1. Volume 155, Issue 6, Pages 1365-1379 (December 2013)
Targeting a Dual Detector of Skin and CO2 to Modify Mosquito Host Seeking 
Genevieve M. Tauxe, Dyan MacWilliam, Sean Michael Boyle, Tom Guda, Anandasankar Ray 
Cell 
Volume 155, Issue 6, Pages (December 2013)
DOI: /j.cell
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 The CO2-Sensitive Mosquito cpA Neuron Detects Human Skin Odor
(A) Schematic of the maxillary palp capitate peg sensillum with three ORNs and electrodes for electrophysiology.
(B) Representative traces and mean change in firing rate of the Aedes aegypti cpA (large amplitude) neuron to foot odor carried on glass beads. n = 6–7.
(C) Chemical structures of known cpA inhibitors and butyryl chloride.
(D) Responses to indicated odorants after pre-exposure to butyryl chloride (10−2) (cpA-off) or solvent (sham treatment).
(E) Mean odor-evoked responses of the cpA neuron in cpA-off and sham-treated mosquitoes and (F) combined odor-evoked responses of the two neighboring neurons, cpB and cpC. (E and F) n = 16.
(G) Sample traces and mean cpA responses to CO2 after treatment.
(H) Sample traces and mean cpA responses to foot odor (mixed beads from persons 1 and 2) after treatment. n = 8–9.
(I) Summed cpB and cpC neuron response; n = 4–8.
(J) Averaged traces and mean normalized EAG responses to foot odor and to a synthetic blend of human odorants. n = 16–18 (foot odor), 8–9 (synthetic blend); analyzed by t test.
(E, F, H and I) Analyzed by ANOVA nested across three to six individuals. ∗p < 0.05, ∗∗∗p < CpA-off mosquitoes were pre-exposed to chemical for 1 min (G) or 3 min (elsewhere). Error bars are SEM. See also Figures S1, S2, and S4.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 2
Activation of cpA Is Required for Navigation of Female A. aegypti toward a Human Skin Odor Source
(A) Schematic of wind tunnel assay for navigation of single female A. aegypti to glass beads worn in socks. Dark circles at the bottom provide visual cues for flight.
(B and C) Proportion of butyryl-chloride-exposed and sham-treated mosquitoes presented with beads with no odor or foot odor that landed on beads (B) or took off from the release cage (C).
(D) Proportion of mosquitoes that did take off that succeeded in landing on the beads.
(B and C) n = 20–23 individuals per condition; analyzed by one-tailed proportion Z test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < See also Figures S2, S3, S4 and Movies S1, S2, S3, and S4.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 3 Specific Odorants in Human Skin Activate the cpA Neuron
(A) Representative traces and mean responses of the cpA neuron to 0.5 s pulses of individual components of skin odor in A. aegypti and A. gambiae. n = 4–7.
(B) Dose responses of A. aegypti cpA to representative activating odorants.
(C) Mean firing frequency of A. aegypti cpA during a 1 s stimulus, counted in 100 ms bins. Responses averaged across n = 4–5 stimuli repeated in 15 s cycles.
(D) CpA responses to combinations of skin odorants (at 10−3) and 0.1% CO2. Airflow was adjusted to maintain concentrations of each odorant. n = 6; one-tailed paired t tests. ∗∗ p < 0.01, ∗∗∗ p < Error bars are SEM. See also Figure S4 and Table S1.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 4
Identification of CO2 Receptor Neuron Activators, Inhibitors, and Ultraprolonged Activator in A. aegypti
(A) Overview of the cheminformatics pipeline used to identify novel cpA ligands from a large untested chemical space.
(B) Representative traces and mean responses of the A. aegypti cpA neuron to 0.5 s pulses of 138 predicted compounds. Responses to solvent have been subtracted. n = 2–6.
(C) Representative traces and mean percent inhibition (compared to solvent) of cpA by a panel of 107 odorants presented as a 1 s stimulus overlaid on a 3 s 0.15% CO2 stimulus. PO = paraffin oil. n = 2, except for ethyl and methyl pyruvate, n = 6.
(B and C) Error bars are SD.
(D) Representative traces from the cp sensillum to 1 s pulses of 0.15% CO2 prior to and following a 3 s exposure to either solvent (paraffin oil) or (E)-2-methylbut-2-enal (10−1).
(E) CpA baseline activity in the 1 s prior to each stimulus after exposure to odorant.
(F) Mean responses of the cpA neuron to 1 s pulses of 0.15% CO2, calculated by subtracting 1 s of baseline activity prior to each stimulus after exposure to paraffin oil (gray) or (E)-2-methylbut-2-enal (10−1) (orange). n = 5–6 individuals; t test, ∗∗∗p <
(E and F) Error bars are SEM. See also Figure S5 and Tables S1 and S2.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

7. Figure 5
CO2 Receptor Neuron Inhibitors Repel and Activators Attract Mosquitoes
(A) Dose response of A. aegypti cpA neuron inhibition by a 1 s stimulus of ethyl pyruvate overlaid on a 3 s stimulus of 0.15% CO2. n = 6.
(B) Representative trace and mean response to a 1 s stimulus of ethyl pyruvate (10−2) overlaid on a 2 s stimulus of foot odor (mixed beads from persons 1 and 2). n = 6.
(C) Representative images of arm-in-cage mesh window and mean number of mosquitoes on the netting over time for ethyl-pyruvate-treated or solvent-treated netting. n = 8.
(D) Dose response of the cpA neuron to cyclopentanone in A. aegypti and C. quinquefasciatus. n = 5–6.
(E) Mean firing frequency during a 1 s stimulus, counted in 100 ms bins during 1 s stimuli of cyclopentanone, CO2, or blank odor cartridges. Responses averaged across n = 4 replicates of 6 repeated stimuli in 20 s cycles. Total baseline activity 5–6 s after each pulse was subtracted from response frequencies.
(F) Representative traces of repeated 1 s stimuli of cyclopentanone and 0.15% CO2.
(G) Schematic of two-choice greenhouse experiments with two counterflow geometry traps.
(H) Mean number of mosquitoes captured out of 50 per trial in baited and control traps. n = 9 trials with CO2, n = 6 for each cyclopentanone treatment.
(I) Preference index for CO2 and cyclopentanone trials (from H) and for similar trials between lactic acid (10−1) and solvent (n = 6) or between CO2 and CO2 with ethyl pyruvate (10−1) (n = 5). t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Error bars are SEM. See also Figure S5.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

8. Figure 6 Encoding of Chemical Space by the Mosquito cpA Neuron
(A) Principle component analysis (PCA) of odorants calculated from 64 optimized molecular descriptor values. Circle size represents cpA activity evoked by each odorant. Dark green = human skin odorants; light green = predicted activators; red = predicted inhibitors; gray = predicted odorants that are inactive; black = odorants that activate A. gambiae olfactory receptors (AgOrs) (Carey et al., 2010).
(B) The same analysis relabeled by chemical functional groups, with circle size representing cpA activity (right).
(C) Hierarchical clustering and sample structures (with associated activity) of cpA-active odorants by activity-optimized descriptors.
(D) Overview of the SVM integrated pipeline to improve computational prediction of novel CpA ligands.
(E) Receiver-operating-characteristic (ROC) curve showing increased predictive accuracy of SVM method (red line) to our previous non-SVM method (black line) in a 5-fold cross-validation.
(F) Mean responses of the A. aegypti cpA neuron to 0.5 s pulses of 19 newly predicted compounds screened as in Figure 4B. Salmon bars correspond to odorants found in human odor. n = 4. See also Tables S1 and S2.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

9. Figure 7 Detection of Structurally Diverse Ligands Depends on Gr63a
(A and B) Sample traces (A) and (B) mean responses of the Drosophila ab1C neuron to a panel of odorants (tested in orco− flies). n = 6.
(C) Mean responses from large basiconics in Gr63a−,orco− flies. n = 24 (six sensilla per individual).
(D) Mean preference index of flies of indicated genotypes to a choice between room air and 0.5% CO2 or cyclopentanone (10−2) in a T-maze. (n = 12–16).
(B–D) Error bars are SEM.
(E) The mosquito CO2 receptor neuron plays a critical role in host seeking by detecting both exhaled CO2 and skin odor. Odor-based intervention strategies include use of inhibitors to block attraction to both CO2 and skin odor (MASK) and activators as lures for traps (PULL).
See also Figure S6.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

10. Figure S1
Attraction to Skin Odor in orco Mutants and Effect of Butyryl Chloride on Antenna, Related to Figure 1
(A) Schematic of the two-choice cage assay. Both wild-type and orco16 mutant mosquitoes showed strong preference for the side of the cage with an odor-laden sock over the side with a clean sock.
(B) Graphs show mean preference over time (left) and cumulative preference averaged across time points from 2–5 min (right). n = 6 trials; arcsine-transformed nested ANOVA.
(C) Butyryl chloride pretreatment has little effect on antennal responses. Averaged traces of EAG responses to 0.5 s stimuli of indicated odorants (10−1 in paraffin oil).
(D) EAG responses normalized to the reference odor 3-methyl-1-butanol. No significant differences due to treatment. n = 9; t test.
Error bars are SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

11. Figure S2
Effect of Butyryl Chloride on Antennal Olfactory Neurons, Related to Figures 1 and 2
(A) Sample traces and (B and C) responses of individual antennal trichoid sensilla to two odor panels (10−2) before and after a 1 s puff of butyryl chloride (50 μl at 10−2) from a standard odor cartridge. (B) In recordings where amplitudes of the A and B neurons could not be clearly resolved, total response is shown in gray and blue (n = 8 sensilla). (C) When amplitudes could be resolved, increase in A (larger) and B (smaller) spikes were counted separately (n = 18 neurons). Double arrows indicate A and B neurons housed in the same sensillum.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

12. Figure S3 Wind Tunnel Behavior Assay, Related to Figure 2
(A–C) Details of wind tunnel behavior assay. (A) Schematic of the wind tunnel showing the mosquito release chamber, foot odor bead stage, and retracted bead cover. (B) Histograms of when mosquitoes took off during the 5 min assay in each of three experimental conditions. Xs mark when a mosquito landed on the odor source. (C) Each row indicates flight behavior of an individual mosquito. Shaded areas on each line represent time between when the mosquito left the release cage and when it landed on the beads or the assay ended at 5 min. Colors correspond to where the mosquito was located in the wind tunnel (as indicated in A) at each moment. Asterisks indicate trials illustrated with Movies S1, S2, S3, and S4.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

13. Figure S4
Heat and Humidity Assay and Electrophysiology with Repeated Odor Stimuli, Related to Figures 1, 2, and 3
(A) Schematic of apparatus used to assay short-range attraction to heat and humidity.
(B) Mosquitoes’ tendency to rest at the top surface of the cage increased in the presence of a warm, humid stimulus; mosquitoes in each treatment group were observed to probe through the mesh with their proboscides when stimulus was present. No significant differences due to treatment. n = 6 trials; Mann-Whitney rank-sum test.
(C) Mean cpA responses to CO2 and skin odorants presented in sequence. There are no significant differences in responses to each odorant. n = 6 replicates; ANOVA. Error bars are SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

14. Figure S5
Dose-Response and Temporal Activity of the Mosquito cpA Neuron, Related to Figures 4 and 5
(A) Mean percent inhibition of A. aegypti cpA response to 0.15% CO2 overlaid with different concentrations of methyl pyruvate. n = 6.
(B) Mean responses of the cpA neuron to different concentrations of thiophene for C. quinquefasciatus and A. aegypti. n = 4.
(C) The temporal response profiles elicited by repeated exposures to cyclopentanone are similar to those elicited by CO2. Individuals were exposed to 6 repeated 1 s pulses of either 0.15% CO2 or cyclopentanone (10−2), spaced 20 s apart. Shown are the mean responses (across 4 animals per treatment) for each sequential pulse. Activity was calculated in 100 ms bins for a total of 6 s following the onset of the stimulus. Values were adjusted by subtracting baseline activity measured between 5 and 6 s after each pulse.
(D) Representative traces and mean responses to 0.5 s pulses of indicated odorants after pre-exposure to butyryl chloride or solvent (sham treatment). n = 16 sensilla from 6 individuals; nested ANOVA. Error bars are SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

15. Figure S6
Effect of Butyryl Chloride Treatment on Drosophila melanogaster, Related to Figure 7
(A and B) Effects of butyryl chloride pre-exposure in Drosophila melanogaster. (A) Odorant-evoked (10−2) responses of ab1C neurons in butyryl-chloride-treated and control flies. n = 25 (treated); n = 6 (control); t test. (B) Mean preference index for 0.5% CO2 in wild-type and orco− (Or83b2) flies. n = 12–14 trials for each condition and genotype (40 flies per trial); t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < Error bars are SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions

16. Figure S7 Odor Delivery Systems, Related to Figures 1, 4, 5, and 6
(A and B) Schematics for (A) human odor delivery system (Figures 1B, 1H–1J and 5B) and (B) cpA activation screens (Figures 4B and 6F). For the activation screens, insertion sites for “blank” and “odor cartridges” were spaced 12 cm apart, and flow rates through the stimulus controller were adjusted for each preparation. Black arrow indicates a switch in airflow that transiently increases the proportion of filtered room air (∼0.04% CO2) during stimulus delivery. This arrangement in (B) increases cpA baseline activity to ∼50 spikes s−1 during odor delivery and allows for identification of inhibitors. Final quantification of odorant response for these panels includes the subtraction of solvent (control) response.
Cell  , DOI: ( /j.cell )
Copyright © 2013 Elsevier Inc. Terms and Conditions