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. 2020 Dec 7:9:e63982.
doi: 10.7554/eLife.63982.

Fruitless mutant male mosquitoes gain attraction to human odor

Nipun S Basrur  1 , Maria Elena De Obaldia  1 , Takeshi Morita  1 , Margaret Herre  1   2 , Ricarda K von Heynitz  1 , Yael N Tsitohay  1 , Leslie B Vosshall  1   2   3
Affiliations

Fruitless mutant male mosquitoes gain attraction to human odor

Nipun S Basrur et al. Elife. .

Abstract

The Aedesaegypti mosquito shows extreme sexual dimorphism in feeding. Only females are attracted to and obtain a blood-meal from humans, which they use to stimulate egg production. The fruitless gene is sex-specifically spliced and encodes a BTB zinc-finger transcription factor proposed to be a master regulator of male courtship and mating behavior across insects. We generated fruitless mutant mosquitoes and showed that males failed to mate, confirming the ancestral function of this gene in male sexual behavior. Remarkably, fruitless males also gain strong attraction to a live human host, a behavior that wild-type males never display, suggesting that male mosquitoes possess the central or peripheral neural circuits required to host-seek and that removing fruitless reveals this latent behavior in males. Our results highlight an unexpected repurposing of a master regulator of male-specific sexual behavior to control one module of female-specific blood-feeding behavior in a deadly vector of infectious diseases.

Keywords: Aedes aegypti; CRISPR-Cas9; behavior; genetics; genomics; mosquito; neuroscience; sexual dimorphism.

Plain language summary

Sexual dimorphism is a phenomenon among animals, insects and plants where the two sexes of a species show differences in body size, physical features or colors. The bushy mane of a male lion, for example, is nowhere to be seen on a female lioness, and only male peacocks have extravagant tails. Most examples of sexual dimorphism, such as elaborate visual displays or courtship behaviors, are linked to mating. However, there are a few species where behavioral differences between the sexes are not connected to mating. Mosquitoes are an example: while female mosquitoes feed on humans, and are attracted to a person’s body heat and odor, male mosquitoes have little interest in biting humans for their blood. Therefore, female mosquitoes are the ones responsible for transmitting the viruses that cause certain blood-borne diseases such as dengue fever or Zika. Determining which genes are linked to feeding behaviors in mosquitoes could allow researchers to genetically engineer females so they no longer bite people, thus stopping the spread of these diseases. Unfortunately, the genes that control mosquito feeding behaviors have not been well studied. In other insects, some of the genes that control mating behaviors that depend on sex have been identified. For example, a gene called fruitless controls courtship behaviors in male flies and silkworms, and is thought to be the ‘master regulator’ of male sexual behavior across insects. Yet it remains to be seen whether the fruitless gene has any effect in mosquitoes, where sex differences relate to feeding habits. To investigate this, Basrur et al. removed the fruitless gene from Aedes aegypti mosquitoes. The genetically altered male mosquitoes became unable to mate successfully, but – similar to unmodified males – still preferred sugar water over blood when feeding. Unlike unmodified males, however, the male mosquitoes lacking fruitless were attracted to the body odor of a person’s arm (like females). These results reveal that fruitless, a gene that controls sex-specific mating behaviors in other insects, controls a sex-specific feeding behavior in mosquitoes. The fruitless gene, Basrur et al. speculate, likely gained this role controlling mosquito feeding behavior in the course of evolution. More research is required to fully understand the effects of the fruitless gene in male and female mosquitoes.

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Conflict of interest statement

NB, MD, TM, MH, Rv, YT, LV No competing interests declared

Figures

Figure 1.
Figure 1.. Sex-specific mosquito attraction to humans and fruitless splicing.
(A, B) Arm-next-to-cage assay schematic A and image B with female (top) and male (bottom) Aedes aegypti mosquitoes. (C) Percent mosquitoes next to arm measured every 30 s. Data are mean ± s.e.m., n = 6 trials, n = 20 mosquitoes/trial; *p<0.05, Mann-Whitney test for each time point. (D, E) Schematic of Aedes aegypti fruitless genomic locus D and sex-specific splicing region with RNA-seq read evidence E. (F) Phylogeny of mosquito species with outgroup Drosophila melanogaster, with conserved fruitless exon structure inferred from de novo transcriptome assembly. In E, F, coding and non-coding exons are represented by filled and open dashed bars, respectively. Toxorhynchites rutilus and Culex salinarius images were used to represent Toxorhynchites amboinensis or Culex quinquefasciatus, respectively. See acknowledgments for photo credits. Cartoons indicate blood-feeding (blood drop) and non-blood-feeding (flower) species. (G, H) Aedes aegypti fruitless exon usage based on male and female RNA-seq data (normalized counts) from the indicated tissue plotted for each exon G and m, f, and c1 exons H n = 3–4 independent RNA-seq replicates (Matthews et al., 2016). (I) Schematic of generation of fruitless∆M and fruitless∆M-tdTomato mutants.
Figure 2.
Figure 2.. Expression of fruitless in the mosquito brain.
(A–F) Confocal images of brains of the indicated genotypes showing fruitless >tdTomato (green) and Brp (magenta) expression. Scale bars, 20 µm. (D–F) Top-to bottom images are optical sections of the same brain, arranged from anterior to posterior.
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. Expression of fruitless in the female mosquito ventral nerve cord.
(A–B) Confocal images of ventral nerve cords of the indicated genotypes showing fruitless >tdTomato (green) and Brp (magenta) expression. All scale bars, 20 µm.
Figure 3.
Figure 3.. Expression of fruitless in the mosquito olfactory system.
(A) Schematic of antennal olfactory sensory neurons and their projections to the antennal lobe of the mosquito brain. (B–D) Confocal images of antennae of the indicated genotypes with fruitless >tdTomato (green) and Orco (magenta) expression. (E) Confocal image of orco mutant antenna, as negative control for Orco and tdTomato antibodies, with DAPI (blue). (F) Number of antennal lobe glomeruli labeled by fruitless >tdTomato in the indicated genotypes. (G–I) Confocal images of antennal lobes of the indicated genotypes with fruitless >tdTomato (green) and Brp (magenta) expression. (J–L) Confocal images of antennal lobes of the indicated genotypes showing fruitless >tdTomato (green) and Brp (magenta) expression. Top-to bottom images are optical sections of the same lobes, arranged from anterior to posterior. All scale bars, 20 µm.
Figure 4.
Figure 4.. Male fruitless mutant mosquitoes gain attraction to a live human host.
(A) Feeding assay schematic. (B) Feeding on indicated meal (n = 8 trials/meal; n = 20 mosquitoes/trial). (C) Insemination assay schematic. (D) Insemination of wild-type females by males of the indicated genotype (n = 6 trials/male genotype, n = 20 females/trial; *p=0.0022, Mann-Whitney test). (E) Schematic of Quattroport assay, highlighting ability to run multiple stimuli and genotypes simultaneously. (F) Side-view schematic of Quattroport, highlighting close-range (attraction) and long-range (activation) metrics (G) Percent activated animals, n = 8–14 trials/group, n = 17–28 mosquitoes/trial. (H, J) Quattroport assay schematic for nectar-seeking H and live human host seeking J. 1% CO2 is added to the airstream in the live human host seeking assay J. (I, K) Percent of attracted animals (n = 8–14 trials per group, n = 17–28 mosquitoes/trial). Data in B, D, G, I, K are mean ± s.e.m. In B, I, G, K, data labeled with different letters are significantly different from each other (Kruskal-Wallis test with Dunn’s multiple comparisons, p<0.05). In B, comparisons are made between genotypes for each meal. In I, K, comparisons are made between all genotypes and stimuli.
Figure 5.
Figure 5.. Olfactory cues selectively drive male fruitless mutant attraction to humans.
(A) Heat-seeking assay schematic. A 20 s pulse of 10% CO2 is added to the assay. (B) Percent of animals on Peltier. Data are mean ± s.e.m., n = 6 trials/temperature, n = 50 mosquitoes/trial. Data labeled with different letters are significantly different from each other, within each temperature. (C) Schematic of human odor host-seeking assay (left) and stimuli (right). (D) Percent of attracted animals. Data are mean ± s.e.m., n = 8–14 trials/group, n = 17–28 mosquitoes/trial. (E–F) Summary of results and model of gain of fruitless function in Aedes aegypti. Photo credit: Aedes aegypti (Alex Wild); D. melanogaster (Nicolas Gompel). In B, D, data labeled with different letters are significantly different from each other (Kruskal-Wallis test with Dunn’s multiple comparisons, p<0.05). In B, comparisons are made between genotypes at each temperature. In D, comparisons are made across all genotypes and stimuli.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. No significant blood-feeding or mating defects in fruitless∆M females.
(A) Percent of females crossed to indicated male genotype blood-feeding on a live human arm. (B) Percent of females crossed to indicated male genotype laying eggs. Data in A, B, are mean ± s.e.m., n = 3–10 trials/group, n = 12–27 mosquitoes/trial. Data labeled with different letters are significantly different from each other (Kruskal-Wallis test with Dunn’s multiple comparisons, p<0.05). Comparisons were made across all genotypes. (C) Number of inseminated females by males of indicated genotype. n = 10 females.
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. Female fruitless∆F mutant mosquitoes have blood-feeding and egg-laying defects.
(A) Schematic of Aedes aegypti fruitless genomic locus. (B) Sex-specific fruitless transcripts and generation of fruitless∆F mutant, with effect of splicing on Fruitless protein. (C) Crossing scheme to generate female mutants and potential male recombinants. (D) Blood-feeding assay schematic. (E) Feeding on live human arm; p<0.0001, Chi-square test. (F) Insemination assay schematic. (G) Insemination of females of indicated genotype by wild-type males; p=0.5464, Fisher’s exact test. (H) Egg-laying assay schematic. (I) Egg-laying by females of indicated genotype; p<0.0001, Fisher’s exact test.
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