Plant Natural Products for the Control of Aedes aegypti: The Main Vector of Important Arboviruses

The mosquito species Aedes aegypti is one of the main vectors of arboviruses, including dengue, Zika and chikungunya. Considering the deficiency or absence of vaccines to prevent these diseases, vector control remains an important strategy. The use of plant natural product-based insecticides constitutes an alternative to chemical insecticides as they are degraded more easily and are less harmful to the environment, not to mention their lower toxicity to non-target insects. This review details plant species and their secondary metabolites that have demonstrated insecticidal properties (ovicidal, larvicidal, pupicidal, adulticidal, repellent and ovipositional effects) against the mosquito, together with their mechanisms of action. In particular, essential oils and some of their chemical constituents such as terpenoids and phenylpropanoids offer distinct advantages. Thiophenes, amides and alkaloids also possess high larvicidal and adulticidal activities, adding to the wealth of plant natural products with potential in vector control applications.


Introduction
The mosquito Aedes aegypti (Diptera: Culicidae) originated in Egypt and it is widely distributed in tropical and subtropical regions, including North America and Europe [1,2]. Ae. aegypti presents complete metamorphosis from immature egg, larva and pupa stages to the adult mosquito itself ( Figure 1). The life cycle varies according to environmental temperature, food availability and quantity of larvae in the same breeding site. Under favorable conditions, after egg hatching, the mosquito transforms into the adult stage within 10 days, even though the eggs can be viable up to 450 days in the absence of water [3].
The female mosquito requires hematophagy for egg maturation. Viral transmission to humans occurs during this process if the mosquito is infected. The lifetime of a female mosquito is approximately 45 days [1]. Ae. aegypti population control is considered the principal measure to combat arboviral diseases as this species is the primary vector of dengue, Zika, chikungunya and urban yellow fever [4,5].
In 2012, dengue was considered the mosquito-borne disease of major importance in the world [5]. According to the World Health Organization, 390 million people are infected annually with the dengue virus, 96 million of which have clinical manifestations [6]. There are various symptoms, the first is usually high fever (39-40 • C) with headache, prostration, arthralgia, anorexia, asthenia, nausea, among others. Some clinical aspects often depend on patient age. There is no specific treatment for dengue and the more complicated cases of the disease can cause hemorrhage, shock and even death [7].
There are several techniques already used to combat mosquitoes, which act both in the immature phases (egg, larva and pupa) and in the adult [13]. Highly toxic synthetic insecticides such as organophosphates, pyrethroids and carbamates have been historically used to combat the mosquito, Ae. aegypti is considered the main Zika virus vector ( Figure 1), but infection can also occur by sexual transmission or blood transfusion [8]. Symptoms are non-specific and self-limited, being easily confused with other arboviral diseases. Some important complications exist, such as microcephaly in fetuses and Guillan Barré syndrome. The virus has been reported in countries in Americas, Europe, Asia and the Pacific region [8]. Like dengue and Zika, chikungunya has no specific treatment. The disease emerged in the Americas in 2013, with about 1.7 million cases identified and 252 deaths reported by August 2015 [9].
In 2013, dengue generated a global cost of US $8.9 billion, with around 58.4 million symptomatic cases (13.5 million fatalities) in the 141 countries and territories. The per capita costs of dengue were $70.1 for hospital treatment, $51.1 for outpatient treatment and $12.9 for cases that did not reach the health system. According to this study, Brazil had an incidence of 751 to 1,000 cases per 100,000 people. The expenses were proportional to the incidence and ranged from $2.5 to $5 for each treated case [10].
A more recent study showed that in 2016 the Brazilian government spent around R $805 million (ca. 160 million US$) to treat diseases caused by the Ae. aegypti mosquito, including direct medical expenses and indirect costs. In addition, about R $1.5 billion were destined to combat the vector, totaling R $2.3 billion, that was 2% of the health budget for that year. More than 2 million cases of Ae. aegypti related diseases were verified. These numbers were underestimated as they did not include complications such as microcephaly and Guillain-Barré syndrome [11].
A study estimated that about 60% of the world population will be at risk of dengue in 2080, which represents over 6.1 (4.7-6.9) billion people [12]. Considering that vector control is the main tool for controlling these expensive arboviruses, investment in techniques to combat the Ae. aegypti mosquito is growing [3]. Investments are particularly focused on techniques with minimal negative impacts on non-target animals and the environment [4,13].

Mosquito Control
There are several techniques already used to combat mosquitoes, which act both in the immature phases (egg, larva and pupa) and in the adult [13]. Highly toxic synthetic insecticides such as organophosphates, pyrethroids and carbamates have been historically used to combat the mosquito, acting mainly on insect larvae [4]. More recently, insecticides with less toxicity which are less persistent in the environment have been developed, including neonicotinoids and oxadiazines [14]. However, these products are still widely used and harmful to living organisms and the environment, and the use of foogers and aerial applications of sintetic insecticides against adults, such as pyrethroids products, contributes to insect resistance problems [4]. Therefore, efforts must be made to ensure newly developed alternative insecticides are more eco-friendly.
Biological tools that control the adult stage are based on behavior, such as the Sterile Insect Technique (SIT), Incompatible Insect Technique (IIT) and Release of Insects carrying a Dominant Lethal gene (RIDL), which involve insect sterilization by chemical irradiation, natural bacteria which are pathogenic for mosquito (highly specific strains of Wolbachia) and genetic modifications to make sterile male mosquitoes, respectively [15,16]. The other technique applied against the adult stage is the use of entomopathogenic fungi, specifically in the orders Entomophthorales, Hypocreales and Pezizales due to their specificity, ability to manipulate and infectiveness to the host [17].
Ae. aegypti control using specific strains of Wolbachia bacteria is currently practiced in different locations around the world through the World Mosquito Program. This program involves the application of the bacteria to laboratory mosquitoes that are released into the local Ae. aegypti population during reproduction. The presence of bacteria in mosquitoes decreases the possibility of arbovirus transmission to people [18].
Biological control tools that act against the immature stages include the application of Bacillus thuringiensis in larvae habitats; products that prevent oviposition and/or inhibit growth and reproduction, including pheromones. There are also natural predators such as fish (especially of the genus Gambusia and Poecilia, family Poeciliidae) [19,20], copepods (including several species of the genus Mesocyclops) [21,22] and the "elephant mosquito" (genus Toxorhynchites) [23,24].
Finally, plant-based insecticides (ovicides, larvicides and pupicides) [25,26] deserve a special mention due to the vast biodiversity of species found in the world, estimated to be approximately 400,000 terrestrial species [27]. Botanical insecticides can be plant extracts, essential oils and/or secondary metabolites [4,14].

Plant Natural Products to Control Mosquitoes
The search for plant natural products to control Ae. aegypti dates back a number of years, with research published since the 1980s [28,29]. However, chemical insecticides are most commonly used, despite their enormous toxicity to non-target organisms, such as: (i) poisoning and death; (ii) cancer, by non-genotoxic mechanisms (immunosuppressants, cytotoxic) or by triggering the carcinogenic process in different ways; (iii) harmful effects on the nervous, renal, respiratory and reproductive systems and (iv) induction of oxidative stress [30].
In addition to toxicity, another concern is the increasing resistance of the mosquito vector to chemical insecticides. One example is the knockdown resistance (kdr) mutation, in which resistance to pyrethroid insecticides occurs, whereby the target site is the sodium channel of the Ae. aegypti nervous system [31,32]. In Brazil, of the five insecticides approved by the Public Health Ministry and recommended by the WHO for adult mosquito control, four belong to the pyrethroid class together with one organophosphate (malathion). However, in 2011 a technical note was issued suspending the use of pyrethroids in Brazil to control Ae. aegypti [33].
The level of resistance is dependent on the insecticide concentration, frequency and duration of application [34]. The resistance mechanisms of mosquitoes may be associated with changes in the insect cuticle resulting in less insecticide absorption [35], changes in insect metabolism involving biotransformation enzymes [36,37] and modifications of the insecticide target site, usually by genetic mutations [38,39].
The main esterases involved in the resistance process are carboxylesterases and cholinesterases. Carboxylesterases are usually resistant to organophosphates, with this resistance relating to both a quantitative mechanism (overproduction of enzyme) and qualitative mechanism (mutations that cause alterations in enzymatic properties) [40]. In the case of cholinesterases, the resistance is mainly caused by gene mutation. The main insecticides resistant to the acetylcholinesterase target site are organophosphates and carbamates [41].
caused by gene mutation. The main insecticides resistant to the acetylcholinesterase target site are organophosphates and carbamates [41].
Another problem associated with chemical insecticides is the damage caused to the environment and living organisms by their degradation products, which may prove more toxic than the original product itself. Examples include the degradation products of temephos, whose effects have already been documented in aquatic environments [42], and malathion, together with its metabolites, in nontarget organisms such as Daphnia magna (Cladocera: Daphniidae) [43].
Insecticides derived from plant natural products therefore offer a promising source of safer new products for mosquito control due to minimal residues from its natural degradation in both the field and in water, minimizing ecosystem disruption [44,45]. There is considerable research on insecticides of natural origin, especially those of microbial and plant origin, due to their innumerable secondary metabolites produced especially as a defense mechanism against natural predators [46]. It is estimated that there are more than 100,000 plant metabolites, with hundreds or more exhibiting some activity against insects [47].
Botanical insecticides are advantageous as they are generally environmentally safe, non-toxic to non-target organisms including homeothermic animals and their residues biodegradable [25,26,30]. The synergic mixture of the active compounds in extracts induce several mechanisms of action and result in less pest resistance [30,48].
The present review focuses on the more recent studies of botanical extracts and active compounds in applications against Ae. aegypti, from immature to adult stages, in addition to their main proposed mechanisms of action. The crude extracts are obtained using different extraction methods with organic solvents or water. Essential oils are obtained by steam distillation or hydrodistillation. The classes of active compounds include terpenes, alkaloids and amides, steroids, flavonoids, furanochromones, phenylpropanoids and phenol derivatives, lignans and neolignans, naphthoquinones, fatty acids and their derivatives. The type of insecticide activity (ovicide, larvicide, pupicide, adulticide) is reported as mortality and lethal concentration values (LC50, LC90 and/or LC99), together with egg hatchability. The other activities tested are mosquito repellency, oviposition deterrence, growth regulation and the antifeedant effect.

Essential Oils
Essential oils deserve special attention as they have yields of 0.5 to 2.0% in the extraction process, contain a high concentratration of secondary metabolites and generally present potent activity due to the synergic effect of the constituents. An important advantage is, with few exceptions, their relatively low, or no, toxicity to mammals ( Figure 2). Some pure compounds constituents of essential oils are moderately toxic to mammals (LD50 800-3000 mg/kg in rodents) while formulated products usually are low or non-toxic to mammals, birds and fish (LD50 above 5000 mg/kg for rodents) [47,49]. These essential oils are mainly obtained from aromatic plants, of which there are more than 3000 species. Approximately 10% of these are already produced in large quantities for other uses, such as flavorings and fragrances, and are therefore readily available at reasonable prices [30]. Essential oils are composed of volatile compounds, which give an important advantage of non-persistence in the environment [49,50].
It is important to note that the same volatility may be a disadvantage in terms of instability. However, this property can be overcome using pharmaceutical technology such as micro and nanoencapsulation [51,52]. Formulation development is therefore critical for essential oils to be used effectively and safely as pesticides. A number of studies have demonstrated that a suitable vehicle prolongs the insecticidal effect [51,53,54].
Of the plant families affording essentials oils, those most tested against Ae. aegypti larvae were Myrtaceae, in particular Eucalyptus species, followed by Fabaceae, Asteraceae, Apiaceae and Lamiaceae. Asteraceae was the most important for the adulticide, repellent and oviposition effects.
Regarding larvicidal activity there is currently no value specified by the WHO to discriminate whether a compound or extract is active against insects. However, researchers usually consider that an LC 50 < 50 µg/mL is very active; an LC 50 50-100 µg/mL is active, and an LC 50 > 100 µg/mL is weak/inactive [55][56][57]. These essential oils are mainly obtained from aromatic plants, of which there are more than 3000 species. Approximately 10% of these are already produced in large quantities for other uses, such as flavorings and fragrances, and are therefore readily available at reasonable prices [30]. Essential oils are composed of volatile compounds, which give an important advantage of non-persistence in the environment [49,50].
It is important to note that the same volatility may be a disadvantage in terms of instability. However, this property can be overcome using pharmaceutical technology such as micro and nanoencapsulation [51,52]. Formulation development is therefore critical for essential oils to be used effectively and safely as pesticides. A number of studies have demonstrated that a suitable vehicle prolongs the insecticidal effect [51,53,54].
Of the plant families affording essentials oils, those most tested against Ae. aegypti larvae were Myrtaceae, in particular Eucalyptus species, followed by Fabaceae, Asteraceae, Apiaceae and Lamiaceae. Asteraceae was the most important for the adulticide, repellent and oviposition effects.
Regarding larvicidal activity there is currently no value specified by the WHO to discriminate whether a compound or extract is active against insects. However, researchers usually consider that an LC50 < 50 µg/mL is very active; an LC50 50-100 µg/mL is active, and an LC50 > 100 µg/mL is weak/inactive [55][56][57]. Considering this classification, this review highlights 11 species with 12 very active essential oils, 11 species with 14 active essential oils and 6 species with weak/no activity for 7 essential oils. Eight species do not have reported LC 50 values and are not considered in this classification. However, these values can change significantly after formulation, as discussed in Section 8 "Limitations and/or Expectations of Plant Natural Product Insecticide Applications".
Some studies made the identification of secondary metabolites in essential oils evaluated for insectidal activities described abouve and its chemical structures are illustrated in Figure 3.
Tables 1 and 2 summarise the publications selected for this review and discussion of the essential oils active against the Ae. aeygpti mosquito. Table 1 describes larvicidal activities, while Table 2 details the adulticidal, repellent and oviposition activities.
Another species is Myristica fragans Houtt. (Myristicaceae), which is popularly known as nutmeg and is used as a flavoring. Essential oil from its seeds demonstrated high toxicity against Ae. aegypti, in both the L3 larval phase (LC 50 28.2 µg/mL) and the adult phase (LC 50 18.5 µg/mg female). The major compounds identified were sabinene (3, 52%), α-pinene (no stereochemistry defined, 4) (13%) and terpinen-4-ol (5) (11%). Regarding neurotoxic effects, this essential oil is non-toxic to humans as its IC 50 values for human acetylcholinesterase and human butyrylcholinesterase are higher than 4000 µg/mL [54]. Nutmeg flower essential oil presented higher larvicidal activity (LC 50 47.42 µg/mL) than the ethanolic extract (LC 50 75.45 µg/mL). This result suggests that the constituents of the essential oil either exhibit higher larvicidal activity, or that the synergy between them favors the toxicity to the mosquito [60].
The results listed and discussed in this section clearly suggest that essential oils present a promising alternative to develop an effective natural and potentially more eco-friendly insecticide for the control of Ae. aegypti, especially during the larval phase. The challenges for these materials are to improve solubility in water and prolong the insecticidal effect. It is also important to understand the synergism and/or antagonism of their constituents, together with the optimum ratio.

Organic/Aqueous Extracts
Concerning organic/aqueous extracts, the plant families with the highest number of species tested against Ae. aegypti larvae were Fabaceae, Asteraceae, Piperaceae and Euphorbiaceae. Similarly, as described by Isman (2015), India was the country with the most publications in this field, followed by Brazil [14].
Of the 20 plant species, at least one organic/aqueous extract showed high larvicidal activity (LC 50 < 50 µg/mL); 12 were active (LC 50 50-100 µg/mL) and 26 had weak activity (LC 50 > 100 µg/mL). Nevertheless, these values can change significantly after formulation in a similar way to essential oils, as described in Section 8 "Limitations and/or Expectations of Plant Natural Product Insecticidal Applications". Figure 4 details the chemical structures of the secondary metabolites identified in the organic extracts.

Organic/Aqueous Extracts
Concerning organic/aqueous extracts, the plant families with the highest number of species tested against Ae. aegypti larvae were Fabaceae, Asteraceae, Piperaceae and Euphorbiaceae. Similarly, as described by Isman (2015), India was the country with the most publications in this field, followed by Brazil [14].
Of the 20 plant species, at least one organic/aqueous extract showed high larvicidal activity (LC50 < 50 µg/mL); 12 were active (LC50 50-100 µg/mL) and 26 had weak activity (LC50 > 100 µg/mL). Nevertheless, these values can change significantly after formulation in a similar way to essential oils, as described in Section 8 "Limitations and/or Expectations of Plant Natural Product Insecticidal Applications". Figure 4 details the chemical structures of the secondary metabolites identified in the organic extracts. Tables 3 and 4 summarise the scientific literature selected for the discussion of insecticidal activities of organic/aqueous extracts against Ae. aeygpti mosquito. Table 3 describes larvicidal  activities, while Table 4 describes adulticidal, pupicidal, ovicidal, repellent and oviposition activities. Tables 3 and 4 summarise the scientific literature selected for the discussion of insecticidal activities of organic/aqueous extracts against Ae. aeygpti mosquito. Table 3 describes larvicidal  activities, while Table 4 describes adulticidal, pupicidal, ovicidal, repellent and oviposition activities.        Piper species (Piperaceae) demonstrated LC 50 ranging from 2.23 to 567 µg/mL for L3 and L4 larval stages [106,109,110]. The most active species extracts were Piper longum L. (fruit ethanolic), followed by P. sarmentosum (entire plant ethanolic LC 50 4.06 µg/mL) and Piper ribesoides Wall. (wood LC 50 8.13 µg/mL) [109]. Piper nigrum L. peppercorn ethanolic extract was active and purified fractions were highly active, with possible toxicity due to oleic acid (18) [110]. P. aduncum and Piper hispidum Sw. displayed weak activity against L3 larvae (LC 50 > 150 µg/mL) [106].
The biological activity of each plant species extract is specific to the plant part(s) and the polarity of the extraction solvent used. Furthermore, activity can differ significantly for the 4 different larval stages [58,86,89,[92][93][94]96,98,100,108,113,115]. This variation is discussed below.
In addition, different parts of Lonchocarpus urucu Killip & A.C. Sm. (Fabaceae) extracted with the same solvent (methanol) showed different toxicity. The root bark extract was more active (LC 50 17.6 µg/mL) than the root medulla extract (LC 50 33.32 µg/mL) against L4 larvae [100]. The toxicity of Heracleum rigens Wall. (Apiaceae) seed extracts was evaluated against different larval stages (LC 50 40.64 to 308.65 µg/mL), with the petroleum ether extract the most toxic to all larval stages and acetone the least toxic [96]. Different organic solvent extracts of Cassia fistula L. (Fabaceae) leaves were evaluated against the mosquito (larvicide, ovicide and repellent). The methanolic extract was the most active for all activities, notably as a larvicidal (LC 50 10.69 µg/mL). Other extracts also demonstrated high activity against larvae: benzene (LC 50 18.27 µg/mL) and acetone (LC 50 23.95 µg/mL). The non-hatching concentration for eggs ranged from 120 to 160 mg/L and the repellent action (100% at 5 mg/cm 2 ) ranged from 6.0 to 4.3 h [86].
Dalbergia brasiliensis Vogel (Fabaceae), commonly known as Jacarandá-da-Bahia in Brazil, is a tree native to the states of Bahia, Minas Gerais, Espírito Santo, Rio de Janeiro and São Paulo. Larvicidal activity of its leaves and trunk bark ethanolic extracts, together with fractions purified by partitioning with n-hexane, ethyl acetate and chloroform, were similar (LC 50 between 24.0 and 44.0 µg/mL) [89].
Studies using the incorporation of inorganic nanoparticles, such as zinc oxide and silver in plant extracts, have shown an increase in their biological activity. They are generally easy to obtain, inexpensive, not to mention non-toxic to humans and animals [53]. All of the plant extracts described below showed higher larvicidal activity when incorporated into nanoparticles [53,82,116].
An aqueous extract of Artemisia herba-alba Asso (Asteraceae) leaves was tested against L4 larvae strains from India and Saudi Arabia. The LC 50 values were 117.18 µg/mL and 614.54 µg/mL for India and Saudi Arabia larvae, respectively. When the extract was incorporated into silver nanoparticles the activity increased significantly to 10.70 µg/mL and 33.58 µg/mL, respectively. Similar results were observed against adult mosquitoes [82].
The activity of a zinc oxide nanoparticle incorporating a Myristica fragans leaf methanolic extract was compared with the crude extract. The activity of the crude extract against the 4 larvae stages (LC 50 162.03 to 273.9 µg/mL) was less than the nanoparticles (LC 50 3.44 to 10.28 µg/mL). Similar activity was reported against the pupa (crude extract LC 50 359.08 µg/mL and nanoparticles LC 50 14.63 µg/mL), and female adult forms (crude extract LC 50 180.26 µg/mosquito and nanoparticles LC 50 15 µg/mosquito) [53].
In another study involving a methanol extract of C. pulcherrima, complete inhibition of egg hatching was reported at 300 ppm. The repellency was the same as the aforementioned study (5 mg/cm 2 ) [117]. Coccinia indica Wight & Arn. (Cucurbitaceae) presented similar insecticidal properties for different extracts, with a methanolic extract the most active in terms of ovicidal activity (zero hatchability at 200 ppm) and a hexanic extract having the more effective repellency (100% of repellency at 1 mg/cm 2 ). For 100% non-hatchability, the concentrations were between 200 ppm and 300 ppm and for 100% of repellency were between 1 and 5 mg/cm 2 [119].
The methanolic extract of Eclipta alba (L.) Hassk (Asteraceae) leaves was also the most active among the solvents of different polarities used to evaluate larvicidal and ovicidal activities of this plant. The LC 50 values against L3 larvae were between 127 and 165 µg/mL. Complete inhibition of egg hatching occurred at 300 ppm for the methanolic extract and 350 ppm for the other solvents [91].
The methanolic extract of Mentha piperita L. (Lamiaceae) and different extracts of Cardiospermum halicacabum L. (Sapindaceae) showed repellent activity [118,120]. Essential oils and aqueous extracts of the red and pink flowers of A. purpurata were investigated for both larvicidal activity and oviposition effect. Similar to the essential oils, the extract of the pink flower was more active than the red, and both disrupted oviposition [67].
In general, organic extracts from different parts of Parthenium hysterophorus (Asteraceae), Pithecellobium dulce (Roxb.) Benth. (Fabaceae) and Solanum xanthocarpum Schrad. & J.C. Wendl. (Solanaceae) showed weak insecticidal action, requiring high concentrations to demonstrate some biological activity [111,114,121]. Other species that were inactive were Helicteres velutina K. However, an important consideration for these materials is the type of extraction solvent employed, such as n-hexane, chloroform, benzene, given their toxicity to humans associated with harmful residues [122].

Terpenes
Terpenoids are a very promising target for the development of products of natural origin to be used in the control of the Ae. aegypti mosquito. These compounds were the most identified in the essential oils, extracts and purified fractions, generally having better results against the mosquito, especially in terms of larvicidal activity. Of the terpenes, monoterpenes are the most active and present great possibilities in bioinsecticide applications due to their low toxicity against mammals and non-target organisms [50]. This significant activity against the mosquito can be explained by the hydrophobicity of this class. Terpene toxicity against Ae. aegypti larvae may be associated with their nonpolar property as reported for other insects [123,124]. This property increases the ability of the compound to penetrate the hydrophobic larvae cuticle and renders them more toxic to the insect in comparison to polar compounds [123]. The chemical structures of the terpenes tested are shown in Figures 5-7.
The triterpenoids ursolic acid (30) and betulinic acid (22) showed larvicidal activity with LC 50 245.24 µM and 310.83 µM, respectively. Their corresponding structures are illustrated in Figure 5. Bioassays of their chemical derivatives, with esterification of the hydroxyl group at the C-3 position, demonstrated less activity, suggesting that the hydroxyl group plays an important role in larvicidal activity [126].

Terpenes
Terpenoids are a very promising target for the development of products of natural origin to be used in the control of the Ae. aegypti mosquito. These compounds were the most identified in the essential oils, extracts and purified fractions, generally having better results against the mosquito, especially in terms of larvicidal activity. Of the terpenes, monoterpenes are the most active and present great possibilities in bioinsecticide applications due to their low toxicity against mammals and non-target organisms [50]. This significant activity against the mosquito can be explained by the hydrophobicity of this class. Terpene toxicity against Ae. aegypti larvae may be associated with their nonpolar property as reported for other insects [123,124]. This property increases the ability of the compound to penetrate the hydrophobic larvae cuticle and renders them more toxic to the insect in comparison to polar compounds [123]. The chemical structures of the terpenes tested are shown in Figures 5-7.      The sesquiterpenes α-costic acid (31) and inuloxin A (32), both isolated from Inula viscosa (L.) Aiton (Asteraceae), demonstrated strong activity against L1 larvae. The concentration of each terpene required for 100% mortality was 4.27 µM and 4.03 µM, respectively [127]. Other sesquiterpene alkaloids with strong larvicidal activity (L3 and L4 instar) were 1-O-benzoyl-1-deacetyl-4-deoxyalatamine (33) (LC 50 9.4 µM) and 1,2-O-dibenzoyl-1,2-deacetyl-4-deoxyalatamine (34) (LC 50 2.3 µM). These sesquiterpenes with a β-dihydroagrofuran skeleton were isolated from M. oblongata stems [101].
Monoterpenes were the most evaluated for larvicidal activity with LC 50 values ranging from 88 to 540 µM. The terpene hydrocarbons: limonene (13) (LC 50  . The (-) enantiomers displayed higher activity than (+), so these results showed that the type of enantiomer and even the racemic mixture could directly interfere with the activity [78].
The monoterpene limonene (13) was incorporated into a nanoemulsion to improve its water solubility and therefore increase its activity [68]. This compound deserves to be highlighted as it presented high oral LC 50 (>4000 mg/kg) and dermatological LC 50 (>5000 mg/kg) values for rodents. It is therefore considered safe and non-toxic to mammals [30].

Phenylpropanoids and Phenolic Derivatives
The chemical structures of the phenylpropanoids and esters discussed in this section are illustrated in Figure 8. Important larvicidal properties were also reported for eugenol (15), a phenolic compound that presents some advantages such as its non-persistence in water and soil, together with its natural degradation in organic acids through the action of Pseudomonas, a soil-dwelling bacterium. Furthermore, it is 1500 times less toxic than pyrethrins and 15,000 times less toxic than azinphosmethyl, an organophosphate [50]. The LC50 value for larvicidal activity was 200.97 µM [64]. The Among the phenylpropanoids and phenolic derivatives classes cinnamaldehyde (48) and cinnamyl acetate (49) can be highlighted as they presented interesting larvicidal activity (LC 50 219.43 and 187.27 µM, respectively) [64]. It is important to note that cinnamaldehyde is present in commercial insect-fighting formulations such as Cinamite ® and Valero ® . This information suggests that these secondary metabolites have a high potential for use against Ae. aegypti due to their possible toxicological safety, given that they have been authorized as insecticides since 2001.
Important larvicidal properties were also reported for eugenol (15), a phenolic compound that presents some advantages such as its non-persistence in water and soil, together with its natural degradation in organic acids through the action of Pseudomonas, a soil-dwelling bacterium. Furthermore, it is 1500 times less toxic than pyrethrins and 15,000 times less toxic than azinphos-methyl, an organophosphate [50]. The LC 50

Alkaloids and Amides
The Piperaceae family has numerous compounds with promising activity against Ae. aegypti. The chemical structures of the aforementioned alkaloids and amides are illustrated in Figure 9. Important larvicidal properties were also reported for eugenol (15), a phenolic compound that presents some advantages such as its non-persistence in water and soil, together with its natural degradation in organic acids through the action of Pseudomonas, a soil-dwelling bacterium. Furthermore, it is 1500 times less toxic than pyrethrins and 15,000 times less toxic than azinphosmethyl, an organophosphate [50]. The LC50 value for larvicidal activity was 200.97 µM [64]. The phenylpropanoid trans-anethole (7) also showed important action against Ae. aegypti larvae (LC50 283.40 µM) [64].
The analysis of the structure-activity relationship for the N-isobutylamide alkaloids 55-61, it is reasonable to hypothesise that the N-isobutylamine moiety is of crucial importance in terms of larvicidal activity, while the methylenedioxyphenyl moiety does not appear to be essential.
The analysis of the structure-activity relationship for the N-isobutylamide alkaloids 55-61, it is reasonable to hypothesise that the N-isobutylamine moiety is of crucial importance in terms of larvicidal activity, while the methylenedioxyphenyl moiety does not appear to be essential.

Thiophenes and Acids
Thiophene and fatty acid chemical structures are illustrated in Figure 10. Nine thiophenes, with different numbers of thiophene rings, isolated from E. transiliensis exhibited strong larvicidal activity and a positive correlation was reported between the number of thiophene rings and larvicidal activity, with thiophene derivatives composed of more rings demonstrating more activity [90]. Nine thiophenes, with different numbers of thiophene rings, isolated from E. transiliensis exhibited strong larvicidal activity and a positive correlation was reported between the number of thiophene rings and larvicidal activity, with thiophene derivatives composed of more rings demonstrating more activity [90].

Furanochromones and Furanocoumarin
The chemical structures of the coumarins are illustrated in Figure 13.

Mechanisms of Action
The mechanisms of action for the Ae. aegypti control relate more to the use of conventional chemical insecticides. Table 5 summarizes the mechanisms of action data of the botanical samples discussed in this section. Ae. aegypti control relies primarily on the use of conventional chemical insecticides which target different critical sites in the mosquito life cycle. Organophosphates and carbamates, for example, target acetylcholinesterase enzyme inhibition. Pyrethroids and some organochlorines target sodium channels. Cyclodienes and polychloroterpenes target gamma-aminobutyric acid (GABA) receptors [34].
Other mechanisms of alternative insecticides authorized by regulatory agencies vary in terms of their action. For example, a biological approach employs the use of entomopathogenic bacteria (Bacillus thuringiensis israelensis and Bacillus sphaericus), which act via the toxic action of their spores damaging the intestinal epithelium of larvae. Insect growth regulators (IGR) differ in that they inhibit insect chitin synthesis, and therefore disrupt the moulting process, while juvenile hormone analogs (JHA) act by interfering with the insect's endocrine system [138].
Further studies are required in order to completely understand the various toxic action mechanisms of botanical insecticides. However, a number of mechanisms have been proposed and proven. The majority of mechanism of action studies have focused on the larval stage, particularly feeding and/or contact. In the case of ingestion, the action is usually through digestive toxicity whereas contact may involve enzymatic inhibition, endocrine disruption (acting especially during the moulting process), toxicity to the nervous system and other mechanisms depending on the target site [143].
The rapid toxic action of essential oils against the insect indicates a possible neurotoxic mode of action [144]. Phytochemicals may act in cholinergic, GABA, mitochondrial and octopaminergic systems [145]. A study of five volatile compounds commonly found in plant essential oils-eugenol, geraniol, coumarin, eucalyptol and carvacrol-investigated docking against octopamine and acetylcholinesterase receptors in Ae. aegypti and Homo sapiens protein models. All compounds were found to dock in both protein models, with some more selectivity for insect proteins [146].
Effects on the larval nervous system were observed after treatment with Piper species extracts. Tremor, convulsion, excitement, followed by paralysis and death were verified after exposure of larvae to P. longum, P. ribesoides and P. sarmentosum extracts. In addition, the larvae showed morphological changes in the anal papillae [109].
Essential oils of I. verum, P. dioica and M. fragrans inhibited acetylcholinesterase causing acetylcholine accumulation in the synapses, with the membrane in a constant state of excitement, culminating in ataxia, lack of neuromuscular coordination and eventual death [54]. The neurotoxic effect was also observed for a nanoemulsion with P. emarginatus essential oil. It probably causes reversible inhibition of acetylcholinesterase and consequently larval death [66].
D. brasiliensis extracts caused external morphological alterations in the larvae, resulting in interference in the moulting process. The authors also reported digestive toxicity and morphological changes in the anal papillae and respiratory siphon of the larvae which interfered with swimming and oxygen flow [89]. Similarly, nilocetin (88) (Figure 14) induced morphological deformations together with moulting symptoms and growth disruption in all mosquito life cycle stages. These compounds also totally ruptured the peritrophic membrane [99].
A Lonchocarpus urucu extract caused disruption in the peritrophic matrix, a medium intestine lining composed of chitin and proteins, whose functions are to protect against abrasion caused by food and micro-organisms, among others such as decreasing the excretion of digestive enzymes through their recycling. In addition, this extract caused extensive damage to the midgut epithelium (Table 5) [100]. Pellitorine (55), an isobutylamide alkaloid, whose structure is illustrated in Figure 9, promoted histological changes in the thorax, midgut and anal gills. These toxic effects probably occur as a result of compound action on the larval osmoregulation system [148]. Already a nanoemulsion with limonene (13) (Figure 7) promoted morphological alterations to the head, siphon, abdomen cuticles and thorax, promoting larvae fragility and low mobility [68].
C. rhamnifolioides essential oil induced toxicity in the larvae by trypsin-like activity. Trypsin is a serine protease that widely occurs in the gut of insects. A decrease in its activity may result in poor nutrient absorption and non-availability of essential aminoacids, causing insect death [72]. A Moringa oleifera (Moringaceae) extract also caused larval toxicity by inhibiting trypsin in the gut [147]. After treatment with grandisin (82) (Figure 12), larvae presented intense tissue destruction and cell disorganization in the anterior midgut [139].

Limitations and/or Expectations of Plant Natural Product Insecticide Applications
As demonstrated in this review, natural products of botanical origin are promising for control of the Ae. aegypti mosquito, although there remain several limitations and challenges to overcome for their application as insecticidal products. From 1998 until early 2011, the number of patents of essential oil-containing mosquito repellent inventions has almost doubled every 4 years [149], but the number of new products does not reflect this. There are several possible reasons for this disparity, such as: (i) the onerous regulatory processes involved in the registration of a pesticide product; (ii) the quantity of raw material biomass required to obtain sufficient extract and/or its isolated active compound, and (iii) most of the research is conducted at the laboratory scale often without field evaluation to confirm the product application [13,47,150].
The complex process of registering an insecticide discourages companies from investing in new products, especially in some places such as Brazil and the European Union. There are numerous criteria, including provision of non-target toxicology and environmental destination data, extensive data to guarantee plant stability and extract standardization, together with physico-chemical and microbiological procedures establishing quality control of the raw material and final product [47,48].
The low availability of raw materials due to limited yields and cultivation usually makes botanical insecticides more expensive than chemicals. The study of bioactive compound synthesis through biotechnology, such as tissue culture in bioreactors, constitutes an alternative to this limitation [150,151]. In moving from laboratory to industrial scale, a number of different factors must be considered: botanical material, analysis technique, formulations, toxicological tests, mechanisms of action, among others.
Considering the botanical material, it is essential to correctly identify the botanical species and determine the chemical composition of the extract (standardization) [13,50]. Chemical composition may vary depending on numerous factors, such as crop period, seasonality, phenological stage, temperature, humidity, luminosity, altitude, pluviometry, ultraviolet radiation, soil and nutrient conditions, geographical locations, collection method, drying and the part of the plant used, among others, and consequently impact insecticidal activity [152][153][154][155].
Regarding larvicidal analysis techniques, important considerations are: (i) larval phase, (ii) analysis time, and (iii) the use of positive and negative controls. In different studies, the younger the larval stage, the more susceptible it is to toxic effects, as reported for Ficus benghalensis, Heracleum rigens, Myristica fragans and Solanum xanthocarpum (Table 3) [53,93,96,114]. This characteristic may relate to the reduced feeding of larvae in the late L4 instar. If the toxic effect of the insecticide is ingestion-dependent, the effect may be less pronounced the closer the larva is to the pupa stage of metamorphosis [156]. Although most studies use 24 h as the contact time to express the mortality result, it is important to note that some materials may have delayed activity, actually causing larvae death after 48, 72 or even 96 h. Thus, during product development, it is necessary to assess the toxic effect at different time intervals. Finally, the use of negative and positive controls is essential to ensure results reliability, although a number of studies did not report this data [157]. Therefore, test non-uniformity makes it difficult to compare the results of different studies. This constitutes another obstacle to overcome for the development of plant natural product insecticides [158].
Understanding the mechanism of action is fundamental in using a material as an inseticidal product. Knowledge of the mechanism of action makes it possible to understand which non-target organisms could be harmed by the use of such products [145,159]. In addition, this information facilitates prospecting other possibly more active materials using biotechnology tools and in silico models [160][161][162]. However, in general it is not easy to understand the mechanisms of action of plant natural products. Normally there are multiple modes of action pertaining to the complex composition of the materials [30,49,145], that usually occur in different target sites, as described for Piper spp, Derris (Lonchocarpus) urucu, Asarum heterotropoides and Dalbergia brasiliensis (Table 5) [89,100,109,148].
During the development stage, it is essential to evaluate toxicity in non-target organisms for promising insecticides using suitable models, such as the fish embryo acute toxicity (FET) test [163]. This model has been proposed to determine the acute or lethal toxicity of materials in the embryonic stages of zebrafish (Danio rerio) and for environmental assessments [163,164]. In addition, it is important to consider other aquatic and terrestrial organisms according to the intended application location, such as fish, amphibians, bees, birds and mammals [159]. Considering that natural product insecticides have natural degradation mechanisms, they possibly present advantages in comparison with insecticides of synthetic origin [26,30,50].
In general, raw materials (essential oils, extracts and isolated compounds) from plant natural products are poorly soluble in water and do not persist in the environment, which complicates the application and reduces the effectiveness of the desired action [26,51,165]. Therefore, the use of pharmaceutical technology is of fundamental importance in the development of formulations. Among the techniques used, nanotechnology, encapsulation and use of hydrophobic matrices with an extended and controlled release system should be highlighted as they can prolong the residual effect of formulations [51,149,[165][166][167] due to controlled release. Formulation development of natural products also poses a challenge for the application of these materials but it is imperative to improve both efficiency and cost-effectiveness [150,165]. Investing in botanical natural product formulations is an important advance in increasing the availability of commercial eco-friendly insecticides for Ae. aegypti control.

Conclusions
Considering the several stages of the insect development, the larvicidal test is the most evaluated bioassay in the search for insecticides to control Ae. aegypti for a number of reasons: (i) the larval phase is the longest in the immature stage; (ii) larvae are generally more sensitive to the toxic effects of compounds, and (iii) larvae breeding sites are localized and usually accessible. The search for ovicidal action is complex, especially due to its composition that hinders the toxic action of compounds. For the adult phase, there are compounds that cause toxicity by contact as well as those with repellent action.
Some very common edible botanical species such as Petroselinum crispum, Foeniculum vulgare, Curcuma longa, Mentha spicata, Ocimum gratissimium and Rosmarinus officinalis are highlighted, especially in the larval phase of Ae. aegypti, due to their possible low toxicity to non-target organisms. However, other non-edible species have shown strong larvicidal extract activity, among them Echinops transiliensis, Piper ssp, Hypericum japonicum and Nerine sarniensis.
Essential oils provide a promising source for insecticidal applications due to their important insecticidal activities and possible toxicological safety for mammals and the environment. Moreover, they generally possess high oral and dermal LC 50 values for these animals and are more readily degraded by natural ecosystem mechanisms.
Among the secondary metabolites, terpenes, especially monoterpenes, and phenylpropanoids are highlighted for larvicidal activity. These compounds are present in large quantities in essential oils. In addition, thiophenes, amides and alkaloids demonstrate high larvicidal and adulticidal activity.
Regarding the mechanisms of action, botanical natural products extracts, and pure compounds have displayed acitivities that include altering insect morphogenesis and therefore impairing the moulting process, respiration, feeding, and self-defense, among others. In addition, they altered biochemical processes and the nervous system.
Despite the limitations and obstacles to overcome, plant natural products are a suitable alternative source of eco-friendly botanical insecticides to control the Ae. aegypti mosquito, popularly known as dengue mosquito. Ever increasing mosquito resistance to conventional chemical insecticides warrants alternative products, which are safer for the environment and pose less risk to human health.