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The mosquito Anopheles gambiae is a major African malaria vector, transmitting parasites responsible for significant mortality and disease burden. Although flight acoustics are essential to mosquito mating and present promising alternatives to insecticide-based vector control strategies, there is limited data on mosquito flight tones during swarming. Here, for the first time, we present detailed analyses of free-flying male and female An. gambiae flight tones and their harmonization (harmonic convergence) over a complete swarm sequence. Audio analysis of single-sex swarms showed synchronized elevation of male and female flight tones during swarming. Analysis of mixed-sex swarms revealed additional 50 Hz increases in male and female flight tones due to mating activity. Furthermore, harmonic differences between male and female swarm tones in mixed-sex swarms and in single-sex male swarms with artificial female swarm audio playback indicate that frequency differences of approximately 50 Hz or less at the male second and female third harmonics (M2:F3) are maintained both before and during mating interactions. This harmonization likely coordinates male scramble competition by maintaining ideal acoustic recognition within mating pairs while acoustically masking phonotactic responses of nearby swarming males to mating females. These findings advance our knowledge of mosquito swarm acoustics and provide vital information for reproductive control strategies.

Background Aedes aegypti mosquitoes are globally distributed vectors of viruses that impact the health of hundreds of millions of people annually. Mating and blood feeding represent fundamental aspects of mosquito life history that carry important implications for vectorial capacity and for control strategies. Females transmit pathogens to vertebrate hosts and obtain essential nutrients for eggs during blood feeding. Further, because host-seeking Ae. aegypti females mate with males swarming near hosts, biological crosstalk between these behaviors could be important. Although mating influences nutritional intake in other insects, prior studies examining mating effects on mosquito blood feeding have yielded conflicting results. Methodology/Principal findings To resolve these discrepancies, we examined blood-feeding physiology and behavior in virgin and mated females and in virgins injected with male accessory gland extracts (MAG), which induce post-mating changes in female behavior. We controlled adult nutritional status prior to blood feeding by using water- and sugar-fed controls. Our data show that neither mating nor injection with MAG affect Ae. aegypti blood intake, digestion, or feeding avidity for an initial blood meal. However, sugar feeding, a common supplement in laboratory settings but relatively rare in nature, significantly affected all aspects of feeding and may have contributed to conflicting results among previous studies. Further, mating, MAG injection, and sugar intake induced declines in subsequent feedings after an initial blood meal, correlating with egg production and laying. Taking our evaluation to the field, virgin and mated mosquitoes collected in Colombia were equally likely to contain blood at the time of collection. Conclusions/Significance Mating, MAG, and sugar feeding impact a mosquito’s estimated ability to transmit pathogens through both direct and indirect effects on multiple aspects of mosquito biology. Our results highlight the need to consider natural mosquito ecology, including diet, when assessing their physiology and behavior in the laboratory., Author summary Controlling mosquitoes and the disease agents they transmit during blood feeding remains a global public heath priority. Some vector control tools in development target the mosquito reproductive system as a means of control. The transfer of semen to a female during mating profoundly impacts her biology by inhibiting re-mating and stimulating egg production and laying. Previous studies in Aedes aegypti that examined potential effects of mating on blood feeding yielded contradictory results. We examined the potential of mating and seminal fluids to modulate female blood feeding and controlled for numerous experimental factors, such as ingestion of sugar prior to blood feeding, that may have led to conflicting results in prior studies. In laboratory studies with field-derived Thai mosquitoes, we show that mating and seminal fluids do not impact blood feeding during a mosquito’s first meal, but prior sugar feeding does. Furthermore, we show that mating, seminal fluids, and sugar feeding can impact blood feeding across multiple consecutive meals. Furthermore, field studies in Colombia show that virgin and mated females were equally likely to contain blood. Our research clarifies the impact of mating on blood feeding and suggests ways to improve our understanding of these behaviors in nature.