Fruitless Mutant Male Mosquitoes Gain Attraction to Human Odor
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bioRxiv preprint doi: https://doi.org/10.1101/2020.09.04.282434; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. fruitless mutant male mosquitoes gain attraction to human odor Nipun S. Basrur1, Maria Elena De Obaldia1, Takeshi Morita1, Margaret Herre1,2, Ricarda K. von Heynitz1,4, Yael N. Tsitohay1, Leslie B. Vosshall1-3* SUMMARY While sexual dimorphism in courtship and copulation behavior is common in the animal kingdom, sexual dimorphism in feeding behavior is rare. The Aedes aegypti mosquito provides an example of extreme sexual dimorphism in feeding, because only the females show strong attraction to hu- mans, and bite them to obtain a blood-meal necessary to stimulate egg production1-8. The genetic basis of this complex, modular, and sexually dimorphic feeding behavior is unknown. The fruitless gene is sex-specifically spliced in the brain of multiple insect species including mosquitoes9-11 and encodes a BTB zinc-finger transcription factor that has been proposed to be a master regulator of male courtship and mating behavior across insects12-17. Here we use CRISPR-Cas9 to mutate the fruitless gene in male mosquitoes. fruitless mutant males fail to mate, confirming the ancestral function of this gene in male sexual behavior. Remarkably, fruitless mutant males also gain strong attraction to a live human host, a behavior that wild-type males never display. Humans produce multiple sensory cues that attract mosquitoes and we show that fruitless specifically controls host- seeking in response to human odor. These results suggest that male mosquitoes possess the neu- ral 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 be- havior to control one module of female-specific blood-feeding behavior in a deadly vector of infec- tious diseases. Key words: Aedes aegypti; mosquito; fruitless; sexual dimorphism; host-seeking behavior; blood-feeding; Zika; dengue; CRISPR-Cas9 INTRODUCTION sexual dimorphism in species-specific behaviors, or do Across animals, males and females of the same species novel genes evolve to control new behaviors? show striking differences in behavior. Male Paradisaeidae birds-of-paradise perform an elaborate courtship dance to Many advances in understanding the genetics of sexually seduce prospective female partners, contorting their bodies dimorphic behaviors have come from the study of Drosoph- in forms resembling flowers, ballerinas, and smiling faces18. ila melanogaster fly courtship, where a male fly orients to- Female Serromyia femorata midges pierce and suck con- wards, taps, and follows a female fly, extending a wing to specific males dry during mating, breaking off his genitalia produce a courtship song before tasting, mounting, and cop- inside her, thereby supplying the female with both nutrition ulating with her12. Courtship comprises behavioral modules, and sperm19. Although an astonishing diversity of sexually which are simple discrete behaviors that must be combined dimorphic behaviors exists across species, most insights to perform a complex behavior and are elicited by different into the genetic and neural basis of sex-specific behaviors sensory modalities and subsets of fruitless-expressing neu- have come from a limited set of model organisms20. Which rons15,21-24. Courtship modules include orienting, which is genes control sexual dimorphism in specialist species that driven by visual information25 and persistent following and have evolved novel behaviors? Do conserved genes control singing, which are triggered by chemical cues on a female 1Laboratory of Neurogenetics and Behavior, The Rockefeller University, New York, NY 10065 USA, 2Kavli Neural Systems Institute, New York, NY 10065 USA, 3Howard Hughes Medical Institute, New York, NY 10065 USA, 4Current address: Technical University of Munich, TUM School of Medicine, 81675 Munich, Germany *Correspondence: [email protected] (LBV) bioRxiv preprint doi: https://doi.org/10.1101/2020.09.04.282434; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. fly15 and guided by vision25,26. Sex-specific splicing of the attracted to the arm (Fig. 1b-c). What is the genetic basis for fruitless gene controls several aspects of courtship behav- this extreme sexual dimorphism? We reasoned that fruit- ior. Male flies mutant for fruitless promiscuously court other less, which is alternatively spliced in a sex-specific manner males and cannot successfully mate with females13,27. Forc- and promotes male courtship and copulation in Drosophila ing male fruitless splicing in females triggers orientation and melanogaster flies13,27, may play similar roles in controlling singing behaviors normally only performed by males14. fruit- sexually dimorphic behaviors in Aedes aegypti. fruitless is a less encodes a BTB zinc-finger transcription factor that is complex gene with multiple promoters and multiple alterna- thought to control cell identity and connectivity during devel- tively spliced exons. A previous study showed11 and we con- opment28,29, as well as the functional properties of neurons firmed that transcripts from the upstream neuron-specific in adulthood30, although its genomic targets and the molec- (P1) promoter in the Aedes aegypti fruitless gene are sex- ular mechanism by which it acts remain unclear. fruitless has specifically spliced (Fig. 1d-h). Both male and female tran- a conserved role controlling courtship in multiple Drosophila scripts include a short male ‘m’ exon, and female transcripts species16,17, and sex-specific fruitless splicing is conserved additionally include a longer female ‘f’ exon with an early across wasps10 and mosquitoes9,11, suggesting that fruitless stop codon, predicted to yield a truncated Fruitless protein may act as a master regulator of sexually dimorphic mating in the female. However, it is unlikely that any Fruitless pro- behaviors across insects. tein is stably expressed in the female. In Drosophila, female fruitless peptides are not detected36, and transformer is Mosquitoes display striking sexually dimorphic mating and thought to inhibit translation by binding to female fruitless feeding behaviors. Only male mosquitoes initiate mating, transcripts37. P1 transcripts of both sexes splice to the first and only females drink blood, which they require to develop common ‘c1’ exon, but only male transcripts are predicted to their eggs. Sexual dimorphism in blood-feeding is one of the encode full-length Fruitless protein with BTB and zinc-finger only instances of a completely sexually-dimorphic feeding domains (Fig. 1e). By analyzing previously published tissue- behavior, since male mosquitoes never pierce skin or en- specific RNA-seq data38, we verified that of all the fruitless gorge on blood. While part of this dimorphism is enforced by exons, only the f exon was sex-specific in Aedes aegypti sex-specific genitalia31 or feeding appendages32, there is brains (Fig. 1g). Moreover, P1 transcripts were specifically also a dramatic difference in the drive to hunt hosts between expressed in the brain and the antenna, the major olfactory males and female mosquitoes2,33. To blood-feed, females organ of the mosquito (Fig. 1h), consistent with fruitless P1 combine multiple behavioral modules2. Female Aedes ae- expression in Drosophila39. gypti mosquitoes take flight when exposed to carbon diox- ide2,6, and are attracted to human olfactory4,5,7, thermal, and To ask if fruitless splicing was conserved across mosqui- visual cues6,34,35, and integrate at least two of these cues to toes, we sequenced RNA from male and female brains of orient toward and land on human skin. Engorging on blood five different species and assembled de novo transcrip- is triggered by specific sensory cues tasted by the female1,8. tomes for each sex. Three of these species are important It is not known which genes have evolved to control this arboviral disease vectors because their females blood feed unique sexually dimorphic and mosquito-specific feeding on humans, whereas adults of the two other species only behavior. Here we generate fruitless mutant Aedes aegypti feed on plants40,41 (Fig. 1f). We identified orthologues of fruit- mosquitoes and show that consistent with observations in less in each species and found that all had conserved ‘m’ Drosophila, fruitless is required for male mating behavior. and ‘c1’ exons and distinct ‘f’ exons. fruitless was sex-spe- Unexpectedly, fruitless mutant male mosquitoes gain the cifically spliced in each of these species with a female-spe- ability to host-seek, specifically driven by an attraction to hu- cific ‘f’ exon and early stop codon, predicted to produce a man odor. Our results demonstrate that sexual dimorphism full-length fruitless protein only in males. in a single module of a mosquito-specific behavior is con- trolled by a conserved gene that we speculate has gained a We used CRISPR-Cas9 genome editing42 to disrupt P1 neu- new function in the course of evolution. ral-specific fruitless transcripts in Aedes aegypti to investi- gate a possible role of fruitless in sexually dimorphic mos- RESULTS quito behaviors. We generated two alleles, fruitless∆M, which introduces a frameshift that is predicted to produce a trun- fruitless is sex-specifically spliced in the mosquito cated protein in males, and fruitless∆M-tdTomato, in which the nervous system fruitless gene is disrupted by a knocked-in We used an arm-next-to-cage assay (Fig. 1a) to monitor at- CsChrimson:tdTomato fusion protein (Fig. 1i). In both al- traction of male and female Aedes aegypti mosquitoes to a leles, the protein is truncated before the downstream BTB live human arm. Consistent with their sexually dimorphic and zinc-finger domains.