Critical analysis of the mosquito repellency evoked by the 10-34 kHz recorded animal sounds: The case of the African female A. gambiae s.s

Animals sounds have been mimicked in electronic mosquito repellents (EMRs) and exploited as a tool in the control of malaria by targeting the vector, the female Anopheles gambiae s.s. The claimed mosquito repellency of 30.3 % due to Anti-Pic®, an electronic mosquito repellent, had failed to be confirmed in subsequent studies. However, studies on mosquito startle based on initial behavioural activities without an attractant yielded 34.12 % repellency elicited by the 10-34 kHz recorded sound of O. tormota. Other malaria intervention measures involving the use of chemicals have been impeded by the pathogen and vector resistance hence slowing down the rate of decline of malaria morbidity and mortality. The research thus focused on the analytical study of the African female A. gambiae s.s repellency evoked by the 10-34 kHz recorded animal sound of male mosquito, Anopheles gambiae and Delphinapterus leucas. Landing rates and behavioural startle responses of the mated female A. gambiae on food attractant evoked by the individual sound of the male mosquito, A. gambiae, O. tormota and D. leucas were determined and analysed. The male and female A. gambiae were bred and reared under controlled laboratory conditions of 60-80 % humidity, 25±2 °C temperature with equal light-darkness hour cycle in KEMRI, entomology laboratories. Isolation of the male and female mosquitoes from a swarm was based on physical features and affinity to blood meal. The sounds of O. tormota and D. leucas were acquired and the sound of the male A. gambiae were recorded from the Kenya Medical Research Institute (KEMRI) entomology laboratory, Kisumu. The sounds were filtered into 10-34 kHz frequency band and analysed using Avisoft-SAS LAB Pro version 5.2 and Raven Pro 1.5 software. The sound of O. tormota was also studied. A fighto-Y glass cage well designed into control, neutral and treatment chambers was used in the study. Both control and treatment chambers were connected to blood meal maintained at 38.60°C. The treatment cage was also connected to the source of sound and a swarm of 50 female mosquitoes into the neutral cage and observed for 1,200 s. The sounds of the A. gambiae, O. tormota and D. leucas yielded 2.10, 2.20 and 3.00 landings/minute respectively associated with adverse behaviour. The protection index (PI) anchored on the number of mosquitoes that landed, probed and fed on the blood meal in the treatment and neutral cage for the sounds of the A. gambiae, O. tormota and D. leucas was 42.73 %, 40.24 % and 10.64 % respectively. The sound of the A. gambiae was characterised by steady and minimally dipped pulsate acoustic power with wide bandwidth. The protection index achieved by the sound of the male A. gambiae did not differ significantly from the sound of O. tormota (0.1740 > 0.05), though differed significantly from the sound emitted from the Anti-Pic® EMR (p = 5.3440 x 10−5). 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.


Highlights of the Worldwide Malaria Situation and Interventions
Adherence to recommended interventions makes malaria a preventable and treatable disease (WHO, 2011). Malaria interventional approaches targeting the vector and which involve the use of insecticide-treated nets (ITNs), indoor residual spraying (IRS) and in some specific settings, larval control are a critical components of the multipronged attack on malaria (Godfray, 2013). The chemoprevention is used for the most vulnerable populations, particularly pregnant women and infants. Confirmation of malaria diagnosis through microscopy or rapid diagnostic tests (RDTs) for every suspected case and timely treatment with appropriate antimalarial medicines is key in malaria control (CDC, 2010;WHO, 2011;Godfray, 2013;WHO, 2013;WHO, 2015;Gething et al., 2016).
Protection by ITNs and IRS has demonstrated greater impact in reducing malaria (WHO, 2013). Hwever, strains of Anopheles mosquitoes developed resistance to DDT, pyrethroid and other insecticides, and the environmental impact of DDT was recognized. Also, the Plasmodium parasites became resistant to chloroquine, the mainstay of antimalarial drug treatment in humans (WHO, 2006;. Sound has over the years been used to scare off pest species, with its humble origins of loud claps and yells in ancient agricultural fields, and now ultrasound producing electronic repellents (EMR) are used in combating mosquitoes (Antonelli et al., 2007;Simeon et al., 2013;Aflitto and DeGomez, 2014). Evaluation experiments with mosquito-repelling devices which included the Anti-Pic®, Mosquito Repeller® DX-600 and Bye-Bye Mosquito® done by exposing human hands to Aedes albopictus (Skuse) adults showed insignificant success in repellency, failing to confirm the 30.3 % repellency due to Anti-Pic® initially determined (Andrade and Bueno, 2001). The electronic mosquito-repelling devices studied ranged from 2kHz to 60 kHz in frequency with harmonic peaks from 4 kHz to 68 kHz (Rutledge et al., 1985;Combemale et al., 1992). Also, experiments with functioning electronic mosquito repellents (EMR) mimicking calls from bats and male Anopheles gambiae in the frequency range of 125 Hz to 74.6 kHz showed that 12 out of 15 field experiments yielded higher landing rate on the human bare body parts than the control experiments (Andrade and Bueno, 2001;Center For the Advancement Of Health, 2007;. The EMRs are used indoors and outdoors and are purported to repel mosquitoes within a range of 2.5 m (CAH, 2007). Recent researches with natural and synthetic sounds have shown startle response in mosquitoes, thus sound was an effective additional tool in the control of mosquitoes, the malaria vector (Mohankumar, 2010;Mang'are et al., 2012). Evasive responses characterized by 58.5 o antenna erection, physical injury, fatigue and falls; attributed to pronounced stress on the nervous system and fear of predation have been reported for both 10-34 kHz and 35-60 kHz sounds of Odorrana tormota based on behaviural activity. An average percentage startle of response of 34.12 % and 46 % was observed in mosquitoes in the 10-34 kHz and 35-60 kHz sounds of O. tormota respectively (Mang'are et al., 2012). Researches based on mosquito landing, probing and feeding determined the percent Protection Index (PI) or Repellency using the equation 1.1 (Rutledge et al., 1985;Combemale et al., 1992) where PH is the number of mosquito bites (or initiated bites) on the supposedly protected blood meal (treatment), and UPH is the same measure for supposedly unprotected blood meal (control).
Other studies have established that mosquitoes detect ultrasound in the range of 38 -44 kHz, regardless of the source, initiating avoidance response since it creates stress on their nervous system, jams mosquitoes' own ultrasound frequency besides immobilizing them (Mohankumar, 2010). The startle response in mosquitoes evoked by the natural sound of the male, Delphinapterus leucas and Tursiops truncatus had not been reported. The sounds of A. gambiae and O. tormota had been studied hence the basis for this study.

Mosquito biology and Audition
The egg, larva and pupa stages in the lifecycle of the A. gambiae are aquatic and last 5-14 days, depending on the species and the ambient temperature (CDC, 2010). 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). Warm-blooded hosts provide mosquitoes with thermal contrast that facilitates the localization of a suitable blood meal (McMeniman et al., 2014;EarthSky in Earth, 2015). An analysis of how the mosquito actually bites, probes for the blood vessels and finally sucks bloods showed that the mean time taken before the mosquito starts probing after landing was 6.5 seconds, the mean probing time was 142 seconds, mean feeding time was 240 seconds (feeding times were between 150 and 329 seconds) giving a total of 389 seconds (6.5 minutes) (Choumet et al., 2012). 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). 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. Adult males and females Anopheles rest with their abdomens sticking up in the air. 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).
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. 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 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. Ultrasound generated artificially or naturally is detected by mosquitoes evoking evasive response (Mohankumar, 2010).

Statement of the Problem
Africa and the world as a whole suffers both economic and health burden due to malaria.
Interventions which includes the use of chemicals targeting malaria pathogens and vectors have led to a decline in malaria mortality and morbidity though at a lower rate due to buildup of resistance. Also the use of electronic mosquito repellents mimicking the sounds of bats or male mosquitoes in the control of mosquitoes has been a debatable issues since the 30.

Sound of the O. tormota, male A. gambiae, T. truncatus and D. leucas.
The  (5 inch x 5 inch) was covered by a mosquito netting. Ends A and B (5 inch x 3 inch)were in contact to the blood feeding chamber to allow for mosquito landing and probing. The Hemotek membrane feeding system was used to feed the blood sucking mosquitoes through an artificial membrane. KEMRI breeds and rears the Anopheles mosquitoes for malaria research. Animal blood (cow blood) used in the study as a meal attractant (Source: the blood stock in Kenya Medical Research Institute (KEMRI) which was used as a regular meal for mosquitoes). The animal blood (cow) was maintained at 38.60 o C, the body temperature of a healthy cow (Kou et al., 2017). The blood chamber which is an aluminium cylindrical container was loaded with fresh blood by means of a pipette through the ports at its back. The ports are covered with a removable rubber material. The mosquitoes pick up cues that indicate presence of animals or humans through vision for spotting the host and thermal sensory information to detect body heat (McMeniman et al., 2014;EarthSky in Earth, 2015).
The loaded blood in the chamber was covered with an artificial membrane (cellulose membrane with pores) and connected to the cage netting as given in Figure 2 and 3.

Figure 2: Mounted blood chamber on the net by means of retort stand
The animal sound (treatment) was allowed through the netting on cage A and B interchangeably as given in Figure 3. landed; landed and probed; or landed, probed, and bit the repellent-treated blood meal (Barnard, 2005). The study was based on the in vitro method and the ASTM E951-94 repellent procedures (Buescher et al., 1983)  The cage with no sound was the control for the bioassay study as given in

(ii). Determination and analysis of the landing rates and behavioural startle responses of the mated female A. gambiae on food attractant evoked by the sound of the male mosquito, A. gambiae
The control bioassay chamber and treatment bioassay chamber were connected to the feeding membrane through the netting. The food attractant on the treatment bioassay cage was

Delphinapterus leucas
The 10-34 kHz frequency band was filtered from the entire spectrum of the sound of D.
leucas and used for the bioassay study. In the control bioassay chamber, the blood meal was not treated with sound. The mosquitoes flew about, to and from the neutral cage with some unfed mosquitoes resting normally on the walls of the cage during the first 120 s. Ten mosquitoes were observed feeding with no signs of disturbance. There was minimum movement by the mosquitoes in the cage after 240s. Two fully fed mosquitoes were observed resting on the cage after 480 s. Notably, one fully fed mosquito flew from the treated chamber to the control chamber then settled in the neutral cage during the 600 th second. Minimum movement and activity with one unfed mosquito rubbing its wings with hind legs was observed in the chamber up to 1080 th second. During the last 120s, two fully fed mosquitoes were seen resting in the cage.
In the treatment cage, the mosquitoes were seen approaching the meal and landing and flying away along the wall, with others flying around the meal without successful landing during the first 120 s. Between 120s and 240s, minimal movement was observed with on unfed mosquito flying into the chamber and resting on the wall of the cage. During the 480-600s duration, two fully fed mosquitoes flew from the treatment bioassay cage to the neutral cage where it rested on the net. One unfed mosquito flew from the neutral chamber into the treated chamber but flew back to the neutral cage immediately. Other unfed mosquitoes rested to the walls and floor with minimum movement during the same duration. Bouncing along the net and on glass walls associated with knocks was observed during the 840-1200s duration.
The number of the female A. gambiae approaching the blood meal in the control bioassay cage were more than the number of the female A. gambiae in the treatment bioassay cage except during the 240 th second where the number of mosquitoes in the treatment cage exceeded the control as given in Figure 10. Instances of attraction were confirmed during the 240 th second giving an instantaneous protection index of -6.67 % due low acoustic energy and power. The sound was least pulsate in nature. The difference in the number of mosquitoes approaching the meal in the control bioassay cage and the treatment bioassay cage was highly significant (p = 4.2749 x 10 -4 < 0.05) and the parameters correlated positively low (r = 0.1250). The overall protection index based on the number of mosquitoes approaching the blood meal in the control and treatment bioassay cages was 23.43 % which was less than the protection index for sound of male mosquito, Anopheles gambiae and Odorrana tormota by 12.81 % and 3.33 % respectively. The number of mosquitoes that landed, probed and fed on the blood meal in the treatment cage were less than the number of mosquitoes that landed, probed and fed on the blood meal in the control cage except during the 240 th , 840 th and 1080 th seconds where they were equal in number as given in Figure 11. The number of mosquitoes that landed, probed and fed on the blood meal in the treatment cage during the 960 th second exceeded the number of mosquitoes that landed, probed and fed on the blood meal in the control cage.   landings/minute. The high protection index was attributed to the pulsate and almost steady acoustic power, wide mean bandwidth (mean entire) and minimal deviation between the maximum and minimum acoustic power within the 10-34 kHz frequency range.