Plasmodium-associated changes in human odor attract mosquitoes

Abstract

Malaria parasites (Plasmodium) can change the attractiveness of their vertebrate hosts to Anopheles vectors, leading to a greater number of vector-host contacts and increased transmission. Indeed, naturally Plasmodium-infected children have been shown to attract more mosquitoes than parasite-free children. Here, we demonstrate Plasmodium-induced increases in the attractiveness of skin odor in Kenyan children and reveal quantitative differences in the production of specific odor components in infected vs. parasite-free individuals. We found the aldehydes heptanal, octanal, and nonanal to be produced in greater amounts by infected individuals and detected by mosquito antennae. In behavioral experiments, we demonstrated that these, and other, Plasmodium-induced aldehydes enhanced the attractiveness of a synthetic odor blend mimicking "healthy" human odor. Heptanal alone increased the attractiveness of "parasite-free" natural human odor. Should the increased production of these aldehydes by Plasmodium-infected humans lead to increased mosquito biting in a natural setting, this would likely affect the transmission of malaria.

Keywords: aldehydes; disease biomarkers; host attractiveness; malaria transmission; parasite–vector–host interactions.

Fig. 1.

Effect of parasitological status on An. gambiae s.s. preference for body odor sampled at two time points, one during Plasmodium infection (T1) and the other following parasite clearance (T2). Blue bars represent attraction to odor from parasite-free samples, and orange bars represent attraction to odor samples from individuals with parasites. Groups of 10 mosquitoes were given a choice between socks worn by each participant at both T1 and T2, in a dual-choice cage assay, with the number of mosquitoes that chose the T1 or T2 odor sample being summed over six replicates per participant. Participants were grouped into those with gametocytes by microscopy or QT-NASBA (at >50 gametocytes per microliter) (n = 23), those with asexual stages only by microscopy (n = 10), or parasite-free (n = 12). Of those with asexual parasites, three had submicroscopic gametocytes (1–34.9 gametocytes per microliter of blood), and three were not tested. Predicted mean proportions from the GLM are plotted with 95 CI. *P < 0.05. (GLM included infection status only as predictor of proportion of mosquitoes attracted to T1 odor samples.)

Fig. 2.

Schematic of protocol (Upper) for odor sampling by air entrainment from Plasmodium-infected individuals, for use in GC-EAG analysis (Lower; here with An. coluzzii) and direct GC analysis of entire odor profile. Children were recruited for odor sampling in groups of three to represent parasite-free, asexual parasite carriers, and gametocyte carriers, if parasite prevalence allowed. Following malaria diagnosis by point-of-care methods and odor sampling, malarious individuals were treated, and the same cohort was resampled on days 8 and 22. Whole blood samples were also taken for retrospective molecular analysis. During GC-EAG, odor samples are injected by syringe at the inlet directly into the column (1), where they are vaporized, and carried through the column by the carrier gas (here, hydrogen) (2). During passage through the (50 m) HP1 column, constituents of the sample are separated by GC, and analytes are split as they elute from the column (3). A proportion is directed, via a heated transfer line (4), into a humidified, purified, airflow (5), which is then directed over the insect antennae (6), simultaneously to the proportion that is detected by a flame ionization detector on the GC (7). GC analytes are represented by peaks (GC trace), while antennal response by nerve cell depolarization causes a perturbation in the electroantennographic detection (EAG trace), indicating entomologically significant analytes. Image courtesy of Iain Robinson (Iain-robinson.com).

Fig. 3.

Amount of IACs produced by individuals of differing parasitological status. (A–C) Heptanal, (D–F) octanal, (G–I) nonanal, (J) (E)-2-octenal, (K) (E)-2-decenal, (L) 2-octanone production (relative to all compounds in odor sample) per group (100-min odor profile sampling). Predicted means (+SE) are given by linear mixed modeling (REML). See Table S3 for details of the models and Table S4 for SE of the difference (SED) values for comparison of predicted means. Sample size is in bar ends. *^,†P < 0.05 (significant pairwise difference in mean amount between two groups indicated, tested by LSD). A, D, G, J, K, and L, total density categorization: Gam, microscopic gametocytes; Neg, negative; lower and higher refer to parasite densities of lesser or greater than 50 p/μL. (B, E, and H) Quartile categorization. Neg and Gam are defined as before. L, low, mean/median parasite density 0.38/0.3, n = 21; M-L, medium-low, mean/median parasite density 16.77/8.3, n = 17; M-H, medium-high, mean/median parasite density 296.60/214.18, n = 19; H, high, mean/median parasite density 102,669.46/13,304.54, n = 23. For bar charts CNL(A), solvent control; CNL(B), empty bag control. C–I show raw GC output for heptanal, octanal, and nonanal. Individual traces represent odor samples, colored according to the parasitological status of the individual from whom the odor sample was taken, Higher-density, lower-density, and negative definitions are as described above. Gametocyte carriers are excluded for clarity, as compound production spanned higher and lower parasite density groups.

Fig. 4.

Comparison of odor profiles from parasite-free individuals vs. those harboring bloodstream parasites. (A) Representative GC traces from an individual with a high-density infection (>50 p/μL blood) and low-density infection (<50 p/μL blood). Compounds found to be associated with infection (other than 2-octanone; not visible due to very small amounts) are annotated. (B) The proportion (percent) that IAC contributed toward the entire odor profile, grouped by parasitological category (total density categories). The average number of non-IAC per group (i.e., all other volatile compounds; gray bar) was 171.27 (SE = 5.23) across all groups.

Fig. 5.

An. coluzzii responses in a dual-port olfactometer to heptanal and a blend of five infection-associated aldehydes, Plas 5. Heptanal (10 µL) at two concentrations (g/mL) was presented with (yellow bars) and tested against (blue bars) odor (socks) from parasite-free study participants (5- to 12-y-old Kenyan children) over eight replicates. Plas 5 [heptanal, octanal, nonanal, (E)-2-octenal, and (E)-2-decenal] at four concentrations (10 µL of 100% approximating the amounts found in the foot odor samples) was presented with (orange bars) and tested against (blue bars) the synthetic lure MB5 [ammonia, l-(+)-lactic acid, tetradecanoic acid, 3-methyl-1-butanol, and butan-1-amine] over 10/11 replicates. Each replicate tested 30 mosquitoes. Predicted mean proportions and 95 CI are presented, from two separate GLMs (for heptanal and Plas 5 assays), assuming a binomial distribution and using a logit link function. *P < 0.05. (See Table S6 for details of the GLMs.)