiOR10 and CquiOR2, narrowly tuned to oviposition attractants and well conserved among mosquito species. They detect skatole and indole, respectively, with reciprocal specificity. We swapped the transmembrane (TM) domains of CquiOR10 and CquiOR2 and identified TM2 as a specificity determinant. With additional mutations, we showed that CquiOR10A73L behaved like CquiOR2. Conversely, CquiOR2L74A recapitulated CquiOR10 specificity. Next, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold and AlphaFold and docked skatole and indole using RosettaLigand. These modeling studies suggested space-filling constraints around A73. Consistent with this hypothesis, CquiOR10 mutants with a bulkier residue (Ile, Val) were insensitive to skatole and indole, whereas CquiOR10A73G retained the specificity to skatole and showed a more robust response than the wildtype receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10A73L, CquiOR2, and CquiOR2L74I were insensitive to 3-ethylindole, whereas CquiOR2L74A and CquiOR2L74G gained activity. Additionally, CquiOR10A73G gave more robust responses to 3-ethylindole than CquiOR10. Thus, we suggest the specificity of these receptors is mediated by a single amino acid substitution, leading to finely tuned volumetric space to accommodate specific oviposition attractants.

A highly expressed odorant receptor from the yellow fever mosquito, AaegOR11, responds to (+)- and (-)-fenchone and a phenolic repellent.

The cornerstone of the reverse chemical ecology approach is the identification of odorant receptors (OR) sensitive to compounds in a large panel of odorants. In this approach, we de-orphanize ORs and, subsequently, measure behaviors elicited by these semiochemicals. After that, we evaluate behaviorally active compounds for applications in insect vector management. Intriguingly, multiple ORs encoded by genes highly expressed in mosquito antennae do not respond to any test odorant. One such case is CquiOR125 from the southern house mosquito, Culex quinquefasciatus Say. To better understand CquiOR125's role in Culex mosquito olfaction, we have cloned a CquiOR125 orthologue in the genome of the yellow fever mosquito, Aedes aegypti (L.), AaegOR11. Unlike the unresponsive nature of the orthologue in Cx. quinquefasciatus, oocytes co-expressing AaegOR11 and AaegOrco elicited robust responses when challenged with fenchone, 2,3-dimethylphenol, 3,4-dimethylphenol, 4-methycyclohexanol, and acetophenone. Interestingly, AaegOR11 responded strongly and equally to (+)- and (-)-fenchone, with no chiral discrimination. Contrary to reports in the literature, fenchone did not show any repellency activity against Ae. aegypti or Cx. quinquefasciatus. Laboratory and field tests did not show significant increases in egg captures in cups filled with fenchone solutions compared to control cups. The second most potent ligand, 2,3-dimethylphenol, showed repellency activity stronger than that elicited by DEET at the same dose. We, therefore, concluded that AaegOR11 is a mosquito repellent sensor. It is feasible that CquiOR125 responds to repellents that remain elusive.

Mosquito odorant receptor sensitive to natural spatial repellents and inhibitory compounds.

Previously, we have identified an odorant receptor (OR) from the southern house mosquito Culex quinquefasciatus, CquiOR32, which responded to both odorants (agonists) and inhibitory compounds (antagonists). CquiOR32/CquiOrco-expressing oocytes responded to methyl salicylate and other odorants with inward (regular) currents but gave currents in the reverse direction when challenged with eucalyptol and other inhibitors. To determine whether hitherto unknown ORs show this intrareceptor inhibition, we have now examined two other receptors in the same cluster, CquiOR27 and CquiOR28. We cloned and tested four variants of CquiOR28, but none of the 250 compounds in our panel of odorants, including an Orco ligand candidate (OLC12), elicited inward or upward deflections of the current traces. By contrast, CquiOR27/CquiOrco-expressing oocytes gave robust, dose-dependent inward currents when challenged with γ-octalactone and other odorants. On the other hand, octylamine and other phenolic compounds elicited dose-dependent currents in the reverse direction. When stimulatory and inhibitory compounds were presented in binary mixtures, γ-octalactone-elicited inward currents were attenuated in a dose-dependent manner according to the concentration of octylamine. As part of our chemical ecology approach, we tested the repellency activity of the most potent ligands in the surface landing and feeding assay and a newly reported hand-in cage assay. Protection elicited by γ-octalactone did not differ significantly from that of DEET at the same dose. In the hand-in cage assay, a cream formulation of γ-octalactone showed 97.0 ± 1.3% protection, with 47.6 ± 8.3% and 1.4 ± 0.7% landings per trial in the hands covered with a control and γ-octalactone cream, respectively (N = 8, p = 0.0078, Wilcoxon matched-pairs signed-rank test).

Odorant inhibition in mosquito olfaction mediated by inverse agonists.

The insect repellent methyl salicylate elicits excitatory responses upon interaction with CquiOR32, an odorant receptor (OR) from the southern house mosquito, Culex quinquefasciatus. By contrast, eucalyptol binds to CquiOR32 to generate electrophysiological and behavioral inhibitory responses. In an attempt to identify CquiOR32 variants displaying more robust inhibitory responses for more accurate current-voltage analysis, we sequenced 31 CquiOR32 clones. In the Xenopus oocyte recording system, CquiOR32V2/CquiOrco-expressing oocytes yielded eucalyptol-elicited outward (inhibitory) currents relatively larger than methyl salicylate-generated inward (excitatory) currents. Rescuing experiments showed that two of the amino acid substitutions in CquiOR32V2 located in a predicted transmembrane helix of the receptor are determinants of the outward/inward ratios. These findings, along with co-stimulus assays, suggest that odorant and inhibitor may bind to the same binding pocket. Current-voltage relationships obtained with standard perfusion buffer and those devoid of Na+ or Cl- indicated that both excitatory and inhibitory currents are mediated, at least in part, by cation. We then concluded that eucalyptol is an inverse agonist, which shifts the open ⇔ closed equilibrium of the receptor toward the closed conformation, thus reducing the spontaneous activity. By contrast, the binding of methyl salicylate shifts the equilibrium towards the open conformation and, consequently, leads to an increase in cation influx.

Colletotrichum falcatum modulates the olfactory behavior of the sugarcane borer, favoring pathogen infection.

Some pathogens can manipulate their host plants and insects to optimize their fitness, increasing the attraction of insects to the infected plant in ways that facilitate pathogen acquisition. In tropical American sugarcane crops, the fungus Colletotrichum falcatum, the red rot causal agent, usually occurs in association with the sugarcane borer Diatraea saccharalis, resulting in large losses of this crop. Considering this association, we aimed to identify the effects of C. falcatum on D. saccharalis host preference and performance as well as the effect of this insect on C. falcatum sugarcane infection. Here, we show that the fungus C. falcatum modulates D. saccharalis behavior to its own benefit. More specifically, C. falcatum-infected sugarcane plants showed a dramatic increase in VOCs, luring D. saccharalis females to lay eggs on these plants. Therefore, sugarcane infection by the fungus C. falcatum increased in cooccurrence with insect herbivory, benefiting the pathogen when associated with D. saccharalis.

Fungal phytopathogen modulates plant and insect responses to promote its dissemination.

Vector-borne plant pathogens often change host traits to manipulate vector behavior in a way that favors their spread. By contrast, infection by opportunistic fungi does not depend on vectors, although damage caused by an herbivore may facilitate infection. Manipulation of hosts and vectors, such as insect herbivores, has not been demonstrated in interactions with fungal pathogens. Herein, we establish a new paradigm for the plant-insect-fungus association in sugarcane. It has long been assumed that Fusarium verticillioides is an opportunistic fungus, where it takes advantage of the openings left by Diatraea saccharalis caterpillar attack to infect the plant. In this work, we show that volatile emissions from F. verticillioides attract D. saccharalis caterpillars. Once they become adults, the fungus is transmitted vertically to their offspring, which continues the cycle by inoculating the fungus into healthy plants. Females not carrying the fungus prefer to lay their eggs on fungus-infected plants than mock plants, while females carrying the fungus prefer to lay their eggs on mock plants than fungus-infected plants. Even though the fungus impacts D. saccharalis sex behavior, larval weight and reproduction rate, most individuals complete their development. Our data demonstrate that the fungus manipulates both the host plant and insect herbivore across life cycle to promote its infection and dissemination.

(3S,6E)-nerolidol-mediated rendezvous of Cyclocephala paraguayensis beetles in bottle gourd flowers.

Cyclocephalini beetles of the genus Cyclocephala (Coleoptera: Melolonthidae: Dynastinae) use flowers of some plants as food, shelter, and mating sites. However, little is known about floral scent chemistry involved in this interaction. Here we show that a sesquiterpene alcohol mediates attraction of Cyclocephala paraguayensis Arrow, on bottle gourd flowers, Lagenaria siceraria (Cucurbitaceae). Both males and females started to aggregate on the flowers at twilight; after that, mating began and remained for the entire night. GC-FID/EAD analysis of the L. siceraria floral scent collected in the field revealed that only the major constituent of the airborne volatiles elicited electroantennographic responses on male and female antennae of C. paraguayensis. This compound was identified as (3S,6E)-nerolidol, which was tested in two field trapping trials in Brazil. In the first bioassay, traps baited with nerolidol (mix of isomers) captured significantly more adult C. paraguayensis than control traps. In the second field trial, catches in traps baited with a mixture of isomers or enantiopure nerolidol were significantly higher than captures in control traps, but the treatments did not differ significantly. Analysis from the gut content of adult C. paraguayensis showed the presence of pollen, suggesting that they also use bottle gourd flowers for their nourishment. Taken together, these results suggest that (3S,6E)-nerolidol plays an essential role in the reproductive behavior of C. paraguayensis by eliciting aggregation, mating, and feeding.

CO2 per se activates carbon dioxide receptors.

Carbon dioxide has been used in traps for more than six decades to monitor mosquito populations and help make informed vector management decisions. CO2 is sensed by gustatory receptors (GRs) housed in neurons in the maxillary palps. CO2-sensitive GRs have been identified from the vinegar fly and mosquitoes, but it remains to be resolved whether these receptors respond to CO2 or bicarbonate. As opposed to the vinegar fly, mosquitoes have three GR subunits, but it is assumed that subunits GR1 and GR3 form functional receptors. In our attempt to identify the chemical species that bind these receptors, we discovered that GR2 and GR3 are essential for receptor function and that GR1 appears to function as a modulator. While Xenopus oocytes coexpressing Culex quinquefasciatus subunits CquiGR1/3 and CquiGR1/2 were not activated, CquiGR2/3 gave robust responses to sodium bicarbonate. Interestingly, CquiGR1/2/3-coexpressing oocytes gave significantly lower responses. That the ternary combination is markedly less sensitive than the GR2/GR3 combination was also observed with orthologs from the yellow fever and the malaria mosquito. By comparing responses of CquiGR2/CquiGR3-coexpressing oocytes to sodium bicarbonate samples (with or without acidification) and measuring the concentration of aqueous CO2, we showed that there is a direct correlation between dissolved CO2 and receptor response. We then concluded that subunits GR2 and GR3 are essential for these carbon dioxide-sensitive receptors and that they are activated by CO2 per se, not bicarbonate.

A popular Indian clove-based mosquito repellent is less effective against Culex quinquefasciatus and Aedes aegypti than DEET.

Insect repellents are widely used as the first line of defense against mosquito bites and transmission of disease-causing agents. However, the cost of daily applications of even the most affordable and the gold standard of insect repellents, DEET, is still high for low-income populations where repellents are needed the most. An Indian clove-based homemade recipe has been presented as a panacea. We analyzed this homemade repellent and confirmed by behavioral measurements and odorant receptor responses that eugenol is the active ingredient in this formulation. Prepared as advertised, this homemade repellent is ineffective, whereas 5x more concentrated extracts from the brand most enriched in eugenol showed moderate repellency activity against Culex quinquefasciatus and Aedes aegypti. DEET showed higher performance when compared to the 5x concentrated formulation and is available in the same market at a lower price than the cost of the ingredients to prepare the homemade formulation.

Conspecific and allospecific larval extracts entice mosquitoes to lay eggs and may be used in attract-and-kill control strategy.

One of the strategies of integrated vector management is to lure gravid mosquitoes for surveillance purposes or to entice them to lay eggs in water containing toxins that kill the offspring (attract-and-kill or trap-and-kill). Typically, the major challenge of this approach is the development of a lure that stimulates oviposition plus a toxin with no deterrent effect. Bacillus thuringiensis var. israelensis (Bti) satisfies the latter criterion, but lures for these autocidal gravid traps are sorely needed. We observed that gravid Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus laid significantly more eggs in cups with extracts from 4th-stage larvae (4 L) of the same or different species. No activity was found when 4 L were extracted with hexane, diethyl ether, methanol, or butanol, but activity was observed with dimethyl sulfoxide extracts. Larval extracts contained both oviposition stimulant(s)/attractant(s) and deterrent(s), which partitioned in the water and hexane phases, respectively. Lyophilized larval extracts were active after a month, but activity was reduced by keeping the sample at 4 °C. In the tested range of 0.1 to 1 larvae-equivalent per milliliter, oviposition activity increased in a dose-dependent manner. In field experiments, Ae. aegpti laid significantly more eggs in traps loaded with larval extracts plus Bti than in control traps with water plus Bti.

Laboratory and field evaluation of acetic acid-based lures for male Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP) is a vector of a pathogen associated with greening and thus a major problem in citriculture worldwide. Lures are much needed for improving ACP trapping systems for monitoring populations and surveillance. Previously, we have identified acetic acid as a putative sex pheromone and measured formic acid- and propionic acid-elicited robust electroantennographic responses. We have now thoroughly examined in indoor behavioral assays (4-way olfactometer) and field tests the feasibility of these three semiochemicals as potential lures for trapping ACP. Formic acid, acetic acid, and propionic acid at appropriate doses are male-specific attractants and suitable lures for ACP traps, but they do not act synergistically. An acetic acid-based homemade lure, prepared by impregnating the attractant in a polymer, was active for a day. A newly developed slow-release formulation had equal performance but lasted longer, thus leading to an important improvement in ACP trap capture at low population densities.

DEET and other repellents are inhibitors of mosquito odorant receptors for oviposition attractants.

In addition to its primary function as an insect repellent, DEET has many "off-label" properties, including a deterrent effect on the attraction of gravid female mosquitoes. DEET negatively affects oviposition sites. While deorphanizing odorant receptors (ORs) using the Xenopus oocyte recording system, we have previously observed that DEET generated outward (inhibitory) currents on ORs sensitive to oviposition attractants. Here, we systematically investigated these inhibitory currents. We recorded dose-dependent outward currents elicited by DEET and other repellents on ORs from Culex quinquefasciatus, Aedes aegypti, and Anopheles gambiae. Similar responses were observed with other plant-derived and plant-inspired compounds, including methyl jasmonate and methyl dihydrojasmolate. Inward (regular) currents elicited by skatole upon activation of CquiOR21 were modulated when this oviposition attractant was coapplied with a repellent. Compounds that generate outward currents in ORs sensitive to oviposition attractants elicited inward currents in a DEET-sensitive receptor, CquiOR136. The best ligand for this receptor, methyl dihydrojasmolate, showed repellency activity but was not as strong as DEET in our test protocol.

Odorant receptors from Culex quinquefasciatus and Aedes aegypti sensitive to floral compounds.

Mosquitoes rely heavily on the olfactory system to find a host for a bloodmeal, plants for a source of energy and suitable sites for oviposition. Here, we examined a cluster of eight odorant receptors (ORs), which includes one OR, CquiOR1, previously identified to be sensitive to plant-derived compounds. We cloned 5 ORs from Culex quinquefasciatus and two ORs from Aedes aegypti, ie, CquiOR2, CquiOR4, CquiOR5, CquiOR84, CquiOR85, AaegOR14, and AaegOR15 and then deorphanized these receptors using the Xenopus oocyte recording system and a large panel of odorants. 2-Phenylethanol, phenethyl formate, and phenethyl propionate were the best ligands for CquiOR4 somewhat resembling the profile of AaegOR15, which gave the strongest responses to phenethyl propionate, phenethyl formate, and acetophenone. In contrast, the best ligands for CquiOR5 were linalool, PMD, and linalool oxide. CquiOR4 was predominantly expressed in antennae of nonblood fed female mosquitoes, with transcript levels significantly reduced after a blood meal. 2-Phenylethanol showed repellency activity comparable to that of DEET at 1%. RNAi experiments suggest that at least in part 2-phenylethanol-elicited repellency is mediated by CquiOR4 activation.

Odorant Inhibition in Mosquito Olfaction.

How chemical signals are integrated at the peripheral sensory system of insects is still an enigma. Here we show that when coexpressed with Orco in Xenopus oocytes, an odorant receptor from the southern house mosquito, CquiOR32, generated inward (regular) currents when challenged with cyclohexanone and methyl salicylate, whereas eucalyptol and fenchone elicited inhibitory (upward) currents. Responses of CquiOR32-CquiOrco-expressing oocytes to odorants were reduced in a dose-dependent fashion by coapplication of inhibitors. This intrareceptor inhibition was also manifested in vivo in fruit flies expressing the mosquito receptor CquiOR32, as well in neurons on the antennae of the southern house mosquito. Likewise, an orthologue from the yellow fever mosquito, AaegOR71, showed intrareceptor inhibition in the Xenopus oocyte recording system and corresponding inhibition in antennal neurons. Inhibition was also manifested in mosquito behavior. Blood-seeking females were repelled by methyl salicylate, but repellence was significantly reduced when methyl salicylate was coapplied with eucalyptol.

Use of machine learning to identify novel, behaviorally active antagonists of the insect odorant receptor co-receptor (Orco) subunit.

Olfaction is a key component of the multimodal approach used by mosquitoes to target and feed on humans, spreading various diseases. Current repellents have drawbacks, necessitating development of more effective agents. In addition to variable odorant specificity subunits, all insect odorant receptors (ORs) contain a conserved odorant receptor co-receptor (Orco) subunit which is an attractive target for repellent development. Orco directed antagonists allosterically inhibit odorant activation of ORs and we previously showed that an airborne Orco antagonist could inhibit insect olfactory behavior. Here, we identify novel, volatile Orco antagonists. We functionally screened 83 structurally diverse compounds against Orco from Anopheles gambiae. Results were used for training machine learning models to rank probable activity of a library of 1280 odorant molecules. Functional testing of a representative subset of predicted active compounds revealed enrichment for Orco antagonists, many structurally distinct from previously known Orco antagonists. Novel Orco antagonist 2-tert-butyl-6-methylphenol (BMP) inhibited odorant responses in electroantennogram and single sensillum recordings in adult Drosophila melanogaster and inhibited OR-mediated olfactory behavior in D. melanogaster larvae. Structure-activity analysis of BMP analogs identified compounds with improved potency. Our results provide a new approach to the discovery of behaviorally active Orco antagonists for eventual use as insect repellents/confusants.

An unsettling explanation for the failure of skatole-baited ovitraps to capture Culex mosquitoes.

Culex mosquitoes are primarily found in temperate and tropical regions worldwide where they play a crucial role as main vectors of filarial worms and arboviruses. In Recife, a northeast city in Brazil, high densities of Culex quinquefasciatus are often found in association with human populated areas. In marked contrast to another part of the city, field tests conducted in the neighborhood of Sítio dos Pintos showed that trapping of mosquitoes in skatole-baited ovitraps did not differ significantly from captures in control (water) traps. Thus, classical and molecular taxonomic approaches were used to analyze the Culex species circulating in Sítio dos Pintos. Results obtained from both approaches agreed on the cocirculation of Culex quinquefasciatus and Culex nigripalpus in three different areas of this neighborhood. What was initially considered as an unexpected failure of this lure turned out to be a more unsettling problem, that is, the first report in Recife of Culex nigripalpus, a vector of Venezuelan equine encephalitis virus and West Nile virus. Unplanned urbanization processes close to remnants of the Atlantic forest, such as observed in Sítio dos Pintos, may have contributed to the introduction of Cx. nigripalpus in urban areas.

Reverse chemical ecology-based approach leading to the accidental discovery of repellents for Rhodnius prolixus, a vector of Chagas diseases refractory to DEET.

Rhodnius prolixus is one of the most important vectors of Chagas disease in Central and South America for which repellents and attractants are sorely needed. Repellents like DEET, picaridin, and IR3535 are widely used as the first line of defense against mosquitoes and other vectors, but they are ineffective against R. prolixus. Our initial goal was to identify in R. prolixus genome odorant receptors sensitive to putative sex pheromones. We compared gene expression of 21 ORs in the R. prolixus genome, identified 4 ORs enriched in male (compared with female) antennae. Attempts to de-orphanize these ORs using the Xenopus oocyte recording system showed that none of them responded to putative sex pheromone constituents. One of the them, RproOR80, was sensitive to 4 compounds in our panel of 109 odorants, namely, 2-heptanone, γ-octalactone, acetophenone, and 4-methylcychohexanol. Interestingly, these compounds, particularly 4-methylcyclohexanol, showed strong repellency activity as indicated not only by a significant decrease in residence time close to a host, but also by a remarkable reduction in blood intake. 4-Methylcyclohexanol-elicited repellency activity was abolished in RNAi-treated insects. In summary, our search for pheromone receptors led to the discovery of repellents for R. prolixus.

Functional Characterization of Odorant Binding Protein 27 (RproOBP27) From Rhodnius prolixus Antennae.

Olfactory proteins mediate a wide range of essential behaviors for insect survival. Odorant binding proteins (OBPs) are small soluble olfactory proteins involved in the transport of odor molecules (=odorants) through the sensillum lymph to odorant receptors, which are housed on the dendritic membrane of olfactory sensory neurons also known as olfactory receptor neurons. Thus, a better understanding of the role(s) of OBPs from Rhodnius prolixus, one of the main vectors of Chagas disease, may ultimately lead to new strategies for vector management. Here we aimed at functionally characterize OBPs from R. prolixus. Genes of interest were selected using conventional bioinformatics approaches and subsequent quantification by qPCR. We screened and estimated expression in different tissues of 17 OBPs from R. prolixus adults. These analyses showed that 11 OBPs were expressed in all tissues, whereas six OBP genes were specific to antennae. Two OBP genes, RproOBP6 and RproOBP13, were expressed in both male and female antennae thus suggesting that they might be involved in the recognition of semiochemicals mediating behaviors common to both sexes, such host finding (for a blood meal). Transcripts for RproOBP17 and RproOBP21 were enriched in female antennae and possibly involved in the detection of oviposition attractants or other semiochemicals mediating female-specific behaviors. By contrast, RproOBP26 and RproOBP27 might be involved in the reception of sex pheromones given that their transcripts were highly expressed in male antennae. To test this hypothesis, we silenced RproOBP27 using RNAi and examined the sexual behavior of the phenotype. Indeed, adult males treated with dsOBP27 spent significantly less time close to females as compared to controls. Additionally, docking analysis suggested that RproOBP27 binds to putative sex pheromones. We therefore concluded that RproOBP27 might be a pheromone-binding protein.