Essential Oils from Plants A Review on Eco-Friendly Mosquito Repellents

Essential oils from medicinal plants are widely used all over the world as insect repellents as they are highly safe and beneficial to environment with least ill-effects on animal and public health. Electronic databases including Science direct, PubMed, Scopus, Cochrane library and Scifinder were searched for papers on essential oils from plants with mosquito repellent efficacy. Ethnobotany, phytochemistry and repellent efficacy of plant essential oils were also discussed in this review. This review discusses in detail the mosquito-repellent plant essential oils which would be helpful in effective formulation of different essential oils for efficient control of mosquitoes, thus vector-borne diseases and for pharmacological studies, i.e., drug designing. This review would assist in finding studies on different mosquito-repellent plant essential oils at one stop, since it summarizes a large number of reports on essential oils.

of people all over the world. 856 million people in 52 countries worldwide were threatened by lymphatic filariasis (http://www.who.int/mediacentre/factsheets/fs102/en/). 128 million people over 78 countries were infected with lymphatic filariasis, 25 million people around the world were infected with microfilaria, and 19 million people around the world were infected with filarial disease symptoms [13,14]. Japanese encephalitis virus (JEV) is transmitted by Culex mosquitos. JEV is in the family of Flaviviridae and is the primary pathogen of viral encephalitis in Asia. Its infection rate varies according to the population and age with the death rates of 0.3-60%. Rare outbreaks of JEV in the territories of the US have occurred. In the previous years, JEV was effectively controlled primarily by vaccination. The countries mostly affected are China, Korea, Japan, Taiwan and Thailand. However, some other countries including Vietnam, Cambodia, Myanmar, India, Nepal and Malaysia still have periodic epidemics.
The adult Culex spp. are nocturnal mosquito, with highest activities after 1 h of darkness. They are mainly exophilic and regularly stay in indoors after feeding on blood. Cx. tritaeniorhynchus acts as a vector of filariasis [15,16]. It's biting causes sensitive reactions including skin irritation and universal reactions, like urticarial and angioedema. Temperate zones are more susceptible to parasitic diseases, and the risk of contracting insect-borne illnesses has increased due to environmental alteration and increasing globalization [5,6] Anopheles-Anopheles is the primary vector of malaria. Totally 400 species of Anopheline have been identified, among which over 70 species are reported as actual vectors of human diseases. Anopheles stephensi is responsible for transmission of malaria in urban regions. Currently about 40% of the world population is endemic to malaria manifestation with about 300-500 million clinical cases of malaria and a death rate of 1.1-2.7 million. Malaria is still one of the most important communicable diseases in the world. Of the world population, 85.7% is exposed to the risk of malaria. 300 deaths were reported in malaria-attributable mortality as per reports [17,18,19,20]. No effective vaccine is available for malaria control, so the only efficient approach to control the incidence of this disease is to eradicate Anopheles mosquito vectors [21]. Life cycle of mosquitoes The mosquito life cycle comprises two main stages: aquatic and terrestrial. Aquatic stage includes egg, larvae and pupae; terrestrial stage includes the adult. The entire life cycle, from an egg to an adult, takes approximately 8-10 days. Usually mosquitoes lay eggs in raft or single which may vary according to different mosquito species. Generally, a single female lays 200-300 eggs at a time. Mostly eggs are laid in the standing water, water with regular flood and dirty water. Most of the eggs hatch within 24-48 h into larvae that feed on microscopic organisms such as planktons and organic matter in the water. Young larvae undergo four instar stages, and at each of these stages they shed their skins and finally develop into pupae (http://www.mosquitoworld.net/aboutmosquitoes/life-cycle/). Mosquitos take 4-5 days to develop from larvae into pupae. Pupae are also called as tumblers which are comma shaped, enclosed in cocoons and they do not feed. In 2-4 days, the adult mosquitos emerge out from pupae (http://www.mosquitoworld.net/about-mosquitoes/lifecycle/) which rest on the water and wait to get their bodies dry out. Males take two complete days to develop their reproductive organs, and then seek out a female for mating by the sound of her wing beats. Usually males live upto 3-5 days by feeding on fruit and plant nectar. After mating, females continue laying eggs after every blood meal. Normal female mosquito can live up to 2 months under the best climatic condition (https://www.cdc.gov/dengue/resources/factSheets/Mosquito LifecycleFINAL.pdf).

Mosquito control by using synthetic insecticides
Currently, mosquito-borne diseases are a major threat all over the world, and the synthetic insecticides are playing a major role in controlling mosquito populations. Mosquito control plays a major role in controlling the mosquito-borne diseases. Mosquito control mainly relies on the synthetic insecticides, such as pyrethroids, organophosphates, organochlorines and carbamates. Temephos is one of the most commonly used synthetic insecticide, which is used against the larval stages of mosquitoes [22]. For efficiency, large quantities of these synthetic insecticides are applied in the field, which results in ecological problem, such as damage to the beneficial organisms and resistance development in insects against these chemical insecticides [23,24,25,26,27,28]. Therefore, insecticides from plant sources are efficient alternatives to such synthetic insecticides. Azadirachtin, a limonoid which acts as an insect feeding deterrent, mosquito larvicide, repellent and growth regulator is one example [29,30,31,32,33].
Mosquito repellents-Repelling mosquitoes from their biting is one of the measures to control the transmission of the infectious diseases that are transmitted through the biting of infected mosquitoes. Repellents are substances which act locally or from a distance, in deterring an insect from flying to, landing on or biting human or animal skin [34,35]. As an approach to prevent mosquito bites for individual protection, the using of repellents is generally accepted as it plays an important role in preventing insect-borne diseases by reducing man-mosquito contact. Prevention of mosquito bites is possible through the application of repellents or physical barriers such as bed nets. Protection of humans and animals from biting of mosquitoes has been already accepted as part of an overall integrated insect-borne disease control program [36,37,38,34,35,39]. A variety of substances, including smoke, plant extracts, oils, tars and mud have been used over the centuries to repel mosquitoes [40].

Synthetic Repellents and Their Adverse Effects
Synthetic repellents are efficient in protecting people from blood-feeding insects such as ticks, mites and other arthropods thereby reducing transmission of arthropod-borne diseases. N,N-Diethyl-3-methylbenzamide (DEET) is one of the most well-known synthetic insect repellents and has been used as a repellent against mosquito for more than half a century [40.35,41]. Several studies have been documented on long-lasting protection of DEET against a wide variety of insect vectors. Currently, DEET is the most successful and accessible synthetic repellent in various commercial formulations such as solutions, lotions, gels, creams, aerosols, sticks and impregnated towelettes [42,43,44,45,46]. Furthermore, picaridin (2-(2-hydroxyethyl)-1piperidinecarboxylic acid 1-methylpropyl ester) is shown to be effective at providing protection against biting mosquitoes [47,48]. Synthetic chemicals such as diethyl toluamide, dimethyl phthalate, chlorpyrifos, dichlorvos, cypermethrin and ethohexadiol are also used as repellents against mosquito biting [49,50]. The efficacy of protection by using synthetic repellents relies on the preparation of formulation, application pattern, species and feeding behavior of the insects [47].
DEET is commonly safe for topical use if applied as recommended; however, some studies report adverse effects or toxicity to man, unsafe for children since it possibly causes encephalopathy and affects immune systems, when used incorrectly or in the extended term [47, ,34,35,51,46]. The environment and people's health are vulnerable to DEET and its related compounds [52]. DEET is not readily degradable by hydrolysis at environmental pHs and thus a ubiquitous pollutant of aquatic ecosystems. DEET has adverse effects such as irritation in skin and mucous membranes, serious neurologic effects, and is toxic to central nervous system of different age groups, specifically unsafe for children [43,44,45,46]. Undesirable features of DEET are unpleasant odor, uncomfortable oily or sticky feeling, and danger to plastics and synthetic rubber [53,52,47,2]. To avoid these inconveniences, repellents derived from plant sources are promisingwhich are effective, safe to users and inexpensiveto replace DEET [54].

Essential oil as repellents
Repelling mosquitoes from biting by using natural materials is advantageous since they do not harm the human and other beneficial organisms. On the contrary, synthetic repellents are reported to be harmful, since they cause several side effects to humans and beneficial organisms. Essential oils from aromatic plants are reported as potential repellents against mosquito vectors. Generally essential oils are used in manufacturing fragrances, and as flavoring agents for foods and beverages. Plant volatiles act as multiple and novel target sites to reduce the insecticide resistance of mosquitoes and also act as fumigants, contact insecticides, repellents, and antifeedants. They can adversely affect the growth rate and reproduction behaviour of mosquitoes, and have a longer duration of repellence than the synthetic chemicals.
Volatile oils extracted from plants and their major components like monoterpenes and sesquiterpenes are alternative sources for controlling mosquitoes at their immature stages and giving protection from biting of adult mosquitoes. Plant volatile oils, commonly used as fragrances and flavoring agents for foods and beverages, were recommended as an alternative source constituting numerous bioactive phytochemicals that could be potentially used for insect control [55]. Essential oils from plants act on multiple and novel target sites to reduce the development of resistance in mosquitoes. Because these oils are eluted from natural source, they are quite safe and beneficial to environment with least impact on animal and public health. Essential oils are applied to humans in a similar way to other conservative insecticides and they tend to be selective and have a little or no harmful effects [56,57,30,58,59,60].

Collection of Essential oil from Plants
The plant parts are crushed to release the volatiles. The solid-phase micro-extraction is used to extract volatile oils from the plants. Both dried and fresh plant parts underwent steam distillation by using Clevenger apparatus and then get placed in an extraction column connected to a roundbottomed distillation flask containing distilled water with approximately five times as much water and 10 glass beads. The flask is heated to about 100°C and allowed to boil until distillation is completed. The liquid-formed oil is collected with the help of a separating funnel. The collected mixture (distilled water and oil) is allowed to settle for 1 day. Separate layers of water and essential oil are formed and the water (lower) layer is slowly drawn out and removed to get the essential oil. The isolated oil is dried over anhydrous sodium sulfate and then collected and kept in an ambercolored bottle at 4°C until it is tested for mosquito repellency. This process is repeated until at least 20 ml of oil has been recovered. For repellency estimation, each oil and DEET are prepared in two formulations: 25% (v/v) in absolute ethanol with and without 5% vanillin [61,62,63,64,65,66,67,68].

Repellent Assay Method
Repellent efficiency of essential oil and its formulation added with 5% vanillin is evaluated against different mosquito species by using the human bait method, a previously recommended method [69,70,71,72,73]. For repellent assays, mosquito colonies are reared under laboratory condition. The adult mosquito is maintained at 25±2°C and 80±10% relative humidity (RH) under a photoperiod of 14:10 h (light/dark) by using slightly modified procedures described by Limsuwan et al. (1987). Mostly 5-to 7-days-old females are starved out and used for experimental studies. DEET (25% solution in ethanol with and without vanillin) is used to compare the repellency of essential oil isolated from plants. For repellency, experiments are carried out in 10 m×10 m ×3 m room at 27-35°C and 60-80% RHday biting mosquito experiment in daytime; night-biting mosquito experiment in night time. Aedes aegypti is tested from 08:00 to 16:00 h, while An. stephensi and Culex quinquefasciatus are tested between 16:00 and 24:00 h [74].
Totally 200-300 non-blood-starved female mosquitoes are selected at random and placed in an experimental cage (30 cm ×30 cm ×30 cm) and left to acclimatize for 1 h. Human volunteers wear a plastic glove with a 3 cm ×10 cm window on the ventral part of the forearm, after cleaning with distilled water. Treated area of the skin is exposed to the mosquitoes. The essential oils are applied at 1.0, 2.5 and 5.0 mg/cm 2 separately in the exposed 30 cm 2 marked area of a forearm [75]. The right forearm of each volunteer is allowed to dry for 1 min at room temperature. For control, the left forearm is treated with an equivalent volume of 5% vanillin in ethanol solution. The left arm is put into the cage and kept for 3 min; at least two mosquitoes must land on the test area. The arm is shaken off before the mosquito imbibes any blood and withdrawn from the cage. Consequently, the right arm is introduced into the cage for the same period and the number of mosquitoes landing on and attempting to feed is noted down. If no mosquito bite is evident during the 3-min exposure, the arm is withdrawn from the cage. The control and test arms are interchanged regularly. Arm exposure is continued at 15 min intervals. Tests for the determination of duration of protection are conducted according to the previously described methods [76,77,78]. Each sample is tested five times with different volunteers. One volunteer is used for one sample per day. Each person is assigned randomly and the volunteers are blinded to the repellent applied. No information is provided to the volunteers on the likely duration of action of each repellent [79,66,80,2]. Percentage and average protection time are calculated according to standard procedures described by Ansari and Razdan [81]. % where Ta is the number of mosquitoes in the control group and Tb is the number of mosquitoes in the treated group.

Kunming Mice Used for Repellent Experiment
Kunming mice are placed in the container supinely with their abdomens cleaned and depilated. Hairless area of the mice is 2 cm × 2 cm and for 2 min the mouse is put into a mosquito cage 0 cm ×30 cm ×30 cm containing 200-300 non-fed females. It is observed whether the number of mosquitoes that bite the mouse is more than 20. Essential oils are tested at different concentrations 1-15%. Prepared oil is (5 μl/cm 2 ) smeared on the exposed part of the mouse abdomen and allowed to dry. Mouse is put in the mosquito cage for 2 min and removed, and this is repeated at an interval of 1 h for 7 h. Finally the total number of blood-fed females is noted down. Triplicate is used for each sample. The percentage of repellency is calculated using the following formula [82].
For control, ethanol is smeared on the abdomen and the number of bites are compared with that of treated mouse. The data are analyzed using repeated measures of analysis of variance (ANOVA) and completed by Statistical Analysis Systems (Version 8.2).

Experimental Procedure for Field Condition
For the next stage of field repellent experiment, the formulation is tested against mosquitoes. Different volunteers with one control for each concentration are used in the tests. In every experiment, 2 ml aliquots of repellent materials are applied evenly between the knee and ankle of all volunteer's leg and tested. The experiment is conducted in triplicate on each subject during the study. For reference control, 5% vanillin in ethanol solution is applied in a similar manner. Volunteer's untreated areas are protected from mosquito bites by wearing head-net, gloves, socks, jacket, and long trousers folded up to the knee. Avoid applying any other cosmetic materials. The volunteers are made to sit in a row, at least 20 m apart from each other. Test and control subjects sitting 5 m apart from each other are exposed with both legs for 120 min with an observation of mosquito bite for every10-min period so that 12 biting collections are made on each volunteer. The mosquitoes are collected before imbibing any blood and stored in plastic cups, which are changed at every collecting site and stored in a moisture box until the processing of specimens. For every 10-min period the volunteers are moved to a new site. The collected mosquitoes are identified with stereomicroscope by using the taxonomic keys described by Tanaka [83] and Rattanarithikul and Panthusiri [84]. Percentage of repellency is calculated using exposure period and biting rate of the mosquitoes compared with the control. The volunteers' positions are changed at night to avoid the bias from any variations in host-seeking ability of mosquitoes.

Repellency Test in Large Room
The repellency tests are conducted in a 6 m  6 m  3 m room. The room has fluorescent lamps, door and six glasswindows that are always closed during the tests. 10 min before, 200-300 non-blood-fed females are released into the room. Three different volunteers are used for both control and tests. 3 ml of oil is applied on the area of the assessment -volunteer's leg, from knee to ankle; one leg is used for test and the other serves as control, and the covering surface area is about 782-826 cm 2 . After application, volunteers go inside the room and sit on the chairs in a triangle position with 1.5 m space from each other. For repellency evaluation, the landed mosquitoes are collected. In each repellent test of 6 h, volunteers are allowed every 10 min for every half-anhour interval and mosquito bites are observed. The position of volunteers is changed to allow for any variation. At each break, all mosquitoes are collected and released back into the room to maintain their number. Different days and time are used to test different mosquito species. Every test is carried out for 6 h and the timing of the test depends on the target mosquitoes: 10:00-16:00 h for Ae. aegypti and 18:00-24:00 h for Anopheles and Culex [85].

II. ESSENTIAL OIL AS MOSQUITO REPELLENTS
Plant essential oils are reported to be lipophilic in nature and they interfere with metabolic, biochemical, physiological, morphological and behavioral functions of insects [202]. Essential oils showing repellent properties and containing toxic effects against mosquitoes act as fumigants, contact insecticides, repellents and antifeedants or they can adversely affect the growth rate and reproduction, and have longer duration of repellence than synthetic chemicals on behavior of insect pests [86,63,87,88,89,90,91,92,93,94,60,95,96,97,98,99].
Synergistic effect between different components of the volatile oils may result in a higher bioactivity and increased repellent response. Components in combination present good repellency compared with when it is a single compound. Therefore, the essential oil containing specific main compounds may be an indication of its prospective use but does not warrant on proof of activity. Composition of essential oil may vary significantly among different aromatic plant species and between the same varieties from different ecological areas [100,101].
From, previous reports we conclude that essential oils are effectively repel the mosquitoes from biting. Certain reports strongly exhibit and suggest that essential oil gave 100% repellency against mosquitoes. In future these essential oil can be used to produce various effective products and formulations to control mosquito population therefore controlling vector borne diseases.

III. COMPONENTS OF ESSENTIAL OIL
Composition of the essential oil is variable depending on strain, chemotype, and geographic origin [154,155,156,157,]. Monoterpenes such as α-pinene, limonene, terpinolene, citronellol, citronellal, camphor and thymol, which are common constituents in volatile oils, are described in the literature as presenting mosquito repellent activity [158,59,159,160,161]. β-Caryophyllene is the most cited as a strong repellent from sesquiterpenes against Ae. aegypti [59,104,162].

IV. FUTURE PROSPECTS
Essential oil from aromatic plants have repellent activity to protect humans from mosquito biting. These essential oils can be used to prepare effective formulations like cream, coil, oil evaporator and other products to repel the mosquitoes. Identification of the specific component present in the essential oil, which is responsible for the repellency, paves the way to prepare new effective formulations for mosquito control. An efficient mosquito repellent could be produced using multiple (synergistic) essential oils. It is the best alternative way to replace the synthetic repellents that cause adverse side effects.

V. FUTURE DIRECTIONS OF THE RESEARCH
In this review, we have mentioned the essential oils that are used as eco-friendly mosquito repellents, so this review should be helpful for a larger-scale research. Furthermore, the repellent activity of these essential oils against other anthropophagous insects should also be studied to establish their possible wider application in controlling human-vector contact. Most of the mosquitoes are sensitive when exposed to the essential oil; however, some essential oils act as repellents only against specific mosquitoes. In future, studies would focus on enhancing the repellent efficacy of any essential oil to all the mosquitoes. It may be useful to finding a potential insect repellent. Furthermore, experiments on repellency of essential oil should also simultaneously evaluate its economic aspects and efficacy under different field conditions. This review would refocus the attention of the research community towards the development and application of known plants rather than screen more plants and isolate novel bioactive molecules that are repellents against mosquitoes. Future investigations should aim at testing essential oils and their mode of action, toxicity of the various biologically active essential oils to the target mechanisms involved and their possible effect on non-target organisms. Repellency approaches of essential oil should be multipronged such as in making of fumigants, sprays, paints, varnishes, incense, candles, etc. In domestic settings, fumigation and spraying in outdoor settings, topical repellents, clothes made of repellent fabrics and repellent wristbands are among other available products for individual protection. Thus, expanded use of essential oils in the ecofriendly insect pest management sector could be of both economic and ecological benefits.

VI. MOLECULAR ASPECTS
The repellent-exposed mosquitoes undergo changes in their life stages. Repellents act on specific targets and ultimately upregulate or downregulate specific gene. Using molecular techniques specific genes can be identified for the biting, flying, and other specific action in the life cycle of mosquitoes [179,180,181,182,183,184,185,186,187,188]. Genetic-based technologies are used to identify functional genomes in the insect. One of the most advanced geneticbased technologies is genome editing. Genome editing uses method such as relies on zinc finger nuclease (ZFN), clustered regulatory interspaced short palindromic repeats (CRISPR), CRISPR-associated protein 9 (Cas9) and transcription activator-like effector nucleases (TALEN). These technologies enable the alteration of specific target genes in mosquitoes. Elimination of specific gene responsible for the biting character of mosquito by using these technologies is one of the measures in the mosquito control program [189,190].

VII. CONCLUSIONS
Rich flora of aromatic plants of wild in nature will certainly be helpful in producing eco-friendly and efficient insecticides to control vector mosquitoes and thus the spread of dreadful diseases. Several studies report that the plant based essential oils exhibit promising repellent activity as against mosquito vectors. Essential oils have also been found to disrupt the host-seeking behavior or disorient the hostseeking mosquito. A mixture of such efficient plant based essential oils in an appropriate ratio would further promise the development of efficacious products towards minimizing the vector born diseases.

Conflict of interest statement
The authors declare that there are no conflicts of interest.  An. gambiae 10-1 mg cm 2 100 Negative control [194] Plectranthus marrubioides Hochst. ex Benth.