**1. Introduction**

Mosquitoes have been a big burden to human health for a long time. These insects can invade in different geographic locations and new habitats through global trade and travel [1] which causes millions of people be at risk of the diseases they transmit. In 2019, an estimated 229 million cases and 409 thousand deaths for malaria and 56 million cases for dengue have been reported worldwide [2,3]. While malaria case incidences were reported to decline, the number of malaria endemic countries has increased in the period 2000–2019 [2]. The global incidence of dengue is thought to be increased about thirty times over the last fifty years with emergencies in new countries [4–6]. A recent study also indicates that mosquito species will continue to spread globally over the coming decades, which may cause about 50% of the world's population at the risk of mosquito-borne viral disease transmission by 2050 [7]. Even a more serious problem is at our doorstep as the climate change is expected to increase the burden of mosquito-borne diseases despite the ongoing disease control interventions [8,9].

The most common way of keeping mosquitoes away from their human hosts is to use synthetic insecticides in mosquito nets, fabrics, and indoor sprays. The usage of chemical strategies has brought hope in controlling disease transmission in endemic regions, but emergence of insecticide resistance has been a major problem in reducing the disease burden. The uncontrolled usage of insecticides has led to reemergence and increase in mosquito populations over the years. Between the years 2010–2019, about 28 malaria endemic countries (out of 82) have detected resistance to all four classes of the most commonly used insecticides, and 73 have detected resistance to at least one insecticide class, an issue that continues to increase globally [2]. Thus, insecticide resistance is now considered a serious threat to control mosquito invasion and disease transmission. It is essential that the methods for insecticide monitoring in mosquito populations and interpretation of results are performed adequately, effectively and in a timely manner for improving mosquito control [10,11].

Current research on mosquito control is now focused on understanding the mosquito resistance to synthetic insecticides and developing novel strategies to overcome the resistance issues. Natural compounds that are more effective and less toxic than the synthetic ones continue to ge<sup>t</sup> more attention in the research community. The use of bioinsecticides, composed of botanical or plant-based compounds, has been a perfect alternative due to their minimal hazardous effects on human health and environment. In this review, we provide current knowledge on synthetic insecticides that are actively used in mosquito control and how they impact prevalence of insecticide resistance in mosquitoes. Major plant-based insecticides, their mode of action and the research about their potential mosquitocidal activity are discussed. A comprehensive understanding of how biochemical compounds can be advantageous to synthetic ones and how we can circumvent insecticide resistance issues in the fight with mosquito-borne disease transmission is provided.

#### **2. Insecticide-Based Mosquito Control Strategies**

Insecticide-based mosquito control plays an important role in efforts to reduce the transmission of mosquito-borne diseases worldwide. Two core insecticidal interventions are in use to control mosquitoes: deployment of insecticide-treated mosquito nets (ITNs) and indoor residual spraying (IRS) of insecticides [10]. These interventions have been effectively used to kill mosquitoes or interfere with their host-seeking behavior to prevent disease transmission worldwide [12–20]. The global malaria cases and malaria death rates have declined about 18% and 48%, respectively, between the years 2000 and 2015, and 70% reduction in malaria cases in sub-Saharan Africa was attributed to ITNs, and 10% reduction was due to IRS [21].

Four classes of insecticides are mostly used in mosquito control programs which include pyrethroids (e.g., deltamethrin, permethrin, cypermethrin, lambda-cyhalothrin), organochlorines (e.g., DTT), organophosphates (e.g., malathion, fenitrothion), and carbamates (e.g., propoxur, bendiocarb) [10] (Figure 1). Most synthetic insecticides have physiological or behavioral impact on mosquitoes (Figure 1), and predominantly target the central nervous system of insects. Among them, pyrethroids are the most widely used insecticides for IRS and the only synthetic insecticide currently used in ITNs and fabrics, with irritant or repellent activity on mosquitoes and less mammalian toxicity [2]. They disrupt the voltage-gated sodium channels in neuronal membranes [22]. When pyrethroids bind an open channel, they prevent its closure, thus leading to a prolonged action potential

or disruption of electrical signaling in the nervous system [23–25]. This causes continuous nerve excitation and paralysis (or knockdown) of the insect and eventually its death [26].

**Figure 1.** Classification of insecticides based on mode of action and chemical composition.

While pyrethroids have been effectively used in ITNs to control mosquitoes for a long time, prevalence of pyrethroid resistance in mosquito species causes a major problem to combat disease transmission worldwide [27–29]. Like pyrethroids, some organochlorines are also inhibitors of the insect's voltage-gated sodium channels. Dichlorodiphenyltrichloroethane (DDT) is an example that targets sodium channels, and it is the first and the most commonly used synthetic insecticide of organochlorine in residual spraying. Its low cost and high effectiveness have made it a favorable chemical for indoor wall spraying. However, resistance developed to DDT in various mosquito species and its toxic effects on humans and non-target organisms have imposed limitations or restrictions in its usage [30,31]. Other organochlorines (such as cyclodienes, dieldrin and fipronil) target γ-amino butyric acid (GABA) receptors, which are hetero-multimeric gated chloride channels in the insect's central nervous system [32]. Cyclodiene insecticides act as neurotoxicants and block the GABA receptors causing hyper-excitation of the central nervous system, convulsions, and eventually death of insects [33–35]. Organophosphates (OP) and carbamates are two other insecticides sharing similar mode of action. They inhibit acetylcholinesterase (AChE) enzyme, preventing breakdown of the neurotransmitter acetylcholine, resulting in neuromuscular overstimulation and death of insects [36–38]. Due to pyrethroid and DDT resistance issues worldwide, they have been used as alternative insecticides in IRS, but they have a shorter residual effectiveness, high toxicity to mammals, and are more costly compared to the others that limit their persistent long-term usage.

#### **3. Insecticide Resistance in Mosquitoes**

Short after its first usage in California in 1945, the resistance of mosquitoes to DDT was reported [39,40]. Since then, insecticidal resistance in mosquitoes has been reported, with a substantial increase between 2010 and 2016 [10]. In these years, insecticide resistance was found to be widespread in *Anopheles* vectors in malaria endemic African regions and insecticide resistance frequency has changed over time [10]. Understanding pyrethroid resistance development in *Anopheles* mosquitoes is particularly important because its prevalence can disable pyrethroid-treated ITN-based interventions, which are used successfully for malaria control [41,42]. Pyrethroid resistance was determined to be very high in the WHO African Region (78%), Eastern Mediterranean Region (70%), and in the South-East Asia Region (38%), Western Pacific Region (51%), but was lower in the Region of the Americas (20%). The incidence of organochlorine resistance was also similar in all WHO regions (60–70%). Carbamate resistance prevalence was between 22% and 54%, and organophosphate resistance prevalence varied widely across regions, 14% in the WHO African Region and 65%

in the WHO Western Pacific Region [10]. While resistance frequencies are generally high in most of the endemic regions, those with lower resistance frequencies could be an indication of recent gain of resistance or selection for resistant populations to insecticides [43].

Despite effective use of insecticide-based mosquito control strategies for decades, their prolonged usage is challenged by high cost, toxicity and, more importantly, the development of resistance to the synthetic insecticides. Insecticide resistance is mostly inferred to the ability of insects to survive exposure to a standard dose of insecticide, owing to physiological or behavioral adaptation [44]. Resistance can be developed due to misusage or overdose usage of insecticides and selection pressure on the insect populations [45]. The question "when does the resistance emerge?" depends on the mechanism of resistance, known susceptibility, cost effectiveness and availability [45]. Various resistance mechanisms have been observed in mosquitoes: changes in their metabolism (changes in enzymes leading due rapid detoxification of insecticides), alterations in target-sites (prevention of insecticides to their target sites), penetration resistance (cuticle barrier diminishes insecticide penetration) and behavioral resistance (changes in their response to insecticidal effect) [46–49]. These mechanisms can be determined by using bioassays, biochemical assays, and molecular techniques through assessment of resistance alleles, analyzing whether metabolic enzymes are upregulated, or determination of the percent mortality rate upon exposure to a given insecticide.

In mosquitoes, alterations of target site nerve receptors (e.g., mutations in *kdr*, *Rdl* and *Ace-1R* genes) and detoxification due to increased or modified enzyme activities (e.g., monooxygenases (P450s), glutathione-S-transferases and carboxylesterases) are the two major mechanisms responsible for insecticide resistance. According to the insecticide resistance monitoring data for 2010 to 2016, almost 70% of the assays to test resistance mechanisms included detection of the presence or absence of target-site mutations and their frequencies in WHO regions [10]. Target site alterations in mosquitoes involve knockdown resistance (*kdr*) mutations (L1014F or L1014S) in the voltage-gated sodium channel gene which causes inability of the insecticides to bind their cognate receptors [50–55]. Occurrence of *kdr* mutations causes insensitivity to pyrethroids and DDT [56,57]. A *kdr*-resistant strain of *An. gambiae* has shown to be less affected by pyrethroids than the susceptible strain [58]. In the last few decades, *kdr* resistance mutations in different mosquito populations have expanded significantly which restricts pyrethroid usage in mosquito control [59]. Another target-site mutation, the AChE gene mutation (*Ace-1R*), causes resistance to organophosphates and carbamates. In mosquitoes, a G119S mutation in the *Ace-1R* gene encoding AChE causes resistance to organophosphate and carbamate insecticides and the mutation frequency is increasing in natural mosquito populations [60–63]. A substitution mutation of alanine-to-serine/glycine (A296S/G) mutation, *Rdl*, in the second transmembrane domain of the GABA receptor subunit causes resistance to organochlorine insecticides and insensitivity in mosquitoes [35,64–69].

Mosquitoes have metabolic enzymes, mainly "detoxifying enzymes" that are responsible for biodegradation of insecticides and elimination of their insecticidal effects. Upon exposure to synthetic insecticides, detoxifying enzyme activity increases (due to increased gene amplification or upregulation) which result in insecticide-resistant mosquitoes [46]. Three classes of detoxifying enzymes are involved in insecticide-resistance in mosquitoes: cytochrome P450 monooxygenases (CYP), glutathione-S-transferases (GST) and carboxylcholinesterases (CCE) associated with pyrethroid, organochloride, and OP and carbamate resistances, respectively. Cytochrome P450 enzymes are involved in the metabolism of all four classes of insecticides. It is found that elevated levels of P450 activity resulted in pyrethroid resistant mosquito vectors [70–74]. Several CYPs are identified in mosquitoes and CYP overexpression is reported from insecticide resistant mosquito populations [45,59,75–77]. Knockdown of the CYP through the RNA-interference technique also showed that mosquitoes become sensitive to pyrethroids [78–80]. Glutathione S-transferases comprise a diverse family of enzymes involved in detoxification of insecticides (e.g., pyrethroids and DTT) in mosquitoes [81]. An increase in the gene expression levels of various GSTs has been

detected in DDT-resistant and pyrethroid-resistant mosquitoes [82–88]. Additionally, a GST gene silencing study indicated an increase in the susceptibility to pyrethroid insecticide which shows that GSTs are involved in insecticide-resistance in mosquitoes [86]. Increased esterase detoxification in OP resistance has been studied most extensively in *Culex* mosquitoes [72,89]. These enzymes sequester the insecticide and interfere with its association with the target AChE by rapid binding and slow turning over of the insecticide [90]. The increase in the activity of esterases was due to overproduction of the enzymes, resulting from co-amplification of two esterase genes, *estα2* and *estβ2*, in OP-resistant individuals [91,92].

It is evident that cross-resistance causes major issues in the managemen<sup>t</sup> of insecticide resistance through the approaches discussed above. These mechanisms can cause resistance to more than one class of insecticide (with similar mode of action) due to prolonged and intensive usage of these chemicals. For example, *Culex* mosquitoes that are resistant to a pyrethroid insecticide also show resistance to OP and other insecticides [93,94]. Pyrethroidresistant *Anopheline* mosquitoes also show resistance to OPs due to constitutively elevated P450 levels leading to cross-resistance [95]. Moreover, insecticide resistance is genetically mediated and can be fixed in mosquito populations in such that individuals with the resistance gene will probably have a selective advantage in the presence of the insecticide [96,97]. Furthermore, mosquitoes that survive insecticide exposures possibly have the chance of passing those traits to their offspring which causes an increase in the percentage of resistant individuals in the next generations in those populations [48]. If resistance gene frequency increases in the populations, this can cause more resistant individuals to circumvent insecticidal exposures. Taken together, the emergence and spread of insecticide resistance, cross-resistance, and increased resistance gene frequencies in mosquito populations significantly effects mosquito-borne disease control and elimination and highlights the need for alternative strategies. There has been a grea<sup>t</sup> interest for safe and healthy biological control strategies and development of novel interventions to overcome problems associated with synthetic insecticides. Hence, extensive research for another class of insecticide for mosquito control, named "bioinsecticide", is an ongoing process and novel natural compounds are being investigated to replace conventional synthetic insecticides. In this review, we will focus on plant-based bioinsecticides with potential activity in mosquito control.

## **4. Plant-Based Bioinsecticides**

Bioinsecticides are derived from natural products, such as bioactive compounds of plants, pheromones, and from microorganisms, such as bacteria, fungi, virus, or protozoan. There are four major classes of bioinsecticides based on their nature of origin: phytochemicals, microbial pesticides, plant-incorporated protectants (PIPs), and pheromones [98] (Figure 1). They have been effectively used in pest managemen<sup>t</sup> and generation of sustainable agricultural products [99,100]. They are less toxic, target-specific, highly effective in small quantities and biodegradable, which makes them excellent alternatives to synthetic compounds. More importantly, mosquitoes are developing resistance to synthetic compounds, a burden that needs to be resolved for successful mosquito disease control. Since biopesticides induce less insect resistance [101,102], most studies now focus on discovery of candidate natural compounds with potential effects on mosquitoes to combat mosquito-borne disease transmission.

Plants have evolved to develop many defensive chemical compounds against pathogenic microorganisms and insects. These biologically active chemical compounds, referred to as "phytochemicals", function as repellents, toxins, feeding deterrents, and growth regulators against insects [103]. Various parts of higher plants (leaves, roots, stems, seeds, barks, fruits, peels of fruit and resin), the whole body of little herbs, or mixture of different plants can be used for an effective plant-based insecticide. The activity of a phytochemical can change significantly depending on the plant species, plant part and its age, polarity of solvents used during extraction procedures and mosquito species [104]. Phytochemicals show their effects through targeting important cell components and affecting insect physiology in different ways; via inhibition of AChE and GABA-gated chloride channel activity, disruption of sodium-potassium ion exchange and nerve cell membrane action, blocking calcium channels, and activation of nicotinic acetylcholine receptors and octopamine receptors [105]. Moreover, phytochemicals can cause cellular destruction of epithelial cells in the midgut of mosquitoes and affect metamorphosis [106,107].

Several phytochemicals have been reported for their mosquitocidal activities [104,108]. These chemical compounds are mostly secondary metabolites, such as essential oils, alkaloids, phenols, terpenoids, steroids, and phenolics from different plants. Phytochemicals in plant species are diverse and discovery of those with mosquitocidal activities, which are governed by changes in expression levels of detoxifying enzymes, are of grea<sup>t</sup> importance to control mosquitoes. In the following sections, we provide the current knowledge on mosquitocidal plant-based compounds and their activities for a better understanding of their efficacy to prevent mosquito-borne diseases.

#### **5. Plant-Based Compounds and Mosquito Control**

Plant-based compounds possess larvicidal, ovicidal and repellent activities on early or adult stages of mosquitoes, affecting nervous, respiratory, endocrine, and water balance systems. Ovicidal and larvicidal effects of many plant compounds have been extensively studied since mosquitoes are immobile at these stages and they can be efficiently eliminated before they emerge as adults. Repellent compounds are effective in keeping human hosts from mosquito bites for a blood-meal. Thus, understanding the mosquito olfactory system is vital for determination of repellent compounds. Insect repellents affect the olfactory receptor neurons via modifying or blocking its response, which in turn, elicit avoidance behavior or a change in the host-seeking behavior of mosquitoes [109,110]. There are many plant compounds with repellent activities. Essential oils, alkaloids, and aromatic compounds from various plants are commonly used for plant-based mosquito repellents [111] and they have shown to interfere with the mosquito host-seeking behavior when applied on human skin or used as indoor spraying [112]. Insecticidal and repellent activities of four major plant metabolites (essential oils, neem, pyrethrum, alkaloids) and other plant compounds (flavonoids and rotenone) are discussed in detail (Table 1).

## *5.1. Essential Oils*

Essential oils have been efficiently used against a variety of pests and for crop protection in the world and they are potential alternatives to synthetic insecticides used against mosquitoes. Essential oils are very complex natural mixtures that consist of a variety of volatile molecules, which are hydrocarbons (terpenes and sesquiterpenes), oxygenated hydrocarbons and phenylpropenes (Table 1). Essential oils are synthesized in the cytoplasm and plastids of plant cells through mevalonic acid and 2- *C*-methyl-erythritol 4-phosphate (MEP) pathways, respectively [113]. Essential oils target the insect nervous system and cause neurotoxic effects through several mechanisms by inhibiting the activity of AChE, and blocking octopamine receptors and GABA-gated chloride channels [114,115]. About 90% of essential oils are composed of monoterpenes, which are determined to be active ingredients for potential plant-based larvicides and cause inhibition of AChE activity in insects [116]. Monoterpenes, such as linalool, cuminaldehyde, 1,8-cineole, limonene and fenchone, cause inhibition of AChE and accumulation of acetylcholine in synapses and state of permanent stimulation, which results in ataxia [117,118]. According to Hideyukiu and Mitsuo [119], a mixture of monoterpenoids is a more potent inhibitor of AChE than single monoterpenoid application and acts synergistically.



The octopaminergic system of insects is another target for essential oils that block octopamine receptors and cause acute and sub-lethal behavioral effects on insects. The increase in cyclic AMP levels, induced upon binding of octopamine to octopamine-receptors, can be inhibited by a mixture of essential oils (eugenol, γ-terpineol and cinnamic alcohol). Moreover, octopamine receptor binding is significantly reduced with low doses of eugenol alone [120,121]. Another possible target for essential oils is ligand-gated chloride channels. Essential oils consist of monoterpenes, such as linalool, methyl eugenol, estragole, citronellal, inhibit GABA-gated chloride channels by binding at the receptor site and increase the chloride anion influx into the neurons, which lead to hyper-excitation of the central nervous system, convulsions, and finally death of insects [122,123].

Many plant oils possess ovicidal, larvicidal, pupaecidal and repellent activities against various mosquito species, some of which will be discussed below. Essential oils of plants from the Lamiaceae, Poaceae, Rutaceae and Myrtaceae families are well-known for repellent activity [103]. Essential oils obtained from citronella, lemon and eucalyptus are commercially available and recommended by the U.S. Environmental Protection Agency (US EPA) as repellent ingredients for application on the skin because of their low toxicity. For example, *P*-menthane-3,8 diol (PMD) is an active component of the lemon eucalyptus plant and responsible for the repellency in mosquitoes [124].

Most of the monoterpenes and sesquiterpenes of essential oils are known with repellent activities [125]. Among monoterpenes, α-pinene, γ-pinene, *p*-cymene, eugenol, limonene, thymol, terpinolene, citronellol, camphor and citronellal are responsible for mosquito repellency [126,127]. Representative molecules of sesquiterpenes are guaiol, α-bisabolol, α-cadinol, germacrene D, β-caryophyllene and nootkatone. β-caryophyllene is known to exhibit strong repellent activity against *Aedes* mosquitoes [126]. Repellent and larvicidal activities of monoterpenes from the essential oils of *Thymus* plant against *Cx. pipiens pallens*, *Cx. quinquefasciatus*, and *Cx. pipiens* biotype *molestus* have been determined [128–130]. Larvicidal activities of phenolic terpenes, such as thymol and carvacrol, of *Satureja* species were observed against *Cx. pipiens* biotype *molestus* [131]. Moreover, repellent and larvicidal activities of carvacrol were determined in the field trials against *Ae. albopictus* mosquitoes in Bologna (Italy) [132]. *Cinnamomum osmophloeum* and *Carum copticum* essential oils had larvicidal activity against *Cx. quinquefasciatus* and *Cx. pipiens*, respectively [107,133]. Toxicity of β-citronellol, geraniol and linalool from *Pelargonium roseum* essential oil was also detected in *Cx. pipiens* [134]. High larvicidal and pupaecidal activities of essential oils from *Cinnamomum verum*, *Citrus aurantifolia*, *Cuminum cyminum*, *Syzygium aromaticum*, *Laurus nobilis*, *Lippia berlandieri* and *Pimpinella anisum* were reported from *Cx. quinquefasciatus* [135]. *Artemisia absinthium* essential oils also showed toxic effects against larval populations of *Aedes*, *Anopheles*, and *Culex* mosquitoes [136]. Essential oils isolated from *Tagetes lucida*, *Lippia alba*, *Lippia origanoides*, *Eucalyptus citriodora*, *Cymbopogon citratus*, *Cymbopogon flexuosus*, *Citrus sinensis*, *Swinglea glutinosa*, and *Cananga odorata* plants showed larvicidal activities on *Ae. aegypti* larvae [137]. Oviposition deterrence and ovicidal activity of some of essential oils, peppermint oil, basil oil, rosemary oil, and citronella oil from *Mentha piperita*, *Ocimum basilicum*, *Rosmarinus officinalis*, *Cymbopogon nardus* and *Apium graveolens* were also reported in *Ae. aegypti* [138]. Manh et al. [139] also showed toxicity of essential oils from *Eucalyptus* and *Cymbopogon* aromatic plants to the larvae of *Ae. aegypti*. Essential oils also cause toxicity at different developmental stages and have repellent activities against adult *Anopheles* mosquitoes [140]. Essential oils extracted from *Cymbopogon proximus*, *Lippia multiflora* and *Ocimum canum* had larvicidal and ovicidal activities against *An. gambiae* and *Ae. aegypti* mosquitoes [141]. Besides monoterpenes and sesquiterpenes, phytol (a diterpene alcohol) and coumarin (an aromatic phenol) were both determined to be responsible for the biting deterrence effect in *Ae. aegypti* [142].

Repellent activity of essential oils is generally attributed to individual chemical compounds, but synergistic effects of plant metabolites have been observed when the effect of an active compound is enhanced by other major compounds or modulated by minor compounds. The efficacy of the major compounds is enhanced by minor compounds

through different mechanisms, which may cause higher bioreactivity compared to isolated compounds of essential oils. The synergistic effect is also observed with mixture of oils. The synergistic action of the major compounds in essential oils results in higher repellent and larvicidal activity and toxicity to insects [140,143–145]. A combination of blends assayed on *An. gambiae* mosquitoes indicated that blends of oils showed higher repellency compared to the individual oil used [146]. It has been also reported that essential oils composed of a mixture of active components might reduce resistance in mosquito population by acting at different target sites or with a different mode of action [139].
