Comparative Analysis of Acoustic Propagation Parameters of Natural Sounds of Anopheles gambiae s.s and Odorrana tormota Significant in Mosquito Startle

Acoustics of varied frequency ranges generated naturally by animals or artificially by electronic devices have shown startle effect to insects. It has been shown that mosquitoes use the reactive near-field in antennae communication with negative phonotaxis in male Aedes diantaeus evoked by low frequency acoustic signals of a carrier frequency 140–200 Hz. Also, studies with the 35-60 kHz Odorrana tormota sound recorded a 46 % repellence in female Anopheles gambiae, the malaria vectors. Declining malaria morbidity and mortality is attributed to current vector and pathogen interventions. However, the rate of decline in malaria morbidity and mortality is impeded by buildup of resistance in pathogens and vectors to chemicals. This study therefore characterised animal sounds essential for further investigation in the control of malaria through mosquito startle. The research determined, analysed and compared the acoustic propagation parameters of the recorded natural sounds of the male Anopheles gambiae, female Anopheles gambiae and Odorrana tormota using Avisoft SASLAB Pro and Raven Pro 1.5. All sounds were observed to have frequency modulation with harmonics stretching to ultrasonic levels. Uniquesly, the sound of O. tormota showed constant frequency modulation. The pupae of A. gambiae were reared in vials quarter filled with water and covered with a net at 60-80 % humidity, 25±2 °C temperature and equal light-darkness hour cycle at Kenya Medical Research Institute (KEMRI) entomology laboratories. The parameters showed a significant deference in fundamental frequency (maximum entire), Peak amplitude (maximum), peak amplitude (mean), Peak amplitude (mean entire) and peak amplitude (maximum entire) of the sound of male A. gambiae and O. tormota (p < 0.05). The maximum frequency (minimum entire) of both sexes of A. gambiae was equal (1.90 kHz) with variability being observed in maximum frequency (end), maximum frequency (maximum), maximum frequency (mean), maximum frequency (maximum entire) and maximum frequency (mean entire). Frequency (maximum). A paired samples t-test comparison of the maximum frequency (mean), maximum frequency (maximum), maximum frequency (end), maximum frequency (maximum entire) and maximum frequency (mean entire) of the sound of the female A. gambiae and male A. gambiae indicated no significant difference between the sounds (p > 0.05). The maximum frequency (mean) of the sounds of both sexes of A. gambiae correlated highly negative (r = −0.658). The bandwidth (end), bandwidth (maximum), bandwidth (maximum entire), peak amplitude (mean) and bandwidth (mean entire) of the sound of the male compared with female A. gambiae differed significantly. The signal power for the non-pulsate sounds of the male A. gambiae remained almost constant at 80 dB from 10 kHz to 65 kHz beyond which the acoustic energy declining to 45 dB. Also, the sounds of the female A. gambiae did not exhibit any spikes in power but remained steady at 85 dB from 10 kHz up to 60 kHz beyond which the acoustic energy declined to 50 dB. The signal power of the pulsate sound of O. tormota was 89 dB. The propagation parameters of the male mosquito and O. tormota compared favourably indicating its potential in the startle of the female mosquito. The author summary Philip Amuyunzu Mang’are is a PhD. Physics student in Egerton University. He has authored many papers and books. He is currently a Lecturer of Physics (Electronics), Masinde Muliro University of Science and Technology. He is a member of the Biophysical Society and the current President of Biophysical society (Kenya). Prof. Ndiritu F. Gichuki, is a Professor of Physics Egerton University. Currently he is the Registrar Academic Affairs in Chuka University. His vast experience has seen him supervise many postgraduate students who have taken key positions in the society. Prof. Samwel Rotich is a Profesor of Physics in Moi University specialising in Electronics. He has a wide experience in Physics and Biophysics. He is a registered member of the Biophysical Society and the Patron of Biophysical Society Kenya Chapter. He has published many papers and supervised many postgraduate students. Dr. Makatiani Kubochi is a Lecturer in Moi University with vast experience in entomology. She has published many papers and supervised many postgraduate students. Dr. Rapando Bernard Wakhu is a renown theoretical Physicist with experience in acoustics and Fourier analysis based in Masinde Muliro University of Science and Technology. He has supervised many postgraduate students and published many papers.


Introduction
The Anopheles gambiae and the Odorrana tormota generate sounds naturally whose frequencies stretch to ultrasonic levels. The focus of this study was to comparatively analyse the acoustic propagation parameters of natural sounds of A. gambiae and O.
tormota. Optimal parameters determined from this study were essential in the investigation of the startle responsiveness of the African female A. gambiae to ultrasound and the design of a mosquito repellent.

The biology of the Anopheles gambiae and Odorrana tormota
Mosquitoes have four distinct stages in their life cycle which are recognized by their unique appearance; which include the egg, larva, pupa, and adult (CDC, 2010). The egg, larva and pupa are aquatic stages in the lifecycle of the A. gambiae and last 5-14 days, depending on the species and the ambient temperature (CDC, 2010). The non-drought resistant eggs having floats are between 0.47mm and 0.48 mm long, convex below and concave above, with the surface covered with a polygonal pattern (Gillies and de Meillon 1968;Foster and Walker 2009;CDC 2010). The organic matter and algae feeding larvae ranges between 5mm to 6 mm in size and lie parallel to the water surface in order to breathe due to lack of respiratory siphons (Gillies and de Meillon, 1968;Garros et al., 2008;Foster and Walker, 2009). The non-feeding pupae are comma-shaped when viewed from the side and are very mobile (Foster and Walker 2009;CDC 2010). Adult female Anopheles have palps which are as long as their proboscis, an average wing length varying from 2.8 to 4.4 mm and rest with their abdomen raised into the air (Gillies and de Meillon, 1968;Foster and Walker, 2009). The adult mosquitoes have slender bodies consisting of the head, thorax and abdomen; the head specialized for acquiring sensory information and for feeding. The mosquito antennae detects host and breeding sites odors (CDC, 2010). The head also has an elongated, forward-projecting proboscis used for feeding, and two sensory palps.
The adult stages of many mosquito species are feeders of blood, which has given some disease causing organisms a reliable mode of transmission to animal hosts. It is during the adult stage that the female Anopheles mosquito acts as malaria vector. The adult females can live up to a month or more in captivity but they don't live more than 1-2 weeks in nature (Antonelli et al., 2007). Both male and female adult mosquitoes feed on plant nectar, but the female feed on vertebrates' blood, for nutrients required for egg maturation (Foster and Walker, 2009). Female Anopheles mosquitoes lay eggs on the surface of the water at night and under favorable conditions, hatching occurs within one or two days and develop within the aquatic habitat. The A. gambiae larvae develop in permanent man-made structures and natural pools (Kweka et al., 2012). It is important to understand the lifecycle of the mosquito so as to effectively control them. Anthropophilic biting female mosquitoes not only irritate people and animals, but also transmit malaria. The body parts of the adult stage of the mosquitoes, mainly the antennae serve an important role in communication (Mohankumar, 2010).
The Odorrana tormota belongs to kingdom Animalia, Phylum Chordata, Class Amphibia, Order Anura and of the family Ranidae. The synonyms for Odorrana tormota are Amolops tormotus, Odorrana tormotus and Rana tormotus (Wu, 1977;Huiqing and Ermi, 2004;Frost;2013). The Odorrana tormota shelters in moist rock crevices during the day, and is found in thick brush alongside streams at night (Fei, 1999). The reproductive season begins in June and they lay eggs which are milky yellow in color, and measure about 2.0 mm in diameter (Fei et al., 1999). The existence of tadpoles has not been recorded for this species (Huiqing and Ermi, 2004). The Odorrana tormota is a frog restricted to Huangshan in Anhui Province, and Jiande and Anji counties in Zhejiang Province, China generating ultrasounds through vocal apparati (Arch et al., 2008;Shen et al., 2011). The frog uses the frequency range of up to 128 kHz for communication (Arch et al., 2008). During the reproductive season, males emit a variety of high-pitched calls at night with energy spectrums extending into the ultrasonic range (Feng et al., 2006;Feng et al., 2009). Recent research with the O. tormota calls showed some degree of downward frequency modulation with a subset of calls having a carrier of constant frequency (Feng et al., 2002;Arch et al., 2008). As observed in recent studies, ultrasound from O. tormota can play a critical role in malaria vector control by evoking startle responses in malaria vectors since the call frequencies stretch beyond the startle range of 38 -44 kHz in mosquitoes (Mohankumar, 2010;Mang'are et al., 2012). The sound of O. tormota having exhibited the greatest startle responses in mosquitoes needs to be further investigated besides the sounds of the male Anopheles gambiae (Mang'are et al., 2012).

Mosquito audition, communication and antennae theory
The mosquito has a pair of large, wraparound eyes, and a pair of long, hairy antennae; its ears projecting from the front of its face (Hoy, 2006). The antenna detects the particle velocity component of a sound field, which is restricted to the immediate vicinity of the sound source in acoustic near field. Ultrasound generated artificially or naturally is detected by mosquitoes evoking evasive response (Mohankumar, 2010). The mosquito flight tone is an unusual communication signal in that its production is directly linked to locomotion, only varying the carrier frequency (Arthur et al., 2014). The flight tone is characteristic of a species and can sometimes be used to identify species or count individuals (Raman et al., 2007). Mosquitoes use flight tones as communication signal with the male mosquitoes being attracted to real and artificial female flight tones (Wishart and Riordan, 1959).
The mosquito antennae consists of an elongated flagellum and a chordotonal organ housed within the pedicel at the base of each antenna called the Johnston's organ . The flagellum vibrates when stimulated by periodic air displacements (Göpfert and Robert, 2000). At its base, the flagellum tapers into a structure of 60-80 prongs which resemble the spokes of an upturned umbrella . The prongs are attached to many thousands of mechanosensory sensillae known as the scolopidia of the Johnston's organ.
Insect hearing organs are divided tympanal ears, which are sensitive to the pressure component of sound, and movement receivers, which respond to oscillations of air particles in the sound field (Hoy and Robert, 1996). Movement receivers are usually light structures such as sensory hairs or antennae that are deflected by the sound-induced oscillations of air particles. The antennae are sexually dimorphic, with males having a greater number of sensillae in the Johnston's organ (∼14,000 in males, ∼7,000 in females) and the greater number of fibrilla on the flagellum led to a conclusion that mating behavior involved audition (Gibson and Russell, 2006). The greater surface area of the male flagellum increases their responsiveness to particle displacement (Göpfert et al., 1999).
Mosquitoes are affected by ultrasounds in the range 38 kHz-44 kHz (Mohankumar, 2010). Ultrasound creates stress on the nervous system of mosquito jamming its own ultrasound frequency immobilizing it. Ultrasound also initiates avoidance behavioural response from the source (Mohankumar, 2010). The male's auditory system is selectively tuned to female flight frequencies of approximately 300Hz -400Hz with maximum intensity at 380 Hz for A. gambiae (Gibson and Russell, 2006). The sound is transmitted in air as frequency modulated wave (FM) and activates the antennae (Maweu et al., 2009). The female mosquitoes which detect the presence of male mosquitoes by sensing the 38 kHz ultrasound require blood meal for egg maturity after breeding (Baldini et al., 2013;Imam et al., 2014).
The bred Female Anopheles gambiae become refractory to the males to avoid further breeding. The antenna, with its fine senses movements of air particles impinging acoustic waves that excite the sensory receptors contained within the JOs to generate action potentials that flood into the insect's brain (Göpfert and Robert, 2000;Hoy, 2006). The sensillae mechanoelectrically transduce and amplify the nanometer sound-induced vibrations into electrical signals (Göpfert and Robert, 2000).
Experiments with recorded flight tones of mosquitoes showed that the individual male and female mosquitoes flew at mean fundamental wing-beat frequencies with males flying at significantly higher frequencies (∼700 Hz) than their conspecific females (∼460 Hz) . The auditory range was noted to be ∼2 cm (Pennetier et al., 2010).
The less sensitive female's Johnston's organ, compared to males, responds to antennal deflections of ± 0.00058 induced by ± 11nm air particle displacements in the sound field which surpasses other insects' sensitivity (Göpfert and Robert, 2000). Ultrasound causes neural stress to mosquitoes evoking a startle response. Mosquitoes like many other insects avoid bat ultrasonic sound that the electronic mosquito repellent devices imitate (Mohankumar, 2010).
The focus of this study is to analyse the acoustic propagation parameter of the sounds of the A. gambiae and O. tormota essential for further study on use the responsiveness of the African female A. gambiae to the sound.
Electromagnetic communication between insects has also been observed with the antennae playing the role of the receiver or transmitter (Abdolali et al., 2013). Many insects use antennal or cercal structures to detect air particle vibrations from near-field sound sources (Sane and McHenry, 2009). The antenna theory enhances the understanding of communication in mosquitoes (Maweu et al., 2009). The hairs on the mosquito antennae serve as the dipole lengths and are equally spaced, with the dipole lengths range from 0.05 to 0.14 cm for the A. gambiae, Culex pipiens and Aedes aegypti. It was established that there were 20 dipoles stemming from the 0.26 cm long transmission line (Maweu et al., 2009).
Basically, mosquitoes use the reactive near-field in antennae communication. Male mosquitoes require about 24 hours before their terminalia get rotated and their fibrillae mature enough to become erect and detect females whereas the female mosquitoes need 48-72 hours before they become receptive to males prior to blood feeding in the wild (Clements, 1992). Anopheles males can mate several times, but females become refractory to re-insemination and re-mating is rare (Mohankumar, 2010). Mating in anopheline mosquitoes occur during the early evening, primarily in swarms (Diabaté et al., 2011). The swarming males use their erect antennal fibrillae to detect a nearby female mosquito's wing beat frequencies (Clements, 1992). The audible frequencies received by the male antennae ranges from 150 Hz to 500 Hz and evoking sexual behaviour that involves shifting pitches of their buzzes until they synchronize. Same sex mosquitoes' sounds cannot converge (Nijhout and Craig, 1971). The case study on Toxorhynchites showed that the males harmonized their wing beat with females as they neared, for species recognition, before mating commenced (Gibson and Russell, 2006). Flying mated female mosquitoes produce familiar whining sound when searching for proteins (Maweu et al., 2009). This sound is very critical in this study as an audio switching system.

Electronic mosquito repellents (EMRs)
Electronic mosquito repellents (EMRs) are designed to repel female mosquitoes by emitting high-pitched sounds (Enayati et al., 2010;Ashim et al., 2017). There exist many electronic mosquito repellents on the market which manufacturers argue that they are effective in repelling or attracting mosquitoes, though not common in Africa. The Anti-Pic®, Mosquito Repeller® DX-600 and Bye-Bye Mosquito® electronic mosquito repellents have been studied in order to establish their effectiveness in mosquito repellence but none has supported the claims of their effectiveness in mosquito repellence (Andrade and Bueno, 2001). The electronic mosquito repellents that generates ultrasound at 38 kHz imitating the M ang'are P. A. et al.: Comparative Analysis of Acoustic Propagation Parameters of Natural Sounds of Anopheles gambiae s.s and Odorrana tormota male mosquitoes have been designed. It was also noted that in 12 of the 15 experiments, the landing rates of mosquitoes on the human participants in the groups with functioning EMR was actually higher than in the control groups (Enayati et al, 2010).
Ultrasound causes nervous stress to the mosquito and at the same time evoke fear due to predation or/and further mating depending on the source of the natural ultrasound (Mohankumar, 2010). Recent research findings with recorded natural ultrasound showed evidence of startle responses in mosquitoes (Enayati et al, 2010;Mohankumar, 2010;Mang'are et al., 2012). The studies about the repellence of the female A. gambiae using the hearing mechanism is still a viable venture based on the confirmation that in both males and females, the antennae are resonantly tuned mechanical systems that move as simple forced damped harmonic oscillators when acoustically stimulated, despite the differences in the hearing ability (Göpfert et al., 1999). Recent experiments demonstrated that tonal acoustic signals with a carrier frequency of 140-200 Hz yielded avoidance responses (negative phonotaxis) of male Aedes diantaeus swarming mosquitoes (Culicidae) in the range of 140-200 Hz (Lapshin and Vorontsov, 2018).

Statement of the Problem
High mortality and morbidity in Africa and the world at large is caused by malaria whose vectors are the mated female A. gambiae. Malaria has also caused a large economic burden to both individuals and countries in an effort to control the vectors or treat malaria. There is a slowed achievement of satisfactory reduction levels in mortality and morbidity due to buildup of resistance in chemicals used to treat malaria or control the vectors. Gate (model 112) adjusted to an appropriate level (50 %) to avoid over modulation and the recording level from the computer set to 20 dB, the recording button was pressed to record the sound. The sound was recorded from a swarm of 100 mated, 3 days old male and female A. gambiae at a sampling frequency of 500 kHz at 16 bit from a cylindrical glass cage covered at both ends with a netting as given in Figure 1. An aspirator was used to transfer mosquitoes from the resident cage to the cylindrical cage for sound recording.

The acoustic propagation parameters of sounds of male and female Anopheles gambiae s.s
The sound of both male and female Anopheles gambiae; and the sound of O. tormota were subjected to Fourier transforms and analysis for determination of acoustic propagation parameters. This was achieved through automatic parameter measurements. The following settings were made to the analysis softwares: from the tools option, the calibration was set to SPL with reference to sound and the SPL reference was 20µPa which is the threshold. For the parameter generation which include amplitude and energy, the calibration method was set to SPL with reference sound for Channel 1and at a /gain (dB) set to zero under the tools menu. The full-scale range was calibrated to 19.8936 Pa and 17.6345 Pa for the male and female A. gambiae respectively. The envelope was set to original waveform whereas the pulse detection was set to gate function. The Fast Fourier transform (FFT), an option under the spectrogram parameters was set to 512 and hamming window selected for the display.
The temporal resolution overlap was set to 50% with the colour palette set to graypal. The frame size was set to 100% for real time spectrogram parameters and the black and white box (B/W) checked for display.
The parameters determined using Avisoft SASLab Pro version 5.2 and Raven Pro. 1.5 included call duration, acoustic energy, peak frequency (mean), peak amplitude (mean), minimum frequency, maximum frequency (mean), bandwidth (mean), peak frequency (minimum entire), minimum frequency (minimum entire), maximum frequency (minimum entire), bandwidth (minimum entire), peak frequency (maximum entire), peak amplitude (maximum entire), minimum frequency (maximum entire), maximum frequency (maximum entire), peak frequency (mean entire), peak amplitude (mean entire), minimum frequency (mean entire), and maximum frequency (mean entire). The acoustic energy whose SI unit is Pa 2 s is a product of the square of amplitudes and sample time. The energy produced is the sum of the squared amplitudes multiplied by time . Also, 1 Pascal pressure is equal to SPL of 94 dB. The data obtained was transferred into an excel worksheet for editing and further analysis through the direct data exchange (DDE)/ Logfile settings. The acoustic propagation parameters of the natural sounds were analysed statistically using the same softwares and SPSS.  (Wishart and Riordan, 1959;Raman et al., 2007;Maweu et al, 2009, Arthur et al., 2014. Large modulations in males could indicate fitness in terms of the ability to fly and maneuver at suboptimal wingbeat frequencies (Arthur et al., 2014).  The sound of O. tormota is characterised by frequency modulation (FM) and constant frequency (CF) modulation as shown in Figure 4 as reported in recent findings (Feng et al., 2002;Arch et al., 2008). It is also characterised by harmonics and subharmonics nonlinear spectral features (Feng et al., 2009). There also exists signal breaks in the sounds of male A. gambiae, female A. gambiae and the O. tormota as evidenced in the spectrogram in Figure 2 and 4 associated with change in locomotion.

Acoustic Transmission Parameters of the sounds of A. gambiae
The acoustic parameters were determined and analysed using the Avisoft SasLab Pro 5.2 and Raven Pro 1.5 softwares. The analysed data for the minimum value, maximum value and mean is given in Table 1, 4 and 5.

Fundamental Frequency and Harmonics
The sounds studied involved a sample of 1161, 1496 and 2342 calls for the female A. gambiae, male A. gambiae and O. tormota respectively. The parameters for the sounds of the male and female A. gambiae are given in Table 1. The calls for both male and female A.
gambiae whose formants are given in the spectrogram given in Figure 3  gambiae was higher than that of the male A. gambiae. Both sexes exhibited equal fundamental frequency (mean), Fundamental (mean), Fundamental (Minimum entire) and Fundamental (start) of 900Hz as given in Table 1 The minimum frequency (end), minimum frequency (maximum), minimum frequency (maximum entire), minimum frequency (mean), minimum frequency (mean entire), minimum frequency (minimum entire) and minimum frequency (start) for the sound of the male A.
gambiae was 900 Hz which corresponded to its fundamental frequency. The minimum frequency (maximum entire) of the sound of the female A. gambiae exceeded that of the male A. gambiae by 1.00 kHz as given in Table 1. The peak frequency (end), peak frequency (mean), peak frequency (minimum entire) and peak frequency (start) for the sound of both sexes of the A. gambiae was 900 Hz. However, variation in peak frequency was observation

Bandwidth
The sound of both the male and female A. gambiae yielded values of the minimum, maximum and mean Bandwidth (minimum entire) of 900 Hz. Variation in parameters of the sound of the male and female A. gambiae was observed in the bandwidth (end), bandwidth (maximum), bandwidth (maximum entire), bandwidth (mean) and bandwidth (mean entire). The female A. gambiae was characterised by a wide bandwidth compared to that of the male A. gambiae in terms of Bandwidth (end), Bandwidth (maximum entire), Bandwidth (mean) and Bandwidth (mean entire) as illustrated in Table 4. However, the mean of Bandwidth (maximum) was widest in the male A. gambiae compared to that of the  The bandwidth (maximum entire) and bandwidth (

Amplitude
The study established statistical data for the amplitude parameters which included peak amplitude (end), peak amplitude (maximum), peak amplitude (maximum entire), peak amplitude (mean) and peak amplitude (mean entire) as given in Table 5. The peak amplitude (mean), Peak amplitude (mean entire) and peak amplitude (end) for the sound of the male A. gambiae were greater than that of the female A. gambiae by 0.9758 Hz and 0.9728 Hz respectively as given in Table 5 negative correlation (r = -0.770). The peak amplitude (maximum) and peak amplitude (maximum entire) for the sound of the male and female A. gambiae were equal as given in Table 5. Also, the amplitude (maximum) and peak amplitude (   tormota formants also showed constant frequency (CF). All sounds consisted of several harmonic segments with energy extending into the ultrasonic range, at some instances, exhibiting signal breaks, subharmonics and a chaotic nonlinear characteristics. The sound of O. tormota was also characterised by constant frequency (CF) modulation. The acoustic energy of the female A. gambiae was greater than that of the male A. gambiae with mean of the fundamental frequency (mean entire) of 1.00kHz and 955.56 Hz respectively . Both sexes showed equal fundamental frequency (mean), Fundamental (mean), fundamental (Minimum entire), Bandwidth (minimum entire) and Fundamental (start) of 900Hz and maximum frequency (minimum entire) of 1.90 kHz. The peak amplitude (maximum) and peak amplitude (maximum entire) for both sexes of the A. gambiae was also equal.
However, the male A. gambiae recorded the highest peak amplitude (mean), peak amplitude (mean entire) and peak amplitude (end) compared to the female A. gambiae. The sounds of male A. gambiae and O. tormota differed significantly high in the fundamental frequency (maximum entire), Peak amplitude (maximum), peak amplitude (mean), Peak amplitude (mean entire) and peak amplitude (maximum entire). In a paired t-test comparison of the sounds of the male A. gambiae and O. tormota, the differences in the maximum frequency (maximum entire), bandwidth (end), bandwidth (maximum) and bandwidth (mean entire) was highly significant. However, there existed no significant difference in the maximum frequency (mean entire) for the sounds of the male A. gambiae and O. tormota. The minimum frequency (maximum), minimum frequency (mean), minimum frequency (minimum entire), minimum frequency (mean entire) and minimum frequency (start) for both the male and female A. gambiae was equal to 900 Hz. However, the peak Frequency (maximum) of the sound of male A. gambiae compared by the sound of O. tormota showed no significant differences (p = 0.629 > 0.05). The sound of the male and female A. gambiae did not differ significantly (0.280) with a low negative correlation (r = -0.429). There existed no significant difference in bandwidth (end), bandwidth (maximum), bandwidth (maximum entire), peak amplitude (end), peak amplitude (maximum) and bandwidth (mean entire) of the sound of the male and female A. gambiae with the exception of the acoustic energy, peak amplitude (mean), peak amplitude (mean entire) and bandwidth (mean). The acoustic energy of the sound male A. gambiae was greater than that of O. tormota, but less than that of the sound of female A. gambiae. The maximum signal power in O. tormota, female A. gambiae and male A. gambiae was 89 dB, 85 dB and 80 dB respectively. These parameters of the sound male A. gambiae and O.