Ultra-prolonged activation of CO2-sensing neurons disorients mosquitoes

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Carbon dioxide (CO2) present in exhaled air is the most important sensory cue for female blood-feeding mosquitoes, causing activation of long-distance host-seeking flight, navigation towards the vertebrate host1 and, in the case of Aedes aegypti, increased sensitivity to skin odours2. The CO2 detection machinery is therefore an ideal target to disrupt host seeking. Here we use electrophysiological assays to identify a volatile odorant that causes an unusual, ultra-prolonged activation of CO2-detecting neurons in three major disease-transmitting mosquitoes: Anopheles gambiae, Culex quinquefasciatus and A. aegypti. Importantly, ultra-prolonged activation of these neurons severely compromises their ability subsequently to detect CO2 for several minutes. We also identify odours that strongly inhibit CO2-sensitive neurons as candidates for use in disruption of host-seeking behaviour, as well as an odour that evokes CO2-like activity and thus has potential use as a lure in trapping devices. Analysis of responses to panels of structurally related odours across the three mosquitoes and Drosophila, which have related CO2-receptor proteins, reveals a pattern of inhibition that is often conserved. We use video tracking in wind-tunnel experiments to demonstrate that the novel ultra-prolonged activators can completely disrupt CO2-mediated activation as well as source-finding behaviour in Aedes mosquitoes, even after the odour is no longer present. Lastly, semi-field studies demonstrate that use of ultra-prolonged activators disrupts CO2-mediated hut entry behaviour of Culex mosquitoes. The three classes of CO2-response-modifying odours offer powerful instruments for developing new generations of insect repellents and lures, which even in small quantities can interfere with the ability of mosquitoes to seek humans.

At a glance


  1. Identification of odorants that affect the CO2-sensitive
neurons in mosquitoes.
    Figure 1: Identification of odorants that affect the CO2-sensitive neurons in mosquitoes.

    a, Comparison of percentages of CO2-response inhibition in cpA neurons of A. gambiae (Ag), A. aegypti (Ae), C. quinquefasciatus (Cx) and D. melanogaster (Dm). Functional group is on the primary carbon atom except for ketones on C2, and the length of the carbon chain (carbon number) is indicated. A 1-s stimulus of vapours from odorant diluted 10−2 applied on cotton wool in cartridge, is overlaid on a 3-s stimulus of 0.15% CO2. n = 3. Data for Dm responses, using 0.33% CO2, is taken from ref. 19. b, c, Dose–response of indicated inhibitors in the three species, in a similar overlay assay. n = 5; error barsares.e.m. d, Representative long-term recordings from the cp sensillum in response to repeated 1-s pulses of CO2 (0.15%; black squares) or 2-butanone (4on; 10−1; grey squares). e, Expanded view of the traces for each during the stimulus period in the centre in d. f, Representative trace of A. aegypti peg sensillum to a 1-s stimulus of 2,3-butanedione (d4on; 10−1). g, Mean responses of the cpA neuron to 1-s 2,3-butanedione (black line) or 0.15% CO2 (grey line) on Anopheles, Aedes and Culex over 7s. n = 4; error barsares.e.m. h, Mean baseline activity of the cpA neuron counted every 30-s interval after pre-exposure to a 3-s stimulus of 2,3-butanedione (10−1; black line) or paraffin oil (grey line). n = 4, error barsares.e.m. Baseline activity before initial stimulus subtracted in g and h.

  2. Ultra-prolonged activation disrupts ability to respond to CO2.
    Figure 2: Ultra-prolonged activation disrupts ability to respond to CO2.

    a, Representative action potential traces from cp sensillum in A. gambiae and A. aegypti exposed to a 3-s stimulus of 2,3-butandione (10−1) followed by two 1-s stimuli of CO2 (0.15%). Expanded views of the indicated regions of the traces are shown. b, Mean increase in frequency of response of cpA neurons to repeated stimuli of 1-s CO2 (0.15%) applied approximately every 30s, following a 3-s pre-exposure to 2,3-butanedione (10−1; black bars) or paraffin oil (grey bars) in A. gambiae, A. aegypti and C. quinquefasciatus. c, Mean baseline activity (squares) and increase in activity (triangles) of an A. aegypti cpA neuron responding to 1-s CO2 (0.15%) pulses applied approximately every 30s after a 3-s pre-exposure to a four-odour blend of 2,3-butanedione, 1-butanal, 1-pentanal and 1-hexanol (10−2) (black) or paraffin oil (grey). d, e, Representative traces from cp sensillum (d) and mean responses from cpA neurons (large spike amplitude) to CO2 (0.15%) and a neighbouring neuron (small spike amplitude) to 1-octen-3-ol (10−3) after a pre-treatment of 3min of paraffin oil or the four-odour blend (e). f, Responses of a cpA neuron in C. quinquefasciatus treated identically as in c. For b, c, e and f, n = 5; error barsares.e.m.

  3. Exposure to ultra-prolonged activator causes long-term disruption
of CO2-mediated attraction behaviour of female Aedes mosquitoes.
    Figure 3: Exposure to ultra-prolonged activator causes long-term disruption of CO2-mediated attraction behaviour of female Aedes mosquitoes.

    a, b, Schematics of the experimental strategy (a) and the wind-tunnel apparatus (b). c, d, Percentage of female mosquitoes flying upwind to half-way point (c) and flying through the CO2-emitting glass ring (d) after pre-exposure for 3min to paraffin oil (control) or ultra-prolonged activating blend at indicated concentrations. e, f, Similar experiment as above except three pre-exposure times were tested to the four-odour ultra-prolonged blend (10−1). g, h, Time-course of percentages of female mosquitoes getting to half-way point and reaching the CO2 source after a 1min pre-exposure to paraffin oil or ultra-prolonged blend at the indicated concentrations. i, Percentage of female mosquitoes flying through the CO2-emitting glass ring after pre-exposure to 3min of paraffin oil (control), indicated odorants (10−2) or ultra-prolonged activating blend (10−2). N = 26 individuals for each condition. Pearsons χ2 test, compared to controls, *P<0.05, **P<0.01, ***P<0.001. j, Schematic of experimental strategy, and percentage of A. aegypti mosquitoes escaping repellency chamber within 5min in response to DEET (9.8%) after pre-exposure to paraffin oil (control) or ultra-prolonged activating blend (10−2) as above. N = 7 trials (20 mosquitoes per trial).

  4. Ultra-prolonged activators disrupt attraction behaviour of female Culex
mosquitoes in semi-field conditions
    Figure 4: Ultra-prolonged activators disrupt attraction behaviour of female Culex mosquitoes in semi-field conditions

    a, Schematic of two-choice MalariaSphere experiment with counter-flow CO2 traps placed inside each experimental hut to attract female Culex mosquitoes released from the centre of enclosure. One hut contains ultra-prolonged blend dispensers (1%, treated) while the other dispenses paraffin oil (control). b, c, Mean percentage of released mosquitoes (b) and mean preference index of female mosquitoes captured in trap and captured in trap plus hut in treated versus control huts (c). N = 4 trials, 100 females per trial; error barsares.e.m.; Student’s t-test, *P<0.05. d, Schematic of one-choice MalariaSphere experiment. Trials were conducted with (treated) or without (control) ultra-prolonged blend (3%) dispensers. e, Mean percentage of mosquitoes in MalariaSphere that are trapped in control and treated huts. N = 4 trial nights each for control and treatment, 100 females added every evening; error barsares.e.m.; Student’s t-test, **P<0.005. f, Mean reduction in percentage of Culex females captured in traps from ultra-prolonged blend treated huts in b and d. g, Proposed model of odorant application for mosquito control. Odorants that are antagonists of the CO2 receptor can be used to mask mosquito attraction, agonists can be used as a lure or attractant in a trap application, and ultra-prolonged activators can be used to block CO2 detection through persistent activation of the neuron. Small grey dots represent other human odours, and large black dots represent odours that can be applied to disrupt host seeking.


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Author information

  1. These authors contributed equally to this work.

    • Stephanie Lynn Turner &
    • Nan Li


  1. Cellular, Molecular, and Developmental Biology Program, University of California, Riverside, California 92521, USA

    • Stephanie Lynn Turner &
    • Anandasankar Ray
  2. Department of Entomology, University of California, Riverside, California 92521, USA

    • Nan Li,
    • Ring T. Cardé &
    • Anandasankar Ray
  3. Human Health Division, International Centre of Insect Physiology and Ecology (ICIPE), P.O. Box 30772-00100, Nairobi, Kenya

    • Tom Guda &
    • John Githure


S.L.T. planned the electrophysiology experiments, performed the experiments, collected and analysed the data and helped write the paper. N.L. performed the wind tunnel, repellency, MalariaSphere and greenhouse behaviour experiments and analysed the data. T.G. performed one-choice behaviour experiments. J.G. helped plan MalariaSphere experiments. R.T.C. helped plan the behaviour experiments, and helped write the paper. A.R. helped plan the experiments, analysed the data, managed the project and wrote the paper.

Competing financial interests

A.R and S.L.T. are listed as inventors in a pending patent application, which is filed by the University of California, Riverside. A.R serves as consultant for a corporation that has obtained the license for this pending patent application from University of California, Riverside.

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Supplementary information

PDF files

  1. Supplementary Information (9.1M)

    This file contains Supplementary Figures 1-9 with legends and Supplementary Table 1.


  1. Supplementary Movie 1 (847K)

    The file movie contains a representative video of a mock-treated female A. aegypti mosquito navigating upwind and through a CO2 turbulent-plume releasing ring in real time.

  2. Supplementary Movie 2 (3.2M)

    This movie file contains a representative video of a pretreated (ultra-prolonged odour blend, 10-2) female A. aegypti mosquito, unable to find upwind CO2 source in real time. For the remaining duration of the 300 sec assay the mosquito does not move after coming to rest on the glass wall.

  3. Supplementary Movie 3 (2.5M)

    This file movie contains a representative video of a pretreated (ultra-prolonged odour blend, 10-1) female A. aegypti mosquito, unable to activate upwind flight from the cage and find upwind CO2 source during the entire duration of the 300 sec assay (video speeded up).

Additional data