Protection against mosquito vectors Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus using a novel insect repellent, ethyl anthranilate

Accepted Manuscript Title: Protection against mosquito vectors Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus using a novel insect repellent, ethyl anthranilate Authors: Johirul Islam, Kamaruz Zaman, Varun Tyagi, Sanjukta Duarah, Sunil Dhiman, Pronobesh Chattopadhyay PII: DOI: Reference:

S0001-706X(17)30151-1 http://dx.doi.org/doi:10.1016/j.actatropica.2017.06.024 ACTROP 4355

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

8-2-2017 16-6-2017 19-6-2017

Please cite this article as: Islam, Johirul, Zaman, Kamaruz, Tyagi, Varun, Duarah, Sanjukta, Dhiman, Sunil, Chattopadhyay, Pronobesh, Protection against mosquito vectors Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus using a novel insect repellent, ethyl anthranilate.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2017.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Protection against mosquito vectors Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus using a novel insect repellent, ethyl anthranilate Johirul Islama, b, Kamaruz Zamanb, Varun Tyagia, Sanjukta Duaraha, Sunil Dhimanc, Pronobesh Chattopadhyaya*

a

Defence Research Laboratory, Tezpur, Assam- 784001, India.

b

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam- 786004,

India.

c

Defence Research and Development Establishment, Gwalior, Madhya Pradesh- 474002,

India

* Correspondence Defence Research Laboratory, Tezpur, Assam- 784001, India. Tel: +91-3712258836/258508, Fax: +91-3712258534 Email: [email protected] (P. Chattopadhyay)

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Highlights 

Repellent activity of ethyl anthranilate (EA) was investigated against Ae. aegypti, An. stephensi and Cx. quinquefasciatus.



EA demonstrated promising repellent activity against all three mosquitoes species.



Results suggest that EA could be useful in developing safer and better mosquito repellents.

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Abstract Growing concern on the application of synthetic mosquito repellents in the recent years has instigated the identification and development of better alternatives to control different mosquito-borne diseases. In view of above, present investigation evaluates the repellent activity of ethyl anthranilate (EA), a non-toxic, FDA approved volatile food additive against three known mosquito vectors namely, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus under laboratory conditions following standard protocols. Three concentration levels (2%, 5% and 10% w/v) of EA were tested against all the three selected mosquito species employing K & D module and arm-in-cage method to determine the effective dose (ED50) and complete protection time (CPT), respectively. The repellent activity of EA was further investigated by modified arm-in-cage method to determine the protection over extended spatial ranges against all mosquito species. All behavioural situations were compared with the well-documented repellent N,N-diethylphenyl acetamide (DEPA) as a positive control. The findings demonstrated that EA exhibited significant repellent activity against all the three mosquitoes species. The ED50 values of EA, against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus were found to be 0.96%, 5.4% and 3.6% w/v, respectively. At the concentration of 10% w/v, it provided CPTs of 60, 60 and 30 min, respectively, against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus mosquitoes. Again in spatial repellency evaluation, EA was found to be extremely effective in repelling all the three tested species of mosquitoes. Ethyl anthranilate provided comparable results to standard repellent DEPA during the study. Results have concluded that the currently evaluated chemical, EA has potential repellent activity against some well established mosquito vectors. The study emphasizes that repellent activity of EA could be exploited for developing effective, eco-friendly, acceptable and safer alternative to the

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existing harmful repellents for personal protection against different hematophagous mosquito species. Keywords: Arbovirus; Dengue; Ethyl anthranilate; Malaria; Mosquito repellent; Zika fever.

1. Introduction Mosquitoes (Diptera: Culicidae) represent a key threat to millions of humans and animals worldwide, since they act as vectors for deadly parasites and pathogens, including malaria, yellow fever, filariasis, Japanese encephalitis, dengue, chikungunya, West Nile and Zika virus (Baden et al., 2016; Benelli and Mehlhorn, 2016; Benelli et al., 2016; Mayer et al., 2017; Moulin et al., 2016; Revay et al., 2013; WHO, 2005). These mosquito-borne diseases incapacitate and seriously debilitate millions of people and unfortunately, decimate countless lives annually (Lalthazuali and Mathew, 2017; Leal, 2014; WHO, 2016a). In addition, mild or symptoms-free infections and the lack of effective medical treatments for some of these diseases further alleviate the sufferings and economic consequences among populations (Benelli and Mehlhorn, 2016; Benelli et al., 2016; WHO, 2016b). For instance, a Zika infection transmitted primarily by Aedes mosquitoes typically does not display observable symptoms in healthy individuals, however the infected pregnant woman may still give birth to a baby with microcephaly and other diseases in the developing fetus (Shan et al., 2017). Recently a live-attenuated vaccine has been developed and their activity has successfully been established in mice and non-human primates (Shan et al., 2017). However, further investigations relating to clinical trials are essential for its use in endemic settings. While, dengue is a mosquito-borne viral disease transmitted by Aedes aegypti and Aedes albopictus, probably the fastest spreading mosquito-borne disease with an estimated

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390 million cases per year globally (Benelli and Mehlhorn, 2016; Lees et al., 2014). There is no specific treatment for dengue, however Dengvaxia, a tetravalent live-attenuated vaccine developed by Sanofi Pasteur is at the most advanced clinical stage of evaluation (phase III) (Benelli and Mehlhorn, 2016; Murrell et al., 2011; Vannice et al., 2016). Although it is presently available in some parts of the world, but its implications have been restricted due to various reasons including the higher rates of hospital admissions in vaccinated seronegative individuals (due to Dengvaxia raising non-protective dengue infection enhancing antibodies) and others (Aguiar et al., 2016; Halstead and Russell, 2016; Vannice et al., 2016). Recent research has also highlighted the potential of plant-synthesized nanoparticles as inhibitors of dengue growth (Benelli, 2016a,b; Benelli and Mehlhorn, 2016). Studies have shown that green-synthesized silver nanoparticles inhibit the production of DEN-2 viral envelope (E) protein in Vero cells, thereby down regulating the expression of dengue viral E gene (Murugan et al., 2016; Murugan et al., 2015; Sujitha et al., 2015). Furthermore, plantderived metal nanoparticles have also been exploited as effective ovicides, larvicides, pupicides, adulticides, and oviposition deterrents against different vector mosquito species, however limited information is available about such formulations which could be employed effectively against majority of mosquito vectors in different endemic countries (Benelli, 2015a,b; Benelli, 2016a,b; Benelli et al., 2016; Govindarajan and Benelli, 2016; Kumar et al., 2016; Pavela, 2015). Currently control of mosquito vectors use integrated approach employing many intervention methods simultaneously. These methods primarily include, synthetic insecticides, long lasting insecticidal nets (LLIN), sterile insect technique, transgenic mosquitoes and synthetic repellents, which have produced encouraging results, however none of these strategies used singly has been proved to be fully successful (Benelli, 2016b; Lees et al., 2014; Pavela and Benelli, 2016a; Spitzen et al., 2017). Moreover, the applicability

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of many commercially available synthetic anti-mosquito formulations has abridged noticeably, and this can be attributed to their toxic effects, both on the environment and also on different non-target organisms (Chattopadhyay et al., 2015; Deletre et al., 2016). Additionally, emerging problem of developing resistance

in mosquitoes to these anti-

mosquito formulations has been a serious concern (Bigoga et al., 2012; Hemingway and Ranson, 2000; Naqqash et al., 2016). Botanical essential oils (e.g., citronella oil, eucalyptus oil, and other plant oils) and their derivatives such as para-menthane-3,8-diol, a derivative of Eucalyptus citriodora, (currently used in Europe) on the other hand, also play a promising role in preventing disease transmission (Pavela and Benelli, 2016b; Phasomkusolsil and Soonwera, 2011; Soonwera, 2015; Soonwera and Phasomkusolsil, 2014; Soonwera and Phasomkusolsil, 2015). However, the number of essential oil based commercial biopesticide remains low chiefly due to strict legislation, low persistence of effects, lack of proper quality control procedure and sufficient quantities of materials for affordable prices (Demirci et al., 2013; Pavela and Benelli, 2016b; Tisgratog et al., 2016). Additionally, the presumed toxic effects such as allergenicity, genotoxicity, mutagenicity, and negative effects on the human endocrine system has negated the commercial availability (Pavela and Benelli, 2016b; Pohlit et al., 2011; US EPA, 1970). Ethyl anthranilate (EA, CAS: 87-25-2), a new member in the realm of entomology, has drawn significant attention in repellent research in the recent years and is being considered as an improved alternative to DEET (Islam et al., 2017; Kain et al., 2013; Ray and Boyle, 2015). Of late, EA was found to interfere with both host seeking behaviour and oviposition of Aedes aegypti mosquitoes(Afify et al., 2014). EA was also found to activate an ionotropic receptor Ir40a in the antenna of fruit fly Drosophila melanogaster, thereby possibly suggesting a different mechanism to incapacitate the target insect (Kain et al., 2013). Ir40a receptor proteins are highly conserved across several agricultural pests and insects,

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such as mosquitoes, head lice, and Tribolium. (Kain et al., 2013), which makes EA an explorable repellent candidate against different species of mosquitoes and other insects as well. Additionally, EA fulfils all the requirements of an ideal repellent (Table 1) and, in comparison with other common repellents available in the market, EA has the advantage of being approved by Food and Drug Administration (FDA), World Health Organization (WHO) and European Food Safety Authority (EFSA) (Api et al., 2015; Kain et al., 2013). Furthermore, EA has been listed in the ‘generally recognized as safe’ (GRAS) list by the Flavour and Extract Manufacturer’s Association (FEMA) (Flavors and Fragrances, 2007-08; Kain et al., 2013; Opdyke, 1979). In the view of the above, an extensive investigation on the repellent activity of EA against three well known vector species of mosquitoes namely, Aedes (Ae.) aegypti, Anopheles (An.) stephensi and Culex (Cx.) quinquefasciatus was carried out under laboratory conditions as per standard procedure and guidelines.

2. Materials and methods 2.1. Reagents and chemicals Ethyl anthranilate and N,N-diethylphenyl acetamide (DEPA) were purchased from Sigma Aldrich (Sigma Aldrich Chemical Co., St. Luis, USA). Acetone (HPLC grade) was purchased from Merck (Merck Pvt. Ltd., Mumbai, India). 3MTM double coated polyethylene tape 9766

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was received from 3M (3M, St. Paul, USA) as a gift sample. All reagents and solvents used were of analytical grade. 2.2. Mosquitoes The laboratory reared 5-7 days old adult female Ae. aegypti, An. stephensi and Cx. quinquefasciatus mosquitoes were obtained from the laboratory resources, Division of Medical Entomology, Defence Research Laboratory, Tezpur, Assam, India. All mosquito species were maintained at the laboratory insectary at 27±2 °C, 75±5% RH and 14L:10D h of light-dark alternative cycles in standard-sized wooden cages (75 cm X 60 cm X 60 cm) with a sleeve opening on one side as described previously (Chattopadhyay et al., 2015). They were provided with adequate nutrition with 10% sucrose solution ad libitum. Before testing, the mosquitoes were starved for 24 h. 2.3. Volunteers Adult human (male/female) volunteers of age 25-35 years were registered for the present study. The volunteers participated in the study were instructed to avoid alcohol, caffeine, and fragrance products (e.g., perfume, deodorant, lotion, and other cosmetics) during the study. All participants were fully made aware of the nature and purposes of the test and the possible health risks owing to exposure to chemicals and insect bites. Informed written consents were obtained from the volunteers participated in the repellent trials. The study design was approved by the Human Ethical Review Committee of Tezpur Medical College & Hospital (TMCH), Tezpur, Assam, India (approval number: IEC03/TMCH). 2.4. Standard solutions preparation Standard stock solution of EA (20% w/v) was prepared by dissolving an appropriate amount of EA in high performance liquid chromatography (HPLC) grade acetone. The solution of standard stock was stored at 4 °C (± 0.5). Working solutions were prepared fresh everyday by properly diluting the standard stock solution with HPLC grade acetone. DEPA

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(10% w/v), which was used as the positive control in the trial, was also prepared by dissolving an appropriate amount in HPLC grade acetone. 2.5. Repellent bioassay The repellency testing was conducted between 1000 to 2300 h IST. Repellency testing for Ae. aegypti was conducted between 1000 h to 1600 h, while for both An. stephensi and Cx. quinquefasciatus mosquitoes, the tests were conducted between 1900 to 2300 h, maintaining temperature (27±2 °C) and relative humidity (75±5% RH), as per the standard procedure and guidelines (WHO, 2009). 2.5.1. Dose response study To determine the initial effective dose (ED) of the repellent, Klun and Debboun (K & D) module was used by adopting the method as described previously with slight modifications (Klun and Debboun, 2000; Klun, 2005). K & D module is a very useful technique in the quantitative behavioural evaluation of arthropod-repellent compounds over a range of doses and considered as the standard method for testing non-commercial repellent formulations on human skin employing laboratory reared mosquitoes. The module was made of Plexiglas and consisted of 5 continuous cells (5 cm X 4 cm X 5 cm). Each cell has a circular stoppered access hole (1 cm) at one side for introduction of mosquitoes to the cell and a bottom with a rectangular (3 cm X 4 cm) window that can be opened and closed by a sliding door. The bottom of K & D module is slightly concave, conformed to the curvature of a human thigh. The frontal region of the thigh of human volunteer was shaved (with razor-blade) 24 h prior to the experiment. Before initiation of the experiment, the volunteer was allowed to be seated on a chair (any chair) with extended legs. The K & D module was then placed over the shaved region and outlined with water-soluble marker into 5 test areas. Meanwhile, aliquots of EA (2, 5, 10% w/v) were prepared fresh by diluting standard stock solution of EA in HPLC grade acetone. The first marked area was treated with 25 μL acetone only (HPLC

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grade) and used as the solvent control. The other 4 marked areas were treated with 25 μL final volume of solvent in corresponding EA concentration and used as treatment. In each experiment, only one concentration was used for repellency testing in order to reduce the complexity of the experiments. Each marked area was allowed to stand for about 2-3 min for complete evaporation of solvent. The K & D module was washed thoroughly with luke-warm water prior to performing new set of experiments. The repellent activity of positive control (DEPA, 10% w/v) was assessed in different set of experiments. A total of ten female Ae. aegypti (5-7 days old, non blood-fed) mosquitoes were placed inside each cell of the K & D module (total 50 mosquitoes in five cells) and kept for 5 min for acclimatization. The module was then secured in the marked area on the volunteer thigh and the sliding door was opened for in-vivo repellency testing by allowing the mosquitoes to access the treated areas. Tests were repeated several times on different days for each concentration and the number of mosquitoes landing/biting in each cell within a 5 min exposure period was recorded. Different batches of mosquitoes were used for replications. The percentage repellency was then calculated to determine the effective dose (ED 50) of EA by the following formula as described elsewhere (Auysawasdi et al., 2016). 𝑅𝑒𝑝𝑒𝑙𝑙𝑒𝑛𝑐𝑦 (%) =

Ta −Tb Ta

X 100.......................... (1)

Ta = No. of mosquitoes landing/biting in the control group Tb = No. of mosquitoes landing/biting in the treated group Similar procedures were also employed to determine the ED50 of EA against An. stephensi and Cx. quinquefasciatus mosquitoes. 2.5.2. Complete protection time Complete protection time (CPT) of a repellent is the time between the application of the repellent and the first mosquito landing and/or probing. The CPT has been considered important to determine the duration of efficacy of any given concentration of repellent (Das 10

et al., 2015; Delong et al., 2016; Phasomkusolsil and Soonwera, 2011; Soonwera, 2015; WHO, 2009). Repellent test chamber with specific size (40 cm X 40 cm X 40 cm) was used to evaluate the repellency of EA against mosquitoes. Approximately 50-60 non blood-fed adult female Ae. aegypti mosquitoes were introduced into the repellent chamber through the hole on the top. Before conducting each test, the untreated forearm was inserted into the cage for up to 30 seconds to confirm the readiness of the mosquito to bite. The repellency test was carried out if at least 10 mosquitoes landed or attempted to probe the arm. Aliquots of EA (2, 5, 10% w/v) were prepared fresh by diluting standard stock solution of EA in HPLC grade acetone. One mL of the test sample was applied to the test hand (wrist to fingertip) of volunteers (n = 3). After about 5 min, the test hand was exposed to mosquitoes for 5 min by inserting inside the test chamber. The procedure was repeated at an interval of 30 min and continued up to 4 h. DEPA (10% w/v) solution was used as positive control, whereas acetone was used as solvent control for present experiment. The experiment was repeated many times and every time fresh batch of mosquitoes was introduced into the test chamber. The CPT was determined against all the test species of mosquitoes as per the equation no. 1.

2.5.3. Spatial repellency Spatial repellents create a mosquito-free space, thereby preventing the contact between insects and humans. They can be used to protect more than one person at a time by dispersing active ingredients into the surrounding air that interferes with the mosquito’s ability to find a host. Repellent in space may interfere with host detection through excito-repellency, causing insects to fly in an undirected manner until they eventually move away from the source of repellent vapour (Maia et al., 2015).

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3. The spatial repellency of EA was evaluated by the method as described previously with slight modifications (Guda et al., 2015; Kain et al., 2013). Briefly, about 50-60 unfed adult female Ae. aegypti mosquitoes were introduced into the repellent chamber (40 cm X 40 cm X 40 cm) through the hole on the top. Different concentrations of EA (2, 5, 10% w/v) were then prepared fresh by diluting standard stock solution of EA in HPLC grade acetone. An untreated high density polyethylene (HDPE) rectangular net (4 cm X 3 cm) was used in this assay and was placed on top of the glove window positioned 6 mm above skin surface. The net was held at the top of a suitable wooden frame, while the lower face of the frame was attached to the glove with an adhesive (Supplementary file S1). A new hexane washed and oven dried (60 °C; 1 h) filter paper (1 cm X 1 cm) was used to adsorb the test chemical. Acetone (100 µL) treated filter paper was used as solvent control, while DEPA (10% w/v) was used as positive control. One hundred µL of test sample was applied on the filter paper followed by subsequent evaporation of the solvent for about 2 min. The treated filter paper was then attached to double coated adhesive tape and pasted over the skin surface in a way that the upper and free face of the filter paper remains exactly at the middle of rectangular area of the glove. The hand was inserted into the glove and placed inside the test cage for up to 5 min to calculate the number of mosquito landing on the net surface. The experiment was carried out for each concentration of EA and fresh batch of mosquitoes was used in each trial.Data analysis The data obtained in dose response study and complete protection time were subjected to percentage repellency calculation by the formula as described in the method section (equation no. 1). The replicated data have been expressed as mean ± SD (standard deviation). The data were compared using analysis of variance (ANOVA) followed by the Tukey’s test of multiple comparison and considered significant when p<0.05 at 95% confidence interval. Spatial repellency data were also analyzed using ANOVA. However, the difference between

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mean of control and treatments were examined by Dunnett's multiple comparison tests. All the statistical analysis was performed by GraphPad InStat statistical software (version-3.05).

4. Results 4.1. Dose response study EA elicited a dose dependent response with increasing doses and this has been indicated by percentage repellency (Fig. 1). All female Ae. aegypti mosquitoes (100%) were repelled by 10% w/v dose of EA, whereas 78% and 90% female mosquitoes were repelled by 2% and 5% dose respectively. An. stephensi also exhibited a dose dependent response, in which 68%, 80% and 96% female mosquitoes were repelled by 2%, 5%, 10% w/v dose of EA respectively. Furthermore, 64%, 82% and 88% female Cx. quinquefasciatus mosquitoes were

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repelled by 2%, 5%, 10% w/v dose of EA, respectively. The repellency data obtained using 10% w/v EA did not vary significantly when compared with the data of DEPA (10% w/v) (p˃0.05). The ED50 values of EA, against Ae. aegypti, An. stephensi and Cx. quinquefasciatus were found to be 0.96%, 5.4% and 3.6% w/v, respectively. 4.2. Complete protection time Arm-in-cage method was employed to estimate the protection time and biting rate under laboratory conditions, which indicated a dose dependent response for all the tested species of mosquitoes. The results obtained have been shown in Table 2. EA elicited a complete protection (100%) i.e. all female Ae. aegypti were repelled completely for a period up to 30, 60 and 60 min with 2%, 5%, 10% w/v dose respectively. Further, at least 57.3%, 72.0%, and 84.0% Ae. aegypti mosquitoes were repelled by 2%, 5%, and 10% w/v of EA for up to 120 min of exposure, respectively (Fig. 2). At the highest dose of EA (10% w/v), altogether 100% protection from An. stephensi was obtained for up to 60 min, whereas a complete protection up to 30 min was obtained with both 2%, and 5% w/v dose respectively. At least, 47.3%, 52.0% and 90.0% female An. stephensi were repelled by 2%, 5% and 10% dose of EA respectively for a period of 120 min. (Fig. 3). Similarly, Cx. quinquefasciatus also exhibited dose dependent protection for 30 min with 2%, 5%, 10% w/v dose of EA. However, at least, 69.3%, 74.6% and 98.0% female Cx. quinquefasciatus were repelled by 2%, 5%, 10% w/v dose of EA respectively up to a period of 90 min (Fig. 4). DEPA (10% w/v), which was used as the positive control, produced almost similar protection (82.6-90.6%) for up to 120 min against all the three species of mosquitoes. The repellency obtained using 2% and 5% EA and positive control (DEPA, 10% w/v) differ significantly , however, the repellency achieved using 10% w/v EA did not differ statistically (p˃0.05). 4.3. Spatial repellency

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Modified arm-in-cage experiments were used to evaluate the spatial repellency against all three species of mosquitoes. The experiment was based on the estimation of number of mosquito landings on the net surface in 5 min time period, which indicated a dose dependent response to the different doses of EA (Fig. 5). However, the repellent action of EA was found to be comparatively higher for Ae. aegypti than the other tested mosquito species. The repellency data generated for EA treatment differ statistically to the control treatment (p<0.05). Although, EA exhibited better response as compared to DEPA (10% w/v) but, no significant variations could be observed (p˃0.05).

5. Please note: The box is actually a ‘greater than’ symbol. While building PDF in online submission (EVISE), the symbol automatically gets converted into the box. Please consider. Discussion The use of mosquito repellents is an obvious, practical and economical way of preventing the transmission of severe mosquito-borne diseases (Tawatsin et al., 2001). However, the continued use of synthetic and natural repellents has resulted in mammalian toxicity, residue contamination of human food, environmental pollution and resistance among the insect vectors (Bharati and Saha, 2017; Qualls et al., 2012; Roy et al., 2017). Therefore, in view of growing concern on the application of existing repellents, eco-friendly alternatives of synthetic and natural counterparts have gained tremendous importance in vectors management in the recent years. In the present investigation, the repellent activity of EA was explored for the first time against three well known mosquito vectors to confirm its suitability and acceptability, as an insect repellent. In this contribution, the effective dose (ED50) of EA was estimated by adopting K & D module that has been well used to estimate the ED of various insect repellent against hematophagous mosquitoes since many years (Klun and Debboun, 2000). Most often, 15

ED50 values are used to assess the effectiveness of different repellents against a single species of mosquito or a repellent against different species of mosquitoes (Klun and Debboun, 2000; Klun, 2005). The ED50 values of EA against Ae. aegypti, An. stephensi and Cx. quinquefasciatus were encouraging and elucidate its importance as broad spectrum mosquito repellent. The ED50 value determined presently were found to be comparable to many essential oils, as reported in the previous studies (Phasomkusolsil and Soonwera, 2011 ; Misni et al., 2009). These investigations have reported that the ED values obtained for the essential oils of Piper aduncum, Cympobogon citrates and Ocimum basilicum against different vector mosquito species under laboratory conditions advocate their use in repellent formulations. Therefore, the low levels of effective dose attained in the present investigation could facilitate the development of ultra low volume active molecule based anti-mosquito formulations. The optimum concentration of the EA was also estimated by the arm-in-cage experiment and was found to be 10% w/v. At 10% w/v concentration, it provided similar repellent activity as of DEPA against all three known vector species of mosquitoes under laboratory conditions. Many previous studies have demonstrated that plant based essential oils singly as well in combination provide acceptable protection against variety of mosquitoes under laboratory and field conditions. However, presently EA was found to exhibit better protection against mosquitoes in cage bioassay than many essential oil based formulations as reported in the previous studies (Das et al., 2015; Phasomkusolsil and Soonwera, 2011). Das et al. (2015) have demonstrated the repellent activity of Curcuma longa, Pogostemon heyneanus and Zanthoxylum limonella essential oils based formulation against laboratory reared Ae. albopictus mosquitoes. In their experiments, Ae. albopictus mosquitoes were significantly repelled by all essential oils for up to 120 min at the dose of 20%, however optimised mixture of three essential oils could provide some improved efficacy. Similarly, essential oils extracted from Cananga odorata, Cymbopogon citrates and Cymbopogon

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nardus were found to provide 116.67 and 128.33 min of protection against Ae. aegypti and Cx. quinquefasciatus mosquitoes respectively with a dose of 0.33μl/cm2 (Soonwera and Phasomkusolsil, 2014). It is important to note that concentrations used in these studies were quite higher (20-30%) and the results showed very high variability between the values obtained for each concentration. In present investigation, EA provided promising repellent activity against all three species of mosquitoes for up to 120 min at the dose of 10% w/v and the response obtained were dose dependent hence did not display much variability, thus establishing its superiority over many essential oils. Besides, depending on the presence of mosquito vectors and severity of disease transmission, higher concentration of EA could be used to obtain effective protection for extended period. Although, the anti-mosquito protection achieved using EA was not as much as DEET, which provided 3 to 6 h protection using human volunteers (Lupi et al., 2013), but it could be considered as viable alternative to DEET. Unlike DEET (undesirable properties), its application as a potential insect repellent may be well accepted due to its high chemical stability under use conditions, good aesthetic reactions and affordable cost per use of the final product, which has been regarded essential criteria in preliminary product development. More significantly, considering the usability parameters of EA, such as safety, smell, and vapour pressure (Table 1), the possibilities of formulation development can be expanded by incorporating EA in sprays, creams, lotions and aerosols based anti-mosquito formulations . Additionally, EA does not damage synthetic fabrics, plastics and, painted and varnished surfaces which further widen the utility of EA in bed nets, cloths and different surfaces in the enedmic settings. Short duration of protection time observed currently is a drawback of EA as mosquito repellent. However, with the new technologies currently accessible, it is possible to increase the residual efficacy of EA by polymer encapsulation or synergised by other

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commercially available compound like vanillin (Barradas et al., 2015; Chattopadhyay et al., 2015; Islam et al., 2016; Lupi et al., 2013; Solomon et al., 2012). In spatial repellency evaluation, EA was found extremely effective in repelling all the three mosquito species, hence displaying superior efficacy than many commercial repellents. Recent study has suggested that the spatial repellency of commercially available mosquito repellent DEET does not extend to even 1 cm (Guda et al., 2015). Furthermore the anthranilate class of compounds contains a large diversity of natural chemicals, hence present many structural substitutes that could be exploited as repellents. Previous studies have found that EA activate the olfactory receptor neurons (ORNs) and gustatory neuron, therefore it was predicted that EA act similar to DEET and activate the similar sensory pathway (Kain et al., 2013). Our findings are similar to the previously reported observations in which EA was found to exhibit promising repellency against Ae. aegypti mosquitoes (Afify et al., 2014; Kain et al., 2013). However, repellency tested in those experiments was performed by using Y-tube olfactometer and modified arm-in-cage methods. The positive control (DEPA) used in the study also exhibited spatial repellency to some extent against mosquitoes, but its spatial repellency was found well below the repellency of EA (Fig. 5). During the experiments, a small proportion of the mosquitoes landed on the net, but escaped immediately, which may be due to quick perception of EA by ORNs, housed in the antenna, maxillary palps and proboscis (Kain et al., 2013; Ray, 2015). Owing to the spatial repellent activity of EA, the applicability can also be further expanded by incorporating it in passive emanators in tropical endemic areas without using electric heating. The non-contact version of repellent assay used in estimation of spatial repellency in the present study can be adopted for preliminary screening of newly developed repellents against both laboratory reared and wild mosquitoes.

6. Conclusion

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The present investigation provided promising anti-mosquito activity against Ae. aegypti, An. stephensi and Cx. quinquefasciatus mosquitoes under laboratory conditions. However, this study is not conclusive, since the present findings relate to the application of the EA activity against laboratory reared Ae. aegypti, An. stephensi and Cx. quinquefasciatus mosquitoes only, therefore more detailed, multi-centric and well replicated field studies are essential to establish long-term efficacy, practicability, affordability and acceptability of the EA against different vector mosquitoes. The present report will benefit entomologists, scientists and managers in the healthcare and pharmaceutical industry to develop EA based repellents that could be used to control various mosquito-borne diseases both at rural and urban settings. Finally, by taking the advantage of availability of IR40a receptor neurons in insects, EA may also have important implications for control of several agricultural pest insects that cause enormous crop loss.

Conflicts of Interest The authors declare no conflicts associated with this work. Acknowledgements One of the authors, Johirul Islam is thankful to University Grant Commission, Govt. of India, for providing financial support in the form of research fellowship. Authors are thankful to Defence Research Laboratory, Tezpur, Assam, India, and Dibrugarh University, Dibrugarh, Assam, India, for providing necessary support for carrying out the research work. Authors also extend gratitude to Dr. D. R. Bhattacharyya, Scientist-E, Indian Council of Medical Research (ICMR), Dibrugarh, Assam, India, for supplying the colony of Anopheles stephensi. All anonymous reviewers are also gratefully acknowledged for their specific comments that help a lot in improving this manuscript.

19

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Figure legends Figure 1: Comparison of dose response of ethyl anthranilate against Ae. aegypti, An. stephensi and Cx. quinquefasciatus (**p˃0.05, *p˂0.001). Please note: The box between **p and 0.05 is a ‘greater than’ symbol, while the box between *p and 0.001 is a ‘less than’ symbol. Figure 2: Ethyl anthranilate induced protection against Ae. aegypti. Figure 3: Ethyl anthranilate induced protection against An. stephensi. Figure 4: Ethyl anthranilate induced protection against Cx. quinquefasciatus. Figure 5: Spatial repellent activity of ethyl anthranilate against Ae. aegypti, An. stephensi and Cx. quinquefasciatus (***p˂0.001, **p˂0.01, *p˂0.05). Please note: The box is actually a ‘less than’ symbol. While building PDF in online submission (EVISE), the symbol automatically gets converted into box. Please consider.

29

Fig.1

30

Fig.2

31

Fig.3

32

Fig.4

33

Fig.5

34

Table 1. Summary of novel insect repellent ethyl anthranilate Synonyms

Benzoic acid, 2-amino-, ethyl ester, Ethyl o-aminobenzoate, Ethyl 2-aminobenzoate

Chemical formula

H2NC6H4CO2C2H5

Structure

Molecular weight

165.19

CAS number

87-25-2

Vapour pressure (mmHg @ 25 °C)

0.01

Boiling point (mmHg @ 25 °C)

260 °C

Melting point (mmHg @ 25 °C)

66.17 °C

Water solubility

413.6 mg/L

Appearance/Organoleptic

A colorless liquid which has a sweet-fruity, grape like odor, milder and less harsh than the methyl ester

Approved for consumption

Yes

Dissolves plastic

No

Repellency spectrum

Aedes aegypti, Drosophila melanogaster

Oral toxicity

3,750 mg/kg (Rat)

Dermal toxicity

0.0012 mg/kg/day (Rabbit)

References

Afify et al., 2014; Api et al., 2015; Flavours and Fragrances, 2007-08; Guda et al., 2015; Islam et al., 2017; Kain et al., 2013; Opdyke, 1979; Ray and Boyle, 2015

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Table 2: Repellent activity of ethyl anthranilate (at 2, 5, 10% w/v) against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus mosquitoes. % Repellency Mosquitoes

Concentration (% w/v)

30 min

60 min

90 min

120 min

150 min

180 min

210 min

240 min

100.0±0.0a 100.0±0.0a 100.0±0.0a 100.0±0.0 a

90.6±4.1a 100.0±0.0a 100.0±0.0a 100± 0.0a

69.3±3.0b 88.6±5.0a 98.6±2.3a 100± 0.0a

57.3±6.4b 72.0±4.0b 84.0±4.0a 90.6±3.0a

35.3±12.2c 66.6±4.1b 76.0±5.2b 82.6± 3.0 a

20.6±9.0c 50.0±10.0b 62.6±6.1b 70.0±11.1b

6.0±5.2d 33.0±6.5c 49.3±4.1c 64.6±6.4b

4.0±4.0 d 13.3±4.1d 32.0±9.1c 63.3±7.0b

100.0±0.0a 100.0±0.0a 100.0±0.0a 100.0±0.0 a

87.3±2.3a 94.6±6.1a 100.0±0.0a 100± 0.0a

62.0±6.0b 78.0±7.2b 98.6±2.3a 100± 0.0a

47.3±3.0c 52.0±9.1b 90.0±2.0a 86.6±3.5a

40.6±13.3c 47.3±9.0c 58.6±3.0b 71.3±3.0 a

32.0±10.0c 35.3±8.3c 41.3 ±7.0c 68.0±8.0b

14.0±12.1d 33.0±6.5c 39.3±20.5c 60.6±9.4b

3.3±4.1d 13.3±9.8d 14.6±11.7d 56.0±16.0c

2.0 100.0±0.0a 82.0±2.0a 69.3±9.4b 49.3±13.3c 40.6±13.0c 24.0±6.0c 12.0± 6.0d 5.0 100.0±0.0a 87.3±6.1a 74.6±7.0b 55.3±9.8b 49.3±5.0c 32.0±11.1c 22.6±5.0c Culex a a a b b c 10.0 100.0±0.0 96.6±4.1 98.0±3.4 68.0±6.0 56.0±6.0 49.3±15.1 31.3±6.1c quinquefasciatus DEPA (10% w/v) 100.0±0.0a 100.0±0.0a 100.0±0.0a 82.66±3.0a 68.6±5.0a 66.0±9.1b 58.0±3.4c In the column, mean % repellency ± SD; ANOVA followed by Tukey’s test of multiple comparison. The values in columns followed by same letters are not significantly different (p > 0.05). ; DEPA (10% w/v) used as positive control.

2 .0±3.4d 12.0±6.0d 15.3±7.0d 54.6±15.5c

Aedes aegypti

Anopheles stephensi

2.0 5.0 10.0 DEPA (10% w/v) 2.0 5.0 10.0 DEPA (10% w/v)

36