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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.

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 seven 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.

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

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 (OCL12), elicited inward or outward currents. 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 best 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).

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. AlphaFold models showed that AaegOR11 and CquiOR125 share structural homolog cores with MhraOR5, the only insect OR structure (PDB: 7LID) elucidated to date. 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.

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.

Summary 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., Graphical Abstract, Highlights • We found dual inhibitory/excitatory odorant receptors (ORs) in mosquitoes • Inhibitory and endogenous ORs coexpressed in flies showed lateral inhibition • The bipolar nature of these inhibitory ORs was displayed in electrophysiology • The duality excitation/inhibition was also manifested in mosquito behavior, Biological Sciences; Neuroscience; Sensory Neuroscience

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.

Chemosensory proteins (CSPs) have been considered to play a key role in chemoreception in insects. As stated in our earlier study, three CSP genes from rice leaf folder Cnaphalocrocis medinalis have been identified and showed potential physiological functions in olfaction. Here, we conducted western blot, immunolocalization, competitive binding assay and knockdown assay by RNA interference both in vitro and in vivo to reveal the functions of these three CSPs in C. medinalis. Results showed that both CmedCSP1 and CmedCSP2 are housed in sensilla basiconica and showed high binding affinities to a wide range of host-related semiochemicals. On the other hand, CmedCSP3 is highly expressed in sensilla trichodea of males and sensilla basiconica of females. It showed binding affinities to plant volatiles, especially terpenoids, as well as two of the C. medinalis sex pheromone components, Z11-16:Ac and Z11-16:Al. The transcript expression level of the three CSP genes significantly decreased after injecting target double-stranded RNAs and resulted in remarkably down-regulation on electroantennogram responses evoked by host-related semiochemicals and one sex pheromone compound, which have high binding affinities with CmedCSPs. In conclusion, the three CmedCSPs tested are involved in C. medinalis reception of semiochemicals, including host attractants and sex pheromones.

SUMMARYCarbon 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 tertiary 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.

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.

In addition to its primary function as an insect repellent, DEET has many “off-label” properties, including a deterrent effect on 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.

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.

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 8 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 2 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.

Insect repellents are important prophylactic tools for travelers to and populations living in endemic areas of malaria, dengue, encephalitis, and other vector-borne diseases, and the first line of defense against emerging arboviruses. 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.

Background Zika virus (ZIKV) has been isolated from many mosquito species in nature, but it is believed that the main vectors in urban environments are species of the genus Aedes. Here, we detected and isolated ZIKV in samples from Aedes aegypti, Aedes taeniorhynchus and Culex quinquefasciatus, collected during the Zika epidemic in Vitória, southeast Brazil. Using quantitative real-time polymerase chain reaction, ZIKV detection was performed in mosquito samples collected from February to April 2016. Results Overall, six pools of mosquitoes were positive for ZIKV: four of Cx. quinquefasciatus, one of Ae. aegypti and one of Ae. taeniorhynchus. Their genomes were sequenced. Conclusions These results support and strengthen the hypothesis that other mosquito species can also be involved in ZIKV transmission.

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.

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 (4L) of the same or different species. No activity was found when 4L 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.

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.

Tissue samples from mosquitoes artificially infected with Zika virus and shown to be positive by RT-qPCR were reexamined by RT-PCR. Using these samples we compared the two methods employed in virus RNA detection for vector competence studies. Results demonstrated that, albeit useful, RT-PCR gave false negatives with low viral loads (< 106 RNA copies/ml).

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.

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.

The body louse, Pediculus humanus humanus, is an obligate blood-feeding ectoparasite and an important insect vector that mediates the transmission of diseases to humans. The analysis of the body louse genome revealed a drastic reduction of the chemosensory gene repertoires when compared to other insects, suggesting specific olfactory adaptations to host specialization and permanent parasitic lifestyle. Here, we present for the first time functional evidence for the role of odorant receptors (ORs) in this insect, with the objective to gain insight into the chemical ecology of this vector. We identified seven putative full-length ORs, in addition to the odorant receptor co-receptor (Orco), and expressed four of them in the Xenopus laevis oocytes system. When screened with a panel of ecologically-relevant odorants, PhumOR2 responded to a narrow set of compounds. At the behavior level, both head and body lice were repelled by the physiologically-active chemicals. This study presents the first evidence of the OR pathway being functional in lice and identifies PhumOR2 as a sensitive receptor of natural repellents that could be used to develop novel efficient molecules to control these insects.

Under laboratory conditions, mating activity in Asian citrus psyllid (ACP) started 4 days after emergence, peaked at day 7, and showed a clear window of activity starting 8 h into the photophase and extending through the first hour of the scotophase. We confirmed that ACP males are attracted to emanations from conspecific females. Traps loaded with a candidate compound enriched with female extract, lignoceryl acetate (24Ac), at various doses were active only after being deployed for several weeks in the field, suggesting that a degradation product, not the test compound, was the active ingredient(s). Lignocerol, a possible product of 24Ac degradation, was not active, whereas acetic acid, another possible degradation product, was found in the airborne volatile collections from lures matured under field conditions and detected in higher amounts in volatiles collected from females at the peak of mating activity than in male samples. Acetic acid elicited dose-dependent electroantennographic responses and attracted ACP males, but not females, in Y-type and 4-way olfactometers. Field tests showed that acetic acid-baited traps captured significantly more males than control traps. Surprisingly, captures of females in acetic acid-baited traps were also higher than in control traps, possibly because of physical stimuli emitted by captured males.

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. Intrareceptor 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.One Sentence SummaryIntrareceptor inhibition was observed in mosquito odorant receptors expressed in heterologous systems, in vivo, and manifested in behavioral responses.

Insect repellents are widely used to fend off nuisance mosquitoes and, more importantly, to reduce or eliminate mosquito bites in areas where viruses and other vector-borne diseases are circulating. Synthesized more than six decades ago, DEET is the most widely used insect repellent. Plant-derived compounds are used in a plethora of commercial formulations and natural recipes to repel mosquitoes. They are also used as fragrances. We analysed Bombshell® to identify the constituent(s) eliciting a previously reported "off- label" repellence activity. The two major fragrance ingredients in Bombshell, i.e., methyl dihydrojasmonate and lilial, demonstrated strong repellence against the southern house mosquito, Culex quinquefasciatus, in laboratory assays. Both compounds activated a previously identified DEET-sensitive odorant receptor, CquiOR136. These compounds were also major constituents of Ivanka Trump eau de parfum. The methyl dihydrojasmonate content was higher in the Ivanka Trump perfume than in Bombshell, the reverse being true for lilial. Both Bombshell and Ivanka Trump eaux de parfums retained activity for as long as 6 hours in laboratory assays. Although wearing these perfumes may repel nuisance mosquitoes, their use as "off-label" repellents against infected mosquitoes is not recommended.

Significance Surveillance of mosquito populations is essential for determining the best timing for intervention as well as for monitoring circulation of new and previously detected pathogens. Trapping gravid females is more effective because they are more likely to be infected than nongravid females. However, better gravid trap attractants are sorely needed to replace fermentation recipes, which are cumbersome, have poor quality control, generate an offensive smell, and do not provide a long-term and consistent source of attractants. By using a reverse chemical ecology approach based on odorant receptors from the southern house mosquito, we have identified that acetaldehyde in a wide range of doses both attracts gravid female mosquitoes and stimulates them to lay eggs in oviposition trays., Pheromones and other semiochemicals play a crucial role in today’s integrated pest and vector management strategies. These semiochemicals are typically discovered by bioassay-guided approaches. Here, we applied a reverse chemical ecology approach; that is, we used olfactory proteins to lead us to putative semiochemicals. Specifically, we used 7 of the top 10 odorant receptors (ORs) most expressed in the antennae of the southern house mosquito, Culex quinquefasciatus, and which are yet to be deorphanized. We expressed these receptors in the Xenopus oocyte recording system and challenged them with a panel of 230 odorants, including physiologically and behaviorally active compounds. Six of the ORs were silent either because they are not functional or a key odorant was missing. CquiOR36, which showed the highest transcript levels of all OR genes in female antennae, was also silent to all odorants in the tested panel, but yielded robust responses when it was accidentally challenged with an old sample of nonanal in ethanol. After confirming that fresh samples were inactive and through a careful investigation of all possible “contaminants” in the old nonanal samples, we identified the active ligand as acetaldehyde. That acetaldehyde is activating CquiOR36 was further confirmed by electroantennogram recordings from antennae of fruit flies engineered to carry CquiOR36. Antennae of female mosquitoes also responded to acetaldehyde. Cage oviposition and dual-choice assays demonstrated that acetaldehyde is an oviposition attractant in a wide range of concentrations and thus of potential practical applications.

Chemical ecology is the study of the chemical languages, cues, and mechanisms controlling interactions among living beings, including communication among individuals of the same species and between organisms and their environment. Organisms use chemicals to lure their mates, associate with symbionts, deter enemies, and fend off pathogens (1). Since the identification of the silkworm moth sex pheromone almost six decades ago (2), chemical ecologists have been deciphering hundreds of these “Rosetta Stones” (3) by using bioassay-guided protocols. This conventional chemical ecology approach is based on an invasive process of extracting secretions from chemical signal (semiochemical) senders (e.g., female moths), separating extracts into fractions, using receivers (e.g., male moths) to assist in the identification of active ingredients and, finally, by elucidating chemical structures and synthesis. The state-of-the-art techniques in chemical ecology have reduced analysis to even single individuals in many cases, but it is still too invasive for studying endangered or vulnerable species. In PNAS (4), a multidisciplinary group of scientists from China, Italy, and France apply tools of reverse chemical ecology (5) to study chemical communication in the giant panda, Ailuropoda melanoleuca , a vulnerable species endemic to China. The giant panda has an obligate bamboo diet and a carnivorous digestive system (6), which leads to a sedentary life with a limited reproduction rate, resulting in only a single offspring every other year. This mismatch of lifestyle and physiology coupled with fragmented habitats in its native environment in southwest China placed the giant panda on a list of … [↵][1]1Email: wsleal{at}ucdavis.edu. [1]: #xref-corresp-1-1

Anecdotes related to preferential mosquito bites are very common, but to date there is no complete explanation as to why one out of two people systematically receives more mosquito bites than the other when both are equally accessible. Here we tested the hypothesis that two constituents of skin emanations, 6-methyl-5-heptan-2-one (6-MHO) and geranylacetone (GA), are natural repellents and may account for differential attraction in different ratios. We studied skin emanations from two human subjects, confirmed in behavioral assays that female southern house mosquitoes are significantly more attracted to subject A (attractant) than to subject N (non-attractant), and tested their 6-MHO/GA ratios in a dual-choice olfactometer. Although repelling at high doses, 6-MHO/GA mixtures were not active at the levels emitted by human skin. We found, however, differential attraction elicited by the aldehydes in the ratios produced by subjects A and N. When tested in a dose commensurate with the level released from human skin and in the ratio produced by subject A, the aldehyde mixture significantly attracted mosquitoes. By contrast, an aldehyde mixture at the same ratio released by subject N did not attract mosquitoes. We, therefore, hypothesized that aldehydes may play a role in the commonly observed differential attraction.