Ripple effects in a communication network: anti-eavesdropper defence elicits elaborated sexual signals in rival males.

Emitting conspicuous signals into the environment to attract mates comes with the increased risk of interception by eavesdropping enemies. As a defence, a commonly described strategy is for signallers to group together in leks, diluting each individual's risk. Lekking systems are often highly social settings in which competing males dynamically alter their signalling behaviour to attract mates. Thus, signalling at the lek requires navigating fluctuations in risk, competition and reproductive opportunities. Here, we investigate how behavioural defence strategies directed at an eavesdropping enemy have cascading effects across the communication network. We investigated these behaviours in the túngara frog (Engystomops pustulosus), examining how a calling male's swatting defence directed at frog-biting midges indirectly affects the calling behaviour of his rival. We found that the rival responds to swat-induced water ripples by increasing his call rate and complexity. Then, performing phonotaxis experiments, we found that eavesdropping fringe-lipped bats (Trachops cirrhosus) do not exhibit a preference for a swatting male compared to his rival, but females strongly prefer the rival male. Defences to minimize attacks from eavesdroppers thus shift the mate competition landscape in favour of rival males. By modulating the attractiveness of signalling prey to female receivers, we posit that eavesdropping micropredators likely have an unappreciated impact on the ecology and evolution of sexual communication systems.

Tiny spies: mosquito antennae are sensitive sensors for eavesdropping on frog calls.

Most mosquito and midge species use hearing during acoustic mating behaviors. For frog-biting species, however, hearing plays an important role beyond mating as females rely on anuran calls to obtain blood meals. Despite the extensive work examining hearing in mosquito species that use sound in mating contexts, our understanding of how mosquitoes hear frog calls is limited. Here, we directly investigated the mechanisms underlying detection of frog calls by a mosquito species specialized on eavesdropping on anuran mating signals: Uranotaenia lowii. Behavioral, biomechanical and neurophysiological analyses revealed that the antenna of this frog-biting species can detect frog calls by relying on neural and mechanical responses comparable to those of non-frog-biting species. Our findings show that in Ur. lowii, contrary to most species, males do not use sound for mating, but females use hearing to locate their anuran host. We also show that the response of the antennae of this frog-biting species resembles that of the antenna of species that use hearing for mating. Finally, we discuss our data considering how mosquitoes may have evolved the ability to tap into the communication system of frogs.

Recording and Analysis of Mosquito Acoustic-Related Mating Behavior.

In mosquitoes, courtship and mating sounds are produced by the movement of the wings during flight. These sounds, usually referred to as flight tones, have been studied using tethered and free-flying individuals. Here, we describe a general approach for recording and analyzing mosquito acoustic-related mating behaviors that can be broadly adapted to a variety of experimental designs.

Acoustic-Related Mating Behavior in Tethered and Free-Flying Mosquitoes.

Acoustics play an essential role in mosquito communication, particularly during courtship and mating. Mosquito mating occurs in flight and is coordinated by the perception of wingbeat tones. Flight tone frequencies have been shown to mediate sex recognition in Aedes, Anopheles, Culex, and Toxorhynchites genera and are thus a conserved feature of mating across the mosquito family (Culicidae). Upon recognizing a flying female, males respond phonotactically by lunging toward the female and initiating a precopulatory courtship flight interaction. During this interaction, males and females often harmonize their flight tones in a behavior known as harmonic convergence, and male acoustics display rapid frequency modulation. These acoustic phenomena have been characterized both in tethered and free-flying mosquitoes using similar audio recording and analysis methods. Further, the manipulation of mosquito acoustic-related mating behavior shows great promise as a tool for reproductive control strategies. In this brief methodological introduction, we provide an overview of the biological and technical concepts necessary for understanding the recording and analysis of mosquito mating acoustics.

Flight tone characterisation of the South American malaria vector Anopheles darlingi (Diptera: Culicidae).

BACKGROUND: Flight tones play important roles in mosquito reproduction. Several mosquito species utilise flight tones for mate localisation and attraction. Typically, the female wingbeat frequency (WBF) is lower than males, and stereotypic acoustic behaviors are instrumental for successful copulation. Mosquito WBFs are usually an important species characteristic, with female flight tones used as male attractants in surveillance traps for species identification. Anopheles darlingi is an important Latin American malaria vector, but we know little about its mating behaviors. OBJECTIVES: We characterised An. darlingi WBFs and examined male acoustic responses to immobilised females. METHODS: Tethered and free flying male and female An. darlingi were recorded individually to determine their WBF distributions. Male-female acoustic interactions were analysed using tethered females and free flying males. FINDINGS: Contrary to most mosquito species, An. darlingi females are smaller than males. However, the male's WBF is ~1.5 times higher than the females, a common ratio in species with larger females. When in proximity to a female, males displayed rapid frequency modulations that decreased upon genitalia engagement. Tethered females also modulated their frequency upon male approach, being distinct if the interaction ended in copulation or only contact. MAIN CONCLUSIONS: This is the first report of An. darlingi flight acoustics, showing that its precopulatory acoustics are similar to other mosquitoes despite the uncommon male:female size ratio, suggesting that WBF ratios are common communication strategies rather than a physical constraint imposed by size.

Flight tone characterisation of the South American malaria vector Anopheles darlingi (Diptera: Culicidae)

BACKGROUND Flight tones play important roles in mosquito reproduction. Several mosquito species utilise flight tones for mate localisation and attraction. Typically, the female wingbeat frequency (WBF) is lower than males, and stereotypic acoustic behaviors are instrumental for successful copulation. Mosquito WBFs are usually an important species characteristic, with female flight tones used as male attractants in surveillance traps for species identification. Anopheles darlingi is an important Latin American malaria vector, but we know little about its mating behaviors. OBJECTIVES We characterised An. darlingi WBFs and examined male acoustic responses to immobilised females. METHODS Tethered and free flying male and female An. darlingi were recorded individually to determine their WBF distributions. Male-female acoustic interactions were analysed using tethered females and free flying males. FINDINGS Contrary to most mosquito species, An. darlingi females are smaller than males. However, the male's WBF is ~1.5 times higher than the females, a common ratio in species with larger females. When in proximity to a female, males displayed rapid frequency modulations that decreased upon genitalia engagement. Tethered females also modulated their frequency upon male approach, being distinct if the interaction ended in copulation or only contact. MAIN CONCLUSIONS This is the first report of An. darlingi flight acoustics, showing that its precopulatory acoustics are similar to other mosquitoes despite the uncommon male:female size ratio, suggesting that WBF ratios are common communication strategies rather than a physical constraint imposed by size.

A new approach to improve acoustic trapping effectiveness for Aedes aegypti (Diptera: Culicidae).

Monitoring mosquito populations is essential to designing and implementing control strategies. Recent strategies based on releasing biologically modified mosquitoes have increased the need to effectively monitor mosquito abundance. Unfortunately, existing surveillance traps are of limited value due to their high cost and low capture rates. Here, we report the results of experiments designed to evaluate the effectiveness of an acoustic trap prototype. Stimuli synthesized from recordings of Ae. aegypti wingbeat signals and pure tones were evaluated as attractants to males in indoor and semi-field conditions. Overall, the acoustic trap´s efficacy differed significantly between indoor and semi-field conditions. After two hours of indoor recapture, ∼69% of males were collected from acoustic traps broadcasting pure tones while ∼78% of males were collected using synthesized wingbeat signals. Under semi-field conditions, however, acoustic traps collected less than ∼1.7% of the males released. Increasing the intensity of the signals up to 90 dB (SPL re. 20 uPa at 1 m from the trap) did not improve the capture rate under semi-field conditions. Overall, our results indicate that acoustic signals synthesized from recordings of wingbeats can be used to enhance capture of male Ae. aegypti.

Precopulatory acoustic interactions of the New World malaria vector Anopheles albimanus (Diptera: Culicidae).

BACKGROUND: Anopheles albimanus is a malaria vector in Central America, northern South America and the Caribbean. Although a public health threat, An. albimanus precopulatory mating behaviors are unknown. Acoustics play important roles in mosquito communication, where flight tones allow males to detect and attract potential mates. The importance of sound in precopulatory interactions has been demonstrated in Toxorhynchites brevipalpis, Aedes aegypti, Culex quinquefasciatus and Anopheles gambiae; convergence in a shared harmonic of the wing beat frequency (WBF) during courtship is thought to increase the chance of copulation. To our knowledge, An. albimanus precopulatory acoustic behaviors have not been described to date. Here, we characterized An. albimanus (i) male and female flight tones; (ii) male-female precopulatory acoustic interactions under tethered and free flight conditions; and (iii) male-male acoustic interactions during free flight. RESULTS: We found significant increases in the WBFs of both sexes in free flight compared to when tethered. We observed harmonic convergence between 79% of tethered couples. In free flight, we identified a female-specific behavior that predicts mate rejection during male mating attempts: females increase their WBFs significantly faster during mate rejection compared to a successful copulation. This behavior consistently occurred during mate rejection regardless of prior mating attempts (from the same or differing male). During group flight, males of An. albimanus displayed two distinct flying behaviors: random flight and a swarm-like, patterned flight, each associated with distinct acoustic characteristics. In the transition from random to patterned flight, males converged their WBFs and significantly decreased flight area, male-male proximity and the periodicity of their trajectories. CONCLUSIONS: We show that tethering of An. albimanus results in major acoustic differences compared to free flight. We identify a female-specific behavior that predicts mate rejection during male mating attempts in this species and show that male groups in free flight display distinct flying patterns with unique audio and visual characteristics. This study shows that An. albimanus display acoustic features identified in other mosquito species, further suggesting that acoustic interactions provide worthwhile targets for mosquito intervention strategies. Our results provide compelling evidence for swarming in this species and suggests that acoustic signaling is important for this behavior.