Odours of Plasmodium falciparum-infected participants influence mosquito-host interactions

Malaria parasites are thought to influence mosquito attraction to human hosts, a phenomenon that may enhance parasite transmission. This is likely mediated by alterations in host odour because of its importance in mosquito host-searching behaviour. Here, we report that the human skin odour profile is affected by malaria infection. We compared the chemical composition and attractiveness to Anopheles coluzzii mosquitoes of skin odours from participants that were infected by Controlled Human Malaria Infection with Plasmodium falciparum. Skin odour composition differed between parasitologically negative and positive samples, with positive samples collected on average two days after parasites emerged from the liver into the blood, being associated with low densities of asexual parasites and the absence of gametocytes. We found a significant reduction in mosquito attraction to skin odour during infection for one experiment, but not in a second experiment, possibly due to differences in parasite strain. However, it does raise the possibility that infection can affect mosquito behaviour. Indeed, several volatile compounds were identified that can influence mosquito behaviour, including 2- and 3-methylbutanal, 3-hydroxy-2-butanone, and 6-methyl-5-hepten-2-one. To better understand the impact of our findings on Plasmodium transmission, controlled studies are needed in participants with gametocytes and higher parasite densities.


Mosquito colony maintenance
The colony of Anopheles coluzzii Coetzee & Wilkerson sp. n. (formerly referred to as the M-form of An. gambiae s.s. Giles, 1 ) used originated from Suakoko, Liberia, and has been kept at the laboratory of Entomology in Wageningen since 1987. Mosquitoes were fed on human blood (Sanquin Blood Supply Foundation, Nijmegen, The Netherlands) daily using a membrane feeding system (Hemotec® PS5, Discovery Workshops, UK) covered with Parafilm® and set at 38°C. A sock releasing human odour and 5% CO2 were offered during blood-feeding to expose mosquitoes to natural host-derived cues. Adult mosquitoes were kept in 30-cm cubic gauze-covered cages (Bugdorm ® , Megaview, Taiwan) in a climatecontrolled room (27±1°C, 80±5% RH, LD 12:12) and had access to 6% (w/v) glucose solution offered on filter paper. Larvae were reared on Liquifry No. 1 (Interpet, Dorking, U.K.) and Tetramin® baby fish food (Tetra GmbH, Melle, Germany) and pupae were collected daily. Groups of 30 females (5-8 days old and presumably mated but not blood-fed) were collected approximately 16-22 h. before experiments and provided with tap water on damp cotton wool.

Dual-port olfactometer
In the dual-port olfactometer, charcoal-filtered, moisturized air was blown into poly-methyl-methylacrylate traps, which held the odour samples (cotton pads, see below). Data loggers (MSR145S, MSR Electronics, GmBhm, Switzerland) were used to monitor temperature, humidity and air pressure in the room, and in each of the three flight chambers. Air entered the traps at a temperature of 29±0.5°C, and humidity of more than 80%, and was released into the flight chambers at a speed of approximately 0.15-0.25 m/s. CO2 (5%) was released below the entrance to each trap at a rate of 175 ml/min in all trials.
Temperature inside the flight chambers was 26.5±0.5°C, and humidity inside the flight chambers was 73±5°C in series 1, and 66±5°C in series 2. A dim light (<1 lux) was on during trials but the room was otherwise kept dark. Experiments were performed during the last four h. of the scotophase, when An. gambiae s.l. is known to be highly responsive to host odour (e.g. 2 ).
Mosquitoes that remained in the flight chamber after 15 min were removed with a vacuum cleaner before starting the next trial. Mosquitoes that remained in the release cages were counted and removed. Each trial started with new groups of mosquitoes, clean traps and odour samples with the exception of worn cotton pads that were re-used a maximum of three times on different days and frozen at -20°C between experiments. This level of replication does not lead to depletion of skin odour present on the cotton pads 3 . Cotton pads with ammonia were only used once.
Over the six replicates, the sequence of worn cotton pads was randomized in such a way that each sample was tested once in each position of the olfactometer, i.e. on the left and right side of each of the three flight chambers. The total number of samples in CHMI2 was too high to test them all on the same day, so we divided participants into two groups. Groups of participants were randomized between experimental replicates so that a particular participant was tested on the same day as each of the other participants at least once. Cotton pads worn by the same participant at different time points (i.e. Before, During and After) were always tested on the same experimental day.

Settings for GC and GC-MS
High resolution GC analysis was carried out using Agilent GC instruments that were fitted with a cool-oncolumn injector, flame ionization detector, non-polar HP1 column (50 m x 0.32 mm with film thickness of 0.52 μm) and used hydrogen as carrier gas. The oven temperature was maintained at 40 °C for 0.5 min and then programmed to increase by 5 °C per min to 150 °C, held for 0.1 minute and then raised by 10 °C per minute to 230 °C, where it was held for 40 min.
For coupled GC-MS analysis, a Waters Autospec Ultima instrument (Manchester, UK) was fitted with a non-polar HP1 column (50m length x 0.32mm and 0.52µm film thickness, J & W Scientific). Ionization was by electron impact (70 eV, 220°C). The GC oven temperature was maintained at 30°C for 5 min and then programmed to rise at 5°C/min to 250°C. Samples were analysed by thermal desorption (PTV unit programmed to start at 30°C then to rise to 250°C at 16°C/sec). The carrier gas was helium. Figure S1. Overview of participants in the clinical malaria studies. CHMI1 had six participants (1A-1F), and CHMI2 had 11 participants (2A-2K). On day 0, participants were challenged with P. falciparum strain NF54 (CHMI1), NF135.C8 or NF166.C10 (CHMI2) by bites of infectious An. stephensi mosquitoes (5 mosquitoes per participant in CHMI1, and 1, 2 or 5 mosquitoes in CHMI2). For each participant, sex and immunization status are indicated. Parasitological status was monitored twice per day by qPCR between days 7 to 13 post challenge for CHMI1, and between days 6 and 13 for CHMI2; red squares refer to qPCR-positives and green squares to qPCR-negatives. Antimalarial treatment was started following two subsequent positive qPCRs, and is indicated with orange triangles. Sampling time points for air entrainment of foot headspace are indicated by circles for each participant; cotton pads were collected at the same time points overnight. In CHMI1, two participants received treatment on the afternoon just prior to headspace entrainment (1A and 1C). They were considered positive in all analyses because they had two consecutive positive qPCR-results prior to headspace entrainment and cotton pad collection. The third participant in this group that was considered positive (1D) had a positive qPCR in the mornings of the days before and after headspace entrainment. It should be noted that these three participants were qPCR-negative on the evening of headspace entrainment. In CHMI2, two participants received treatment during headspace entrainment and one just after headspace entrainment (while wearing the cotton pad) (2C, 2H and 2I). Of these, participant 2I had a fever at this time point. All three had positive qPCR-results on the evening of headspace entrainment and on the following morning and were thus considered positive in all analyses. Five participants became positive for the first time in the morning of day 7 post challenge, and were thus considered positive for behavioural analyses but negative for analyses of the odour profile.