Humming in Tune: Sex and Species Recognition by Mosquitoes on the Wing
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Mosquitoes are more sensitive to sound than any other insect due to the remarkable properties of their antennae and Johnston’s organ at the base of each antenna. Male mosquitoes detect and locate female mosquitoes by hearing the female’s flight tone, but until recently we had no idea that females also respond to male flight tones. Our investigation of a novel mechanism of sex recognition in Toxorhynchites brevipalpis revealed that male and female mosquitoes actively respond to the flight tones of other flying mosquitoes by altering their own wing-beat frequencies. Male–female pairs converge on a shared harmonic of their respective fundamental flight tones, whereas same sex pairs diverge. Most frequency matching occurs at frequencies beyond the detection range of the Johnston’s organ but within the range of mechanical responsiveness of the antennae. We have shown that this is possible because the Johnston’s organ is tuned to, and able to detect difference tones in, the harmonics of antennal vibrations which are generated by the combined input of flight tones from both mosquitoes. Acoustic distortion in hearing organs exists usually as an interesting epiphenomenon. Mosquitoes, however, appear to use it as a sensory cue that enables male–female pairs to communicate through a signal that depends on auditory interactions between them. Frequency matching may also provide a means of species recognition. Morphologically identical but reproductively isolated molecular forms of Anopheles gambiae fly in the same mating swarms, but rarely hybridize. Extended frequency matching occurs almost exclusively between males and females of the same molecular form, suggesting that this behavior is associated with observed assortative mating.
Keywordsmosquito hearing frequency tuning distortion products difference tones frequency matching sexual recognition species recognition Johnston’s organ
Anopheles gambiae s.l. has been described as the most dangerous animal in the world, responsible for the majority of malaria deaths worldwide, killing 1–2 million people annually and ranked the second highest contributor to the global Disability Adjusted Life Year in Africa (WHO 2002). If we take into account the populations of all species of mosquito that transmit pathogens and parasites to humans and their domestic livestock worldwide, mosquitoes easily cause more human suffering than any other organism. Their sensory physiology is finely tuned to locate host animals mainly by olfactory cues, and their visual system is well adapted to locating hosts mainly in the dark at times of day when hosts are most quiescent and least able to defend themselves from bites, making them one of the few insects that routinely fly at night (Gibson and Torr 1999; Land et al. 1999). It is beginning to become apparent that their auditory system is one of the most highly developed among the insects.
The males of most mosquito species of medical importance are known to locate mates by sound; males aggregate over conspicuous markers and fly continuously in swarming flight, keeping station over the marker. Virgin females are also attracted to the same markers, which are generally, but not universally species-specific. When a male detects the flight tone of a female, he chases her by localizing the source of her flight tone. This mating chase normally leads to the formation of a mating copula. The mechanisms by which male mosquitoes detect and locate females has been reasonably well understood for many years (Charlwood and Jones 1979; Belton 1994; Clements 1999). It has been proposed that the male auditory organ acts as an acoustic filter for female flight tones, but the evidence so far suggests that the filter is too broad and flight tone frequencies alone are not sufficiently species-specific to be the basis for male identification of con-species females (Tripet et al. 2001). Audition is, therefore very important to mosquitoes for sexual and perhaps species recognition.
In recent years, interest in the mating behavior of mosquitoes has increased for two key reasons; (1) to understand the basis of complex patterns of reproductive isolation in sympatric populations of closely related molecular forms of the malaria mosquito A. gambiae s.s. that do not hybridize even though they occur in the same mating swarms (della Torre et al. 2002), and (2) to investigate the likely efficacy of using genetically modified mosquitoes to control wild populations. The approach is only possible if we can ensure that laboratory reared populations will respond appropriately to the mating behavior of wild mosquitoes. Hence, we have undertaken an extended investigation of the auditory behavior of mosquitoes in relation to mating interactions.
In many mosquito species, the antennae are sexually dimorphic, with males having a greater number of sensillae in the JO (∼14,000 in males, ∼7,000 in females, Göpfert et al. 1999) and a greater number of fibrillae (fine hair-like structures) on the flagellum (Fig. 1A), which led Johnston to speculate that for these mosquitoes, mating behavior involved audition (Johnston 1855). It is likely that the greater surface area of the male flagellum increases their responsiveness to particle displacement.
The sensillae mechanoelectrically transduce and amplify the nanometer sound-induced vibrations of the antennal flagellum into electrical signals (Göpfert and Robert 2000).
The sources of extracellular electrical compound potentials from the JO of mosquitoes are believed to be extracellular currents associated with the generation of receptor potentials and action potentials in the JO (Fig. 1C–E; Tischner 1953; Keppler 1958; Wishart et al. 1962; Warren et al. 2009). As with recordings of the cochlear microphonic from the round window of the mammalian cochlea (Patuzzi et al. 1989), these potentials are overwhelmingly dominated by the receptor currents from the sensory cells closest to the extracellular electrode. Acoustically elicited receptor potentials recorded from the JO are composed of a tonic (DC) component, a phasic F1 component, and an F2 component that is twice the frequency of the applied acoustic stimulus (Fig. 1C and D). The bandwidth of the F2 component has been used to determine the frequency limits of hearing in mosquitoes because it dominates the electrical response of the JO (Tischner 1953; Keppler 1958; Wishart et al. 1962; Belton 1974; Warren et al. 2009). The frequency doubling of the F2 component is thought to be due to the summed outputs of the nonlinear electrical responses of two populations of sensory cells in the scolopidia with opposite polarities of morphological and functional symmetry (Tischner 1953; Wishart et al. 1962; Belton 1974; Clements 1999; Warren et al. 2009), as has also been proposed for the frequency doubling of the microphonic potential of the lateral line receptors of fishes (Flock 1965). The phasic F1 component of the receptor potential dominates at high frequencies and low sound intensities, probably because potentials from only the scolopidial population nearest the electrode are recorded. Potentials from populations of scolopidia further away from the recording site, that are of opposite polarity and which would summate to produce a potential with twice the frequency of the stimulating frequency, are attenuated before reaching the electrode. A tonic component or direct current (DC) shift of the potential occurs upon and during presentation of a tone (Fig. 1C) but its precise origin has yet to be determined (Cator et al. 2009).
Efforts to establish the upper frequency limit of hearing by mosquitoes have led to a wide range of differing results. Discrepancies in published findings may be due to the use of the different components of the extracellular potentials of the JO to assess the bandwidth of hearing in mosquitoes (Belton 1974; Clements 1999; Pennetier et al. 2010; Warren et al. 2009; Arthur et al. 2010), and to differences in the maximum particle velocity level used for acoustic stimulation, differences in location of the electrode in the JO and differences in criteria of the thresholds used to derive tuning curves. Our most recent approach was to construct a threshold behavioral audiogram from motor potentials (“M”, Fig. 1F) recorded from the thorax (Warren et al. 2009), which we found to be less sensitive than those derived from the phasic and DC potentials recorded from the JO of the same mosquito, but its high-frequency limit corresponded to that set by the F1 phasic receptor potentials (“F1”, Fig. 1F).
Frequency Matching at Fundamental Wing-Beat Frequencies; Toxorhynchites brevipalpis
Behavioral Frequency Tuning Curves
The frequency convergence and transient responses of the mosquitoes to pure tones enabled us, for the first time, to derive mosquito behavioral auditory tuning curves for each sex (Fig. 3A). A response was scored if the mosquito either altered its wing-beat frequency upward or downward in response to the stimulus tone frequency. The overall shape of the male and female behavioral auditory threshold curves (Fig. 3A) show that there is a relatively sharp transition in behavior and sensitivity of response at ∼350 and ∼500 Hz. At stimulus frequencies between 350–500 Hz (indicated by the blue region in Fig. 3A) mosquitoes converged on the stimulus tone, even when it was delivered at remarkably low dB, whereas for stimulus frequencies outside this range (200–345 and 500–800 Hz), mosquitoes responded with a transient change in their wing-beat frequencies, but only if the stimulus was delivered at a relatively high dB. Mosquitoes appeared to be unable to converge on stimulus tones outside the 400–500 Hz range.
The behavioral tuning curves of males and females are similar in shape, most sensitive at frequencies close to 400 Hz, and show males to be approximately seven times more sensitive than females, responding to tone levels which displace their antennae by 0.8 nm at 400 Hz compared with 5.5 nm for females.
Mechanical Antennal Frequency Tuning
Mechanical tuning curves were constructed from laser-diode interferometer (Lukashkin et al. 2005) measurements of the antennal flagellum vibrations, made close to the pedicel (inset Fig. 3B). We found that the mechanical frequency tuning of the antennae of male and female T. brevipalpis (500 and 400 Hz, respectively; Fig. 3B) are more closely matched than previously reported (420 Hz and 240 Hz, respectively) for this species (Göpfert and Robert 2000). The reasons for the discrepancy between these results are unknown, but resonance frequency can be influenced, for example, by the size of the antennae and by the stiffness of the cuticle, due to factors such as age, physiological state, and ambient temperature.
Nonetheless, it is interesting to note that our behavioral tuning curves (Fig. 3A) are more sharply tuned than our mechanical tuning curves (Fig. 3B). From our mechanical measurements, the male antenna is more effectively coupled to particle displacements than the female antenna by a factor of 2.3, which is in agreement with previous measurements (Göpfert and Robert 2000). Thus, behaviorally, males are more sensitive than females to antennal displacement by a factor of about 3, which could be related to the observation that there are about twice as many sensillae in the JO of males than of females (Boo and Richards 1975), and, of more novel significance, behavioral thresholds in both sexes are about 10 times more sensitive than neural thresholds (Gibson and Russell 2006). These differences could be explained by neural processing (Stumpner and Helversen 2001; Hennig et al. 2004; Wyttenbach and Farris 2004).
To investigate the auditory interaction between two flying mosquitoes, we recorded the flight sounds of pairs of tethered mosquitoes held within acoustic range of each other (5 cm apart). In the case of male–female pairs, their wing-beat frequencies converged within a 0.5 s (Fig. 2F and G). The wing-beat frequencies of the two mosquitoes often exactly coincided within the resolution of the frequency spectrum (0.7 Hz), as shown in Figure 2F.
When pairs of the same sex were flown together (Fig. 2H and I), the wing-beat frequencies of the two individuals diverged to give frequency separation between the two flight tones of ∼70 Hz (Gibson and Russell 2006). In free flight, the divergence of their flight tones would probably lead to spatial separation because it has been hypothesized that in cruising insects flight velocity should remain proportional to wing-beat frequency unless amplitude increases (Taylor et al. 2003). This auditory behavior of male mosquitoes is not unlike visual interactions between other male Diptera during the course of mate-chasing flights such as the visually driven flight oscillations of male hoverflies (Syritta pipiens); males repeatedly and rapidly respond to each other leading to an unstable interaction that quickly results in separation (Collett and Land 1975). The stereotyped frequency separation behavior observed in same sex pairs is also reminiscent of the “jamming avoidance response” found in fish electrolocation and bat echolocation (Heiligenberg 1977; Ulanovsky et al. 2004).
Auditory interaction between the sexes is not uncommon amongst arthropods, although it is normally based on sequences of calls and responses produced for the sole purpose of communication (Bailey 2003). Our studies of the acoustic-motor behavior of T. brevipalpis is, however, the first demonstration of a mating interaction in flying insects based on acoustically controlled feedback between sound input (flight tones of both mosquitoes) and motor output (changes in wing-beat frequencies) in flight muscles, which are otherwise also engaged in flight maneuvers that stabilize flight and bring the two mosquitoes closer together. It is also the first to demonstrate auditory behavior by female mosquitoes and to report a pattern of behavioral responses that separates the sexes.
Frequency Matching at Higher Harmonics; Culex and Anopheles Species
Mechanical and Electrical Johnston’s Organ Tuning Curves
The minima of the receptor potential tuning curves are similar to those of the mechanical tuning curves but the low- and high-frequency slopes of receptor potential tuning curves are steeper than those of the mechanical tuning curves. Accordingly, the receptor potential tuning curves appear more narrowly tuned with band-pass characteristics (Fig. 5C). The rapid decrease in sensitivity above the minima of the receptor potential tuning curves means that, by contrast with mechanical frequency range of the flagellum, the frequencies at which the mosquitoes match their flight tones is outside the bandwidth of the JO phasic receptor potentials and thus outside the auditory range of C. quinquefasciatus and A. gambiae mosquitoes. It has been reported for A. aegypti (Cator et al. 2009) that the auditory range of the DC component of the JO receptor potential extends far above that of the phasic response and encompasses the frequency matching range. We measured DC components of the receptor potential and plotted DC frequency tuning curves (insets to Fig. 5C for A. gambiae and Fig. 1F for C. quinquefasciatus). It is clear from our findings that DC component frequency tuning curves are bounded by the phasic receptor potential tuning and do not extend the auditory range of the JO.
Changes in the frequency tuning and sensitivity of the JO during extension of fibrillae are complex and may not entirely be due to mechanical changes in the flagellum. The electrical responses of the JO and mechanical responses of the flagellum are metabolically vulnerable when the fibrillae are extended, and can collapse within 5 min when disturbed by experimental procedures. It would be interesting to discover if there is metabolic enhancement of the sensitivity of the JO during the increased hydrostatic pressure that causes erection of the fibrillae.
We conclude that C. quinquefasciatus and A. gambiae match their flight tones at frequencies that are outside the bandwidth of the JO’s phasic responses to acoustic stimulation but within the frequency response of the vibrations of the antennal flagellum.
Difference Tone Generation by the Johnston’s Organ
Frequency matching of flight tones between pairs of flying, tethered, opposite sex mosquitoes has now been demonstrated in four genera, including Toxorynchites (Gibson and Russell 2006), Culex (Warren et al. 2009), Anopheles (Pennetier et al. 2010) and Aedes (Cator et al. 2009) species. We suggest that mosquitoes detect the beat frequencies or difference tones between their respective wing-beat frequencies, and use these to maintain a relatively fixed ratio of wing-beat frequencies. The auditory information provided by the difference tone informs a mosquito not only that another mosquito is nearby, but provides exact information (an error signal) about the relative frequency ratio of their respective wing-beat frequencies. If both mosquitoes maintain a ratio that keeps them near a common frequency, then the pair are certain to be of opposite sex and the male has a means of tracking the position of the female, while frequency avoiding other males that may be nearby. The female may exert a degree of mate choice by changing her flight tone to see how well the male follows her, or change frequencies if she wants to “lose” him.
Mosquitoes are unusual, and perhaps unique (Gerhardt and Huber 2002; Kössl et al. 2008) in that they make use of their ability to detect low-frequency distortion products, to bring behavior that is played out at frequencies beyond the range of the phasic responses of their auditory receptors to within the scope of their hearing organ. This represents the first demonstration of an insect exploiting and responding to distortion products generated by its own auditory system for eliciting auditory behavior. There is also recent evidence that neurons in the midbrains of mammals respond to acoustic distortion products (Holmstrom et al. 2010; McAlpine 2004; Abel and Kössl 2009; Portfors et al. 2009), and that they may have a role in perceiving important characteristics of a complex tone, such as the pitch (Smoorenburg 1970; Goldstein et al. 1978). Portfors and her colleagues in their studies on the central processing and significance of ultrasonic vocalizations by mice discovered that many of the vocalization responsive neurons that they recorded in the inferior colliculus did not respond to the individual ultrasonic frequencies contained within the vocalizations, but they did respond to combinations of ultrasonic tones if the difference between the tones was within the excitatory frequency tuning curve (Portfors et al. 2009). As with the mosquito hearing organ, nonlinear interactions of frequencies, due at least in the mosquito JO to distortions in the system, may be used to mediate, or to enhance sensitivity to behaviorally important stimuli.
Species Recognition in A. gambiae s.s.
Given the novel nature of the physiological mechanism by which mosquitoes harmonize, what can be said about the significance of this unusual form of communication in the wider context of mosquito behavior? Does it enable mosquitoes to identify more than just the sex of the other mosquito? In the case of the malarial mosquito A. gambiae s.s., it appears that frequency matching may provide a breakthrough in the long-standing mystery as to how new species can arise out of sympatric populations of cryptic sub-species (Ritchie and Immonen 2010).
A. gambiae s.s. is a member of a species complex that consists of seven morphologically identical, yet reproductively isolated, species and several chromosomal/molecular forms, thought to be incipient species. The complexity of malaria epidemiology and the extreme resilience with which malaria has established itself in human populations is due, in part at least, to the remarkable genetic plasticity of certain members of species complex, enabling them to adapt rapidly to an ever widening range of human-influenced habitats. This leads to rapid ecological speciation when reproductive isolation mechanisms develop (Coluzzi 1982; Powell et al. 1999; della Torre et al. 2001; 2002; Ayala and Coluzzi 2005; Costantini et al. 2009). Although reproductive isolation is essential for speciation, little is known about how it occurs in sympatric populations of incipient species (Costantini et al. 2009).
The A. gambiae s.l. complex has become a focus of research on the evolution of species complexes to understand how populations diverge and become distinct species (Ayala and Coluzzi 2005). Within the A. gambiae complex, several degrees of reproductive isolation among its members can be observed in field populations. On one hand, formally recognized species such as A. gambiae s.s. and Anopheles arabiensis have evolved strong reproductive isolation, although a permeable species barrier still exists leading to a small degree of introgressive hybridization (Powell et al. 1999; Besansky et al. 2003). On the other hand, within A. gambiae s.s. cryptic incipient speciation has led to the recognition of several genotypic forms, which are distinguishable only on the basis of molecular markers and/or differences in chromosomal banding patterns. Two molecular forms, named “M” and “S” (della Torre et al. 2001), hybridize at different frequencies across different eco-geographical settings due to differing degrees of reproductive isolation (Tripet et al. 2001; della Torre et al. 2002; Caputo et al. 2008). These two forms are thought to have evolved through selection for populations adapted to different types of breeding site, e.g., rain-fed pools v. impounded irrigation ponds.
Although it is possible to distinguish particular molecular/chromosomal forms phenotypically by their association with one or another type of breeding water, there is no clear understanding as to how local sympatric forms became reproductively isolated in the first instance, although different patterns of mating behavior have been observed locally. In Mali, for example, unknown behavioral cues used by the two forms to identify mating swarm sites have diverged and, since they mate in segregated swarms, hybrids are rarely produced (Diabate et al. 2009); in effect, these forms are (locally) geographically separated with respect to mating behavior. However, in Burkina Faso, only 500 km away, M and S form mosquitoes can be found in the same swarm (della Torre et al. 2001; Tripet et al. 2001; Diabate et al. 2004; Diabate et al. 2006) and yet hybrids are notably rare, indicating the potential existence of a close-range barrier to interbreeding. There are no published reports of close-range mechanisms of mate recognition, and attempts to demonstrate mate recognition in the field with volatile pheromones have not been successful (JD Charlwood, personal communication, 2009). We have discovered, however, that the sexual recognition mechanism described above, also confers the capability of form-specific mate recognition (Pennetier et al. 2010).
This is the first report of a phenotypic difference in close-range mating behavior between M and S form A. gambiae s.s., and on the basis of these findings, we conclude that M and S form mosquitoes can discriminate between mosquitoes of the “same” and “other” form.
Previous attempts to detect potential mate recognition characteristics in the mean wing-beat frequency characteristics mosquito species may have failed because wing-beat frequencies have always been measured only in solo flying mosquitoes. Our findings demonstrate that the mean wing-beat frequency and its variance alter significantly when a mosquito detects the flight tones of another mosquito, and hence assessing wing-beat frequency in solo mosquitoes provides misleading and unreliable information about their flight tone characteristics. Also, the potential significance of the relative wing-beat frequencies of males and females at higher harmonic ratios had not yet been appreciated (Clements 1999; Tripet et al. 2004).
It is striking that this form of communication is based on no fixed “signal” and “response”. The absolute mean matching frequency is variable, unique to each interaction, and can change during a matching sequence with one mosquito frequency tracking the other. For example, in Figure 7A and B the pairs of mosquitoes frequency-match at a ratio of 3:2 for a few seconds at a time (light-colored regions), reducing the variability in their respective WBFs when the ratio between them is close to 1.5, but when they come back together after breaking apart, the mean matching frequency has generally changed. Fine time-scale interactions are shown in Figure 7C and D to illustrate the ability of mosquitoes to respond to changes in each other’s wing-beat frequency on a moment-to-moment basis with a brief (∼50–60 ms) delay. It is worth noting that both males and females actively respond to the other during these interactions, a finding that overturns the “accepted wisdom” that males only respond to the flight tones of females by chasing them, and that females are generally passive during these chases (Clements 1999).
The interactive aspect of frequency matching appears to be essential; presentation of pure tones or pre-recorded mosquito flight tones to individual tethered-flying Anopheles mosquitoes did not elicit frequency matching in either form, unlike the case of courtship song in Drosophila melanogaster, a dipteran species for which courtship song is well documented (Ritchie et al 1999; Ritchie and Immonen 2010). Analysis of factors controlling frequency matching and subsequent mating behavior must now be undertaken in free-flight experiments.
Frequency matching may have evolved due to a selected advantage of mating in free flight; frequency matching at close-range would enable the relatively small male to form a copula with the larger female in mid-flight by synchronizing with the potentially turbulent air stream generated by her wing beats (Sane 2003; Lehmann 2008). Our findings represent the first breakthrough in furthering our understanding of mosquito mating interactions since Belton’s analysis of male mate localisation by sound >35 years ago (Belton 1974). They are also the first documentation of form-specific close-range interactions related to mating behavior since Coluzzi first put forward his theory of the evolution of reproductive isolation in diverging populations (Coluzzi 1982; Ayala and Coluzzi 2005).
This research is supported by a grant from the BBSRC. BW is supported by a BBSRC studentship.
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