Rearing of Experimental Mosquitoes

For detailed information see Supplementary Information.

Behavioural Bioassays

We ran all behavioural bioassays in mesh cages (77 × 78 × 104 cm) wrapped with black cloth except for the top to allow illumination from ambient fluorescent light. We kept cages at 23–26 °C, 40–60% RH, and a photoperiod of 14 L:10D. For each 24-h bioassay, we released 50 virgin, 1- to 3-day-old (unless otherwise stated), 24-h sugar-deprived females of C. pipiens or A. aegypti from a Solo cup (see Supplementary Information: Mosquito Rearing) into a cage. We randomly assigned the treatment and the control stimulus to adhesive-coated (The Tanglefoot Comp., Grand Rapids, MI 49504, USA), custom-made delta traps (9 cm × 15 cm) placed on each of two stands spaced 30 cm apart inside the cage. We wore latex gloves (Microflex Corporation, Reno, NV 89523, USA) during preparation of test stimuli.

Effect of Olfactory and Visual Inflorescence Cues on Mosquito Attraction

We collected blooming inflorescences from in-situ tansies on the Burnaby campus of Simon Fraser University (SFU) and from potted, greenhouse-grown plants. The treatment stimulus consisted of one tansy inflorescence with 10–15 composite flowers cut from the plant bearing it. The control stimulus consisted of the stem of an inflorescence (with composite flowers excised and removed) cut from another plant. Because cut surfaces emanate “green leaf volatiles” and control plants had additional cuts due to the excision and removal of composite flowers, we inflicted cuts also on the stem of treatment plants. We covered all cut surfaces with petroleum jelly to minimize the release of green-leaf volatiles, inserted the treatment and the control plant into separate water-filled, parafilm-covered 4-mL vials, and placed each vial horizontally into a trap. We ran two experiments in parallel to rigorously study the effects of olfactory and visual inflorescence cues on mosquito attraction. To test the effect of olfactory cues, we occluded both the treatment and the control inflorescence by three layers of cheesecloth (VWR International, Radnor, PA 19087, USA) with a mesh size sufficiently wide to permit odorant dissemination. To test for an interactive effect between olfactory and visual cues, we occluded one inflorescence with three layers of cheesecloth and placed the other on top of the cheesecloth layers. To compare head-to-head the relative attractiveness of inflorescences presenting both visual and olfactory cues, or just olfactory cues, we occluded one of the two inflorescences with cheese cheesecloth.

Capture and Attractiveness of Headspace Floral Odorants

We inserted 5–10 inflorescences into a 250-mL water-filled beaker which we then placed into a Pyrex® glass chamber (34 cm high × 12.5 cm wide). A mechanical pump drew charcoal-filtered air at a flow of 1 L min−1 for 24–72 h through the chamber and through a glass column (6 mm outer diameter × 150 mm) containing 200 mg of Porapak-Q™ adsorbent64. We desorbed floral odorants captured on Porapak-Q with 2 mL each of pentane and ether and bioassayed aliquots of Porapak-Q headspace volatile (HSV) extract for mosquito attraction. The treatment stimulus consisted of a 1-mL HSV extract aliquot [equivalent to the amount of odorants emanating from one or two blooming tansy plants per hour for 24 h, or approximately 240 inflorescence-hour-equivalents (IHE); 1 IHE = the amount of odorants released from one inflorescence during 1 h of odorant capture], emanating from a horizontally-placed 4-mL glass vial with a 2-mm hole in its lid. In the control stimulus, the HSV extract aliquot was replaced with the corresponding amount of pentane and ether (1:1 mix).

Identification of Floral Odorants in HSV Extracts

After adding octyl acetate as an internal standard to HSV extract, we analyzed 2-µl aliquots by gas chromatography-mass spectrometry (GC-MS), operating a Saturn 2000 Ion Trap GC-MS fitted with a DB-5 GC-MS column (30 m × 0.25 mm i.d.; Agilent Technologies Inc., Santa Clara, CA 95051, USA) in full-scan electron impact mode. We used a flow of helium (35 cm s−1) as the carrier gas with the following temperature program: 50 °C (5 min), 10 °C min−1 to 280 °C (held for 10 min). The temperature of both the injector port and ion trap was 250 °C. To reveal the presence of low-molecular-weight carboxylic acids (which chromatograph poorly), we converted carboxylic acids to the corresponding silylated derivatives (which chromatograph well). To this end, we treated a 100-µL aliquot of HSV extract with BSTFA (10 µl; N,O-bis(trimethylsilyl)trifluoroacetamide) and TMCS (10%; trimethylchlorosilane; both Pierce Chemical Co., Rockford, IL 61101, USA) and after 5 min without any work-up analyzed 2-µl aliquots by GC-MS. We identified odorants in HSV extract by comparing their retention indices (RI; relative to n-alkane standards65) and their mass spectra with those reported in the literature66 and with those of authentic standards (Table 1).

Preparation of a Synthetic Floral Odorant Blend

We prepared a synthetic blend of floral odorants (Table 1) including all those odorants present at >1.25% in floral HSV extract. The quantity and ratio of odorants in this synthetic blend matched those found in HSV extract. Moreover, we prepared a second synthetic blend (Table 1) consisting of only those floral odorants that are also found in headspace volatiles of human skin, breath, or skin microbiota.

Attractiveness of Synthetic Floral Blends to Mosquitoes (1- to 3- or 5- to 6-day-old)

We tested the attractiveness of synthetic floral blends using the two-choice general bioassay design described above. In three sets of two parallel experiments, we tested a complete synthetic blend (CSB) of all floral odorants (Table 1) or a partial synthetic blend (PSB) comprising only those floral components also found in headspace volatiles of human skin, breath, or skin microbiota (Table 1) each versus a solvent control. We prepared the complete blend at approximately 240 IHEs dissolved in pentane/ether (1 mL; 1:1), and disseminated it from a horizontally-placed, 4-mL glass vial with a 2-mm hole in the lid. The control stimulus consisted of the equivalent solvent mixture (1 mL) disseminated from the same type of dispenser.

Measurements of Tansy CO 2 Emissions in the Field and Laboratory

We measured CO 2 concentrations from a single cut tansy inflorescence weighing 3.6 g with a Q-Trak 7575-X air quality monitor (TSI Inc., Shoreview, MI 55126, USA) set to take readings every second and to average them in 1-min intervals. To track changes in ambient CO 2 around in-situ tansies, we placed the monitor circa 5 cm above ground in a patch of tansies on the Burnaby campus of SFU, taking measurements from 20:30 to 22:30 h on 18 August 2015, with civil dusk occurring at circa 21:00 h.

Effect of Trace CO 2 on Mosquito Attraction

Using the two-choice general bioassay design described above, and running two experiments in parallel with both C. pipiens and A. aegypti, we tested the effect of CO 2 on mosquito attraction. To provide a neutral stimulus, both traps in each experiment were fitted with a horizontally-placed, 4-mL glass vial containing pentane and ether (1 mL; 1:1) which were dispensed through a 2-mm hole in the lid. The test variable in one experiment consisted of a mixture of medical-grade air containing 1% CO 2 (Praxair Inc., Mississauga, ON L5B 1M2, Canada) which amounts to a CO 2 concentration about 10 × that near a single cut tansy inflorescence (see above), or comparable to that near a single intact tansy plant at the time when it is a net CO 2 producer. To make sure that mosquitoes were not just responding to the flow of a gas mixture, the test variable in the parallel experiment consisted of medical grade air (Praxair Inc.). We delivered each test variable at the same flow rate [5000 μL min−1] through copper tubing (1.5 m × 2 mm i.d.) and aluminum tubing (0.5 m × 0.5 mm i.d.) to the respective delta trap and recorded the number of mosquitoes captured in each trap after 2 h.

Effect of Tansy Floral Odorant Blend on Attraction of Mosquitoes to CO 2

Using the two-choice general bioassay design described above, we tested whether floral odorants enhance attraction of C. pipiens and A. aegypti to CO 2. In each experiment, we delivered a mixture of medical-grade air containing 1% CO 2 to both the treatment and the control trap (as described above), baited the treatment trap with the complete blend of floral odorants (CSB; as described above), and fitted the control trap with a solvent control (as described above).

Statistical Analyses of Data

We used SAS statistical software version 9.4 (SAS Institute Inc., Cary, NC 27513, USA) for data analyses, excluding from analyses experimental replicates with no mosquitoes responding. We used a binary logistic regression model with a logit link function and a Firth bias correction factor to compare mean proportions of responders between test stimuli, with overdispersion corrected for using the Williams method where appropriate (Exp. 9). We analyzed differences between experiments using non-adjusted least squares means. We worked with back-transformed data to obtain means and confidence intervals. We analyzed vegetative CO 2 emission with autocorrelated linear regression to obtain concentration changes over time.

Ethics approval and consent to participate

The research on plants performed in this study conforms with institutional, national, and international guidelines.