The electronic song “Scary Monsters and Nice Sprites” reduces host attack and mating success in the dengue vector Aedes aegypti

Acta Tropica 194 (2019) 93–99

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The electronic song “Scary Monsters and Nice Sprites” reduces host attack and mating success in the dengue vector Aedes aegypti

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Hamady Dienga,b, , Ching Chuin Thec, Tomomitsu Sathod, Fumio Miaked, Erida Wydiamalae, Nur Faeza A. Kassimf, Nur Aida Hashimg, Ronald E. Morales Vargash, Noppawan P. Moralesi a

Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, Malaysia Mosquito Research and Control Unit, Grand Cayman, Cayman Islands c Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota Samarahan, Malaysia d Faculty of Pharmaceutical Sciences, Fukuoka University, Japan e Faculty of Medicine, Lambung Mangkurat University, South Kalimantan, Indonesia f School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia g School of Food Science and Technology, Universiti Malaysia Terengganu, Kuaka Terengganu, Malaysia h Faculty of Tropical Medicine, Mahidol University, Thailand i Faculty of Science, Mahidol University, Thailand b

A R T I C LE I N FO

A B S T R A C T

Keywords: Dengue vector Music Visitation Host attack Blood feeding Copulation

Sound and its reception are crucial for reproduction, survival, and population maintenance of many animals. In insects, low-frequency vibrations facilitate sexual interactions, whereas noise disrupts the perception of signals from conspecifics and hosts. Despite evidence that mosquitoes respond to sound frequencies beyond fundamental ranges, including songs, and that males and females need to struggle to harmonize their flight tones, the behavioral impacts of music as control targets remain unexplored. In this study, we examined the effects of electronic music (Scary Monsters and Nice Sprites by Skrillex) on foraging, host attack, and sexual activities of the dengue vector Aedes aegypti. Adults were presented with two sound environments (music-off or music-on). Discrepancies in visitation, blood feeding, and copulation patterns were compared between environments with and without music. Ae. aegypti females maintained in the music-off environment initiated host visits earlier than those in the music-on environment. They visited the host significantly less often in the music-on than the musicoff condition. Females exposed to music attacked hosts much later than their non-exposed peers. The occurrence of blood feeding activity was lower when music was being played. Adults exposed to music copulated far less often than their counterparts kept in an environment where there was no music. In addition to providing insight into the auditory sensitivity of Ae. aegypti to sound, our results indicated the vulnerability of its key vectorial capacity traits to electronic music. The observation that such music can delay host attack, reduce blood feeding, and disrupt mating provides new avenues for the development of music-based personal protective and control measures against Aedes-borne diseases.

1. Introduction Despite advances in control technologies, diseases transmitted by Aedes mosquitoes—i.e., Zika, chikungunya, and dengue—are still serious public health problems worldwide (Wilder-Smith et al., 2017; Roiz et al., 2018). As with most mosquito-borne diseases, efforts to prevent or reduce the incidence of Aedes-borne viral illnesses have mainly focused on the application of insecticides (Caetano and Yoneyama, 2001; WHO, 2010; Abad-Franch et al., 2017). Although this strategy has historically been useful in managing mosquito-borne diseases (WHO,

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2014), there have been no major successes in recent decades (Rivero et al., 2010; Corbel and N’guessan, 2013; Patterson et al., 2016). In addition to the increased resistance to all four classes of insecticide used to date (Dia et al., 2012; Chanda et al., 2016; David et al., 2018), these agents have a number of other disadvantages, including biodiversity loss and adverse effects on the health of animals (Lawler, 2017) and humans (Peterson et al., 2006), which have led to anti-pesticide activism (Logomasini, 2004; Nicolopoulou-Stamati et al., 2016) and calls for the development of more environmentally friendly strategies (Patterson et al., 2016; Poulin et al., 2017).

Corresponding author at: Mosquito Research & Control Unit, 99 Red Gate Rd., P.O. Box 486, Grand Cayman KY1-1106, CAYMAN ISLANDS. E-mail address: [email protected] (H. Dieng).

https://doi.org/10.1016/j.actatropica.2019.03.027 Received 26 January 2019; Received in revised form 24 March 2019; Accepted 24 March 2019 Available online 25 March 2019 0001-706X/ © 2019 Published by Elsevier B.V.

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harmonium (a keyboard instrument) or an audio-oscillator (an instrument that produces radio frequency signals) interrupted acoustic and mating communications of insects and suggested that music has the potential to disrupt mating. Both leafhoppers and planthoppers communicate with their sexual partners by sound vibrations (Gillham, 1992; Eriksson et al., 2011). Although mosquitoes, including dengue vectors, have the most acute sense of hearing known among invertebrates (Göpfert and Robert, 2000) and also use acoustic vibrations for sexual recognition and mating (Robert, 2009; Vigoder et al., 2013), the role of music-derived vibrations as they affect mosquito behaviors remains unexplored. Any nearby source of vibrations resonating at frequencies different to the harmonic frequencies needed by male and female mosquitoes to listen to each other via adjustment of their flight tones (Robert, 2009) may alter sexual recognition and mating success. Although it has been reported that dengue mosquitoes can create and listen to love songs (Cator et al., 2009; Robert, 2009), this is typical of humans (Feng, 2012), who throughout history have produced a diversity of musical genres (Ahrendt, 2006). Despite of differences in pitch, rhythm, and dynamics of genres, one common factor is that music consists of a mixture of sounds with discrete and rational frequencies with a discernible dominant frequency (Nisha and Sorupani, 2018). Although music is uniquely human (Feng, 2012), devices for playing music are increasingly been used in insect studies (Saxena and Kumar, 1980; Ikeshoji and Yap, 1990; Mankin, 2012; Barton et al., 2018). Recently, it has been shown that rock music can alter interspecific interactions and cause negative effects on insect feeding activity (Barton et al., 2018). Despite evidence that dengue mosquitoes have an acute sense of hearing (Göpfert and Robert, 2000), are responsive to sound frequencies beyond the fundamental range (Ikeshoji and Yap, 1990; Cator et al., 2009), and that both sexes exert effort to hear each other (Robert, 2009), whether music has behavioral effects on these insects remains unknown. The present study was performed to examine whether electronic music influences the foraging activity, blood feeding and mating successes of the dengue vector, Aedes aegypti.

The majority of non-chemical insect pest management strategies involve manipulation of the target organism’s behavior by direct attraction and repellence, or indirectly by disrupting key behavioral traits, such as host seeking, blood feeding, and mating (Foster and Harris, 1997; Polajnar et al., 2014). Polajnar et al. (2014) suggested that effective containment requires incorporation of knowledge about the target’s sensory modalities. In mosquitoes, blood meal uptake and mating are closely related and play key roles in both population persistence and disease occurrence (Dieng et al., 2018a). Blood feeding is the principal process by which disease transmission occurs (Foster, 1995; Lehane, 2005), as it facilitates the transfer of infectious agents to and from the host (Dieng et al., 2018a). In dengue vectors, such agents can be transferred via mating (Martins et al., 2012), a prerequisite for the production of subsequent generations (Dieng et al., 2006) and increasing population density (Dieng et al., 2018a). In fact, in addition to their strong preference for human hosts (Takken and Knols, 1999; Oliva et al., 2013), the effectiveness of mosquitoes as disease vectors is closely related to their multi-gonotrophism. Male mosquitoes transfer sperm to the female during mating (Helinski and Harrington, 2011; Alfonso-Parra et al., 2016). After a single successful insemination, female mosquitoes store sperm for their whole lifetime, using it to fertilize eggs whenever a blood meal is ingested (Clement, 1992; Childs et al., 2016). Such mating acts necessitate a sequence of behavioral rituals (Klowden and Zwiebel, 2005) in which flight is an essential component (Belton, 1994; Spitzen and Takken, 2018). In aedines, mating is initiated in swarms—an assembly of males displaying flight patterns (Klowden and Zwiebel, 2005) or single male–female pairing (Oliva et al., 2013) in the vicinity of a marker that is mostly a warm-blooded organism (Anderson, 1974; Jeanson, 1985). Both males and females produce sounds through the beating of their wings (Belton, 1994), which generates vibrations (Alexander, 1957). For successful mating to occur, the male must harmonize its flight tone with that of its partner using auditory sensitivity, mediated by the antennae and Johnson’s organs (Robert, 2009; Gibson et al., 2010). In dengue vectors, males and females possess different fundamental frequencies (male: 600 Hz; female: 400 Hz, Cator et al., 2009). There is evidence that both sexes can hear (Robert, 2009) and share harmonic frequencies beyond the fundamental frequency of their respective flight tones (Clement, 1999; Cator et al., 2009). Although sound has been reported to play an important role in the sexual interactions and survival of many insects (Alexander, 1957; Polajnar et al., 2014), including mosquitoes (Clement, 1999; Robert, 2009; Cator et al., 2009), there is evidence that sound has detrimental effects on insect acoustic signal perception, courtship, and feeding activities. Costello and Symes (2014) reported that male tree crickets were less likely to begin calling in the presence of road noise, which they suggested decreases the ability of females to assess and compare males. Lee et al. (2011) examined the feeding activity on plant hosts in the presence of acoustic stimuli with different frequencies and intensity levels by the aphid Myzus persicae. A broad range of frequencies (100–10,000 Hz) appeared to effectively suppress phloem feeding in this insect. Barton et al. (2018) investigated the direct effects of anthropogenic sounds on the growth of plants and their herbivorous pests, as well as predation activity of an insect biocontrol agent. Some sounds were shown to decrease predation rates of Coccinellidae lady beetles, thereby indirectly increasing aphid abundance and decreasing plant biomass. There is also evidence that incidental vibrations trigger startle responses in many insects. Dropping a metal weight on a plant or a surface connected to the plant has been reported to induce cessation of activity by the Colorado potato beetle (Polajnar et al., 2014). In situations similar to those described previously by these authors, cessation of blood feeding and flying away from human hosts are often observed after vibrational cues emitted by strong noise or slapping close to a blood feeding female (Dieng et al., unpublished data). In experiments using leafhoppers and planthoppers, Saxena and Kumar (1980) reported that sound vibrations generated by a

2. Materials and methods 2.1. Colony maintenance and experimental mosquitoes The Ae. aegypti strain used in this study originated from a colony established in 2017 at the Entomology Unit of the Faculty of Resource Science and Technology (University Malaysia Sarawak, Kota Samarahan) under controlled environmental conditions (temperature 26 °C–28 °C; relative humidity 75%–85%; and light:dark photoperiod of 14:10 h). Samples of eggs were flooded in aged tap water for 24 h and the resulting newly hatched larvae (NHLs) were reared at densities ranging from 50 to 100 per plastic tray containing 1.2 L of tap water (As One Corporation, Osaka, Japan). They were supplied with diet consisting of cat food pellets as follows: one pellet was supplied to NHLs in powdered form by spreading onto the water surface; developing larvae were provided two pellets every 2 days. When necessary, the rearing medium was replaced once before completion of larval development. Pupae were collected in small plastic vials, which were transferred into mosquito cages. Emerging adults had permanent access to a sugar diet (10% of sucrose solution). To produce experimental adults, pupated individuals were singly placed into 1.5-μl of Eppendorf tubes and upon emergence the gender of adults was determined under a stereomicroscope (SZ-LED; Kenis, Osaka, Japan). Males and females were placed in two separate cages (30 × 30 × 30 cm, BugDorm; MegaView Science Co, Ltd, Taichung, Taiwan) labeled “virgin male cage” and “virgin female cage,” respectively, to avoid any mating events prior to bioassays requiring virgin adults. For convenience, “virgin males” and “virgin females” are referred to as VMs and VFs, respectively.

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2.4. Testing To examine whether the music had an impact on the behaviors of Ae. aegypti, 10 VFs that had been starved for 12 h were placed in the OA containing one virgin conspecific male (VM) and a restrained hamster. Immediately upon collective release of the VFs, an assistant turned the player on to play the song and an observer began recording the time to first visit and feeding attempt, number of visits, feeding events, and number of copulas within 10 min. On a different day, another OA was set up with the same experimental features, procedures, and data collection process as outlined above, but with no music being played (music-off, control). Ten additional replicates of the treatment [one OA with 10 VFs + 1 VM + restrained hamster + music-on] and eleven other replicates of the control [one OA with 10 VFs + 1 VM + restrained hamster + music-off] were set up on the same or other days and monitored as described for the first OAs. All observations were conducted during the day (10:00 and 17:00) under controlled environmental conditions identical to those described above. The observer was requested to avoid producing any sound during the 10-minute observation period.

Fig. 1. Audio file of the song “Scary monsters and nice spirits” used in the study.

2.2. Characteristics of the experimental song Electronic music was used in this study and the song “Scary Monsters and Nice Spirit” (duration, 4 min 5 s) by the American electronic music producer Skrillex was selected. The dynamics of the song were analyzed using a digital audio workstation (FL Studio 20; Image-Line, Gent, Belgium). The song was characterized as noisy based on the resulting vibragram and strong sound pressure/vibration with constantly rising pitches (Fig. 1).

2.3. Experimental features All bioassays were carried out essentially according to the same experimental design as described previously with some modifications (Dieng et al., 2017; Barton et al., 2018). The observation arena (OA) consisted of a standard mosquito cage (30 × 30 × 30 cm; BugDorm) placed on a fixed virtual square with a hamster restrained within a mesh device using round metal purse paper clips. The mesh device was positioned at the bottom center of the cage with the clips and half covered with hard plastic material. A speaker connected to a woofer equipped with a volume controller (2.1 Stereo speaker, Model F40; Sensonic, Johor, Malaysia) was placed close to the cage with the cone facing but not touching one of the netted sides. This positioning of the speaker was done to make sound audible within the cage while avoiding any vibration effects on the cage via the mesh netting. Music was played using Groove Music Software 2018 version (Microsoft Corp., Redmond, WA). The volume level of our music treatment (25 = 1 quarter of the maximal volume) was obtained by manipulating only the volume controller of the computer (Model A456U; AsusTek Computer Inc., Taiwan). The changes in sound pressure level during the playing of the song analyzed using the 2nd Gen Audio Interface (model: Scarlett 18i20, UK) are displayed in Table 1. All observations, each lasting for 10 min, were performed between 10:00 and 18:00 under controlled laboratory conditions (26 °C–28 °C, 75%–85% relative humidity, and 14:10 h light:dark photoperiod). For each observation, fresh adults experimental mosquitoes starved for 12–14 h (10 VFs and 1 VM) were used. The timing of selected mosquito female biting behavior rituals was recorded using a stopwatch on a smartphone (Huawei P9 lite; Huawei Technologies Co., Ltd., Shenzhen, Guangdong, China) with lap time features and tally counters (Daiso Co., Ltd., Hiroshima, Japan).

2.5. Data collection and analysis In both test and control bioassays, the time to first visit attempt by any one of the VFs on the mesh device was recorded immediately using a stopwatch after transfer of the VFs into the cage. A visit attempt was defined as a VF landing on the mesh device, as described previously (Dieng et al., 2018b). The time between the end of the collective transfer of the 10 VFs and a visit attempt was noted for each bioassay replicate of each bioassay type (test and control). In each of these cases, the mean ( ± SE) of these times (expressed in seconds) was considered as a measure of response time to the host. Any landing event where the VF remained on the mesh device for at least 5 s was taken as a visit. The mean number of visits was calculated and used as the visitation score. A VF landing on the uncovered part of the hamster and actively attempting to probe its skin was defined as a biting attempt, as described previously (Dieng et al., 2017). The time between the end of release of VFs into the cage and a biting attempt was recorded for each of the tested VFs in both test and control bioassays. The mean ( ± SE) of these times (in seconds) was used as a measure of the time to first biting attempt. A blood feeding event was defined essentially as described previously (Dieng et al., 2018b), but with slight differences: here, it was considered as the contact between the hamster skin and the mouthparts of the VF that lasted for at least 10–20 s. The subsequent numbers of feeding events were utilized to compute the mean values. Copulation was considered as any effective genital contact of a formed copula, which lasted for at least 10 s (Roth, 1948; Dieng et al., 2018c). The numbers of such sexual encounters were recorded for each replicate and bioassay type, and were used to calculate the means. The discrepancies in response time, visitation, time to first biting attempt, blood feeding events, and occurrence of copulation between test (music-on) and control (music-off) conditions were compared using the non-parametric Mann–Whitney U test (http://astatsa.com/WilcoxonTest). In all analyses, P < 0.05 was taken to indicate statistical significance.

Table 1 Variations of sound pressure level (in decibels: dB) during the playback of the song “Scary monsters and nice spirits” at low volume. Time length

SPL (dB)

0:00–0:30 0:30–1:00 1:00–1:30 1:30–2:00 2:00–2:30 2:30–3:00 3:00–3:30 3:30–4:05

−25 −22 −23 −24 −23 −24 −20 −26

to to to to to to to to

3. Results 3.1. Response times

−15 −14 −12 −15 −14 −11 −13 −15

Ae. aegypti females showed significant variations in response time to the presence of a host related to audio player status (Mann–Whitney U test, z = 2.98, P = 0.001). Females showed a delayed response to the presence of a host when the music was playing (131.30 ± 19.04 s, range 17–235 s) compared to when the player was turned off (35.22 ± 11.06 s, range 12–115 s) (Fig. 2). 95

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Fig. 2. Response times by Ae. aegypti females when provided chances to take blood meals from a vertebrate host when the audio player loaded with the electronic music was on or turned off.

3.2. Timing of first biting attempt

Fig. 4. Host visitation by Ae. aegypti females with reference to the audio player status (ON or OFF) when loaded with the electronic song.

The time to first biting attempt of Ae. aegypti varied appreciably according to the audio player status (Mann–Whitney U test, z = 2.57, P = 0.005). The time taken by females to initiate probing on the host skin in the music-off environment was 82.44 ± 23.11 s (range 21–235 s), while the corresponding time for the music-on environment was 191.70 ± 29.69 s (range 69–372 s) (Fig. 3).

levels (music-on and music-off conditions), but at different rates. The mean proportion of feeding events was significantly lower in the musicon environment compared to the music-off environment (4.00 ± 0.55 (range 2–7) vs. 6.63 ± 0.85 (range 1–11), respectively; Mann–Whitney U test, z = 2.28, P = 0.011) (Fig. 5).

3.3. Visitation responses

3.5. Copulation activity

Although Ae. aegypti females visited the host regardless of whether the music was on or off, the visitation level differed significantly between the two conditions. The mean number of visits to the host in the music-off environment was 11.72 ± 1.29 (range 3–8) visits, while the corresponding value when the audio player was turned on was 7.00 ± 1.19 (range 2–12) visits (Mann–Whitney U test, z = 2.14, P = 0.015) (Fig. 4).

Copulas were observed when Ae. aegypti females and lone males were allowed to cohabit in both the music-off and music-on environments, but their occurrence differed between the two conditions. Copulation was extremely rare when adults were exposed to music (mean number of copulation events: 0.90 ± 0.40, range 0–4), while the mean number was 5.18 ± 0.99 (range 1–12) in the music-off environment. This difference was significant (Mann–Whitney U test, z = 3.20, P < 0.001) (Fig. 6).

3.4. Patterns of blood feeding activity Ae. aegypti females provided with an opportunity to take blood meals from a live host fed under both conditions with different sound

Fig. 5. Mean ( ± SE) number of blood feeding events by Ae. aegypti with reference to the audio player status (ON or OFF) when loaded with the electronic music.

Fig. 3. Mean ( ± SE) timing of first biting attempt of Ae. aegypti with reference to the audio player status (ON or OFF) when loaded with the electronic music. 96

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respectively), and attacked the hosts much later (191.70 vs. 82.44 s, respectively) than those maintained in the noiseless environment. In addition, there was a clear link between the presence of sound and blood feeding activity, with more feeding events in the silent environment than that where music was being played. The behavior patterns in blood feeding insects, including dengue vectors, leading to blood meal uptake are divided into the phases of foraging (Walker and Edman, 1985) and actual feeding (Labuda and Kozuch, 1989). Foraging consists of appetitive searching, activation/ orientation, and attraction (Lehane, 2005), whereas blood uptake incorporates a searching sequence (Moreira et al., 2009), stylet penetration (Ribeiro, 1987), overpowering host defenses (Walker and Edman, 1985), and seizing capacity (Clement, 1992). These behavior patterns are not arranged hierarchically (Lehane, 2005) and when a host is present, hunger has often been linked to the instant activation of appetitive searching (Klowden, 1986; Lehane, 2005; Dieng et al., 2016). In the present study, females tested in both the presence and absence of music had the same starvation period, as such behavioral responses due to differences in hunger status are unlikely. The reports cited above and the results of the present study suggested that the sound vibrations from the speaker during playback of the electronic music reduced stimulation of the early phases of host location, i.e., appetitive searching, activation/orientation, and detection. It is plausible that the presence of sound vibrations and continuous pitches induced stress and fear, thus resulting in a quiescent state in the insects. As special care was taken to ensure that the experimental hosts were restrained with no defensive reactions, the decreased blood feeding activity observed in the music-on environment suggested possible interference with the probing process by sound vibrations—e.g., exploration of suitable blood vessel, insertion of stylet, or tarsal gripping ability. Epidemiologically, delayed female mosquito host attack will likely result in unsuccessful blood meal uptake, a decreased likelihood of virus pickup or transfer, and thus reduce the probability of disease occurrence. In addition to biting by females during a blood meal, Aedes-borne arboviruses that cause dengue can be transmitted to other mosquitoes during mating (Rosen, 1987; Fontaine et al., 2018; Sánchez-Vargas et al., 2018). Exposure to electronic music markedly reduced the copulation success of Ae. aegypti. Adults maintained in an environment with music copulated far less often than those kept without music. Similar observations have been reported in many arthropods, including insects. Saxena and Kumar (1980) exposed leafhoppers and planthoppers to sounds with a frequency of 200 Hz via a harmonium or an audiooscillator and observed interruption of their mating communication. Therefore, they suggested that music is a potentially effective mating disruptor. Costello and Symes (2014) reported delayed starts and reduced incidence of mating calls by male crickets associated with the occurrence of road noise. They suggested that the increase in courting latency occurred due to the inability of conspecific females to assess and evaluate the quality of males in noisy environments. Hartbauer et al. (2012) examined noise signal perception in crickets (Mecopoda elongata) in which males exhibit synchronous mating signaling. They reported that loud noise perturbed the synchrony of the male chorus, and they attributed this effect to masking interference by the noise that obscured conspecific signals. Similar to other reports (Brumm and Slabbekoorn, 2005), they also suggested that noise can impede discernment of conspecific signals when there is overlap in the beat content and the intensity exceeds a critical threshold. Gordon and Uetz (2012) investigated the effects of acoustic noise on sexual communication and mating success in an arthropod (spider). They showed that noise affected the capacity to recognize partners, and reduced female responsiveness to male courtship, resulting in fewer mating events and a reduced level of mating success. Mosquitoes also use acoustic communication and songs to find mates. Males must go through a range of stereotypical actions before sperm and seminal material transfer into the female, including pursuit, tarsal contact, ventral orientation, terminalial contact, and grasping on the female (Jones and Wheeler, 1965;

Fig. 6. Copulation activity of Ae. aegypti in two environments with different musical sound occurrence levels (no-sound and music being played).

4. Discussion To our knowledge, this is the first study to test the impacts of music, which is unique to humans (Feng, 2012), on the behaviors of a dengue vector. Experiments using the popular electronic song “Scary Monsters and Nice Sprites” by Skrillex using the Groove Music audio player and a speaker indicated that playing this song near a warm-blooded vertebrate host delayed attack, reduced blood feeding, and disrupted mating by Ae. aegypti. We used this electronic song in the experiments due to its loudness and pitch, which are two factors considered to contribute to noisiness (Nisha and Sorupani, 2018). Although we did not assess its amplitude and frequency, the strong sound pressure/vibration and constantly rising pitches emanating from the speaker placed close to the experimental cages indicated that the song was loud and noisy. Females exposed to such music began foraging later than their counterparts in the music-off environment. There has been a great deal of work regarding the effects of sound on arthropods, including insect disease vectors. Noise is associated with detrimental effects on the feeding activities of insects (Acheampong and Mitchell, 1997; Lee et al., 2012). Using different music treatments at varying volumes (sound-off and sound-on) and computer speakers, Barton et al. (2018) reported that exposure to a rock song (“Back in Black” by AC/DC) and a mix of urban sounds was associated with reduced feeding on prey by the lady beetles, resulting in a marked increase in the aphid population. They suggested that sound vibrations emanating from speakers struck the beetles and provoked a reduction in predatory activities. Such delayed latency to respond to nutritional sources and reduced feeding response in the presence of noise has been reported in many other animals. Villalobos-Jiménez et al. (2017) reported that noise decreased feeding behavior of aquatic insects compared to quiet conditions, even when food resources were readily available. Some groups have suggested that noise causes stress (Simpson et al., 2015) and masks acoustic signals, thus making detection of food difficult (Siemers and Schaub, 2011). Female mosquitoes are bloodsucking animals (Lehane, 2005), and bloodsucking itself represents a form of predation with humans as the prey (Weeks and D’Antonio, 2001). In the present study, in which the experimental procedures were similar to those reported by Barton et al. (2018), all female mosquitoes were starved for 12–14 h, and hunger has been often linked to instantaneous appetitive searching when a host is present (Klowden, 1986; Lehane, 2005; Dieng et al., 2015). Following exposure of Ae. aegypti to the electronic music, all behavioral responses involved in host location and contact were delayed: females exposed to music initiated foraging significantly later, visited hosts 3.72-fold later (131.30 vs. 35.22 s, respectively) and less frequently (7 vs. 11.72 times, 97

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Klowden and Zwiebel, 2005; Ponlawat and Harrington, 2009). Most of these behavior patterns involve flight (Belton, 1994) during which the wing beats generate tones, referred to as songs (Arthur et al., 2014). For mating to occur, courting partners must adjust their flight tones to produce a common frequency (Robert, 2009; Arthur et al., 2014). Although considered stereotyped, such sexual behaviors are not fixed and can respond to other stimuli, as mosquitoes can modify their flight tones to converge or diverge (Robert, 2009). Adult mosquitoes of the same sex have been reported to avoid synchronizing (Vigoder et al., 2013). In addition, the frequency range of mosquito hearing is highly variable and can include high-frequency sound signals (2000 Hz) in dengue mosquitoes (Cator et al., 2009). Moreover, sound impact studies in arthropods that exhibit auditory courtship have shown that noise can render conspecific signals ambiguous (Hartbauer et al., 2012), diminish partner recognition ability (Gordon and Uetz, 2012), interrupt mating communication (Saxena and Kumar, 1980), or reduce mating chances (Costello and Symes, 2014). Any strong incidental vibration that resonates at frequencies higher than those required for normal acoustic sexual communication will likely alter the probability of mating success. Thus, the low copulation success of Ae. aegypti observed in our study may have been due to the interference by the electronic music with either VM courtship performance or VF receptivity to courtship. It is possible that some musical vibrations would have added to those of wing beats, thus offsetting attempts to synchronize flight tone. The vibrations emitted from the speaker may have had inhibitory effects on tarsal contact, orientation, and seizure of VFs by the VMs. Reduced flight activity of either VFs or lone VMs may also explain the reduced copulation success, as acoustic maneuvers for mating generally occur in the air. In support of this suggestion, noise has been shown to cause quiescence in insects that use acoustic communication for their sexual behaviors (Polajnar et al., 2014; Barton et al., 2018).

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5. Conclusions This was the first formal attempt to document the effects of music on the behaviors of a mosquito vector. The music tested in the present study was clearly detrimental to Ae. aegypti; females exposed to music showed a longer response time to the mammalian host and reduced blood feeding activity compared to their non-exposed counterparts. Furthermore, exposure to music resulted in a very low incidence of copulation. Thus, the electronic music acted both as an anti-mosquito attack factor and as a mating disrupting agent. These properties suggest the potential for development of music-based mosquito vector control strategies. As music is loved by many people, the development of musicbased anti-mosquito control measures may represent an appealing alternative to strategies involving the use of harmful chemical insecticides. Acknowledgments The authors are grateful to Rahah Binti Mohammad Yakup of the Institute of Biodiversity and Environmental Conservation, University Malaysia, Sarawak. This work was financially supported by the Department of Microbiology, Faculty of Pharmaceutical Sciences, Fukuoka University, Japan. References Abad-Franch, F., Zamora-Perea, E., Luz, S.L.B., 2017. Mosquito-disseminated insecticide for citywide vector control and its potential to block arbovirus epidemics: entomological observations and modeling results from Amazonian Brazil. PLoS Med. 14 (1), e1002213. Acheampong, S., Mitchell, B.K., 1997. Quiescence in the Colorado potato beetle, Leptinotarsa decemlineata. Entomol. Exp. Appl. 82, 83–89. Ahrendt, P., 2006. Music Genre Classification Systems—A Computational Approach. Dissertion of Technical University of Denmark, Kongens Lyngby, Denmark. Alexander, R.D., 1957. Sound production and associated behavior in insects. Ohio J. Sci. 57 (2), 101.

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