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Behavioral and molecular responses of Aedes aegypti to ultrasound

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Highlights

  • This study characterized behavioral and molecular responses of Aedes aegypti to ultrasound.

  • Ultrasound disrupted CO₂-oriented olfactory behaviors of Aedes aegypti.

  • Long time exposure to ultrasound reduced host finding ability of mosquito.

  • Ultrasound changed gene expression level of CO2 receptor and putative hearing related gene.

  • We suggest that ultrasound can be useful repellent for mosquito control.

Abstract

Mosquito-borne infectious diseases cause mortality and global infectious disease burden worldwide. There are several electronic mosquito repellents (EMRs) based on ultrasound have been developed and commercialized to reduce human-mosquito contacts. However, the efficacy of EMRs against mosquitoes is still unclear. In this study, we present experimental evidence that ultrasound of different frequency and sound pressure differentially affects the host-seeking behavior of Aedes aegypti females. Behavioral tests were accompanied by molecular experiments to check whether mosquitoes respond to ultrasound and are there any changes in specific mRNA expression. Experiments in bioassays revealed that the ultrasound of 100 kHz frequency and 90–110 dB pressure significantly disrupted CO₂-oriented olfactory behaviors and blocked indoor invasion. Furthermore, a long time (>24 h) exposure to 100 kHz frequency/90 dB pressure of ultrasound decreased attractive behaviors to human skin. At the molecular level, there was no change in expression of odorant receptor co-receptor (AaOrco) in ultrasound treated animals, while one of the CO2 receptor genes, AaGr3, and putative hearing-related gene, AAEL009258, were down-regulated and up-regulated, respectively. Our study indicates that high frequency (100 kHz) and pressure (90–110 dB) of the ultrasound has repellent effects to olfactory–driven behaviors of mosquitoes.

Introduction

Aedes aegypti L. (Diptera: Culicidae) is a primary vector of dengue virus, a cause of morbidity and mortality in tropical and subtropical areas of the world (Guzman and Harris, 2015, Kurane, 2007). This virus increases recent decades with 3.9 billion people in 128 countries at risk for infection and 390 million people infected annually (Brady et al., 2012). Ae. aegypti also is a vector of yellow fever, chikungunya and zika virus (Epelboin et al., 2017). Global climate change, travel, and migration cause the expansion of Ae. aegypti resulting in increased public health threat (Liu-Helmersson et al., 2014). There are several environmental, biological, chemical and electronic methods to reduce human-mosquito contacts (Enayati et al., 2007, World malaria report, 2018). Strong demand for personal anti-mosquito equipment is coupled with a supply of chemical and electronic mosquito repellents (EMRs) (Enayati et al., 2007, Tavares et al., 2018). Although chemical repellents demonstrate a comparatively strong anti-mosquito effect (Tavares et al., 2018), they also have a side effect on human health, apparel, and architecture (Barradas et al., 2013, The Medical Letter, 2016). EMRs, in contrast, are a more convenient, safe and eco-friendly method of mosquito control (Enayati et al., 2007, Okorie et al., 2015). However, the efficacy of ultrasound-based EMRs, has not been verified.

Ultrasound is a sound waves with frequencies higher than 20 kHz that are not audible to the human (Heffner and Heffner, 2007). Mosquitoes auditory organ is well described (Albert and Kozlov, 2016, Belton, 1994, Nadrowski et al., 2011, Na et al., 2016, Römer, 2018) and it is suggested that Ae. aegypti hears up to 2 kHz sound (Cator et al., 2009, Menda et al., 2019). But the sensitivity of Ae. aegypti to high-frequency ultrasound is still unclear. Interestingly, the mosquitoes predators (Gonsalves et al., 2013) bats use ultrasound to navigate and hunt insects (Schnitzler and Kalko, 2001, Simmons et al., 1996). Some insects species observed avoidance behavior against bat-ultrasound cries, due to the coevolutionary arms race with insectivorous (Conner and Corcoran, 2012, Yager, 2012). For malaria vector mosquito Anopheles gambiae the startle response to 35–60 kHz cry of African sheath-tailed bat Coleura afra was observed (Mang’are, 2015, Okorie et al., 2015). We note that Ae. aegypti is a day-time mosquito, consequently, its coevolutionary relationship with bats is questionable. Meanwhile, this is not a reason to rule out the possibility that Ae. aegypti may respond to ultrasound. EMR manufacturers use two rationales to explain the repellency of ultrasound on mosquitoes: (1) inseminated females avoid the high-frequency wing beat of males; (2) mosquitoes avoid the ultrasound cries of bats (Foster and Lutes, 1985). Actually, the mosquitoes flight sound frequency is much lower than ultrasound. This raises serious doubts about the consistency of the first explanation (Chapman, 1982, Michelsen and Larsen, 1985). The second explanation is also disputed by Mankin (Mankin, 2012) and by Enayati et al. (2007).

To date, EMRs efficacy to mosquitoes is not approved (Ahmad et al., 2007, Andrade and Bueno, 2001, Foster and Lutes, 1985, Lewis et al., 1982) and disputed (Enayati et al., 2007, Mankin, 2012). The devices used in these studies had sound frequencies of 2–100 kHz and sound pressure up to 115 dB. Also, constant and random sound patterns at various frequency range were tested. These studies suggest that sound alone is not effective against mosquitoes. Studies on Ae. aegypti showed that the blend of stimuli gains higher response than these stimuli alone (Pang et al., 2018, van Breugel et al., 2015). In the wild mosquitoes integrate an array of attracting and startling sensory information while host-seeking. We tested the hypothesis that the ultrasound in combination with wind and CO₂ may gain higher response enough to disorder host-seeking behavior of Ae. aegypti females. It was recently shown (Pang et al., 2018, van Breugel et al., 2015) that a combination of different stimuli shifts the ratio of upwind turns of mosquito's host-seeking fly, that also supports our hypothesis. To test this hypothesis we carried out various behavioral experiments with the ultrasound of different frequency and pressure in combination with wind and CO₂. The behavioral experiments were accompanied by molecular tests to check whether mosquitoes respond the ultrasound and are there any changes in host-seeking or hearing-related mRNA expression.

Section snippets

Mosquito culture

Ae. aegypti non-blood-fed inseminated 5–7 days females were used in all experiments. The stock cultures of Ae. aegypti (originated from the National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul, Korea) maintained in temperature-controlled insect rearing rooms of Seoul National University. Larvae were reared in 24 × 35 × 5 cm plastic trays containing 0.5 g of sterilized diet (40-mesh chick chow powder/yeast, 1/1 by weight). Adults were maintained on a 10% sucrose

Ultrasound has no effect on mosquitoes survival rate

Before behavioral experiments, we checked whether the ultrasound of high pressure has an insecticidal effect on Ae. aegypti. Using ultrasound of 90 dB that is the human-safe pressure we compared the survival rate of mosquitoes treated for 24 h at a different frequency. Our data demonstrate that high-pressure ultrasound of any frequency doesn't have an insecticidal effect on Ae. aegypti females (Fig. 1b).A subtle effect of 45 and 80 kHz waves was observed on mosquitoes survival rate, but the

Ultrasound in wind tunnel breaks up mosquitoes CO₂-oriented behaviors

We asked whether the ultrasound combined with a wind disorders Ae. aegypti female's CO₂-oriented flight. To test this hypothesis we created bioassay (Fig. 2a) combining CO₂, ultrasound, and wind in close to real manner. Our results show that 50 dB ultrasound has a subtle effect on Ae. aegypti females. The moderate repellency observed in all other tests with higher sound pressure. It is notable that the test with the ultrasound of the highest acoustic energy (100 kHz and 90 dB) demonstrated best

Discussion

Several studies demonstrated a frustrating inability of commercial ultrasound based EMRs to repel mosquitoes (Ahmad et al., 2007, Enayati et al., 2007, Foster and Lutes, 1985, Jensen et al., 2000, Mankin, 2012, Schreck et al., 1984). In contrast to them the data provided by Hadi et al., 2009, Okorie et al., 2015 indicates that ultrasound has a significant repellent effect on mosquitoes. These contradictory findings tell us that the ultrasound of different frequency and pressure in different

Author contributions

D.K., R.I. and S.L. and H.W.K. designed the experiment. D.K. performed overall experiments. D.K. and U.Y. carried out data analysis. D.K., R.I., U.Y., and H.W.K wrote the main manuscript text. All authors reviewed the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This research was supported by the Postdoctoral Fellowships in the Incheon National University (2018).

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    These authors contributed equally to this work.

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