Male swarming aggregation pheromones increase female attraction and mating success among multiple African malaria vector mosquito species

Abstract

Accumulating behavioural data indicate that aggregation pheromones may mediate the formation and maintenance of mosquito swarms. However, chemical cues possibly luring mosquitoes to swarms have not been adequately investigated, and the likely molecular incitants of these complex reproductive behaviours remain unknown. Here we show that males of the important malaria vector species Anopheles arabiensis and An. gambiae produce and release aggregation pheromones that attract individuals to the swarm and enhance mating success. We found that males of both species released significantly higher amounts of 3-hydroxy-2-butanone (acetoin), 6-methyl-5-hepten-2-one (sulcatone), octanal, nonanal and decanal during swarming in the laboratory. Feeding males with stable-isotope-labelled glucose revealed that the males produced these five compounds. A blend composed of synthetic analogues to these swarming odours proved highly attractive to virgin males and females of both species under laboratory conditions and substantially increased mating in five African malaria vectors (An. gambiae, An. coluzzii, An. arabiensis, An. merus and An. funestus) in semi-field experiments. Our results not only narrow a conspicuous gap in understanding a vital aspect of the chemical ecology of male mosquitoes but also demonstrate fundamental roles of rhythmic and metabolic genes in the physiology and behavioural regulation of these vectors. These identified aggregation pheromones have great potential for exploitation against these highly dangerous insects. Manipulating such pheromones could increase the efficacy of malaria-vector control programmes.

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Fig. 1: Swarming odours of An. arabiensis (Dongola and KGB) and An. gambiae (Keele).
Fig. 2: Behavioural responses of Anopheles mosquitoes to swarming odours.

Data availability

The sequencing data are available at the GEO database (SRA accession no. PRJNA590389). The data presented in this publication have been deposited in the National Centre for Biotechnology’s Gene Expression Omnibus and are accessible through GEO series accession no. PRJNA590389. The raw data, including the amounts of swarming odours, are presented in Supplementary Table 3a–e.

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Acknowledgements

We thank the Swedish Research Council (Vetenskapsrådet) for funding to S.N.E. (grant no. VR/2017-01229) and (grant no. VR/2017-05543 UFNW) network. We thank Jeanssons Stiftelser (SJ-2018) for the valuable support for setting up the 3D wind-tunnel facility. This project was initially supported by funding awarded to A.-K.B.-K. from the International Atomic Energy Association, grant nos 13733/R1, 13733/R2 and 13733/R3; by Lithuanian state grant “Research into Functions, Responses and Adaptations of Biological Systems and Application Prospects”, Research Program III to R.M.; by L.L.K. and J.W.Z. through the Department of Science and Innovation (DSI)/National Research Foundation (NRF) competitive programme for rated researchers and DSI/NRF South African Research Chairs Initiative Grant (grant no. 171215294399); and Illumina sequencing and Bioinformatics Access Facility to M.H. supported by M.R.F. We thank B. Knols for valuable discussions in the initial stage of the project. We thank L. Ignatowicz and J. Paleovrachas for their support and M. Coetzee for language review and comments.

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Contributions

A.-K.B.-K., R.M., L.L.K., K.P., M.R.F. and S.N.E. conceived the study. K.P., R.M., M.H., V.S., I.B. and J.S. carried out the laboratory experiments. R.M., L.L.K., J.W.Z. and S.N.E. designed the field experiments. J.W.Z. and S.N.E. collected the field data. R.M. and S.N.E. analysed the data with help from M.H., K.P. and J.S. R.M. and S.N.E. wrote the manuscript. J.S. arranged the figures. J.K.B. and all coauthors edited the manuscript.

Corresponding author

Correspondence to S. Noushin Emami.

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Competing interests

S.N.E., R.M. and A.-K.B.-K. are inventors on a patent application (Sw patent application no. ZSE1077999) submitted by the main applicant, S.N.E. (Zacco, Stockholm University), that covers the attraction effects of the aggregation pheromone and the synthetic attractant odour blend. S.N.E. is a cofounder at Molecular Attraction AB. The authors declare no other competing interests.

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Extended data

Extended Data Fig. 1 Effect of swarming odours of Anopheles arabiensis (Dongola) and (KGB) as well as Anopheles gambiae (Keele).

a, The correlation between the number of swarming mosquitoes and amount of odours trapped, males of An. arabiensis (Dongola). b, Amounts of five swarming odours (VOCs) collected during the control, i.e. photophase (C) and transition periods from scotophase to photophase (TSP) and from photophase to scotophase (TPS). The values are taken from the General Linear Mixed Model estimations (GLMM), including the random effect of experimental replication. Significantly different comparisons are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). In panel b, top of the columns are medians and vertical bars represent standard errors.

Extended Data Fig. 2 Behavioural responses of mosquitoes to control vs control.

Responses of male and female mosquitoes [An. arabiensis (Dongola) and An. gambiae (Keele)] in two-choice olfactometer bioassay were evaluated. The yellow bars show An. arabiensis, and green bars represent An. gambiae panel. It is shown the comparison of Control vs Control: [An. arabiensisi (Dongola) male: χ 21= 0.26, P= 0.601; An. arabiensis (Dongola) female: χ 21= 0.34, P= 0.566; An. gambiae (Keele) male: χ 21= 0.33, P= 0.453; An. gambiae (Keele) female: χ 21= 0.40, P= 0.330].

Extended Data Fig. 3 Behavioural response of Anopheles mosquitoes upon reception of the pheromones.

(1) Stimulated Anopheles males secrete an aggregation pheromone (Chemoemiter) which is a mixture of five volatile compounds including acetoin, sulcatone, octanal, nonanal and decanal. (2) The pheromone mediates formation and sustenance of swarm comprised of flying males. (3) Males respond to the pheromone through antennal sensory organs (Chemoreceiver) with a peak of swarming activity during the photoperiod transition (through the diel and circadian gene regulation). (4) After the male swarm a critical swarm size is initiated achieved, the pheromone enhances female attraction to the swarm and increases mating activity (the section between two arrows). Females respond to the male pheromone (our data), and acoustic signal as an essential signal for coupling (previous literature) with characteristic agitated flight, which serves as attraction stimulus to males in the swarm, inducing males to copulate with females while flying. The graphical illustration is made by Emami group.

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Supplementary Information

Supplementary Figs. 1 and 2, Tables 1–3, gene expression profiling of An. gambiae males, results, discussion, methods and references.

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Mozūraitis, R., Hajkazemian, M., Zawada, J.W. et al. Male swarming aggregation pheromones increase female attraction and mating success among multiple African malaria vector mosquito species. Nat Ecol Evol 4, 1395–1401 (2020). https://doi.org/10.1038/s41559-020-1264-9

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