Mosquito biology. Evolution of sexual traits influencing vectorial capacity in anopheline mosquitoes

Abstract

The availability of genome sequences from 16 anopheline species provides unprecedented opportunities to study the evolution of reproductive traits relevant for malaria transmission. In Anopheles gambiae, a likely candidate for sexual selection is male 20-hydroxyecdysone (20E). Sexual transfer of this steroid hormone as part of a mating plug dramatically changes female physiological processes intimately tied to vectorial capacity. By combining phenotypic studies with ancestral state reconstructions and phylogenetic analyses, we show that mating plug transfer and male 20E synthesis are both derived characters that have coevolved in anophelines, driving the adaptation of a female 20E-interacting protein that promotes oogenesis via mechanisms also favoring Plasmodium survival. Our data reveal coevolutionary dynamics of reproductive traits between the sexes likely to have shaped the ability of anophelines to transmit malaria.

Fig. 1. Mating plug phenotypes across anophelines

Considerable divergence in mating plug phenotypes is observed across nine geographically dispersed anopheline species that span three major subgenera (vertical branch labels). The African A. gambiae, A. arabiensis, and A. funestus and the Indian A. stephensi species transfer a structured, fully coagulated plug, whereas the east and southeast Asian species A. farauti, A. dirus, and A. sinensis, along with the European species A. atroparvus, present a less coagulated, amorphous plug phenotype. No mating plug transfer was recorded in the New World species A. albimanus. Species are placed according to their approximate geographical distribution, and the scale bar present in A. farauti applies to all. For accurate branch lengths, see fig. S2.

Fig. 2. Synthesis and transfer of 20-hydroxyecdysone (20E) and its effect on oviposition and refractoriness to mating

(A) 20E levels in male accessory glands (MAGs) differ significantly across the nine anophelines [analysis of variance (ANOVA) F_8,73 = 227.8, P < 0.0001]. Species are color-coded according to 20E levels (high, red; intermediate, blue; negligible, green). (B) 20E levels in the female atrium from five selected species dissected 1 hour postmating. A. gambiae and A. arabiensis receive significantly higher levels of 20E from males compared with the other species analyzed (ANOVA F_4,32 = 80.52, P < 0.0001). In both (A) and (B), results are presented as mean values ± SEM, and letters indicate post hoc significance (Tukey's post hoc test). (C and D) 20E injections in virgin females induce oviposition after blood feeding (C) and refractoriness to mating (D) in a species that sexually transfers 20E (A. arabiensis, P < 0.0001), but not in 20E-less A. albimanus (P = ns) when compared with ethanol-injected controls (Fisher's exact test). A. gambiae results are taken from previously published data (6). Both oviposition and refractoriness data are presented as percentage relative to control females.

Fig. 3. Maximum parsimony multistate ancestral state reconstructions of mating plug and 20-hydroxyecdysone (20E) characters

Mating plug phenotypes (left) and 20E titers (right) from nine anopheline species and two related dipteran species (A. aegypti and D. melanogaster) were each coded as multistate categorical characters. Reconstructions reveal that the most common ancestor to all species (indicated by *) was one that lacked a mating plug and did not produce 20E in their male accessory glands (MAGs). Labeled nodes (A to D) are discussed in the text.

Fig. 4. Phylogenetic analysis of MISO and AGAP002621

A maximum likelihood phylogeny of protein sequences from nine anophelines and two outgroup dipteran species (D. melanogaster and A. aegypti) using randomized axelerated maximum likelihood (RaxML). Across the anophelines, high sequence conservation (shorter branch lengths) is seen in the MISO paralog AGAP002621 (2621), whereas greater divergence, reflected in a higher d_N and ω ratio, is exhibited by MISO. Colors are coded according to 20E levels in Fig. 2A.