Sensory coding of borneol repellency in culicine mosquitoes via the Or49 pathway

Domain: chemistry
Published: 2026-02-20
Editorial outcome: major_revision

Abstract
Plant-derived repellents containing borneol and camphor have been used for centuries, yet the sensory mechanisms that mediate their effects in mosquitoes have remained obscure. Here, we show that culicine mosquitoes possess a dedicated olfactory pathway for detecting bicyclic monoterpenoids. We identify odorant receptor 49 (OR49) as a conserved and highly selective borneol receptor expressed in a distinct neuron of the maxillary palp. This neuron forms a defined projection to the MD3 glomerulus in the antennal lobe, establishing a labeled line for plant-derived repellents. Genetic disruption of Or49 abolishes electrophysiological responses to borneol and markedly diminishes avoidance behavior during human host-seeking. These results reveal that female mosquitoes detect borneol through a specialized sensory channel distinct from those mediating attraction to human cues. Our findings provide a mechanistic framework for understanding how plant volatiles shape mosquito behavior and offer a foundation for the rational design of next-generation olfactory repellents.

Introduction
The use of plants to ward off insects has been a human practice since prehistoric times1 and is still used in many parts of the world2,3. Plant-based essential oils (EOs), including lemon eucalyptus leaf oil4, citronella oil5, and coconut oil6 exhibit different degrees of mosquito repellency due to the presence of pyrethrins7, phenol derivatives, and terpenoids8. The latter includes oxygen-containing compounds with open-chain hydrocarbons, such as linalool, citronellol, geraniol, and bicyclic derivatives such as cineole, fenchone, camphor, and borneol (Fig. 1a). The EO of the camphor tree Cinnamomum camphora is composed of over 70 volatile organic compounds (VOCs), most of which are oxygenated monoterpenes dominated by camphor and other related compounds9. Camphor and borneol extracts are believed to have originated from the camphor tree in the island of Borneo (Fig. 1a), where they were initially traded with China and then introduced to the West due to their therapeutic, refreshing, and repellent effects against mosquitoes10. How these terpenoids molecules exert repelling effects against mosquitoes is not well-understood but is likely mediated by their olfactory system.

The capitate-peg (cp) sensillum located on the mosquito maxillary palp comprises three olfactory neurons, each distinguishable by size, olfactory receptor gene expression profile, and odor response characteristics (Fig. 1b). In both culicine and anopheline mosquitoes, the largest olfactory sensory neuron (cpA) expresses three gustatory receptors (Grs) that specifically detect CO2 11,12. The medium-sized cpB neuron of Anopheles gambiae and Aedes aegypti expresses the 1-octen-3-ol odorant receptor Or8 and its co-receptor Orco13–15. Both CO2 and 1-octen-3-ol elicit attraction and signal the presence of animal hosts in anopheline16 and culicine17 mosquitoes. This cellular and functional organization has remained remarkably conserved over 180 million years of mosquito evolution (Fig. 1c).

In anophelines, the small cpC neuron expresses the Or28 gene13, which responds to plant volatile organic compounds (pVOC)18. In culicines, the cpC neuron expresses the Or49 gene19, which is unrelated to AgamOr28 (Fig. 1b). Using a pharmacological approach, we expressed Or49 in a heterologous expression system and exposed it to odorant mixtures, EOs, and individual VOCs. Our findings provide strong evidence that the OR49 receptor and the cpC neuron respond to plant-derived bicyclic monoterpenoids with a marked selectivity towards borneol, traditionally used as a mosquito repellent. To directly assess its olfactory function, we generated an Or49 null mutant line in Aedes aegypti, which completely lacked electrophysiological responses to borneol and displayed reduced avoidance behavior in the context of human-host seeking. Furthermore, we show that borneol selectively activates the MD3 glomerulus in the antennal lobe of Ae. aegypti. Together, these results provide a framework for dissecting the molecular and neural mechanisms underlying olfactory integration and host-seeking behavior in mosquitoes.

Results
Odorant receptor 49 is an evolutionary conserved and selective borneol receptor
The odorant receptor 49 gene 19 is represented by conserved 1-to-1 orthologs in Aedes albopictus, Culex quinquefasciatus, Toxorhynchites amboinensis, and Aedes aegypti (Supplementary Fig. 1). Phylogenetic analyses support previous studies19,20 describing the OR49 family as Culicinae-specific with no conserved orthologs detected in Anopheles (Supplementary Fig. 1c–d). However, related Or49-like genes are located on chromosome 2 in both Aedes and Anopheles mosquitoes (Supplementary Fig. 1e). Clear evidence of conserved synteny with neighboring genes is observed between Ae. aegypti and Ae. albopictus (Supplementary Fig. 1e–f).

The Or49 gene confers sensitivity to borneol
As a continuation of our work on the functional identity of the cp sensillum in culicine mosquitoes19,21, we investigated the receptive field of Ae. albopictus OR49 (AalbOR49) using two-electrode voltage clamp recordings (Fig. 1d) and a panel of 81 odorants representing diverse chemical classes (Supplementary Data 1). Xenopus laevis oocytes expressing functional receptor complexes were strongly activated by blends containing ketonic and alcoholic terpenes (Fig. 1d bottom). Testing of single compounds from these two active blends revealed the efficacy of bicyclic monoterpenoids. (1R)-(+)-camphor emerged as the most efficacious ligand, followed by 1,8-cineole (also called eucalyptol) and both stereoisomers of fenchone (Fig. 1d).

In an independent set of experiments, we investigated the potential of Cannabis EOs (Supplementary Fig. 2, Supplementary Data 1) to activate OR49 from the nectar-feeding species Tx. amboinensis (TambOR49) (Fig. 2a-b). We tested eight Cannabis EOs (Fig. 2c) containing a total of 42 plant VOCs, dominated by terpenes (Supplementary Fig. 2). Among these, the most active were Pineapple Haze, OG Kush, and Adom #9, while Master Kush and Jack Herer showed much lower efficacy. Furthermore, Alien OG, Forbidden Fruit and Kush Note did not activate the receptor (Fig. 2c). All active mixtures evoked consistent current responses (Supplementary Fig. 3). Successive fractionations of these EOs into 6 sub-mixtures and 4 single compounds revealed that racemic borneol is the most potent compound (Fig. 2d), consistent with its content in the original Cannabis mixtures (Fig. 2c, Supplementary Fig. 4a) and sub-mixtures (Supplementary Fig. 4b). TambOR49 and AaegOR49 did not exhibit enantioselectivity towards the (+) and (–)-borneol enantiomers (Fig. 2e), as indicated by their EC50 values in the one-digit micromolar range. We therefore focused subsequent oocyte analyses on the (+) enantiomers. OR49 orthologs from Ae. albopictus, Ae. aegypti, Cx. quinquesfasciatus and Tx. amboinensis all responded robustly to the two most potent ligands, (+)-borneol and (+)-camphor, although with varying selectivity. We established concentration-response relationships with these ligands in TambOR49, Cx. quinquefasciatus OR38 (CquiOR38), Ae. aegypti OR49 (AaegOR49), and Ae. allbopictus OR49 (AalbOR49) (Fig. 2f). (+)-Borneol was 19-32 times more potent than (+)-camphor in all cases except for TambOR49 where both compounds were equally active in the low micromolar range (Fig. 2f). However, when comparing maximum response magnitudes, (+)-borneol consistently elicited higher currents than (+)-camphor across all four culicine species, including the nectar-feeding Tx. amboinensis (Fig. 2g, Supplementary Fig. 3). These results indicate that OR49 shows strong selectivity for borneol and closely related compounds.

By contrast, in An. gambiae, the cpC neuron expresses OR28 instead of OR49 (Fig. 1b). This receptor is activated by plant VOCs, with acetophenone and 2,4,5-trimethylthiazole being the most effective ligands in the Xenopus oocyte expression system13,18. The EC50 values of acetophenone, α-pinene, and α-terpineol were in the low millimolar range, indicating that more potent ligands remain to be identified (Supplementary Fig. 5). Notably, borneol did not elicit any response at the tested concentrations suggesting that AgamOR28 is tuned to VOCs belonging to a different chemical class.

The Or49 gene confers sensitivity to borneol
To investigate the olfactory effect of camphor and borneol in vivo, we conducted electropalpogram (EPG) recordings on three culicine (Ae. albopictus, Ae. aegypti, Cx. pipiens) and one anopheline (An. gambiae) species (Fig. 3a). 1-Octen-3-ol, which served as a positive control, induced significant responses in all mosquitoes tested. Camphor and borneol elicited consistent palp responses in Culicine mosquitoes, including Ae. albopictus, Ae. aegypti, and Cx. pipiens but not in An. gambiae, which expresses Or28 in the palp instead of Or49 (Fig. 3b). Camphor and borneol evoked lower responses than 1-octen-3-ol in the culicine palps. In Ae. albopictus, (–)-borneol was more active than (+)-camphor, while in Ae. aegypti both borneol enantiomers elicited significantly greater EPG responses than camphor enantiomers. In Cx. pipiens, borneol and camphor elicited comparable EPG responses. We established dose-response relationships with (+)-camphor and (+)-borneol in Ae. albopictus, Ae. aegypti, and Cx. pipiens (Fig. 3c). Aedine spp. exhibited significantly larger responses to (+)-borneol in doses higher than 0.1 µg. While Cx. pipiens exhibited statistically significant larger responses in response to (+)-camphor in doses higher than 1 µg.

To test a potential causal relationship between Or49 and the borneol response, we used CRISPR-Cas9 to knockout the Or49 gene in Ae. aegypti, which serves as a more genetically tractable model than Ae. albopictus (Supplementary Fig. 6). This knockout line exhibits a 442 bp deletion spanning the start codon and the first exon of the Or49 gene, along with multiple SNPs upstream at the promoter sequence (Supplementary Fig. 6).

To investigate the genetic mechanism determining borneol sensitivity in the palp, we conducted single-sensillum recordings (Fig. 4a) from the cp sensillum of Ae. aegypti (Fig. 4c), An. gambiae (Fig. 4d), Ae. albopictus (Fig. 4e), and Cx. quinquefasciatus (Fig. 4f). The response of the cpA and cpB neurons to CO2 (data not shown) and R-(–)−1-octen-3-ol23, respectively, has been well characterized in previous studies13–15; note that two previous studies have identified cpC as the R-(–)−1-octen-3-ol responding neuron in Ae. aegypti14,24, which may be due to differences in genotypes used. (+)-Borneol elicited a dose-dependent response in the cpC neuron of Ae. aegypti (Supplementary Fig. 7), Ae. albopictus, Cx. quinquefasciatus but not in An. gambiae s.s. (Fig. 4b). Single sensillum recordings identified the spontaneous activity of three sensory neurons housed in the capitate peg sensilla of wild-type Ae. aegypti (Fig. 4c) and An. gambiae, which are clearly distinguishable by differences in spike amplitudes (Fig. 4d). No SSR responses were recorded from the cpC neuron in the Ae. aegypti Or49 null mutant line in response to (+)-borneol (Fig. 4c), indicating that Or49 is necessary to confer the cpC neuron sensitivity to borneol. Although recordings were not obtained from the cpA and cpB neurons in the Or49 null mutant, these neurons displayed normal spontaneous activity (Supplementary Fig. 8). We note that the reduced longevity of the mutant line could potentially affect neuronal function; however, no such effect was observed. The lack of response of the cpC neuron in Anopheles (Fig. 4d) is consistent with the expression of Or28 instead of Or49, which is not activated by borneol (Supplementary Fig. 5). The absence of OR28 activation and An. gambiae palp EPG responses by borneol and camphor support this conclusion.

Borneol and camphor specifically activate the MD3 glomerulus in the antennal lobe
To examine how Ae. aegypti processes borneol in the antennal lobe (AL), two-photon imaging experiments were conducted using pan-neuronal GCaMP-expressing Ae. aegypti mosquitoes. We utilized existing mosquito lines that contained a QUAS-GCaMP7s transgene crossed with the brp-QF2 driver line25, allowing the directed expression of the calcium indicator GCaMP7s in all neurons of the AL. Mosquitoes were glued to holders that permitted two-photon imaging of calcium responses in the AL (Fig. 5a), allowing repeatable registration of the AL glomeruli between preparations (Fig. 5b, S10) and recording of glomerular responses (Fig. 5c)26. Glomeruli from our two-photon imaging results were mapped to AL atlases27,28, allowing accurate identification of the glomeruli of interest in their spatial context (video rendering of the antennal lobe in Supplementary Movie 1). We focused on the mediodorsal (MD) glomeruli, using the most recently published AL atlas27, as these glomeruli receive input from the capitate peg sensilla of maxillary palps (Fig. 5b, c, S10) and are responsive to host odors, including 1-octen-3-ol (MD2) and CO2 (MD1)28.

To determine the odor coding of the mediodorsal glomeruli, and identify the cognate glomerulus representing borneol, we concentrated on recording from the MD3 glomerulus and stimulating with borneol (10-4 dilution). Next, we examined responses of the MD1 and 2 glomeruli if they occurred in the same imaging plane while stimulating with CO2 (5%), 1-octen-3-ol (10-4 dilution), borneol (10-4 dilution), and the solvent control. For the MD3 glomerulus, (+)-borneol elicited strong, tonic responses that were significantly greater than the solvent control (Kruskal-Wallis test, P=0.006) (Fig. 5d, g). By contrast, responses of the MD1 and MD2 glomeruli to borneol were not significantly different from the control (P=0.18 and 0.31, respectively). Similarly, the MD2 glomerulus showed the strongest response to 1-octen-3-ol and the MD1 glomerulus to CO2 (Supplementary Fig. 9). There was no significant difference between borneol enantiomers (Kruskal Wallis test with multiple comparisons, P>0.93), although borneol evoked significantly greater responses than the solvent (hexane) control (Kruskal Wallis test with multiple comparisons, p<0.05) (Fig. 5g, S9). These findings are consistent with previous studies demonstrating that the three MD glomeruli receive input from the maxillary palp cp neurons, whereby the MD1 glomerulus receives CO2 input from the cpA neurons29 while the medium-sized MD2 glomerulus (previously labeled as MD328) receives 1-octen-3-ol input from the cpB neurons28.

Borneol repellency in human-host-seeking female Ae. aegypti is largely mediated by Or49
As the mutant lines initially used for SSR exhibited reduced behavioral activity, we generated a new homozygous Or49 knockout line after backcrossing for six generations and focused on behavioral validation to assess borneol’s repellency. To examine whether (+)-borneol’s repellency to blood-seeking Ae. aegypti mosquitoes is mediated by Or49, we conducted an arm-in-a-cage assay. Fifteen WT and Or49 K.O. females were exposed to human skin odor in the presence and absence of (+)-borneol, for 10 minutes (Fig. 6a). A protective glove covered the hand, allowing mosquitoes to detect the odor through a dorsal open area, which was protected with a screen and equipped with a chemical holder for the deposition of (+)-borneol (1M) (Fig. 6a, Supplementary Fig. 10). Although the setup allows for potential contact between mosquitoes and the borneol-soaked filter paper, we did not observe any instances of direct physical interaction.

Our results show that under (+)-borneol exposure, wild-type females exhibited an average reduction of 54% in visits to the ROI compared to the vehicle control, whereas Or49⁻/⁻ mutants displayed a more modest reduction of 25% compared to their respective vehicle control (Fig. 6b). Representative behavioral traces and detections are shown in Fig. 6e and Supplementary video 2. The repellent effect of (+)-borneol in WT mosquitoes was observed mainly at the first three minutes of the experiment. Contrary to this, Or49 null mutants behaved similarly to the control conditions (i.e., without (+)-borneol) (Fig. 6c). To assess the mixed effects of visit rates, time intervals, and their interaction, we performed a Generalized Linear Mixed Model (GLMM) with biological replicates included as a random effect. Under control conditions (Hand + diethyl ether), there was no significant difference in the number of visits to the region of interest (ROI) between genotypes (p=0.492), and no significant interaction between genotype and time (p=0.309). However, both WT and KO mosquitoes exhibited a significant reduction in visit rates over time (p<0.001), consistent with a gradual decline in exploratory activity (Fig. 6d top Supplementary Data 2). In the presence of (+)-borneol, visit rates diverged significantly between strains. WT mosquitoes displayed consistently low visit rates, while KO mosquitoes made significantly more visits (strain effect: p<0.0001), with a significant strain × time interaction (p<0.0001), indicating differential behavioral trajectories over the course of the assay (Fig. 6d, bottom; Supplementary Data 2). Wild type mosquitoes showed no significant change in the number of visits over time (p=0.668), indicating a stable avoidance response. In secondary behavioral metrics, WT and Or49⁻/⁻ mosquitoes showed no significant difference in walking distance (p=0.09) or visit duration (p=0.55) when exposed to the hand alone (Data S4). In contrast, Or49⁻/⁻ mosquitoes exhibited a significant increase in visit duration (p=0.005) under borneol exposure (Supplementary Fig. 11; Supplementary Data 2). Full trajectory data for all treatments are provided in Supplementary Fig. 12.

Methods
Insects
Our mosquito colonies were reared according to previously published protocols48. Ae. aegypti originated from a colony established by Prof. Joel Margalit. The Ae. aegypti Liverpool strain was provided by the Akbari lab. Aedes albopictus was the FPA Foshan strain collected (Foshan, China) reared at the insectary of the University of Pavia since 2013 (Palatini et al., 2017). Anopheles gambiae was the G3 strain originally isolated from West Africa (MacCarthy Island, The Gambia) in 1975 (Federica Bernardini et al., 2017). Culex pipiens pipiens originated from a wild type population that was provided by Dr. Laor Orshan (Ministry of Health, Israel).

Phylogenetic and genomic analyses
The AaegOR49 protein sequence (AAEL001303-PA) was used as a query to identify homologs in other mosquito species. Amino acid sequences of homologous ORs were obtained from the Vectorbase database (vectorbase.org) and the Tx. amboinensis genome assembly49. The multiple sequence alignment was conducted using ClustalW50. The Maximum-likelihood phylogenetic tree was constructed using MEGAX software (Model: G, bootstraps: 5000). Exon-intron structure analysis was conducted manually by aligning predicted amino-acid sequences to genomic regions. Approximate mapping of the genes in chromosome location for each mosquito species and construction of synteny maps were done using Geneious Prime software51 using the latest genome assemblies available for each mosquito species20,52–55.

Genomic DNA (gDNA) was isolated from whole bodies of Tx. amboinensis adults. gDNA was diluted to 25ng/µL in nuclease free water and used as a template in polymerase chain reactions to amplify odorant receptors (TambOR) using Taq polymerase and the following primer sets: TambOr6.F1 (ATGCGCTTCTACGAGAAATAC), TambOr6.R1 (TCAGAAATTATCCTTCAGGATC); TambOr12.F1 (ATGCCATCGGTTTTCTTGGTT), TambOr12.R1 (CTAAAACACTCGCTTCAATATC); TambOr13.F1 (ATGTTCTGCTTCAGGAAGATC), TambOr13.R1 (CTAGAAGTGGTTTTTCAATATAA); TambOr49.F1 (ATGTTGTTCAAGAACTGTTTCC), TambOr49.R1 (TTAATAATTGAATCTTTCCTTCAG); TambOr71.F1 (ATGGGCAGCAGTGATGGTGAC), TambOr71.R1 (CTACTGGTTGATTTTACTGAGG). Cycling conditions were 94°C for 60s; 30 cycles of 94°C for 20s, 56°C for 20s, 72°C for 30s; and a final extension of 72°C for 5 minutes. Amplicons were analyzed by electrophoresis on a 1% agarose gel, cloned into the TOPO-TA pCR2 plasmid, heat-shock transformed into TOP10 competent E. coli cells, and grown overnight at 37°C on LB+ampicillin+X-gal agar plates. Ampicillin-resistant, white colonies were isolated using a sterile pipette tip and grown overnight at 37°C in 3 mL of LB+ampicillin. Plasmids were isolated by DNA miniprep, and the Sanger method was used to determine the DNA sequence in both directions using standard T7 and M13.rev primers. TambOR nucleotide sequences were compiled into contigs and intron/exon regions were inferred by comparing to coding sequences described in a previous publication49.

Chemical reagents
The chemicals used for the deorphanization of receptors were obtained from Acros Organics (Morris, NJ, USA), Alfa Aesar (Ward Hill, MA, USA), ChemSpace (Monmouth Junction, NJ, USA), Sigma Aldrich (St. Louis, MO, USA), TCI America (Portland, OR, USA), Thermo Fisher Scientific (Waltham, MA, USA), and Penta Manufacturing Corp. (Livingston, NJ, USA) at the highest purity available. Cannabis EOs and sub-mixtures were formulated and supplied by Eybna Technologies (Kfar Saba, Israel) (Supplementary Data 1 and Supplementary Fig. 2, 4).

Two-electrode voltage clamp of Xenopus laevis oocytes
In vitro transcription and two-microelectrode voltage clamp electrophysiological recordings were carried out as previously described56. Experimental procedures for the Ae. albopictus receptor clone was performed as previously described57. AalbOr49 and AalbOrco templates were synthesized by Twist Biosciences (San Francisco, CA, USA) and cloned into the pENTRTM vector using the GatewayR directional cloning system (Invitrogen Corp., Carlsbad, CA, USA) and subcloned into the X. laevis expression destination vector pSP64t-RFA. Several of these genes were codon-optimized (Supplementary data 2). The EC50 values were calculated using Graphpad Prism 8. Briefly, after data collection, the data of each oocyte was normalized against its highest response. This was done to overcome the high variance between individual oocytes. After normalization, the data of each oocyte was analyzed using the function “Nonlinear regression” with “Dose – response – Stimulation” equation “log(agonist) vs. normalized response”. This function yields the EC50 value for each repetition (at least five oocytes). The EC50 values were then analyzed for the average and S.E.M values (Supplementary Fig. 13). For pairwise current comparisons of (+)-borneol and (+)-camphor, two separate perfusion systems were assembled, one for each compound. The outlet from each system was connected to a 2 to 1 perfusion manifold. At each tested concentration, (+)-borneol and (+)-camphor were administered consecutively. Representative current traces can be seen in Supplementary Figs. 3 and 5. Odorant receptor 49 and Orco clones and recording measurements can be found in Supplementary Data 2 and 3, respectively. The use of Xenopus laevis frog eggs was carried out according to The Hebrew University of Jerusalem Ethics committee (NIH approval number: OPRR-A01-5011). The list of Or nucleotide sequences for injection in Xenopus oocytes can be accessed in Supplementary data 2.

Electropalpogram recordings
Based on the voltage clamp data, responses from the maxillary palps of Ae. albopictus, Ae. aegypti, Cx. pipiens and An. gambiae were recorded with camphor or borneol. Electopalpogram (EPG) assays were performed according to published procedures58 with a few modifications. Within the Pasteur pipette, 20 μL of odorants diluted in hexane were deposited at the desired dose onto a 4 × 20 mm Whatman filter paper strip. The odorants were delivered into a consistent humidified air-stream (at a flow rate of 50cm/s) at approximately 2cm from the maxillary palps. The pulse duration was 0.1s, and the recording time was set for 5s. A 2-min gap was allowed between stimuli to recover the EPG sensitivity. At least 5 individual females were tested, for each individual, 3 technical replicates were performed with doses ranging from 0.01 ug to 100 μg. Negative controls (hexane and air) and positive controls (1-octen-3-ol) were performed at both the beginning and end of each recording session to monitor the decline in sensitivity of the maxillary palps. EPG peak responses were normalized to the response of hexane.

Generating Or49 knockout line in Ae. aegypti and isolating an Or49-/- homozygous line
Genomic DNA was extracted from whole bodies of Ae. aegypti Liverpool individuals using the DNeasy Blood & Tissue Kit (Qiagen, Redwood City, CA). Homology arms of the Or49 gene flanking 1kb upstream and downstream of the desired insertion site (Supplementary Fig. 6a) were amplified with the Q5 High Fidelity DNA polymerase (New England Biolabs, Ipswich, MA), using primer pairs Left_Arm_Or49_FWD and Left_Arm_Or49_REV as well as Right_Arm_Or49_FWD and Right_Arm_Or49_REV, respectively (Supplementary Data 3). The QF2-ECFP DNA cassette was amplified using the V1117A plasmid as template and the primer pair QF2_OpIE_ECFPFWD and QF2_OpIE_ECFP REV. PCR bands were cut out from agarose gels and purified with Zymoclean Gel DNA Recovery Kit (Zymo Research, Irvine, CA). Upstream and downstream homology arms, as well as the QF2-ECFP cassette PCR products, were assembled into the backbone of the V1117A plasmid using Gibson assembly reaction, following the manufacturer's recommendations. Gibson’s reaction-derived plasmid product was used to transform JM109 cells (Zymo Research), and colonies were individually grown overnight for minipreps with Zyppy Plasmid Miniprep Kit (Zymo Research). The sequence of the plasmid V1117F-Or49 (Supplementary Fig. 6b) was confirmed by restriction enzyme digestion using the enzymes ScaI-HF and AvrII as well as by whole plasmid sequencing (Primordium Labs, Monrovia, CA). V1117F-Or49 plasmid was used to retransform JM109 cells, which were grown for maxiprep purification using the PureLink Expi Endotoxin-Free Maxi Plasmid Purification Kit (Thermo Fisher Scientifics, Waltham, MA).

For guide RNA synthesis, non-template reactions were carried out with the gRNA_Left_Or49_F and gRNA_Left_Or49_R forward primers and the universal guide RNA reverse primer (Universal-sgRNA_R). PCR bands were isolated from agarose gel and purified as described above (Supplementary Fig. 6c). Guide RNAs were synthesized with the Ambion MEGAscript kit (Thermo Fisher Scientifics) for 4 hours at 37°C, using 300 ng of purified PCR product. Guide RNAs were further purified with the Megaclear Kit (Thermo Fisher Scientifics).

To assess the cleavage activity of the synthesized guide RNAs, in vitro Cas9 cleavage assays were carried out. A DNA fragment spanning 1,009 bp overlapping the cleavage sites of the guide RNAs was amplified with primers Or49_cleav_F and Or49_cleav_R, DNA bands were isolated from agarose gel, purified, and 100 ng of which was used in cleavage assays with 300 ng of recombinant Cas9 (PNABIO, Thousand Oaks, CA) and 100 ng of each guide RNA upon incubation at 37°C for 1 hour.

For embryo microinjection, an injection mix was prepared with the plasmid V1117F-Or49 at 500 ng/ul, gRNAs left and right at 100 ng/ul each, and recombinant Cas9 at 300 ng/ul. Mix was filtered with the Ultrafree-MC Centrifugal Filter UFC30GUOS (Millipore, Burlington, MA). Freshly harvested Ae. aegypti embryos (Liverpool strain) were injected with the transformation mix using a custom-made insect embryo microinjector (Hive Technologies, Cary, NC) at 100 psi pulse and 3 psi back pressures, using quartz needles pulled with a P-2000 needle puller (Sutter Instrument Co., Novato, CA). Genomic DNA of the G1 fluorescent mosquitoes (Supplementary Fig. 6d) was individually extracted with the Qiagen blood kit. For whole DNA cassette sequencing (Primordium), 2.5 kb DNA fragments were amplified from the DNA of G1 individuals using the primer pair Or49_diag_up_F and Or49_diag_down_R (Supplementary Fig. 6e).

For isolation of homozygous knockout individuals, pupae were sex sorted, and single pairs were transferred into Narrow Drosophila Vials (Genesee Scientific, El Cajon, CA), filled with 10 mL of deionized water (DI) and closed with cotton stoppers. Upon emergence, water was drained, and sugar cottons were replaced into the vials. Mosquitoes were allowed to mate for 5 days in the vials, and then all individuals were transferred to a single cage for blood feeding. Three days after blood feeding, females were individually transferred to a fresh vial containing a piece of brown paper towel wetted with 1 mL DI water. Females were allowed to lay eggs, and each egg batch was individually hatched in 250 mL Clear Pet Cups (9oz). Larvae were screened for fluorescence, and the batches that resulted in 100% fluorescent individuals were grown to adulthood and intercrossed to confirm homozygosity in the following generation. Two out of thirty egg batches resulted in homozygous offspring. To further confirm the homozygous status of these individuals, genomic DNA of three pools of 10 males, 10 females, and a mix of males and females were PCR amplified with the primer pair Or49_diag_up_F and Or49_diag_down_R, which resulted in a single band for homozygous individuals and two bands for heterozygous individuals (Supplementary Fig. 6f). To solve the low initial performance of this mutant line in behavioral assays, this line underwent 6 generations of backcross to the wild type line, intercrossed for 3 generations, and a homozygous line was selected as described.

Three primer pairs (Or49_diag_offsite_F/R, Or49_amp_F/R, and Or49_M13_F/R) that target different regions of the plasmid backbone (Supplementary Fig. 6a) were used to verify whether the plasmid was incorporated into the genome of the Or49 knockout line (Supplementary Fig. 6g). The Or49_diag_up_2F and Or49_diag_down_2R primer pair, which base-pair with genomic sequences outside of the homology arms, on the other hand, were used to amplify the homology arms along with the DNA cassette, in order to amplify the endogenous Or49 gene (Supplementary Fig. 6h), rather than the sequence of the Or49 gene of the plasmid that was inserted into the genome.

Single-sensillum electrophysiology
Single sensillum recordings from capitate peg sensilla on the maxillary palp of wild-type female Ae. aegypti (Liverpool), Ae. albopictus (FPA), Cx. quinquefasciatus (Thai) and An. gambiae sensu stricto (G3) and Ae. aegypti Or49-/- mutant mosquitoes were performed using an established protocol59. Spikes were quantified offline using the established nomenclature for the sensory neurons60. The number of spikes counted during a 0.5s stimulus delivery interval was subtracted from the number of spikes counted during a 0.5s prestimulus period, and the result was multiplied by 2 to obtain the activity of individual sensory neurons housed in the capitate peg sensilla as spikes/s measurement.

Discrimination between the cpB and cpC neurons is based on spike amplitude, spontaneous activity and their respective functional responses. In our study, the cpB neuron, which is sensitive to 1-octen-3-ol, consistently exhibited larger action potential amplitudes than the cpC neuron, which is activated by borneol. This is in line with several other studies in Aedes, Culex, and Anopheles species that have reported that the cpB neuron is responsive to octenol13,23,59,61–63. While other investigations in Aedes aegypti have identified the cpC neuron, characterized by the smallest spike amplitude, as the octenol-sensitive neuron the results are ambiguous. The reason for this discrepancy remains unclear, but likely stem from either a misidentification of the sensory neurons14 or differences in experimental methods or strain variation24,64.

To investigate the physiological activity of the A, B and C neurons housed in single capitate peg sensilla, CO2, R-(–)−1-octen-3-ol and (+)-borneol were used: gas cylinders containing metered amounts of CO2 (300, 600, 1200, 2400, or 4800 ppm) and oxygen (20%), balanced by nitrogen (Strandmöllen AB, Ljungby, Sweden) were used to assess the activity of the A neuron; serial decadic dilutions of R-(-)−1-octen-3-ol (CAS: 3687-48-7, Penta Manufacturing, Livingston, USA), diluted in paraffin oil, were used to assess the activity of the B neuron; and serial decadic dilutions of (+)-borneol (CAS: 464-43-7, Sigma-Aldrich, St. Louis, MO, USA), diluted in diethyl ether (SupraSolv, Billerica, MA, USA) were used to assess the activity of the C neuron. Stimulation with gaseous CO2 was by filling Pasteur pipettes with metered amounts of CO2 (150, 300, 600, 1200, 2400, 4800 ppm) and oxygen (20%), balanced by nitrogen (Strandmöllen AB, Ljungby, Sweden) similar to previously described methodology65. After filling, both ends of the pipettes were sealed with Parafilm. For delivery, the Parafilm was removed, and the pipette was immediately connected to the odor delivery system, allowing CO2 to be administered in the same manner as other odor stimuli. Pasteur pipettes were refilled with CO2 after each puff to ensure consistent stimulus delivery. A 15μL aliquot of each dilution of R-(–)−1-octen-3-ol and (+)-borneol was pipetted onto a filter paper (5mm × 15mm) inserted inside a Pasteur pipette, and the diethyl ether was allowed 15 min to evaporate, before being used for stimulus delivery. All stimuli were delivered into the air stream passing over the maxillary palp preparation.

Calcium imaging in the Ae. aegypti antennal lobe (AL)
Odor-evoked responses in the Ae. aegypti antennal lobe (AL) were imaged using the brp-QF2>QUAS-GCaMP7s progeny from the brp-QF2 and QUAS-GCaMP7s parental lines25. A total of six 6–8-day-old female mosquitoes were used for all calcium experiments. Each mosquito was cooled on ice and transferred to a Peltier-cooled holder that allowed the mosquito head to be fixed to a custom stage using ultraviolet glue. The stage permits the superfusion of saline to the head capsule and space for wing and proboscis movement26,66. Once the mosquito was fixed to the stage, a window in its head was cut to expose the brain, and the brain was continuously superfused with physiological saline67. Calcium-evoked responses in the AL were imaged using the Prairie Ultima IV two-photon excitation microscope (Prairie Technologies) and Ti-Sapphire laser (Chameleon Ultra; Coherent; at 1910 mW power). Experiments were performed at 75 μm depth from the dorsal surface of the AL, allowing characterization of the mediodorsal glomerular responses to olfactory stimuli and allowing these glomeruli to be repeatedly imaged across preparations. Scanning other AL regions26 did not show discernible responses to borneol, although future work will need to thoroughly characterize the glomerular tuning throughout the AL. To record odor-evoked responses in the mediodorsal glomeruli, images were collected from a 110 μm × 83 μm plane every 513 ms (line period of 1 ms), and for each odor stimulus, images were acquired for 25s, starting 10s before the stimulus onset. Image data were imported into Matlab (v2017; Mathworks, Natick, Massachusetts) for Gaussian filtering (2×2 pixel; σ=1.5–3) and alignment using a single frame as the reference at a given imaging depth and subsequently registered to every frame to within ¼ pixel. The one-frame offset between odor onset and image acquisition was aligned during post-processing. Odor stimuli were diluted to 10-3 and 10-4 ethanol (>100%; Sigma), with ethanol used as the solvent control. During an experiment, odor stimuli were separated by intervals of 120s to avoid receptor adaptation, and odor syringes were used once per preparation to prevent decreased concentration within the cartridge. Calcium responses are calculated as the change in fluorescence and time-stamped and synced with the stimulus pulses. After an experiment, the AL was sequentially scanned at 0.5 μm depths from the ventral to the dorsal surface to provide glomerular assignment and registration between preparations. Glomeruli (down-sampled to 1 μm3 voxel) were mapped and registered based on the positions and odor-evoked responses of the putative AL3, MD1-3, and AM2 glomeruli, using the most recent AL atlases27,28 and the Amira software (v. 6.5, FEI Houston Inc.). Our prior identification of the 1-octen-3-ol sensitive glomerulus used a different atlas 47, which caused us to tentatively identify the glomerulus as MD328. However, here in this study, for each preparation, we outlined and mapped the glomeruli in Amira (v. 6.5, FEI Houston Inc.), and subsequently compared the glomerulus positions to the new atlas27, allowing us to identify the borneol-sensitive glomerulus as MD3, and the 1-octen-3-ol glomerulus as MD2. Data collected from preparations 1 and 4 were excluded from the analysis due to strong 3D movement and inconsistent imaging plane in those samples.

Behavioral assay
The role of borneol in human host-seeking female mosquitoes was examined with an arm-in-a-cage assay (Fig. 6a) described previously48 with minor modifications in an air-conditioned room (26±2°C, 60±5% RH). Briefly, the experimenter’s hand was presented to fifteen 5–10 days post emergence adult females. A three-dimensional-printed interlocking ring with a diameter of 55 mm was used in this experiment (Supplementary Fig S13). The ring was placed over the dorsal side of a nitrile glove (powder-free latex). To evoke human odor and prevent mosquito bites, we replaced the nitrile glove between the two ring components with a plastic net. The interlocking ring included a central odorant delivery platform comprising a 10-mm-diameter cover glass and two 5-mm-diameter filter discs (WHA10016508; Merck) for the addition of VOCs (see Supplementary Data 6 & 7). The platform has two black circles with a gap of 20mm in between; to convert the distance from pixels to cm. Plastic net, nitrile glove, and odorant delivery platform were replaced between repetitions to avoid contamination. Mosquitoes were placed in a 20.3 cm3 metal cage located in an experimental room with a vent, under a video camera (EOS 70D, lens: MACRO 0.25/0.8 ft; Canon Inc., Tokyo, Japan) and a light ring. Mosquitoes were allowed to acclimate for at least ten minutes before recording. Mosquito behavior was recorded for 10 minutes at 25 frames per second. The number of mosquito detection on the screen and ring were automatically counted using a custom YOLOv11l model. We used the solvent diethyl ether (DEE) as a vehicle. On a blank filter disc, 25 μL of DEE, and DEE with (+)-borneol (1M, 3.58mg) were deposited on the filter paper and allowed to evaporate for 2 min prior to mosquito exposure outside the experimental room to avoid contamination. To enhance attractiveness, at the beginning of each experiment, the experimenter first rubbed the ring-mounted glove against the shirt and skin for 1 minute. Additionally, the experimenter blew twice, once into the cage and once into the glove. All experiments were conducted during the first 4 hours of the diurnal period and lasted 10 min. This schedule was chosen for practical reasons and because mosquitoes consistently exhibited attraction to the human hand. Mosquito trajectories were counted and summed for each time interval (30 seconds). We excluded all trajectories that were located more than 2 to 4 mm away from the ring. Statistical comparisons of trajectory visits per time interval were carried out with Mann-Whitney U test (n=3). For visits variation across repetitions, see Supplementary Fig 12. Statistical comparisons between groups were performed using two-sided Mann–Whitney U tests, and generalized linear mixed models (GLMMs) were fitted in R (v3.6.1; see Supplementary Data 8).

Mosquito detection and software
To investigate the behavioral role of (+)-borneol in mosquitoes, we used YOLito68: a YOLOv11l model69. We evaluated YOLito on our setup using a test dataset of 180 annotated images (15 images/replicate). YOLito achieved a recall of 0.98, indicating a strong ability to detect true positives, and a precision of 0.86, reflecting accurate identification of mosquitoes. Detections were filtered using a minimum confidence and intersection over union (IoU) threshold of 0.5. All detections are illustrated in Supplementary Fig 12. Model predictions were analyzed with the YOLito toolkit, producing two primary data frames. The first frame contained frame-level data, including frame number, 2D coordinates (x,y), treatment group, and trajectory ID (Source data are provided as a Source Data file). The second frame, summarized behavioral data (Source data are provided as a Source Data file) for each 30 second interval, including (i) cumulative mosquito visits (Fig. 6b), (ii) normalized distance (total distance traveled divided by number of visits, in cm) and (iii) normalized duration (total time spent divided by number of visits, see Source Data file, Supplementary Fig. 11). All custom R code used for statistical analyses is available in the Source Data files.

Statistics & Reproducibility
Except when specifically stated in the figure captions, no data were excluded from the analyses. Unless stated otherwise, the experiments were randomized (e.g., mosquito cohorts were randomly assigned to odor or solvent control, mosquitoes did not experience multiple treatments). The Investigators were blinded to the identity of the composition and formulations of the EOs, sub-mixtures, and single compounds until the final outcome of the functional characterization of OR49.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Vainer et al. identifies a mosquito OR that responds to Borneol and camphor, two plant-derived mosquito repellants with a long history of human use. Expressing Expressing OR49 from various culicine mosquitos in a heterologous oocyte system, they show that these ORs respond to both borneol and camphor. They next measure responses to these same compounds in culcine mosquitos, but not Anopheles gambiae, which does not express this receptor, using both electropalpograms and single sensillum recordings. In the single sensillum recordings they further show that mutant Aedes aegypti that lack Or49 do not respond to borneol. Using pan-neuronal calcium imaging, they identify a glomerulus that responds to borneol. Finally they show that borneol can reduce attraction of Aedes aegypti mosquitos to humans. The results presents potentially of high interest as they identify a potential neural and molecular substrate for a set of commonly used mosquito repellents. However, some conclusions in the Discussion are not fully supported by the data, in particular the claim that the repellent properties of camphor are largely mediated by activation of OR49. Additional behavioral experiments in mutant Aedes aegypti and Anopheles gambiae in the presence and absence of borneol and camphor could strengthen the causal claims of the manuscript. In addition, data on the full anaotmical distirbution of responses to borneol and camphor should be shown.\nMajor comments:\n1) An exciting claim made by the paper is that the molecular mechanism identified for borneol/camphor sensitivity in OR49 is causally invovled in the mosquito-repellent properties of these compounds. However, this claim is not tested directly, although methods to do so appear to be present. It would greatly strengthen the manuscript to perform behavioral experiments in OR49 mutant Aedes aegypti with and without borneol and/or camphor to test the hypothesis that this OR is required for the repellent properties of these compounds. In addition, it would be helpful to perform experiments examining attraction of Anopheles gambiae to human in the presence and absence of borneol or camphor. Based on the molecular studies, one would predict that this species might either not be repelled by borneol/camphor or that repellence in this species would be independent of OR49 and mediated by some other mechanism. Both of these experiments would greatly expand the significance of the exciting results presented here. An additional variant might be to measure behavior in Aed. aegypti and/or Anopheles from which the palps have been removed.\n2) It would helpful to include results of imaging for other glomeruli? Are there any significant responses to borneol in any other glomeruli?\nMinor:\nline 87: since not all readers will have read the previous papers cited (19,21) it would be helpful to include a brief description of the recording setup (2-electrode voltage clamp in oocytes, odor presentation methods, perhaps an example of raw data) at the beginning of figure 2.\nagain, since not all readers may be familiar with the mosquito species used and which express which receptors, it might be helpful to provide a reminder that An. gambiae does not have OR49 around line 105 and line 127, as well as in the related figures.\nline 222-224: “the produce of chemical overstimulation” this seems like a fairly strong statement. Showing data on whether any other glomeruli respond to borneol might allow the authors to make stronger statements about whether OR49 is the only borneol sensor.\nline 238: “largely mediated by” see major comment 1 above. I think with the behavioral data currently presented this statement is too strong.\nline 545: figure legend panel b: phylogenetic tree is not referenced here and is critical for understanding the logic of some of the experiments. please explain.\nline 549: figure legend panel c: which ketones and alcohols are measured here? with what method? It is unclear what these data represent.", "reviewer_2": "“A conserved odorant receptor underpins borneol-mediated repellency in culicine mosquitoes” by Vainer et al provides an analysis of the responses of the maxillary palp neurons in mosquitoes, specifically focusing on terpenoid molecules. They show that the third neuron on the capitate peg sensillum (besides the CO2 and 1-octen-3-ol sensitive neurons), which is previously known to express Or49, responds to borneol and camphor. Overall, the manuscript is written well, with an interesting story-like introduction.\nThe authors used the oocyte recordings, EPG and SSR recordings for showing that Or49 responds to borneol and camphor. They also created transgenic mosquitoes with Or49 null mutations, although not much could be concluded from these experiments as I describe below. Based on the available data and also conceptually, I find it odd to infer that the primary ligand for Or49 is borneol, which happens to be just one of the hundreds of plant-derived compounds that repel mosquitoes. Herre et al 2023 have recently shown that each of the capitate peg neurons expresses multiple types of receptors, so it is likely that many other ligands, not yet tested, can activate the third neuron. Even in the current study, it seems that many compounds activate Or49 in vitro, but only two were tested in vivo and they caused comparable activity.\n“Antennal activation by borneol requires doses 10,000 times greater than in the maxillary palp (Supplementary Fig. 7)” – the supplementary figure only shows the amount of odorant tested in the palps. For the dose required on the antenna, I believe the authors are referring to the previous paper by Wood et al, which used higher concentrations. However, this does not prove that the antenna cannot detect lower concentrations. To make the claim that the antenna cannot detect lower concentrations, the authors need to perform electroantennogram experiments for a range of concentrations in the same manner as done for EPG. Moreover, the doses used by the authors here in behavioral experiments are as high as the doses used by Wood et al for antennal recordings – which means that the any repellence observed in the current study can possibly be explained by the action of the antenna. This puts a question mark on the main conclusion of the study, highlighted in the title, that borneol repellency is mediated by Or49.\nAnother problem with the statement about the “10,000 times” dosage is that the dose here is calculated in terms of the weight of the odorant applied on the filter paper, but not the final concentration of the odorant in the air that reaches the animal, and that ultimately matters for the olfactory receptor. Can the authors provide any data or estimate on how a 10000 times change in the dose on the filter paper will affect the final vapor concentration? Otherwise it is possible that the actual difference in concentrations in the two experiments is much smaller than 10,000.\nFigure 3a: Were t-tests conducted on all pairs of responses out of (+C, -C, +B, -B), or only hand-picked pairs? In Cx pipiens, -B responses look weaker than +C; please report the significance (as done in Ae albopictus).\nFigure 3b. In Aedes aegypti, EPG responses to camphor are not smaller than borneol, at any of the test concentrations (if anything, they are slightly higher – the authors should mention the n-value and show the error bars. If the error bars are shown but are too small to be visible, then I am surprised that the differences are not significantly different!).\nIt seems that while in vitro oocyte recordings show higher responses to borneol than camphor, in vivo EPG responses at different concentrations do not show any major difference between camphor and borneol in any of the mosquito species. One possible explanation for this discrepancy could be that borneol is more soluble in the medium in the in vitro experiments, so it is better delivered in those artificial experiments, while in the natural conditions, both chemicals are equally effective in activating the neuron. To provide a more clear answer, the authors should quantify the SSR responses to both camphor and borneol. Otherwise, based on the available data (including Figure 5, see my comment below), it would be wrong to highlight borneol over camphor as the ligand for OR49.\nFigure 4b: The recording trace for OR49 null mutants shows some B and C spikes – this seems inconsistent with the 0 response reported in Figure 4f. The should authors show some more traces in a supplementary figure, and also report the tabulated source data for this figure (and other figures of the paper, as recommended by the Nature Publishing Group).\nFigure 4 shows very small difference between the sizes of B and C spikes. The authors should provide details of how they differentiated between the two neurons while analyzing SSR data. One of the publications of by the senior author (Bohbot et al., Front. Physiol., 2013) mentions cpC neurons as 1-octen-3-ol sensitive. In the current manuscript, cpB neurons are assigned for sensing 1-octen-3-ol. As the size difference is critical for the identification of the neurons, authors should explain the difference in the previous and the current study that resulted in the reversal of identity.\nIn line 210, it is mentioned that OR49 mutants had “severe reduction in fitness” – this puts a question mark over the SSR data from these mutants. The authors should provide more details on the nature of the reduction in fitness and the possible reasons for this unusual outcome as OR or GR mutants reported in previous studies have been fit enough to be used for behavioral analysis (authors can check if the guide RNAs used have off-target activity, and if so, considering using a different guide RNA). To convince that the reduced SSR response in OR49 mutants is specific to the mutation and not because of the poor health of the animals, the authors need to provide a side-by-side comparison of the CO2, octenol and borneol responses in these mutants (CO2 and octenol will serve as positive control).\nIn calcium imaging experiments, the authors mention that the glomeruli were identified based on the odor-evoked responses. This creates a problem of circularity: if the region reported as MD3 was identified based on whichever region showed high brightness to borneol, then using this data to show that MD3 responds to borneol is problematic. Ideally, given that the authors have the ability to insert transgenes (as done for the null mutant), they should have created an Or49-QF2 line to drive GCaMP expression, which would have allowed unambiguous recordings from the corresponding glomerulus. In the absence of that, the authors should at least provide a supplementary figure showing the imaged plane in each of the brains used for calcium imaging, with the used ROIs marked as MD1, MD2 and MD3. This will help the readers in assessing the reliability of the identification of glomeruli by checking their consistency across the animals. The authors should also show the responses of MD1 and MD2 glomeruli to the tested odors in Figure 5g to convince that the responses are specific to MD3 glomerulus -– this should be straightforward from their existing data (currently only some examples are shown in panel d).\nIn behavioral studies, it is unclear why this study shows an indirect measure (length of the trajectories) rather than the more direct measures of attraction or aversion: the frequency of landing on the hand. The previously cited paper for the same assay (Dekel et al 2022) had shown landing percentage for a similar task. The landing statistics should be reported here for a more complete picture of the results.\nKeeping in line with the current practices for transparency, please replace bar graphs throughout the paper with some other representation (e.g., the one used in Figure 6e) that shows the data points in the bar.\nLine 130: “Camphor and borneol evoked lower responses than 1-octen-3-ol in the culicine palps, which is consistent with the smaller size of the cpC neuron in comparison to the cpB neuron” – this statement is unjustified for two reasons: (1) the authors have not shown if the octenol response is statistically larger than the borneol, and (2) EPG magnitude likely depends on many variables, such as how strongly the neurons are activated by the respective ligands by their respective ligands at the tested concentrations.\nIn figure 2a, authors do not explain the empty circles on the bars. It would be easier to understand if they mention that the empty circles correspond to borneol weight % in the respective blends.\nIn figure 2d, the values of effective concentration at 50% response need some explanation. For example, in TambOR49, the concentration at 50% response when borneol is given is 6 while for camphor is 10. Authors might wish to provide a brief explanation on how these values were computed, and what are the units of these numbers (6 and 10, etc.)\nMinor comments:\nThe figure numbers mentioned in line 153 are incorrect. Wildtype Aedes aegypti – fig 4B (not fig 4C). Wildtype Anopheles gambiae – Fig 4C (not fig 4D).\nThe figure numbers mentioned in line 155 are incorrect. Response of Aedes aegypti or49 null mutant line to (+)-borneol is fig 4B (not fig 4C).\nIn figure 4 b-e, EPG traces can be extended by at least 1s pre and 1s post stimulation so that we can appreciate the changes in firing rate during odor stimulation.\n“From this panel, (–)-borneol elicited the greatest response in the MD3 glomerulus (Fig. 5g), closely followed by (+)-borneol, enantiomers of camphor, and geosmin.” This sentence is phrased to give the impression that MD3 shows stronger responses to (+) and (-) borneol than camphor. The authors should report the statistical comparison between the odorants, and if there is no statistical difference, then rephrase the above sentence accordingly.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports.", "reviewer_4": "SUMMARY\nVainer et al. examine the tuning and behavioral function of a conserved odorant receptor expressed in one of three olfactory neurons housed on the mosquito maxillary palp. It has long been recognized that the mosquito palp is home to a single type of sensory hair that houses 3 neurons. One of these neurons is famous for its role in detecting carbon dioxide, a critical mosquito activator and attractant. The second expresses a conserved receptor that is exquisitely sensitive to 1-octen-3-ol, likely to play a role in host attraction across diverse mosquito species. The third neuron is also known to express a receptor that is conserved across the Culicine subfamily of mosquitoes, but its tuning and function were previously unclear. Vainer and colleagues begin to fill this gap. They show that the receptor, called Or49 in most species, is sensitive to a number of related terpenoid compounds that are abundant in plant essential oils and were historically used as mosquito repellents. Data suggest that the best ligand is a compound called borneol. The authors use targeted mutagenesis to show that Or49 is required for borneol responses of the neuron and claim that the receptor also mediates borneol repellency.\nThis is an important advance, especially given the impact of mosquito behavior on human health and the conserved nature of the studied olfactory neuron and receptor. The work is notable for the number of different species included and the breadth of the electrophysiological testing and approaches. We also applaud the authors for extending their work all the way to behavior and for generating a mutant that could allow a direct link back to the receptor. However, we feel that a few claims are not well substantiated – requiring either clarification in the text or additional experiments to support them. We will discuss these issues below.\nMAJOR COMMENTS\n1) As discussed above, identifying a biologically relevant ligand and behavioral function for the conserved orphan palp neuron is a significant advance. If fully substantiated, this work may also help corroborate a pattern of valence opponency among cohoused olfactory neurons that was recently suggested for insects (Wu, Shiuan-Tze, et al., 2022).\n2) The authors show convincingly that Or49 is sensitive to a number of related terpenoid compounds, including camphor, eucalyptol, and borneol. However, it is less clear to us that, among these related compounds, the best ligand is borneol. In order to show such selective tuning, the authors would need to measure the response of the receptor to the different compounds delivered at the same concentration. If we understand correctly, concentrations were tightly controlled in the liquid phase in the Xenopus oocyte assay (Fig. 2). We know little about working with odorants in the Xenopus system. Can the authors be sure that the compounds were fully soluble? We ask in part because the difference in sensitivity to borneol vs. camphor seems to go away in the EPG assay (Fig. 3). How can the authors explain this difference? At what vapor phase concentration were the two compounds delivered in the EPG assay and in the AL imaging assay, where only liquid phase dilutions are reported? If the authors cannot say for sure that Or49 is most sensitively tuned to borneol when exposed to equal vapor phase concentrations of each compound, those claims should be revised. (Note that we don’t think this would take away from the significance of the work.)\n3) There are several places where we suggest clarifications in the text. One is in the description of the behavior assay. It was not clear to us from the main text or methods whether the mosquitoes are able to touch the borneol/solvent held in the odor delivery platform. If mosquitoes are able to touch the chemical, then the demonstrated effects could be mediated by the taste system rather than the olfactory system. We are not familiar with the literature on borneol repellency. It may be that volatile borneol has already been convincingly shown to repel these mosquitoes. Nevertheless, the presented assay would not be adequate to do that unless the mosquitoes were fully prevented from touching the borneol.\n4) It’s great that the authors took the step of developing an Or49 knock-out line. It allows them to show that the receptor is required for cpC neuron response to borneol. We understand that efforts to test whether Or49 was also required for borneol repellency via behavioral testing were stymied by the reduced fitness of homozygous mutants mentioned in line 210-211. This is a huge bummer. Without this, the authors cannot verify a link between Or49 and borneol repellency. Even if unlikely, It is possible that borneol is also detected by antennal neurons and that it is the antennal neurons/receptors that drive repellency rather than Or49/cpC.\nAs an aside, we have found that the OpIE2 promoter can cause fitness problems in Aedes aegypti mosquitoes and suspect that this may underlie the fitness issues of the KO line — especially since it is very unusual for KO of a single odorant receptor to cause such problems. We would suggest remaking the strain using 3xP3 to promote expression of the selective marker, rather than OpIE2. Alternatively, since the authors have inserted the transcription factor QF2 into the Or49 locus, they should be able to cross their KO line to a QUAS-toxin/silencer line in order to kill or silence the neurons in heterozygotes. They would have to generate the QUAS-toxin/silencer line, but this is generally much easier (with pBac mediated transgenesis) than remaking the mutant (with CRISPR/Cas9). A final option would be to use the same CRISPR guides to generate a small, frame-shifting NHEJ mutation in the Or49 locus.\nIt is obviously up to the authors how much more effort they want to devote to this. However, the current experiments do not prove a link between Or49 and borneol repellency. Without further investigation, any text claiming or implying that Or49 is known to drive borneol repellency should be revised (including title).\nMINOR COMMENTS\n- Paragraph starting at line 78: We found the text on conservation/expression of Or49 across mosquitoes confusing, especially because Anophelines have receptors named Or48/49 that are related, but not orthologous to the Culicine Or49. We think that less text might be helpful here, leaving details for the supplementary figure and its legend. Any mention of Anopheles Or48 is bound to confuse.\n- We found the explanation of some of the ephys testing of various compounds and mixes somewhat confusing as the text jumps back and forth among species and among testing of single compounds vs mixtures and sub-mixtures. Anything the authors can do to clarify or justify the choices made in this section would be helpful. For example, the shift to Toxorhyncitis Or49 and the shift to using cannabis blends in line 95 felt sudden. A sentence or two explaining the rationale would be helpful given that the section starts with Ae. albopictus Or49 and with a different blend mixture. At the least, a paragraph break would be appropriate here. Generally, paragraph breaks whenever a new species is introduced would help with paper clarity.\n- Consider removing Fig. 1c or moving to supplement. For a non-chemist, it is confusing that the ketone response in panel c ends up being explained by a group of related terpenoids, not all of which are ketones.\n- Line 90: Specify ‘Xenopus’ frog oocytes on first mention.\n- Fig. 1c/d: Not clear in figure itself or in figure legend that the results in panels c/d come from the Xenopus oocyte assay. In general, a simple icon indicating the type of ephys assay would help the reader keep track of the assays being used across figures 1,2,3. Also, the legend says there are * over statistically significant compounds, but no asterisks are visible on the figure.\n- Supplementary figure 1e and 1f: The Culex quinquefasciatus genes are confusing, since according to the scale bar they are longer by at least an order of magnitude than the Anopheles and Aedes genes, which are apparently only blips on chromosomal portions named A, B, C, etc. The meaning of the names A, B, C... are not clear from either the image or legend. The arrows at the ends of these blocks suggest they are exons but they are too long. Are they syntenic blocks? Also pls fix minor typographical errors in species names.\n- Fig. 2a: Not clear which axis corresponds to the points/line and which to the bars.\n- Fig. 2d: What are the units on the circled effective concentrations for camphor and borneol? There is no obvious connection to the x-axis scale in the figure.\n- Why does the difference between borneol and camphor seem so much larger for TambOr49 in Fig. 2e than in Fig. 2d?\n- Line 143: eCFP was not ‘ubiquitously expressed’ but rather expressed under the control of OpIE2\n- Line 155: The experiment shows that Or49 is required for cpC sensitivity to borneol but it does not show that it is necessary. Pls revise text accordingly.\n- Figure 3a legend: 1-octen-3-ol is orange, not blue.\n- Figure 4b: It is a bit confusing as to why Ae. aegypti is suddenly at the fore when albopictus normally was the featured mosquito. This may be worth a brief comment in the figure legend or the text.\n- Line 184: It would have been interesting to have a bit more elaboration on the odorants here and the rationale for choosing them over other odorants used in the paper.\n- Figure 5C: Suggest indicating the stimulus (borneol) somewhere in this panel and labelling the white circle MD3.\n- Figure 5d-f: Why is a range given for n (n=4-8) in this experiment. If each odorant and glomerulus was tested in each individual imaged, then n should be identical across panels.\n- Lines 249-250: It was already known that the three palp glomeruli are adjacent in the antennal lobe – first documented by Ignell et al. 2005 in the original Aaeg antennal lobe atlas but also subsequently by others.\n- Supp Fig. 6a schematic does not make it clear that QF2 was inserted directly at the start codon. Recommend adjusting figure and stating this in the legend.\n- Supp Fig. 6d appears to show results from 2 PCR reactions used to confirm proper integration of the construct into the Or49 locus. But the reactions amplify stretches of DNA that are found in the donor plasmid, which could have inserted unexpectedly in a different genomic location. To show proper integration, the authors must place one primer in a genomic location (just) outside of the homologous arm. We could also not understand what was being shown in panel f nor what primers were being used for panel g.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "This manuscript by Vainer et al. identifies a mosquito OR that responds to Borneol and camphor, two plant-derived mosquito repellants with a long history of human use. Expressing OR49 from various culicine mosquitos in a heterologous oocyte system, they show that these ORs respond to both borneol and camphor. They next measure responses to these same compounds in culicine mosquitos, but not Anopheles gambiae, which does not express this receptor, using both electropalpograms and single sensillum recordings. In the single sensillum recordings they further show that mutant Aedes aegypti that lack Or49 do not respond to borneol. Using pan-neuronal calcium imaging, they identify a glomerulus that responds to borneol. Finally, they show that borneol can reduce attraction of Aedes aegypti mosquitos to humans. The results present potentially of high interest as they identify a potential neural and molecular substrate for a set of commonly used mosquito repellents. However, some conclusions in the Discussion are not fully supported by the data, in particular the claim that the repellent properties of camphor are largely mediated by activation of OR49. Additional behavioral experiments in mutant Aedes aegypti and Anopheles gambiae in the presence and absence of borneol and camphor could strengthen the causal claims of the manuscript. In addition, data on the full anatomical distribution of responses to borneol and camphor should be shown.\nMajor comments:\n1) An exciting claim made by the paper is that the molecular mechanism identified for borneol/camphor sensitivity in OR49 is causally involved in the mosquito-repellent properties of these compounds. However, this claim is not tested directly, although methods to do so appear to be present. It would greatly strengthen the manuscript to perform behavioral experiments in OR49 mutant Aedes aegypti with and without borneol and/or camphor to test the hypothesis that this OR is required for the repellent properties of these compounds. In addition, it would be helpful to perform experiments examining attraction of Anopheles gambiae to human in the presence and absence of borneol or camphor. Based on the molecular studies, one would predict that this species might either not be repelled by borneol/camphor or that repellence in this species would be independent of OR49 and mediated by some other mechanism. Both of these experiments would greatly expand the significance of the exciting results presented here.\nAn additional variant might be to measure behavior in Ae. aegypti and/or Anopheles from which the palps have been removed.\nAuthors’ response: We thank the reviewer for highlighting this important point regarding the causal role of Or49 in mediating repellency to borneol and camphor. In response, we generated and validated an Ae. aegypti Or49 knockout (KO) line and conducted behavioral assays comparing wild-type and KO mosquitoes. As shown in the revised Figure 6 and Supplementary Figure 10, Or49 KO mosquitoes exhibited a significantly reduced behavioral response to borneol, confirming that Or49 contributes substantially to the repellency effect of this compound in Ae. aegypti. These data strengthen our central claim that Or49 is a key mediator of behavioral avoidance of borneol in culicine mosquitoes.\nRegarding Anopheles gambiae, we appreciate the suggestion to explore interspecies comparisons. Our study primarily focused on culicine mosquitoes, particularly Ae. aegypti. Bioinformatic analysis did not identify a clear ortholog of Ae. aegypti Or49 in An. gambiae, and our preliminary molecular screening did not detect borneol-responsive olfactory receptors in this species. While the exploration of borneol’s effect in An. gambiae is beyond the current scope, it represents an interesting direction for future comparative studies, especially to test whether repellency in An. gambiae is mediated via distinct molecular mechanisms or sensory appendages.\nWe agree that palp ablation experiments may help delineate the contribution of palp-associated receptors such as Or49. However, such surgical manipulations can introduce confounding effects on general olfactory behavior and are technically challenging to execute in a high-throughput and reproducible manner. Nevertheless, they remain an intriguing approach for future functional dissection of the capitate peg sensillum.\n2) Would it be helpful to include results of imaging for other glomeruli? Are there any significant responses to borneol in any other glomeruli?\nAuthors’ response: We appreciate the reviewer’s suggestion to evaluate potential responses in glomeruli beyond the MD cluster. In our calcium imaging recordings, we did not observe significant activation in other glomeruli in response to borneol. However, we acknowledge that our scan coverage of the antennal lobe (AL) was not exhaustive. Our imaging efforts were deliberately focused on the region containing the MD glomeruli—particularly those previously implicated in processing maxillary palp input—due to the expected projection site of Or49-expressing neurons.\nTo partially address this concern, we analyzed the ROIs within the accessible imaging depth surrounding the MD glomeruli and confirmed that no other glomeruli showed borneol-evoked activity. This analysis has now been included in Supplementary Figure 9, and the figure caption has been updated accordingly in the revised manuscript: “a. 3D reconstruction of the left antennal lobe of a female Aedes aegypti highlighting the MD glomeruli of interest (MD1, MD2, and MD3) in blue, orange, and green respectively. This reconstruction is based on calcium imaging data and depicts the spatial organization of these glomeruli. b. Representative images of the antennal lobe of six mosquitoes, captured using two-photon microscopy. Each image shows color-coded glomerular regions of interest (ROIs) corresponding to the MD1, MD2, and MD3 glomeruli. Adjacent to each image, a 3D illustration displays the relative orientation of the imaging plane with respect to the MD glomeruli within the reconstructed antennal lobe. c. Time series of the calcium activity in the MD3 glomerulus (green trace) and two additional randomly selected regions of interest (ROI-4 and ROI-5, shown in red and purple, respectively). Responses are presented as changes in fluorescence (ΔF/F ) over time in response to a 2-second exposure to (-)-borneol (indicated by the grey-shaded area). MD3 shows a robust increase in neural activity upon borneol exposur₀e, whereas ROI-4 and ROI-5 display no significant changes in fluorescence.“\nMinor:\nline 87: since not all readers will have read the previous papers cited (19,21) it would be helpful to include a brief description of the recording setup (2-electrode voltage clamp in oocytes, odor presentation methods, perhaps an example of raw data) at the beginning of figure 2.\nAuthors’ response: We have added a schematic of the TEVC set-up and the perfusion system (Figure 2a and 2b). The caption was edited accordingly. We provided all the current traces in supplementary figure S3.\nAgain, since not all readers may be familiar with the mosquito species used and which express which receptors, it might be helpful to provide a reminder that An. gambiae does not have OR49 around line 105 and line 127, as well as in the related figures.\nAuthors’ response: We have added such reminders in line 104: “In An. gambiae, the cpC neuron does not express OR49 (Figure 1b), but rather OR28…” and in line 129: “…An. gambiae, which expresses OR28 in the palp instead of OR49 (Fig. 1b).\nline 222-224: “the produce of chemical overstimulation” this seems like a fairly strong statement. Showing data on whether any other glomeruli respond to borneol might allow the authors to make stronger statements about whether OR49 is the only borneol sensor.\nAuthors’ response: We agree with the reviewer’s comment. We have surveyed the activation of glomeruli in ROI adjacent to the MD region and did not detect any other borneol-activated glomeruli (Supplementary Fig. 9). We have added this observation in support of our statement: “…camphor tree EO are mediated in part by the activation of OR49 by bicyclic monoterpenoids and more specifically by…”.\nline 238: “largely mediated by” see major comment 1 above. I think with the behavioral data currently presented this statement is too strong.\nAuthors’ response: The text was changed accordingly: “…camphor tree EO are mediated in part by the activation of OR49 by bicyclic monoterpenoids and more specifically by…” (line 243).\nline 545: figure legend panel b: phylogenetic tree is not referenced here and is critical for understanding the logic of some of the experiments. please explain.\nAuthors’ response: This is correct, we unintentionally missed citing the phylogenetic tree. A reference was added to the figure legends: “… b, Phylogenetic relationship and assigned ontology across six mosquito species…” (line 561-562).\nline 549: figure legend panel c: which ketones and alcohols are measured here? with what method? It is unclear what these data represent.\nAuthors’ response: We re-labeled these odorant mixtures to reflect their chemical nature. The two active mixtures have now been named “ketonic” and “alcoholic” terpenes.\nWe have also added a schematic of the oocyte expressing OR49 and exposed to odorant mixtures and single compounds.\nReviewer #2 (Remarks to the Author):\n“A conserved odorant receptor underpins borneol-mediated repellency in culicine mosquitoes” by Vainer et al provides an analysis of the responses of the maxillary palp neurons in mosquitoes, specifically focusing on terpenoid molecules. They show that the third neuron on the capitate peg sensillum (besides the CO2 and 1-octen-3-ol sensitive neurons), which is previously known to express Or49, responds to borneol and camphor. Overall, the manuscript is written well, with an interesting story-like introduction.\nThe authors used the oocyte recordings, EPG and SSR recordings for showing that Or49 responds to borneol and camphor. They also created transgenic mosquitoes with Or49 null mutations, although not much could be concluded from these experiments as I describe below. Based on the available data and also conceptually, I find it odd to infer that the primary ligand for Or49 is borneol, which happens to be just one of the hundreds of plant-derived compounds that repel mosquitoes. Herre et al 2023 have recently shown that each of the capitate peg neurons expresses multiple types of receptors, so it is likely that many other ligands, not yet tested, can activate the third neuron. Even in the current study, it seems that many compounds activate Or49 in vitro, but only two were tested in vivo and they caused comparable activity.\nAuthors’ response: We thank the reviewer for their thoughtful comments. We agree that inferring a single “primary” ligand for Or49 would be conceptually limiting, especially given the complexity of the mosquito olfactory system and recent findings, including those by Herre et al. (2023), showing that each capitate peg (cp) neuron expresses multiple olfactory receptors.\nTo clarify, our claim is not that borneol is the “primary” ligand for Or49 in a biological sense, but rather that it is the most potent agonist identified in our comprehensive pharmacological screen. The term “potent” is used here in a pharmacological context, referring specifically to the magnitude and sensitivity of the electrophysiological responses observed in our oocyte recording assays.\nWe fully acknowledge the likelihood that additional ligands, particularly among plant-derived volatiles, may activate Or49, and that future studies may identify such compounds. However, within the chemical space screened in this study, Or49 displayed a high degree of selectivity toward bicyclic monoterpenoids, including borneol and camphor. Importantly, Or49 did not respond to a wide range of other chemical classes (e.g., amines, indoles, alcohols, lactones, carboxylic acids) (see Fig. 1d), lending support to our focus on this subset of compounds.\nGiven these results, and the need to balance breadth with mechanistic depth, we focused our downstream in vivo and neurophysiological investigations on borneol and camphor. We agree that further screening, both in vitro and in vivo, will be valuable in elucidating the broader functional role of Or49, as well as additional ligands for the cpC and cpB neurons. The evidence provided by Herre et al. (2023) strengthens the view that multiple receptors and ligands contribute to the functional complexity of the cp sensillum, and our study provides a foundational step toward that broader understanding.\n“Antennal activation by borneol requires doses 10,000 times greater than in the maxillary palp (Supplementary Fig. 7)”– the supplementary figure only shows the amount of odorant tested in the palps. For the dose required on the antenna, I believe the authors are referring to the previous paper by Wood et al, which used higher concentrations. However, this does not prove that the antenna cannot detect lower concentrations. To make the claim that the antenna cannot detect lower concentrations, the authors need to perform electroantennogram experiments for a range of concentrations in the same manner as done for EPG. Moreover, the doses used by the authors here in behavioral experiments are as high as the doses used by Wood et al for antennal recordings – which means that the any repellence observed in the current study can possibly be explained by the action of the antenna. This puts a question mark on the main conclusion of the study, highlighted in the title, that borneol repellency is mediated by Or49.\nAuthors’ response: We thank the reviewer for this important clarification. Upon reevaluation, we agree that our initial statement suggesting that the antenna requires a 10,000-fold higher dose of borneol than the maxillary palp (as shown in now removed Supplementary Fig. 7) was not adequately supported by direct experimental evidence in the current study. Specifically, we did not conduct a dose-response series using electroantennogram (EAG) recordings for the antenna, and therefore cannot conclusively state that the antennal detection threshold is significantly higher.\nThe reference to antennal responses in the Wood et al. study used higher odorant concentrations, but as the reviewer rightly points out, this does not preclude the possibility of antennal sensitivity at lower doses. Moreover, the concentrations used in our behavioral assays overlap with those used for antennal activation in Wood et al., which could confound interpretations regarding the source of borneol-induced repellency.\nTo address this concern and avoid overstatement, we have removed the claim comparing antennal and palp sensitivity from the revised manuscript. Additionally, and in light of the new behavioral findings using the Or49 KO line, we have retained the manuscript title.\nAnother problem with the statement about the “10,000 times” dosage is that the dose here is calculated in terms of the weight of the odorant applied on the filter paper, but not the final concentration of the odorant in the air that reaches the animal, and that ultimately matters for the olfactory receptor. Can the authors provide any data or estimate on how a 10000 times change in the dose on the filter paper will affect the final vapor concentration? Otherwise, it is possible that the actual difference in concentrations in the two experiments is much smaller than 10,000.\nAuthors’ response: We agree with the reviewer’s comment and have removed this aspect of the discussion.\nFigure 3a: Were t-tests conducted on all pairs of responses out of (+C, -C, +B, -B), or only hand-picked pairs? In Cx pipiens, -B responses look weaker than +C; please report the significance (as done in Ae albopictus).\nAuthors’ response: We only conducted a one-way anova and reported the pairs with statistical significance, meaning that pairs with no significant differences are not labeled in Figure 3b (see corresponding figure caption). For example, In Ae. albopictus, the only statistical significance was detected between +C and -B. Consequently, we have added a short statement in the caption of figure 3 (“Only significant statistical differences are shown” line 595-596).\nFigure 3b. In Aedes aegypti, EPG responses to camphor are not smaller than borneol, at any of the test concentrations (if anything, they are slightly higher – the authors should mention the n-value and show the error bars. If the error bars are shown but are too small to be visible, then I am surprised that the differences are not significantly different!).\nAuthors’ response: We are thankful for the reviewer to have picked up on this significant mistake, which after revision was due to an analysis error. For each species, the palp responses to borneol and camphor of the 5 tested specimens were normalized to one individual and we performed a new statistical analysis. We have also plotted standard deviations instead of SEM error bars. The results are discussed in the revised manuscript (lines 135-139). The n-values (n=15) are shown in the figure caption (lines 598-600).\nIt seems that while in vitro oocyte recordings show higher responses to borneol than camphor, in vivo EPG responses at different concentrations do not show any major difference between camphor and borneol in any of the mosquito species. One possible explanation for this discrepancy could be that borneol is more soluble in the medium in the invitro experiments, so it is better delivered in those artificial experiments, while in the natural conditions, both chemicals are equally effective in activating the neuron. To provide a more clear answer, the authors should quantify the SSR responses to both camphor and borneol. Otherwise, based on the available data (including Figure 5, see my comment below), it would be wrong to highlight borneol over camphor as the ligand for OR49.\nAuthors’ response: We thank the reviewer for this insightful comment. We agree that it is essential to reconcile any discrepancies between in vitro and in vivo results to accurately assess the relative potencies of borneol and camphor.\nUpon reanalysis of our EPG data (see revised Figures 5 and 6), we discovered a data processing error that had previously obscured the true response magnitudes. The corrected analysis now shows that borneol elicits consistently stronger responses than camphor across both Ae. aegypti and Ae. albopictus, confirming the trend observed in our two-electrode voltage clamp (TEVC) recordings in Xenopus oocytes. This alignment across two independent methodologies strengthens our conclusion that borneol is the more potent agonist of OR49.\nWe also appreciate the reviewer’s suggestion regarding solubility. Borneol and camphor have similar solubility in DMSO (16 mg/mL vs. 20 mg/mL, respectively), making differential solubility an unlikely explanation for the in vitro results. However, a notable distinction lies in their vapor pressures: borneol (0.0035 mmHg @25°C) is significantly less volatile than camphor (0.65 mmHg @25°C), implying that camphor may evaporate and disperse more readily under assay conditions. Despite this, borneol still elicited stronger physiological responses, further supporting its potency as an OR49 ligand in both liquid and vapor phases.\nFor the purposes of the current manuscript, the concordance between TEVC and EPG data provides strong, multi-modal evidence that OR49 preferentially responds to borneol over camphor.\nFigure 4b: The recording trace for OR49 null mutants shows some B and C spikes – this seems inconsistent with the 0 response reported in Figure 4f. The authors should show some more traces in a supplementary figure, and also report the tabulated source data for this figure (and other figures of the paper, as recommended by the Nature Publishing Group).\nAuthors’ response: All neurons of the cp sensillum exhibit a basal level of spontaneous firing, even after the abolishment of OR49. This is in line with previous studies in Drosophila melanogaster and Anopheles coluzzii, after the knock out of an endogenous tuning OR (Dobritsa et al., 2003). This pattern is observed across all tested species versus negative control and at low doses of borneol. As different neurons have different basal firing rates, averaging and normalization of the data yields rates close to 0. Source data for all figures was added as extended data figures. We have also added a new supplementary figure with additional traces for Or49 null mutants and the wild type line (Supplementary Figure 8). The source data for this figure is provided in “Extended Data Fig. 6 SSR”.\nFigure 4 shows very small difference between the sizes of B and C spikes. The authors should provide details of how they differentiated between the two neurons while analyzing SSR data. One of the publications by the senior author (Bohbot et al., Front. Physiol., 2013) mentions cpC neurons as 1-octen-3-ol sensitive. In the current manuscript, cpB neurons are assigned for sensing 1-octen-3-ol. As the size difference is critical for the identification of the neurons, authors should explain the difference in the previous and the current study that resulted in the reversal of identity.\nAuthors’ response: Discrimination between the cpB and cpC neurons is based on spike amplitude, spontaneous activity and their respective functional responses. In our study, the cpB neuron, which is sensitive to 1-octen-3-ol, consistently exhibits larger action potential amplitudes than the cpC neuron, which is activated by borneol. This is in line with several other studies in Aedes, Culex, and Anopheles species that have reported that the cpB neuron is responsive to octenol (Syed & Leal, 2007; Lu et al., 2007; Cook et al., 2011; Omondi et al., 2015; Majeed et al. 2016; Herre at al., 2022). While other investigations in Aedes aegypti have identified the cpC neuron, characterized by the smallest spike amplitude, as the octenol-sensitive neuron these results are ambiguous. The reason for this discrepancy remains unclear, but likely stem from either a mis-identification of the sensory neurons (Grant & Dickens, 2011) or differences in experimental methods or strain variation (Bohbot et al., 2013; Singh et al., 2022).\nWe incorporated this explanation and references to previous works in the M&M section of the revised manuscript (lines 571-575).\nIn line 210, it is mentioned that OR49 mutants had “severe reduction in fitness” – this puts a question mark over the SSR data from these mutants. The authors should provide more details on the nature of the reduction in fitness and the possible reasons for this unusual outcome as OR or GR mutants reported in previous studies have been fit enough to be used for behavioral analysis (authors can check if the guide RNAs used have off-target activity, and if so, considering using a different guide RNA). To convince that the reduced SSR response in OR49 mutants is specific to the mutation and not because of the poor health of the animals, the authors need to provide a side-by-side comparison of the CO2, octenol and borneol responses in these mutants (CO2 and octenol will serve as positive control).\nAuthors’ response: We appreciate the reviewer’s insightful suggestion regarding the importance of positive controls to rule out general health defects in the OR49 mutants. At this stage, however, we are not in a position to perform additional SSR experiments comparing CO , octenol, and borneol responses in the mutant line. We fully agree that such data would strengthen the conclusions, and we hope to explore this direction in future work. In ₂the current version, we have revised the text (result section of behavioral assay) to clearly acknowledge this limitation and to present our findings with appropriate caution.\nIn calcium imaging experiments, the authors mention that the glomeruli were identified based on the odor-evoked responses. This creates a problem of circularity: if the region reported as MD3 was identified based on whichever region showed high brightness to borneol, then using this data to show that MD3 responds to borneol is problematic. Ideally, given that the authors have the ability to insert transgenes (as done for the null mutant), they should have created an Or49-QF2 line to drive GCaMP expression, which would have allowed unambiguous recordings from the corresponding glomerulus. In the absence of that, the authors should at least provide a supplementary figure showing the image plane in each of the brains used for calcium imaging, with the used ROIs marked as MD1, MD2 and MD3.This will help the readers in assessing the reliability of the identification of glomeruli by checking their consistency across the animals. The authors should also show the responses of MD1 and MD2 glomeruli to the tested odors in Figure 5g to convince that the responses are specific to MD3 glomerulus -– this should be straightforward from their existing data (currently only some examples are shown in panel d).\nAuthors’ response: We thank the reviewer for raising this important point about the potential circularity in glomerulus identification based solely on functional responses. We agree that glomerular identification must be anatomically and functionally validated to ensure accurate interpretation.\nWe have included a new supplementary figure (Fig. S9) that displays the image plane used for each preparation, along with the location of the AL glomerular ROIs identified as MD1, MD2, and MD3. In our experiments, we performed 3D imaging in 0.5 µm increments along the z-axis and registered each dataset to a previously published antennal lobe (AL) atlas (see Supplementary Figure 9). This allowed us to anatomically assign ROIs to glomerular structures with a high degree of confidence, independent of the odor-evoked responses.\nWe also agree that including functional data from neighboring glomeruli strengthens our conclusion regarding the specificity of the borneol response. To that end, we have quantified and now report the odor-evoked responses of MD1 and MD2 across all preparations (see Supplementary Figure 9). These glomeruli showed no significant activation in response to borneol, supporting the selective responsiveness of MD3.\nWhile generating an Or49-QF2>GCaMP line would indeed offer a powerful validation tool, the timeline for generating such a transgenic line was outside the scope of the current study. Nonetheless, our multipronged approach, combining high-resolution anatomical alignment with functional response mapping, provides a strong basis for identifying MD3 as the glomerulus associated with OR49 input.\nIn behavioral studies, it is unclear why this study shows an indirect measure (length of the trajectories) rather than the more direct measures of attraction or aversion: the frequency of landing on the hand. The previously cited paper for the same assay (Dekel et al 2022) had shown landing percentage for a similar task. The landing statistics should be reported here for a more complete picture of the results.\nAuthors’ response: We conducted a new set of behavioral experiments with the mutant line and as a result, Figure 6 has been completely revised. We now report number of visits, mean distance and mean duration (revised Figure 6 & Fig S10). P-values are now provided either in the figure captions. In addition, we have included a supplementary table (Table S5) detailing all p-values for each pairwise comparison.\nKeeping in line with the current practices for transparency, please replace bar graphs throughout the paper with some other representation (e.g., the one used in Figure 6e) that shows the data points in the bar.\nAuthors’ response: All relevant bar plots in Figure 2 are now showing individual data points.\nLine 130: “Camphor and borneol evoked lower responses than 1-octen-3-ol in the culicine palps, which is consistent with the smaller size of the cpC neuron in comparison to the cpB neuron” – this statement is unjustified for two reasons: (1) the authors have not shown if the octenol response is statistically larger than the borneol, and (2) EPG magnitude likely depends on many variables, such as how strongly the neurons are activated by the respective ligands by their respective ligands at the tested concentrations.\nAuthors’ response: We agree with this point, we removed “which is consistent with the smaller size of the cpC neuron in comparison to the cpB neuron” from the revised manuscript.\nIn figure 2a, authors do not explain the empty circles on the bars. It would be easier to understand if they mention that the empty circles correspond to borneol weight % in the respective blends.\nAuthors’ response: We have added an explanation in the figure legends lines 574-575: “borneol content is indicated by white circles, values correspond to the right Y-axis.”\nIn figure 2d, the values of effective concentration at 50% response need some explanation. For example, in TambOR49, the concentration at 50% response when borneol is given is 6 while for camphor is 10. Authors might wish to provide a brief explanation on how these values were computed, and what are the units of these numbers (6 and 10, etc.)\nAuthors’ response: We have added such an explanation in the Materials & Methods section: “The EC values were calculated using Graphpad Prism 8. Briefly, after data 50 collection, the data of each oocyte is normalized against its highest response. This is done to overcome the high variance between individual oocytes. After normalization, the data of each oocyte was analyzed using the function “Nonlinear regression” with “Dose – response – Stimulation” equation “log(agonist) vs. normalized response”. This function yields the EC value for each repetition (at least five oocytes). The EC values were 50 50 then analyzed for the average and S.E.M values (Fig. S8)”.\nIn addition, a new supplementary figure has been added (Figure S7), which plots and reports the EC values. Figure captions were updated as well: “Bar plot of Culicine 50 OR49 EC values vs. (+)-borneol and (+)-camphor. Raw data are shown in the 50 adjacent table. Data indicates the means ± SEM, n = 5-7.“\nMinor comments:\nThe figure numbers mentioned in line 153 are incorrect. Wildtype Aedes aegypti – fig 4B (not fig 4C). Wild type Anopheles gambiae – Fig 4C (not fig 4D). The figure numbers mentioned in line 155 are incorrect. Response of Aedes aegypti or49 null mutant line to (+)-borneol is fig 4B (not fig 4C).\nAuthors’ response: We have corrected the legends in figure 4 accordingly.\nIn figure 4 b-e, EPG traces can be extended by at least 1s pre and 1s post stimulation so that we can appreciate the changes in firing rate during odor stimulation.\nAuthors’ response: We appreciate the reviewer’s suggestion to extend the EPG traces in Figure 4b–e by at least 1 second before and after odor stimulation. We fully agree that longer traces can help visualize baseline activity and recovery dynamics following odor exposure.\nHowever, in our attempts to incorporate extended time windows in these panels, we found that the increased trace length significantly reduced the visual clarity of individual action potentials. Since these panels are intended to illustrate fine-scale temporal firing patterns, especially in relation to spike amplitude discrimination between neurons, we opted to retain the current format with a shorter window.\nTo address the reviewer’s concern while maintaining clarity, we have added a new example trace to panel 4a showing the EPG response of the cpC neuron in Aedes aegypti to 0.1% borneol, with 1 second of pre- and post-stimulation recording. This trace highlights the full temporal profile of the response, including baseline and recovery, and complements the cropped traces in panels b–e. We believe this compromise effectively balances readability and informative value. This addition is reflected in the revised Figure 4 and its updated caption.\n“From this panel, (–)-borneol elicited the greatest response in the MD3 glomerulus (Fig. 5g), closely followed by (+)-borneol, enantiomers of camphor, and geosmin.” This sentence is phrased to give the impression that MD3 shows stronger responses to (+) and (-) borneol than camphor. The authors should report the statistical comparison between the odorants, and if there is no statistical difference, then rephrase the above sentence accordingly.\nAuthors’ response: In the revised manuscript, we have updated the sentence to reflect the results of appropriate statistical analysis:\n“Among the tested odorants, there was no significant difference in the calcium responses of the MD3 glomerulus between (+)-borneol, (–)-borneol, and either camphor enantiomer (Kruskal-Wallis test with multiple comparisons, P > 0.93). However, all borneol and camphor isomers elicited significantly greater responses than 1-octen-3-ol, CO , or the solvent control (P < 0.05).”\nThis revised phrasing avoids implying a ranking where one was not supported sta₂tistically and more accurately reflects the equivalence in response magnitude among the structurally related monoterpenoids. The updated results are now shown in Fig. 5g and discussed in lines 183–190 of the revised manuscript.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nReviewer #4 (Remarks to the Author):\nSUMMARY\nVainer et al. examine the tuning and behavioral function of a conserved odorant receptor expressed in one of three olfactory neurons housed on the mosquito maxillary palp. It has long been recognized that the mosquito palp is home to a single type of sensory hair that houses 3 neurons. One of these neurons is famous for its role in detecting carbon dioxide, a critical mosquito activator and attractant. The second expresses a conserved receptor that is exquisitely sensitive to 1-octen-3-ol, likely to play a role in host attraction across diverse mosquito species. The third neuron is also known to express a receptor that is conserved across the Culicine subfamily of mosquitoes, but its tuning and function were previously unclear. Vainer and colleagues begin to fill this gap. They show that the receptor, called Or49in most species, is sensitive to a number of related terpenoid compounds that are abundant in plant essential oils and were historically used as mosquito repellents. Data suggest that the best ligand is a compound called borneol. The authors use targeted mutagenesis to show that Or49 is required for borneol responses of the neuron and claim that the receptor also mediates borneol repellency.\nThis is an important advance, especially given the impact of mosquito behavior on human health and the conserved nature of the studied olfactory neuron and receptor. The work is notable for the number of different species included and the breadth of the electrophysiological testing and approaches. We also applaud the authors for extending their work all the way to behavior and for generating a mutant that could allow a direct link back to the receptor. However, we feel that a few claims are not well substantiated – requiring either clarification in the text or additional experiments to support them. We will discuss these issues below.\nMAJOR COMMENTS\n1) As discussed above, identifying a biologically relevant ligand and behavioral function for the conserved orphan palp neuron is a significant advance. If fully substantiated, this work may also help corroborate a pattern of valence opponency among cohoused olfactory neurons that was recently suggested for insects (Wu, Shiuan-Tze, et al., 2022).\nAuthors’ response: We thank the reviewer for highlighting this point. We agree that the identification of a biologically relevant ligand (borneol) and behavioral function for the conserved orphan palp neuron represents a significant advance. Our findings are indeed consistent with the emerging concept of valence opponency among co-housed olfactory neurons, as described by Wu et al. (2022). In our system, the cpC neuron (borneol-responsive) and its neighboring cpA and cpB neurons, which are activated by CO and octenol respectively, may form a functional unit in which conflicting odor valences are integrated early in the olfactory pathway. While our current study focused on rece₂ptor-ligand relationships and behavioral responses, further investigation into the mechanistic basis of potential valence opponency in this sensillum will be an exciting direction for future work.\n2) The authors show convincingly that Or49 is sensitive to a number of related terpenoid compounds, including camphor, eucalyptol, and borneol. However, it is less clear to us that, among these related compounds, the best ligand is borneol. In order to show such selective tuning, the authors would need to measure the response of the receptor to the different compounds delivered at the same concentration. If we understand correctly, concentrations were tightly controlled in the liquid phase in the Xenopus oocyte assay (Fig. 2). We know little about working with odorants in the Xenopus system. Can the authors be sure that the compounds were fully soluble? We ask in part because the difference in sensitivity to borneol vs. camphor seems to go away in the EPG assay (Fig. 3). How can the authors explain this difference? At what vapor phase concentration were the two compounds delivered in the EPG assay and in the AL imaging assay, where only liquid phase dilutions are reported? If the authors cannot say for sure that Or49 is most sensitively tuned to borneol when exposed to equal vapor phase concentrations of each compound, those claims should be revised. (Note that we don’t think this would take away from the significance of the work.)\nAuthors’ response: The use of DMSO to solubilize VOC is customary across existing pharmacological assays, including the TEVC system. The solubilities of borneol (16 mg/mL) and camphor (20 mg/mL) in DMSO (standard carrier molecule when working with hydrophobic molecules in the TEVC system) are comparable suggesting that the receptor is exposed to likewise similar concentrations of both compounds.\nThis issue, which is a recurring question in the field, is complex when operating in the gas phase. When operating an air delivery system, it is not known how much of the VOCs go into the gas phase, how much crosses the distance between the pipette and the preparation, and how much enters the sensilla. Considering all these unknowns being equal between treatments, we know that borneol (0.0035 mmHg @25°C) is 185 times less volatile than camphor (0.65 mmHg @25°C). Yet, borneol elicits comparable or larger EPG responses than camphor (see revised figure 3) suggesting that the receptor is more sensitive to borneol, which is consistent with the pharmacological data.\nOur main claim is that borneol is a significantly more potent ligand than camphor (see revised heading: “Odorant receptor 49 is an evolutionary conserved and selective borneol receptor” line 78 & 567) based on our pharmacological data, which documents the molecular mechanism of monoterpenoid-sensing via OR49. We have shown OR49 selective ligand tuning as well as ligand efficacy and ligand sensitivity towards borneol. This pharmacological survey is the main reason why we restricted our subsequent analyses to camphor and borneol. By definition, the EPG, SSR and brain imaging measurements result from complex biochemical environments and neuronal circuitries, which provide a lower resolution understanding of the relative activity of borneol and camphor.\n3) There are several places where we suggest clarifications in the text. One is in the description of the behavior assay. It was not clear to us from the main text or methods whether the mosquitoes are able to touch the borneol/solvent held in the odor delivery platform. If mosquitoes are able to touch the chemical, then the demonstrated effects could be mediated by the taste system rather than the olfactory system. We are not familiar with the literature on borneol repellency. It may be that volatile borneol has already been convincingly shown to repel these mosquitoes. Nevertheless, the presented assay would not be adequate to do that unless the mosquitoes were fully prevented from touching the borneol.\nAuthors’ response: We thank the reviewer for this important clarification. In our behavioral assay, the odorant delivery platform consists of a perforated lid that sits atop a container holding a filter paper soaked in either borneol or solvent. While the mosquitoes could potentially reach the center, direct contact is physically discouraged by the design, which recesses the soaked filter paper slightly below the lid surface. To assess the possibility of gustatory interaction, we systematically reviewed the high-resolution video recordings of all behavioral trials. In no instance did we observe any mosquito making direct contact with the treated filter paper. Furthermore, the behavioral avoidance typically occurred before the mosquito reached the platform, consistent with a response to volatile cues.\nAlthough we cannot entirely exclude the possibility of transient contact in a few undetected cases, we believe the combination of assay design and observed behavior strongly supports an olfactory rather than gustatory basis for repellency. For full transparency, we have revised the Methods section (lines 628-640) to clarify the physical setup and video analysis, and we now mention this caveat in the Discussion section.\n4) It’s great that the authors took the step of developing an Or49 knock-out line. It allows them to show that the receptor is required for cpC neuron response to borneol. We understand that efforts to test whether Or49 was also required for borneol repellency via behavioral testing were stymied by the reduced fitness of homozygous mutants mentioned in line 210-211. This is a huge bummer. Without this, the authors cannot verify a link between Or49 and borneol repellency. Even if unlikely, It is possible that borneol is also detected by antennal neurons and that it is the antennal neurons/receptors that drive repellency rather than Or49/cpC.\nAs an aside, we have found that the OpIE2 promoter can cause fitness problems in Aedes aegypti mosquitoes and suspect that this may underlie the fitness issues of the KO line — especially since it is very unusual for KO of a single odorant receptor to cause such problems. We would suggest remaking the strain using 3xP3 to promote expression of the selective marker, rather than OpIE2. Alternatively, since the authors have inserted the transcription factor QF2 into the Or49 locus, they should be able to cross their KO line to a QUAS-toxin/silencer line in order to kill or silence the neurons in heterozygotes. They would have to generate the QUAS-toxin/silencer line, but this is generally much easier (with pBac mediated transgenesis) than remaking the mutant (with CRISPR/Cas9). A final option would be to use the same CRISPR guides to generate a small, frame-shifting NHEJ mutation in the Or49 locus.\nAuthors’ response: We thank the reviewer for the question. In fact, we have created other lines with the OpIE2 promoter, and these other lines performed well in behavior assays. In order to address the low performance of the Or49 KO line, we backcrossed this line with the wild type Liverpool for six generation to remove potential background mutations and then isolated a new homozygous line by intercrossing for 3 generations and pinpointing homozygous offsprings by PCR as done previously and described in the methods. This \"genetically purified\" line exhibited great performance in the behavioral assay, as showed in the new version of the manuscript, ruling out any concern with fitness load (overstated in the previous version of the manuscript).\nAdditionally, we appreciate the suggestion to leverage the QF2/QUAS system for conditional silencing, and we are currently exploring this direction for future experiments to achieve spatial and temporal control over Or49-expressing neurons.\nIt is obviously up to the authors how much more effort they want to devote to this. However, the current experiments do not prove a link between Or49 and borneol repellency. Without further investigation, any text claiming or implying that Or49 is known to drive borneol repellency should be revised (including title).\nAuthors’ response: We appreciate the reviewer’s thoughtful feedback and fully agree that establishing a mechanistic link between odorant receptor activation and behavior is a significant challenge. Since the initial submission, we have taken additional steps to strengthen our claims. Specifically, Ae. aegypti Or49 knockout line now exhibit improved performance in behavioral assays comparing wild-type and Or49 mutant females in response to borneol. These results, now included in the revised manuscript (Figure 6 and Supplementary Figure 9), demonstrate that Or49 mutants show a significantly reduced behavioral aversion to borneol compared to controls.\nWhile we acknowledge that behavioral phenotypes can sometimes involve redundant or parallel sensory inputs, the combination of our in vitro, electrophysiological, and now behavioral data provides converging evidence that Or49 plays a key role in mediating borneol-induced repellency. We have carefully revised the manuscript text to clarify that Or49 is a major contributor to borneol repellency, rather than the sole determinant. The title remains unchanged as it reflects this refined conclusion supported by our updated dataset. We hope these additions address the reviewer’s concern and strengthen the manuscript’s overall contribution.\nMINOR COMMENTS\n- Paragraph starting at line 78: We found the text on conservation/expression of Or49 across mosquitoes confusing, especially because Anophelines have receptors named Or48/49 that are related, but not orthologous to the CulicineOr49. We think that less text might be helpful here, leaving details for the supplementary figure and its legend. Any mention of Anopheles Or48 is bound to confuse.\nAuthors’ response: To avoid this confusion, we removed the statement in question.\n- We found the explanation of some of the ephys testing of various compounds and mixes somewhat confusing as the text jumps back and forth among species and among testing of single compounds vs mixtures and sub-mixtures. Anything the authors can do to clarify or justify the choices made in this section would be helpful. For example, the shift to Toxorhynchites Or49 and the shift to using cannabis blends in line 95 felt sudden. A sentence or two explaining the rationale would be helpful given that the section starts with Ae. albopictus Or49 and with a different blend mixture. At the least, a paragraph break would be appropriate here. Generally, paragraph breaks whenever a new species is introduced would help with paper clarity.\nAuthors’ response: We have rewritten this section in the revised manuscript to clear this matter.\n- Consider removing Fig. 1c or moving to supplement. For a non-chemist, it is confusing that the ketone response in panel c ends up being explained by a group of related terpenoids, not all of which are ketones.\nAuthors’ response: Because of the clear confusion, we have reorganized figure 1 by re-labeling the odorant mixtures (ketonic and alcoholic terpenes) so their identification is consistent between the two functional screens. Table S1 and the corresponding text have been edited accordingly.\n- Line 90: Specify ‘Xenopus’ frog oocytes on first mention.\nAuthors’ response: This statement was edited accordingly.\n- Fig. 1c/d: Not clear in figure itself or in figure legend that the results in panels c/d come from the Xenopus oocyte assay. In general, a simple icon indicating the type of ephys assay would help the reader keep track of the assays being used across figures 1,2,3. Also, the legend says there are * over statistically significant compounds, but no asterisks are visible on the figure.\nAuthors’ response: We added an icon of the oocyte, a schematic of the TEVC system (as requested by reviewer 1), and an icon of the EPG system to Fig. 1d., Fig. 2a, and Fig. 3a, respectively. We have added statistical significance to Figure 1 and edited the caption accordingly.\n- Supplementary figure 1e and 1f: The Culex quinquefasciatus genes are confusing, since according to the scale bar they are longer by at least an order of magnitude than the Anopheles and Aedes genes, which are apparently only blips on chromosomal portions named A, B, C, etc. The meaning of the names A, B, C... are not clear from either the image or legend. The arrows at the ends of these blocks suggest they are exons but they are too long. Are they syntenic blocks? Also pls fix minor typographical errors in species names.\nAuthors’ response: Scale bars were revised. The caption was updated accordingly (lines 659-663). Originally A,B,C represented genes around the OR49 gene and were removed to avoid confusion. Now, orthology between genes is represented by color and dashed lines. We did not find typographical errors.\n- Fig. 2a: Not clear which axis corresponds to the points/line and which to the bars.\nAuthors’ response: Figure caption of revised Fig. 2c was edited accordingly. Borneol content is indicated by white circles (lines 572-573).\n- Fig. 2d: What are the units on the circled effective concentrations for camphor and borneol? There is no obvious connection to the x-axis scale in the figure.\nAuthors’ response: We have added the units in the caption and legends accordingly (lines 579-580).\n- Why does the difference between borneol and camphor seem so much larger for TambOr49 in Fig. 2e than in Fig.2d?\nAuthors’ response: In Fig. 2f (new panel label), the TambOR49 concentration-response curves have been normalized to more easily compare the EC values. The normalization 50 equalizes the maximum responses (plateaus) thereby masking differences in absolute current responses but highlights differences in EC values (sensitivity). In Fig. 2g (new 50 panel label), the current responses are absolute and not normalized, which highlights differences in ligand efficacy.\n- Line 143: eCFP was not ‘ubiquitously expressed’ but rather expressed under the control of OpIE2\nAuthors’ response: Reviewer is right. The text has been modified to reflect that “…eCFP fluorescent marker under the control of the OpiE2…” (lines 145-146)\n- Line 155: The experiment shows that Or49 is required for cpC sensitivity to borneol but it does not show that it is necessary. Pls revise text accordingly.\nAuthors’ response: We modified the text according to the reviewer’s recommendation (line 154).\n- Figure 3a legend: 1-octen-3-ol is orange, not blue.\nAuthors’ response: The color coding in the figure caption was corrected accordingly.\n- Figure 4b: It is a bit confusing as to why Ae. aegypti is suddenly at the fore when albopictus normally was the featured mosquito. This may be worth a brief comment in the figure legend or the text.\nAuthors’ response: We highlighted Ae. aegypti because it is a significantly better genetic model with well-established genetic engineering tools than Ae. albopictus. We added a short sentence to this effect in the result section “To test a potential causal relationship between Or49 and the borneol response, we used homology-directed repair (HDR) to knock in a transgenic construct in Ae. aegypti, which serves as a more genetically tractable model than “(lines 143-144).\n- Line 184: It would have been interesting to have a bit more elaboration on the odorants here and the rationale for choosing them over other odorants used in the paper.\nAuthors’ response: The tested odorants were used based on the responses in nearby glomeruli (e.g., CO2, 1-octen-3-ol) or were enantiomers also tested in the SSR and EAG experiments (+/- camphor, +/- borneol). For the sake of clarity, we now show only responses to (-)borneol, 1-octen-3-ol, CO2, and the mineral oil control for the MD3 glomerulus (Fig. 5g). we also incorporated this explanation in the revised manuscript (lines 184-185).\n- Figure 5C: Suggest indicating the stimulus (borneol) somewhere in this panel and labelling the white circle MD3.\nAuthors’ response: Legends were added accordingly.\n- Figure 5d-f: Why is a range given for n (n=4-8) in this experiment. If each odorant and glomerulus was tested in each individual imaged, then n should be identical across panels.\nAuthors’ response: The n = 4-8 because multiple experiment series were conducted. The first series (n=4) tested borneol, CO2, 1-octen-3-ol, geosmin, and mineral oil (no odor) control. The second series examined whether the MD3 glomerulus also responded to different enantiomers of borneol and camphor. For this reason, there are differences in the number of replicates for the tested odorants.\n- Lines 249-250: It was already known that the three palp glomeruli are adjacent in the antennal lobe – first documented by Ignell et al. 2005 in the original Aaeg antennal lobe atlas but also subsequently by others.\nAuthors’ response: Thanks for pointing to this important omission. We have edited the statement as follows: “Our data provide functional evidence that the 3 MD glomeruli receive afferent fibers form the maxillary palp (Ignell et al., 2005) (line 253-254).”\n- Supp Fig. 6a schematic does not make it clear that QF2 was inserted directly at the start codon. Recommend adjusting figure and stating this in the legend.\nAuthors’ response: We disagree with the Reviewer. In fact, QF2 was not designed to be inserted at the start codon. As illustrated in the figure 5A, the guide RNAs target the 3' end of the Or49 promoter and a site downstream to the Or49 start codon. It was intended to create a small deletion and be replaced by the DNA cassette containing the complete coding sequence of the QF2 gene (including its own start codon) along with OpIE2-ECFP.\n- Supp Fig. 6d appears to show results from 2 PCR reactions used to confirm proper integration of the construct into the Or49 locus. But the reactions amplify stretches of DNA that are found in the donor plasmid, which could have inserted unexpectedly in a different genomic location. To show proper integration, the authors must place one primer in a genomic location (just) outside of the homologous arm. We could also not understand what was being shown in panel f nor what primers were being used for panel g.\nAuthors’ response: We appreciated the reviewer’s inquiry and designed primers as suggested (primer pair P11 & P12; Or49_diag_up_2F/2R). As shown in the new Figure S5 and in the edited new version of the manuscript, the whole plasmid was inserted into a locus tightly linked to the Or49 one (see edit manuscript text for further details), yet the latter showed a large 440bp deletion downstream of the gRNA1 (spanning the coding sequence) along with extensive SNPs upstream in the promoter region, confirming that the transgenic line is actually a knockout for the Or49 gene. This is in accordance with the single-cell electrophysiological results showing lack of responses for the cpC neuron to borneol.\nThe former panel f (currently panel e) is a diagram representing the alignment of the QF2-ECFP cassette obtained from different G1 individuals (G1F1, G1F4, and G1M1; generation 1 female 1, etc.) as in the gel in panel F, along with the sequence of the plasmid itself. This figure was created with the software Geneious Prime.\nPrimers used in the former panel g were Or49_diag_up_F and Or49_diag_down_R.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my major concerns and I support publication of the manuscript.\nI have only a few minor comments:\nline 75: this sentence seems unchanged from the first version. Consider previewing the effects of the OR49 KO here?\nline 119: I agree with the other reviewers that it is too strong to say that OR49 funcitons as a selective borneol receptor. I would say instead that OR49 shows strong selectivity for borneol and closely related compounds.\nline 160: I think I would say here that Or49 is necessary for the cpC neuron sensitivity to borneol, rather than sufficient, based on the KO effect.\nI didn’t see a reference in the text ot the data shown in Fig 6e. These figures seem to show more visits by the KO mosquitos, which seems inconsistent with the quantificaiton in 6b. Can you clarify?", "reviewer_2": "The authors have made several improvements in the manuscript, generating new data and correcting mistakes in analyses at multiple places. However, some issues remain to be addressed.\nI had expressed concerns about the reliability of the SSR data from the Or49-mutant line, which had reduced fitness. The reduced SSR response can also be explained by reduced fitness, and to rule out this possibility, I asked that the authors include SSR data for CO2 and octenol as positive controls, which the authors have declined (these data could have been easily acquired in the same animals along with borneol – it is surprising to me that this was not done). At the very least, the authors should clearly state this limitation (reduced fitness and the lack of a positive control) when interpreting the mutant data in the SSR results section.\nI still have concerns about the imaging results in the revised manuscript. In the revised version, the response to 1-octen-3-ol seems barely rising above the baseline, while earlier studies (Wolff et al) have shown strong responses to octenol in one of three MD glomeruli. Also, strangely, Fig. 5d shows calcium responses in MD3 rising above the baseline even before borneol is given – this was not the case in the previous version of the manuscript).\nFigure 6b shows that even OR49-mutant mosquitoes have reduced but still significant repellency for Borneol (Or49 seems to account for only half of the effect). However, in their writing, the authors have over-emphasized the role of Or49 and under-emphasized the fact that there must be alternate pathway(s) which also contributes significantly (almost equally to Or49) in borneol repellency.\nThe authors mention that a new OR49 mutant was generated for use in behavioral experiments; however, I could not locate any description of how the process of generating this mutant was different from the first mutant. The authors should provide more information on what changes were made in the procedure of generating a new mutant line that produced healthier mutants.\nIn their response to my question about cpB and cpC neurons, the authors wrote: “Discrimination between the cpB and cpC neurons is based on spike amplitude, spontaneous activity and their respective functional responses. In our study, the cpB neuron, which is sensitive to 1-octen-3-ol, consistently exhibits larger action potential amplitudes than the cpC neuron, which is activated by borneol. This is in line with several other studies in Aedes, Culex, and Anopheles species that have reported that the cpB neuron is responsive to octenol (Syed & Leal, 2007; Lu et al., 2007; Cook et al., 2011; Omondi et al., 2015; Majeed et al. 2016; Herre at al., 2022). While other investigations in Aedes aegypti have identified the cpC neuron, characterized by the smallest spike amplitude, as the octenol-sensitive neuron these results are ambiguous. The reason for this discrepancy remains unclear, but likely stem from either a misidentification of the sensory neurons (Grant & Dickens, 2011) or differences in experimental methods or strain variation (Bohbot et al., 2013; Singh et al., 2022). We incorporated this explanation and references to previous works in the M&M section of the revised manuscript (lines 571-575).”\nI could not find this explanation and the references in line 571-575 – this needs to be added. Another reason the authors could add in this explanation is the relatively minor difference in spike sizes of B and C neurons (as is also the case in the traces provided by the authors), which makes the identification difficult. As the authors have also acknowledged, there is ambiguity between B and C neurons, and this needs to be communicated, rather than hiding under the carpet. Another example of this ambiguity can be seen in line 194: “while the medium size MD2 glomerulus receives 1-octen-3-ol input from the cpB neurons (Wolff et al)”. However, Wolff et al actually reports MD3 as the glomerulus responding to 1-octen-3-ol!\nMinor:\nFigure 5: The responses to the solvent controls are missing in the figures – these should be displayed for comparison.\nIn figures 5d, 5e, and 5f, the authors have changed the calcium imaging plots for all odorants (compared to the previous version). But in the main text, the p values have not changed for all three odorants.\nIn their response letter, the authors seem to agree that borneol is not the “Primary” ligand for Or49 in a biological sense. But they still mention in the discussion that OR49 is ‘selectively tuned to this particular compound’ (Line 271) .\nIn line 81, references 18,19 are cited for “The odorant receptor 49 gene”. The reference 18 describes the Ors in Anopheles gambiae and does not mention Or49.\nThe Figure 6d label/title should indicate that the figure is showing predicted values.\nIn Figure 2B, for clarity, ‘Vibration table’ can be changed to “Vibration isolation table”.\nLine 379 Current Traces of Xenopus oocytes are referenced to supplementary figure 4, which should probably be changed to 5.\nLine 488 “Pasteur pipettes were filled with the metered CO2 and used immediately to stimulate the preparation.” It is not clear how Pasteur pipettes were used to deliver CO2, i.e the filling, the delivery etc\nLine 173 “allowing repeatable registration of the AL glomeruli between preparations (Fig. 5b, S10) “. Cited supplementary figure number is wrong; it should be S9.\n“Our results show that under (+)-borneol exposure, wild-type females exhibited an average reduction of 54% in visits to the ROI compared to the vehicle control, whereas Or49⁻/⁻ mutants displayed a more modest reduction of 25% (Fig. 6b)” – Can the authors provide the mean values of the 4 bars to verify these numbers, and also provide ‘n’ for these graphs?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "We found the revised manuscript to be significantly improved over the original submission. Most importantly, the authors now test the behavior of Or49 mutant females and show that they are less repelled by borneol than their wild type counterparts. These new experiments provide the previously missing causal link between Or49 and borneol repellency. We also found the different methods/assays used much easier to follow (the new figure panels help!). We found several areas still or newly in need of clarification (see below), but support publication in Nature Communications.\nTextual clarifications\n- The first paragraph of the results section (line 80) could use a tad more clarification.\nFirst, we suggest changing “is conserved in” to “is represented by conserved 1-to-1 orthologs in”.\nSecond, the sentence on lines 82-84 says that the Or49 family is culicine-specific, while the sentence on lines 84-86 says that Or49 genes are located on the same chromosome in Aedes (Culicines) and Anopheles (Anophelines). This seems contradictory, with the first suggesting Or49 related receptors do not occur in Anopheles and the second suggesting that the do. We suggest revising the first sentence to read, “conserved Or49 orthologs are only found in the Culicines” and modify the second sentence to make it clear that it addresses a larger group of related genes (e.g. “Or49-like receptors”).\nFinally, the last sentence suggests that synteny among Or49-like receptors and neighboring genes is conserved from Aedes to Anopheles, but we did not see evidence of this in Supplementary Figure. 1f. It looked to us like only Aedes aegypti and Aedes albopictus showed clear evidence of synteny.\n- The two sentences spanning lines 114-118 also seem contradictory, with one stating that TamboOr49 responds similarly to camphor and borneol and the other stating that TamboOr49 is selective for borneol. The authors explained in their rebuttal that the focal experiments report distinct response variables (current response as a % of maximum and current response in nA). But the text doesn’t make that clear. Can the authors use more precise language to clarify the apparent discrepancy and its biological interpretation?\n- The pitch of the results section entitled “Borneol and camphor specifically activate the MD3 glomerulus in the antennal lobe” remains somewhat confusing (lines 166-194). The title of the section is appropriate, but three distinct but overlapping goals are listed across the first sentences of each of the two paragraphs: (1) “examine how Ae. aegypti processes borneol in the antennal lobe”, (2) “determine the odor coding of the mediodorsal glomeruli” and (3) “identify the cognate glomerulus representing borneol”. Might be more clear and accurate to simply say that the goal is to confirm that the orphan MD glomerulus (MD3) responds selectively to borneol (as expected given it is the presumed target of CpC neurons based on previous anatomical work – which the authors now cite).\n- It’s fantastic that the authors were able to outcross the Or49 KO line to improve fitness and enable behavioral testing of homozygotes. This should be more clearly explained in the text. The results states that a “new” Or49 knockout line was generated (line 199-200) which falsely implies more injections and an independent transformation. At the same time, the methods are confusing. They mention 6 generations of backcrossing but it comes just after an explanation of homozygosing and, without any further information about why a meticulously homozygosed line would be outcrossed again, it comes across as a mistake/typo. We recommend avoiding mention of a ‘new’ line in the main text and then revising the methods to make it more clear what work was conducted to generate the line used for ephys and then, perhaps in a new paragraph, why and how additional backcrossing and homozygosing was conducted to clean/improve the line for behavior.\nIt would also be helpful for the community if the authors could report in the methods section whether they tested the line to see if it was capable of driving expression of a QUAS effector in CpC neurons.\n- In the summary of results at the end of the introduction, we recommend removing the phrase “suggesting that all three cp neurons project to the same brain area” on line 74-75. As discussed above, this was already known and is therefore not a key result of the current work. Also, the next sentence (starting “Corroborating our pharmacological and electrophysiological results…”) seems to be a holdover from the original manuscript and could be revised to include the exciting new behavioral result that knocking our Or49 significantly reduces the repellent effect of borneol during host-seeking.\nAdditional minor corrections\n- Line 109: Could add “and so we focused our following oocyte analyses on the (+) forms” to improve clarity.\n- Typo on line 113: CquiOr49 should be CquiOr38\n- The authors responded to our concern about the potential for females to contact the borneol in the behavioral assay in their rebuttal but did not include much detail in added text. It would be very helpful to note in the results section (e.g. lines 204-206 or figure 6 legend) that they did not observe mosquitoes touching the borneol during the assay. We were satisfied with the authors response in the rebuttal but readers will not be privy to that.\n- The authors report use of a QUAS-GCaMP6s line for imaging and cite Zhao et al 2022 (ref 24) as the source in both results and methods. However, that paper generated a QUAS-GCaMP7s line, not a 6s line. Is the use of 6s in the paper a typo? Which construct was used and where did it come from?\n- The authors also cite Zhao et al 2022 (ref 24) for the brp-QF2w strain in both results and methods, but should instead cite the earlier paper that generated the line: Zhao et al 2020 Cell Reports Methods (doi.org/10.1016/j.crmeth.2021.100042)\n- line 240: we suggest changing ‘mechanisms’ to ‘mechanism’ and ‘are’ to ‘is’ for readability. ‘more than one’ is often treated as singular even though it refers to plural. Alternatively, could change ‘more than one’ to ‘several’.\n- Figure 3C: The y (mV) scales for borneol and camphor current traces differ, making it hard to compare by eye for a single species.\n- Figure 4b: Why no error bars on some dots?\n- Figure S1d: The numbers in the exons are not fully explained in the figure legend. Does nucleotide position refer to the length of the exon in basepairs?\n- Figure S5: mention in the figure legend that these recordings are from oocytes [line 126]\n- Figure S6i: a bit confused by the alignment—shouldn’t there be an insertion there?\n- Figure S7: mention in the figure legend that these recordings are from sensilla\nWe look forward to seeing this paper in print!"}, "author_response": "Reviewer #1:\nThe authors have addressed my major concerns and I support publication of the manuscript.\nI have only a few minor comments:\nline 75: this sentence seems unchanged from the first version. Consider previewing the effects of the OR49 KO here?\nAuthors’ response: According to the reviewer’s comment, we are now emphasizing that the use of the Or49 null mutant establishes a direct causal link between this gene and the sensory and behavioral phenotypes observed in the wild-type strain. In addition, we have revised the latter part of this paragraph to improve its overall flow and to address the reviewer’s concern.\n“To directly assess its olfactory function, we generated an Or49 null mutant line in Aedes aegypti, which completely lacked electrophysiological responses to borneol and displayed reduced avoidance behavior in the context of human-host seeking”.\nline 119: I agree with the other reviewers that it is too strong to say that OR49 functions as a selective borneol receptor. I would say instead that OR49 shows strong selectivity for borneol and closely related compounds.\nAuthors’ response: As recommended, we have revised the sentence to read: “These results indicate that OR49 shows strong selectivity for borneol and closely related compounds”.\nline 160: I think I would say here that Or49 is necessary for the cpC neuron sensitivity to borneol, rather than sufficient, based on the KO effect.\nAuthors’ response: As suggested, we have revised the wording and now state that Or49 is necessary, rather than sufficient, for cpC neuron sensitivity to borneol.\nI didn’t see a reference in the text ot the data shown in Fig 6e. These figures seem to show more visits by the KO mosquitos, which seems inconsistent with the quantificaiton in 6b. Can you clarify?\nAuthors’ response: In the revised manuscript, we have now cited Fig. 6e in the relevant section of the text.\nRegarding the second point, it is correct that Or49 / female mosquitoes (“Or49 / (+)-borneol”) make more visits than their wild-type counterparts (“WT (+)-borneol”), and this difference is accurately reflected in the quantification shown in Fig. 6b. As the figure is consistent with the data, no revisions were made to Fig. 6.\nReviewer #2:\nThe authors have made several improvements in the manuscript, generating new data and correcting mistakes in analyses at multiple places. However, some issues remain to be addressed.\nI had expressed concerns about the reliability of the SSR data from the Or49-mutant line, which had reduced fitness. The reduced SSR response can also be explained by reduced fitness, and to rule out this possibility, I asked that the authors include SSR data for CO2 and octenol as positive controls, which the authors have declined (these data could have been easily acquired in the same animals along with borneol – it is surprising to me that this was not done). At the very least, the authors should clearly state this limitation (reduced fitness and the lack of a positive control) when interpreting the mutant data in the SSR results section.\nAuthors’ response: We acknowledge the limitation of not including positive control SSR recordings in the Or49 null mutant line; however, we observed that cpA and cpB neurons exhibited normal spontaneous activity (see new supplementary figure 9 below), and despite the reduced longevity of the line, no adverse effect on neuronal function was observed. Accordingly, we added the following statement in the revised manuscript:\n“Although recordings were not obtained from the cpA and cpB neurons in the Or49 null mutant, these neurons displayed normal spontaneous activity (Supplementary Fig. 9). We note that the reduced longevity of the mutant line could potentially affect neuronal function; however, no such effect was observed.”\nI still have concerns about the imaging results in the revised manuscript. In the revised version, the response to 1-octen-3-ol seems barely rising above the baseline, while earlier studies (Wolff et al) have shown strong responses to octenol in one of three MD glomeruli. Also, strangely, Fig. 5d shows calcium responses in MD3 rising above the baseline even before borneol is given – this was not the case in the previous version of the manuscript).\nAuthors’ response: In the first submitted manuscript, we initially used functional responses (glomerular tuning) and coarse neuroanatomical identification to identify glomeruli. Moreover, during our experiments, we concentrated on borneol responses and the associated glomerulus, rather than the simultaneous responses of all MD glomeruli to their cognate ligands, or the responses of the neighboring MD2 glomerulus and its responses to 1-octen-3-ol.\nAt the suggestion of the reviewer, in the last revised manuscript, we used strict neuroanatomical criteria for glomerular registration from our AL reconstruction and registration with an existing atlas from Conor McMeniman’s lab. In the last revised manuscript, the glomerular ROIs were restricted to solely their neuroanatomical position (relative to the atlas) rather than activity, resulting in changing in our ROI assignments and associated calcium responses. Although we received responses to 1-octen-3-ol, they were not always located in MD2, based on the neuroanatomy from Shankar and McMeniman (2020). The criteria we used, as per Shankar & McMeniman (2020): MD3 (cognate ligand: borneol) sits in the medio-proximal corner and has the same size as MD2; MD2 (cognate ligand: 1-octen-3-ol), near MD3; MD1 (cognate ligand: CO2), a large glomerulus that is located under MD2 and MD3.\nRegarding the rise of the calcium before the odor onset, there are two reasons for this phenomenon. The first one is that during the experiments, stimulation happens exactly 10000 ms after beginning the time series. However, frame collection happens every 513 ms due to constraints associated with the resolution of the ROI. This seemingly small difference adds up to an offset of about 250 ms by the time of activation of the solenoid that releases the odor stimuli. Due to that offset, the 20th frame (which should be the time of stimulus release) is recorded 0.25 s after odor release, showing a response that is stronger than what we would expect if the imaging would happen exactly at the time of odor release. In our previous figures we plotted the odor release at frame 20th, which did not account for that offset. We present now new figures in which we adjusted the data points to match the time at which the images were collected, and clarify this in the Methods. The second reason has to do with the smoothing and plotting of the time series. Since there’s no response in frame 19 (pre-stimulation), but there’s a strong one in frame 20 (post-stimulation), it is inevitable to see a rising curve between these two smoothed data points in the series (see figure below).\nFigure 6b shows that even OR49-mutant mosquitoes have reduced but still significant repellency for Borneol (Or49 seems to account for only half of the effect). However, in their writing, the authors have over-emphasized the role of Or49 and under-emphasized the fact that there must be alternate pathway(s) which also contributes significantly (almost equally to Or49) in borneol repellency.\nAuthors’ response: We thank the reviewer for this insightful comment. In the revised Discussion, we have clarified that OR49 is a major, but not exclusive, contributor to (+)-borneol avoidance. The revised section now reads:\n“Although avoidance was not abolished in the KO strain, the attenuated response indicates that Or49 contributes to (+)-borneol detection, while alternative receptors or pathways provide a comparable contribution through overlapping sensitivity to borneol and related terpenoids.”\n“…these findings establish OR49 as a central, though not exclusive mediator of (+)-borneol-induced avoidance…”\nThe authors mention that a new OR49 mutant was generated for use in behavioral experiments; however, I could not locate any description of how the process of generating this mutant was different from the first mutant. The authors should provide more information on what changes were made in the procedure of generating a new mutant line that produced healthier mutants.\nAuthors’ response: The manuscript was modified to indicate that a new Homozygous line (rather than new mutant line) was obtained after backcrossing to wild type for six generations so as to remove any background mutation that might have reduced the behavioral performance of the Or49 mutant line. Such a procedure is described in the Methods section.\nIn their response to my question about cpB and cpC neurons, the authors wrote: “Discrimination between the cpB and cpC neurons is based on spike amplitude, spontaneous activity and their respective functional responses. In our study, the cpB neuron, which is sensitive to 1-octen-3-ol, consistently exhibits larger action potential amplitudes than the cpC neuron, which is activated by borneol. This is in line with several other studies in Aedes, Culex, and Anopheles species that have reported that the cpB neuron is responsive to octenol (Syed & Leal, 2007; Lu et al., 2007; Cook et al., 2011; Omondi et al., 2015; Majeed et al. 2016; Herre at al., 2022). While other investigations in Aedes aegypti have identified the cpC neuron, characterized by the smallest spike amplitude, as the octenol-sensitive neuron these results are ambiguous. The reason for this discrepancy remains unclear, but likely stem from either a misidentification of the sensory neurons (Grant & Dickens, 2011) or differences in experimental methods or strain variation (Bohbot et al., 2013; Singh et al., 2022). We incorporated this explanation and references to previous works in the M&M section of the revised manuscript (lines 571-575).”\nI could not find this explanation and the references in line 571-575 – this needs to be added. Another reason the authors could add in this explanation is the relatively minor difference in spike sizes of B and C neurons (as is also the case in the traces provided by the authors), which makes the identification difficult. As the authors have also acknowledged, there is ambiguity between B and C neurons, and this needs to be communicated, rather than hiding under the carpet.\nAuthors’ response: We thank the reviewer for pointing out this issue. Due to an oversight, the explanation and references were not included in the final edit of the first revision and has now been incorporated into the revised manuscript.\nAnother example of this ambiguity can be seen in line 194: “while the medium size MD2 glomerulus receives 1-octen-3-ol input from the cpB neurons (Wolff et al)”. However, Wolff et al actually reports MD3 as the glomerulus responding to 1-octen-3-ol!\nAuthors’ response: In the Wolff et al article, we used an earlier atlas from Rickard Ignell’s lab. However, here in this manuscript, we use the Shankar and McMeniman (2020) atlas, which is the current state-of-the-art. The Amira atlas was kindly provided by the McMeniman lab, allowing us to register between the atlas and two-photon microscopy images. We now clarify this in the Methods.\nMinor:\nFigure 5: The responses to the solvent controls are missing in the figures – these should be displayed for comparison.\nAuthors’ response: In the revised figure, we now include the blank controls.\nIn figures 5d, 5e, and 5f, the authors have changed the calcium imaging plots for all odorants (compared to the previous version). But in the main text, the p values have not changed for all three odorants.\nAuthors’ response: We included this information in the revised manuscript.\nIn their response letter, the authors seem to agree that borneol is not the “Primary” ligand for Or49 in a biological sense. But they still mention in the discussion that OR49 is ‘selectively tuned to this particular compound’ (Line 271).\nAuthors’ response: The reviewer raises an important conceptual issue related to the biological relevance of receptor-ligand pharmacology. In the context of this study, we showed that borneol is the most efficacious and potent (meaning OR49 is selectively activated by borneol) OR49 ligand when compared to the other tested odorant ligands. Therefore, qualifying borneol as the primary ligand is correct. However, we can’t exclude that another ligand with greater potency remains to be identified. We agree with the initial comment from the reviewer that the cpC neuron may respond to ligands other than borneol via other olfactory receptors (ORs or IRs). In any case, we have revised the sentence: “Why OR49 shows such strong selectivity for borneol, among the many plant-derived volatiles known to repel mosquitoes, remains an open question.”\nIn line 81, references 18,19 are cited for “The odorant receptor 49 gene”. The reference 18 describes the Ors in Anopheles gambiae and does not mention Or49.\nAuthors’ response: This is correct, thanks. Reference 18 does not describe Or49 and has therefore been removed in the revised manuscript.\nThe Figure 6d label/title should indicate that the figure is showing predicted values.\nAuthors’ response: We thank the reviewer for this suggestion. We have clarified this point in the revised figure caption: “Lines correspond to the model’s predictions, and points represent the observed data”.\nIn Figure 2B, for clarity, ‘Vibration table’ can be changed to “Vibration isolation table”.\nLine 379 Current Traces of Xenopus oocytes are referenced to supplementary figure 4, which should probably be changed to 5.\nAuthors’ response: Figure 2b has been updated to read “Vibration isolation table”. In addition, the reference to the current traces of Xenopus oocytes has been corrected to the appropriate supplementary figure (supplementary figures 3 & 5).\nLine 488 “Pasteur pipettes were filled with the metered CO2 and used immediately to stimulate the preparation.” It is not clear how Pasteur pipettes were used to deliver CO2, i.e the filling, the delivery etc\nAuthors’ response: We have included the following description in the material and methods section: “Stimulation with gaseous CO was by filling Pasteur pipettes with metered amounts of CO (150, 300, 600, 1200, 2400, 4800 ppm) and oxygen (20%), balanced by nitrogen (Strandmöllen AB , Ljungby, Sweden) similar to deBruyne et al., 2001; Neuron 30: 537-552). After filling, both end of the pipettes were sealed with parafilm. Upon delivery, the parafilm was removed, and the filled pipette immediately connected to the odor delivery system, and the CO administered in the same manner as for the other odor stimuli. The Pasteur pipettes were refilled with CO after each puff.” ₂ ₂ ₂\nLine 173 “allowing repeatable registration of the AL glomeruli between preparations (Fig. 5b, S10) “. Cited supplementary figure number is wrong; it should be S9.\nAuthors’ response: We thank the reviewer for noting this mistake. The incorrect reference has been corrected, and the sentence now cites Supplementary Fig. S8, which contains the calcium imaging data.\n“Our results show that under (+)-borneol exposure, wild-type females exhibited an average reduction of 54% in visits to the ROI compared to the vehicle control, whereas Or49 / mutants displayed a more modest reduction of 25% (Fig. 6b)” – Can the authors provide the mean values of the 4 bars to verify these numbers, and also provide ‘n’ for these graphs?\nAuthors’ response: We have added the mean values to Fig. 6b and revised the caption accordingly, including the sample sizes (n values).\nReviewer #3 & 4:\nWe found the revised manuscript to be significantly improved over the original submission. Most importantly, the authors now test the behavior of Or49 mutant females and show that they are less repelled by borneol than their wild type counterparts. These new experiments provide the previously missing causal link between Or49 and borneol repellency. We also found the different methods/assays used much easier to follow (the new figure panels help!). We found several areas still or newly in need of clarification (see below), but support publication in Nature Communications.\nTextual clarifications\n- The first paragraph of the results section (line 80) could use a tad more clarification.\nFirst, we suggest changing “is conserved in” to “is represented by conserved 1-to-1 orthologs in”.\nSecond, the sentence on lines 82-84 says that the Or49 family is culicine-specific, while the sentence on lines 84-86 says that Or49 genes are located on the same chromosome in Aedes (Culicines) and Anopheles (Anophelines). This seems contradictory, with the first suggesting Or49 related receptors do not occur in Anopheles and the second suggesting that the do. We suggest revising the first sentence to read, “conserved Or49 orthologs are only found in the Culicines” and modify the second sentence to make it clear that it addresses a larger group of related genes (e.g. “Or49-like receptors”).\nFinally, the last sentence suggests that synteny among Or49-like receptors and neighboring genes is conserved from Aedes to Anopheles, but we did not see evidence of this in Supplementary Figure. 1f. It looked to us like only Aedes aegypti and Aedes albopictus showed clear evidence of synteny.\nAuthors’ response: We thank the reviewer for these constructive suggestions. We have revised the first paragraph of the Results section to improve, clarity and avoid potential contradictions:\n1. We replaced “is conserved in” with “is represented by conserved 1-to-1 orthologs in”, as recommended.\n2. We now state that “conserved Or49 orthologs are only found in the Culicines” and clarified that the subsequent sentence refers to more distantly related Or49-like receptors in Anopheles.\n3. We modified the final sentence to reflect the actual data shown in Supplementary Fig. 1e-f, specifying that conserved synteny with neighboring genes is observed between Ae. aegypti and Ae. albopictus, but not in Anopheles.\n- The two sentences spanning lines 114-118 also seem contradictory, with one stating that TamboOr49 responds similarly to camphor and borneol and the other stating that TamboOr49 is selective for borneol. The authors explained in their rebuttal that the focal experiments report distinct response variables (current response as a % of maximum and current response in nA). But the text doesn’t make that clear. Can the authors use more precise language to clarify the apparent discrepancy and its biological interpretation?\nAuthors’ response: We agree that the original wording could appear contradictory. To clarify, we have revised the text to explicitly distinguish between the two response variables: potency (EC values, Fig. 2f) and maximum response magnitude (nA, Fig. 2g). The text now reads:\n“(+)-Borneol was 19-32 times more potent than (+)-camphor in all cases except for TambOR49 where both compounds were equally active in the low micromolar range (Fig. 2f). However, when comparing maximum response magnitudes, (+)-borneol consistently elicited stronger currents than (+)-camphor across all four culicine species, including the nectar-feeding Tx. amboinensis (Fig. 2g, Supplementary Fig. 3). These results indicate that OR49 shows strong selectivity for borneol and closely related compounds.”\n- The pitch of the results section entitled “Borneol and camphor specifically activate the MD3 glomerulus in the antennal lobe” remains somewhat confusing (lines 166-194). The title of the section is appropriate, but three distinct but overlapping goals are listed across the first sentences of each of the two paragraphs: (1) “examine how Ae. aegypti processes borneol in the antennal lobe”, (2) “determine the odor coding of the mediodorsal glomeruli” and (3) “identify the cognate glomerulus representing borneol”. Might be more clear and accurate to simply say that the goal is to confirm that the orphan MD glomerulus (MD3) responds selectively to borneol (as expected given it is the presumed target of CpC neurons based on previous anatomical work – which the authors now cite).\nAuthors’ response: As suggested, we have clarified the rationale confirming that the orphan MD3 glomerulus responds selectively to borneol, consistent with its predicted role as the target of cpC neurons based on previous anatomical studies. To simplify the text further, we removed redundant phrasing and revised the opening sentences accordingly. The text now reads:\n“To confirm that the orphan MD3 glomerulus in the antennal lobe responds selectively to borneol, we performed two-photon calcium imaging in Ae. aegypti. We utilized existing mosquito lines…”\n“We then tested MD1-3 glomeruli…”.\n- It’s fantastic that the authors were able to outcross the Or49 KO line to improve fitness and enable behavioral testing of homozygotes. This should be more clearly explained in the text. The results states that a “new” Or49 knockout line was generated (line 199-200) which falsely implies more injections and an independent transformation. At the same time, the methods are confusing. They mention 6 generations of backcrossing but it comes just after an explanation of homozygosing and, without any further information about why a meticulously homozygosed line would be outcrossed again, it comes across as a mistake/typo. We recommend avoiding mention of a ‘new’ line in the main text and then revising the methods to make it more clear what work was conducted to generate the line used for ephys and then, perhaps in a new paragraph, why and how additional backcrossing and homozygosing was conducted to clean/improve the line for behavior.\nAuthors’ response: We would like to thank the reviewers for their positive response to a yearlong effort it took the Akabari’s and Bohbot’s lab to generate the KO line and assessing its behavioral phenotype, respectively. We addressed this issue above in response to reviewer 2 on page 3.\nThe manuscript was modified to indicate that a new homozygous line (rather than new mutant line) was obtained after backcrossing to wild type for six generations so as to remove any background mutation that might have reduced the behavioral performance of the first OR49 mutant homozygous line. It now reads:\n\"As the mutant lines initially used for SSR exhibited reduced behavioral activity, we generated a new homozygous OR49 knockout line after backcrossing for six generations and focused on behavioral validation to assess borneol’s repellency.”\nSuch a procedure is described in the Methods section, which was modified to include the above suggestions:\n“To solve the low initial performance of this mutant line in behavioral assays, this line underwent 6 generations of backcrossing to the wildtype line, intercrossed for 3 generations, and a new homozygous line was selected as described.”.\nIt would also be helpful for the community if the authors could report in the methods section whether they tested the line to see if it was capable of driving expression of a QUAS effector in CpC neurons.\nAuthors’ response: Preliminary experiments indicated fluorescence in the corresponding antennal lobe glomerulus, consistent with expression in CpC neurons. However, more detailed experiments are required to confirm and fully characterize this expression. As such, we have not included these preliminary findings in the manuscript, but we plan to report them in a future study once validated.\n- In the summary of results at the end of the introduction, we recommend removing the phrase “suggesting that all three cp neurons project to the same brain area” on line 74-75. As discussed above, this was already known and is therefore not a key result of the current work. Also, the next sentence (starting “Corroborating our pharmacological and electrophysiological results…”) seems to be a holdover from the original manuscript and could be revised to include the exciting new behavioral result that knocking our Or49 significantly reduces the repellent effect of borneol during host-seeking.\nAuthors’ response: In the revised manuscript, we removed the redundant phrase regarding cp neuron projections, as this information is indeed established in prior studies. The paragraph now reads: “Furthermore, we show that borneol selectively activates the MD3 glomerulus in the antennal lobe of Ae. aegypti. Together, these results provide a framework for dissecting the molecular and neural mechanisms underlying olfactory integration and host-seeking behavior in mosquitoes.”\nAdditional minor corrections\n- Line 109: Could add “and so we focused our following oocyte analyses on the (+) forms” to improve clarity.\nAuthors’ response: We have added the recommended clarification in the revised manuscript: “We therefore focused subsequent oocyte analyses on the (+) enantiomers”.\n- Typo on line 113: CquiOr49 should be CquiOr38\nAuthors’ response: The typo has been corrected in the revised manuscript.\n- The authors responded to our concern about the potential for females to contact the borneol in the behavioral assay in their rebuttal but did not include much detail in added text. It would be very helpful to note in the results section (e.g. lines 204-206 or figure 6 legend) that they did not observe mosquitoes touching the borneol during the assay. We were satisfied with the Authors’ response in the rebuttal but readers will not be privy to that.\nAuthors’ response: In the revised manuscript, we have added a clarification to the Results section: “Although the setup allows for potential contact between mosquitoes and the borneol-soaked filter paper, we did not observe any instances of direct physical interaction.”.\n- The authors report use of a QUAS-GCaMP6s line for imaging and cite Zhao et al 2022 (ref 24) as the source in both results and methods. However, that paper generated a QUAS-GCaMP7s line, not a 6s line. Is the use of 6s in the paper a typo? Which construct was used and where did it come from?\nAuthors’ response: Now corrected. It was the GCaMP7s line. We also corrected the legend in Figure 5c accordingly.\n- The authors also cite Zhao et al 2022 (ref 24) for the brp-QF2w strain in both results and methods, but should instead cite the earlier paper that generated the line: Zhao et al 2020 Cell Reports Methods (doi.org/10.1016/j.crmeth.2021.100042)\nAuthors’ response: The citations have been updated to reference Zhao et al., 2021 (Cell Reports Methods, doi:10.1016/j.crmeth.2021.100042) as the original source of the QUAS-GCaMP7s line.\n- line 240: we suggest changing ‘mechanisms’ to ‘mechanism’ and ‘are’ to ‘is’ for readability. ‘more than one’ is often treated as singular even though it refers to plural. Alternatively, could change ‘more than one’ to ‘several’.\nAuthors’ response: We thank the reviewer for this suggestion. In the revised manuscript, we clarified the statement in question: “These findings suggest that multiple olfactory-mediated mechanisms operate in insects, as Or49 homologs have not been identified in these species”.\n- Figure 3C: The y (mV) scales for borneol and camphor current traces differ, making it hard to compare by eye for a single species.\nAuthors’ response: In the revised manuscript, we adjusted the y-axis ranges in Figure 3c to allow direct visual comparison between borneol and camphor traces for each species.\n- Figure 4b: Why no error bars on some dots?\nAuthors’ response: The data points in question do not actually lack error bars; rather, the error bars are very small and thus not easily visible in the figure. These points correspond to Culex quinquefasciatus, for which only two replicates were performed. The corresponding error bar values are shown in Extended Data Figure 6.\n- Figure S1d: The numbers in the exons are not fully explained in the figure legend. Does nucleotide position refer to the length of the exon in basepairs?\nAuthors’ response: In the revised manuscript, we clarified the figure caption: “The length of each exon and intron is indicated by the number within.”\n- Figure S5: mention in the figure legend that these recordings are from oocytes [line 126]\nAuthors’ response: We thank the reviewer for this comment. In the revised manuscript, we clarified this point by changing the figure title to “All recordings were performed using the two-electrode voltage clamp system.”\n- Figure S6i: a bit confused by the alignment—shouldn’t there be an insertion there?\nAuthors’ response: The alignment does not contain an insertion; rather, it shows a large 440 bp deletion downstream of the gRNA1 target site (spanning the coding sequence) along with multiple SNPs in the upstream promoter region, as described in our previous response to the reviewers.\n- Figure S7: mention in the figure legend that these recordings are from sensilla\nAuthors’ response: We thank the reviewer for this comment. Figure S7 in fact summarizes EC data obtained from TEVC assays, not sensillum recordings. To avoid confusion, we have clarified this in the revised figure legend: “This data was collected using the TEVC ₅s₀ystem.”\nWe look forward to seeing this paper in print!"}]

Decision letter