*5.4. Alkaloids*

Alkaloids are nitrogen-containing natural products found in bacteria, fungi, animals, and plants. They are commonly isolated from plants and found in large quantities in many members of the Berberidaceae, Fabaceae, Solanaceae, and Ranunculaceae families. The alkaloids obtained from these plants are used extensively in conventional insect repellents [171–173]. The mode of action of alkaloids varies depending on the type of alkaloids and interferes with major cellular and physiological functions by affecting AChE receptors in the nervous system, regulating hormonal activity, and causing toxicity [174]. Alkaloids are not volatile like essential oils. However, they could be used as repellents against mosquitoes by burning plants to generate an insecticidal smoke that repels insects and directly causes toxicity [124]. In *Ae. aegypti*, the inhibitory effect of natural alkaloids on AChE activity was determined by using molecular docking studies. Among the 25 different alkaloids tested, alpha-solanine has been found to fit into the AChE1 binding pocket and potentially be the best inhibitor of AChE1 [175].

Extracts of the castor bean (*Ricinus communis*, Euphorbiaceae) contain the alkaloid ricinine and have a strong insecticidal effect. It showed strong larvicidal activity against larvae of *An. arabiensis* [176]. Additionally, pyridine alkaloid from *R. communis* showed bioactivity against *An. gambiae* larvae and adults [177]. The larvicidal activity of alkaloids against *Ae. albopictus*, *Cx. pipiens pallens* and *Ae. aegypti* has also been determined [178,179]. Alkaloid from *Arachis hypogaea* plant also had larvicidal toxicity against *An. stephensi* and *Ae. aegypti* mosquitoes [180].

Nicotine is an alkaloid derived from tobacco plant (*Nicotiana tobacco*) that mostly consists of phenolic compounds, such as nicotine and diterpene. Nicotine, nornicotine and anabasine mimic the neurotransmitter acetylcholine, which causes symptoms similar to organophosphate or carbamate insecticides [160]. Extracts of tobacco leaves were mixed with bio-oil and high repellent activity was observed against *Ae. aegypti* [181]. Furthermore, nicotine has been found to be the most dominant compound among the other active compounds of the repellent mixture, including nicotine, d-limonene, indole, and pyridine. In addition, the repellent compound was harmless to human skin as confirmed by sensitivity tests on volunteers.

#### *5.5. Other Plant Compounds*

Besides the most common plant-based bioinsecticides mentioned above, there are other natural plant metabolites that show insecticidal properties. Among them, flavonoids elicit larvicidal activity by inhibiting AChE in mosquito larvae [182]. They could also act as respiratory inhibitors and result in the disturbance of the larval respiratory system. Alkaloids have multiple effects including inhibition of the AChE enzyme, degradation

of cell membranes, and they may act as stomach poisons [182]. It has been shown that flavonoid and alkaloid components of bangle rhizome extract from *Zingiber montanum* act differently against *Ae. aegypti* [183]. Flavonoids from *Derris trifoliata* extract also exhibited larvicidal activity against *Ae. aegypti* [184]. Rotenone is an isoflavonoid extracted from roots and stems of *Derris* (*Derris elliptica*, *Derris involute*), *Lonchocarpus* (*Lonchocarpus utilis*, *Lonchocarpus urucu*) and *Tephrosia virginiana* [160]. It has long been used as a biopesticide due to less harmful effects to the environment. Rotenone has the potential to be used as a larvicide to control mosquitoes and interferes with the cellular respiration system of insects and prevents energy production [185].

#### **6. Assessment of Plant-Based Bioinsecticide Efficacy in Mosquito Control**

It is important that inherent activity of candidate bioinsecticides should be assessed before they can be effectively used against mosquito populations. The World Health Organization has established methods to screen the efficacy and field application acceptability of new compounds as potential mosquito larvicides and adulticides (for IRS and ITNs); they are laboratory studies, small-scale and large-scale field trials [186–188]. Laboratory studies focus on determination of biopotency, efficacy, residual activity, irritant or repellent properties, diagnostic concentration, and possible cross-resistance of candidate larvicides or adulticides. In laboratory bioassays, mosquito larvae are exposed to various concentrations of larvicides, and a mortality rate based on lethal concentration (LC) of the larvicide for 50% and 90% mortality (LC50 and LC90) or for 50% and 90% inhibition of adult emergence (IE50 and IE90) is recorded. LC values are determined and can then be compared with the LC50 or LC90 values of other insecticides to assess the activity of the compound as "sufficiently effective". For adulticides, LC is determined by tarsal contact to treated papers. The "time to first take-off" (FT) for the 50% and 90% of the mosquitoes to take off (FT50 and FT90) after exposure to treated substrates are measured to determine the irritant or repellent activity of an adulticide. Insecticide-treated nets are used for bioassays of adult mosquitoes to determine the efficacy and residual activity of different dosages of the candidate compounds. Moreover, efficacy and wash-resistance of ITNs against susceptible mosquito species should be determined using standard WHO cone bioassays or tunnel tests [188]. The efficacy criteria for cone bioassays are ≥80% mortality or ≥95% knock-down, and for the tunnel test, it is ≥80% mortality or ≥90% blood-feeding inhibition. Candidate larvicides and adulticides are also tested against multi-resistant mosquito strains and a susceptible reference strain to assess the cross-resistance and, if detected, biochemical, immunological, and molecular methods are used to determine the mechanism of resistance [189].

Once candidate compounds are selected from laboratory tests, they are subjected to small-scale field testing in natural breeding sites (such as drains sewage water tanks, ponds, rice plots, etc.) or under simulated field conditions (artificial containers filled with water, experimental huts). Larvicidal efficacy is determined by the level of inhibition of emergence of adults and the percentage reduction in larval and pupal densities, while adulticidal efficacy can be assessed in terms of mortality, residual effect, deterrence, blood-feeding inhibition and induced exophily. These trials elucidate efficacy of candidate compounds against different mosquito species in different breeding sites, determine optimum field application dosage of the compound and possible impact on the mosquito behavior. Abiotic parameters that may influence the efficacy of the product and effect on non-target organisms can also be observed. Those larvicides and adulticides that show promise in small-scale field trials should be validated in larger-scale field trials against natural mosquito populations in natural breeding habitats using optimum field dosages. At this stage, the storage, handling, and application of the insecticide formulation should be considered for proper functioning of application and dispersal of the bioinsecticide in natural ecosystems.

There are also potential limitations to the efficacy of bioinsecticides, such as environmental conditions, mosquito fitness, mosquito resistance as well as the parts of the plants used, solvents used in extraction steps, insecticide dose and exposure time [190,191]. These effects should be considered for successful assessment of novel bioinsecticides in mosquito

control. While efficacy tests provide promising information on possible mosquitocidal effects, new compounds from plant origin, the identification of actual active ingredient for efficacy and their mode of action are still waiting to be resolved.

#### **7. Effective Use of Plant-Based Bioinsecticides in Resistant Mosquito Populations**

Most of the bioinsecticides are now effective alternatives to chemical insecticides and have become an integral part of the integrated mosquito managemen<sup>t</sup> (IMM) programs because the development of resistance to bioinsecticides is low due to their multiple mode of actions [192,193]. The synergic mixture of the active compounds in plant extracts also minimizes resistance development [167]. However, resistance already developed to extensively used chemical insecticides is a major problem that limits the success rate of novel bioinsecticides against mosquito populations. Insecticide resistance should be reduced or reverted (which takes time) in order to apply new and effective bioinsecticides in resistant populations. Surveillance of mosquito resistance and effective resistance managemen<sup>t</sup> strategies should be routinely conducted to determine the levels, mechanisms, and geographic distribution of resistance in field populations of mosquitoes for increasing efficacy of bioinsecticides [44]. Moreover, proper application technologies should be considered as they greatly influence the bioinsecticide efficacy.

Surveillance of resistance development to many different insecticides are determined by dose-mortality bioassays, the World Health Organization tube testing, and Centers for Disease Control and Prevention (CDC) bottle bioassay for mosquitoes [11,44,194,195]. In the dose-mortality assay, the resistance ratio (RR) is determined in a susceptible population to monitor changes in resistance over time. The RR is calculated from LC50 values of the field and susceptible populations, in which an RR lower than five indicates susceptibility or low resistance and an RR value higher than ten indicates high resistance. In the WHO tube testing, the insecticide susceptibility status of the selected mosquitoes is evaluated through susceptibility tests measuring the mortality rate twenty-four hour after exposure [44]. A mortality rate lower than 98% indicates occurrence of resistance and should be confirmed with biochemical and molecular analysis. A mortality rate less than 90% confirms the existence of resistant genes in the tested mosquito populations. The CDC bottle bioassay is a measure of insecticide effectiveness, where diagnostic doses (DDs) and diagnostic times (DTs) are determined for candidate compounds using susceptible mosquitoes prior to testing in field mosquito populations. The DD is a measure of insecticide dose that kills 100% of susceptible mosquitoes within a certain period of time (DT). A mortality rate lower than 97% is an indication of resistance that needs to be confirmed, and below 80% suggests strong resistance at the recommended DT. The DD and DT values for some active ingredients are available for *Anopheles* and *Aedes* mosquito populations and these parameters should be defined for a particular insecticide and mosquito population [195].

It is evident that no single strategy is effective enough to solve insecticide resistance of mosquitoes. According to the WHO [44], one strategy to prevent the resistance problem is rotational usage of different classes of bioinsecticides with different modes of action. There are several new plant-based larvicides with different modes of action (discussed in Section 5) and they could be good alternatives for mosquito control in larval stages. Additionally, multiple interventions that affect different stages of mosquitoes (such as larvae and adults) can be used together to manage insecticide resistance. It is also suggested that different classes of insecticides with different modes of action can be used in neighboring geographic locations. To successfully implement these strategies, knowledge of the mode of action of the novel bioinsecticide is essential. The resistance mechanism developed by the local population of mosquitoes should also be determined to reduce cross-resistance effects.

RNA interference (RNAi) mediated loss-of-function technique has been proposed for pest managemen<sup>t</sup> programs [196,197] and to study insecticide resistance [198]. Genes responsible for resistance development in insects (e.g., genes for DDT or pyrethroid resistance) can be identified and used as a target for the development of novel RNAi based insecticides. Several delivery methods including nonmicrobial and microbial are used

routinely to induce RNAi in mosquito larvae [199]. Nonmicrobial delivery methods consist of soaking, injection, nanoparticles and dehydration and rehydration. Although soaking and injection methods are used as excellent tools in RNAi research, they have no application in the field. Soaking, injection methods and nanoparticles have been effectively used to introduce dsRNA into first-instar *Ae. aegypti* larvae [200] and fourth instars of *Ae. aegypti* [201]. In mosquitoes, a chitosan/dsRNA-based nanoparticle has also been used in feeding the larvae of *An. gambiae* mosquitoes which led to successful gene silencing of two chitin synthase genes and increased susceptibilities to DTT [202]. Such an RNAi-based bioinsecticide can be potentially used as an effective strategy to enhance the efficacy of new bioinsecticides for mosquito control.

Another technology used for the manipulation of insect behavior is "Specialized Pheromone and Lure Application Technology (SPLAT)". SPLAT is a chemical controlledrelease emulsion technology, and it has been used as an alternative managemen<sup>t</sup> strategy to target the aquatic life stages of mosquitoes [203]. SPLAT emulsions can be formulated by using a variety of compounds, such as sex pheromones, attractants, repellents, phagostimulants and insecticides. SPLAT consists of both aqueous and non-aqueous components. The aqueous component of the SPLAT emulsion is involved in the liquid property of the product and evaporates within 3 h upon application. The non-aqueous component of the emulsion is the controlled-release device that releases active ingredients (e.g., semiochemical or pesticides) at a controlled rate for 2 weeks to 6 months by protecting the active ingredients from environmental, chemical, and biological degradation. It has been reported that combination of attractant and larvicidal agents in a single formulation and biodegradable matrices causes significant increase in larval mosquito mortality, specifically *Cx. quinquefasciatus*, compared to formulations consisting of larvicidal agents alone in semi-field trials (e.g., large-screened greenhouse and emulating field conditions) [204]. The major benefits of this technology are a timely-manner release of both pheromone and insecticide, reduced insecticide resistance, and persistence in the field [203].

#### **8. How to Improve Plant-Based Bioinsecticide Efficacy in Mosquito Control Strategies?**

Synthetic chemicals used to control mosquitoes are now causing serious health problems and, more importantly, resistant mosquitoes that lead to search for more effective, healthier, safer, and eco-friendly natural solutions. Phytochemicals derived from plant resources are excellent targets to search for bioactive compounds because plants synthesize these chemicals naturally in response to their environment (such as against insect predators and microbial attacks), thus, plants are indeed natural insecticide sources. While searching the literature for plant-based compounds, we have encountered a tremendous number of efforts to identify and evaluate compounds that could have potential mosquitocidal activity with negative impact on mosquito physiology at different development stages. Since phytochemicals have multiple modes of action and exert their effects on multiple target sites in insects, their efficacy can be enhanced when used as a blend (e.g., mixture of oils) against mosquitoes. In addition, insects are more likely to develop resistance to a single chemical compound rather than a mixture of compounds. Thus, a combinatorial usage of phytochemicals would limit development of resistance in mosquitoes. Phytochemicals have short residual half-life which could be advantageous when synergistically used together with other biological control agents [205]. It is encouraging that these features of phytochemicals make them alternative natural solutions for the development of suitable products to interfere with the mosquito–host interaction and reduce disease transmission.

Among the phytochemicals, essential oils are extensively studied and their repellent activities against mosquitoes makes them favorable natural chemicals. However, they are volatile compounds, and this causes issues in their long-term applications in mosquito control. In recent years, new technologies, such as microencapsulation and nanoemulsion, have been used to overcome this problem by enhancing the duration and efficacy of essential oils [140]. Since ITNs are one of the major intervention methods to control

mosquitoes, the incorporation of plant-based insect repellents in fabrics seems a prompt and alternative way to provide safer protection against mosquito bites. Fabrics treated with microencapsulated citronella essential oil have been reported to provide higher repellent activity and longer lasting protection, up to three weeks, against insects compared to the fabrics sprayed with ethanol solution of the essential oil [206]. Grancaric et al. [207] also reported that microencapsulated immortelle oil had the highest repellent efficacy against *Ae. aegypti* compared to immortelle oil alone on cotton samples. In another study, microcapsules composed of two biopesticides, namely citronella essential oil and citriodiol, were prepared and applied to cotton textiles using a variety of techniques. As a result, citriodiol-treated cotton fabrics had a prolonged durability, and 100% repellent activity for more than 30 days after its application [208]. Additionally, encapsulation of citronella oil into microcapsules of poly ε-caprolactone has been considered as an effective and sustained release system with potential application in protection against mosquitoes [209]. Encapsulated citronella oil nanoemulsions prepared by high pressure homogenization at varying amounts of surfactant and glycerol were tested for mosquito repellency. It has been shown that increasing concentration of glycerol and surfactant improved the stability of the emulsion causing prolonged mosquito protection [210]. These results clearly indicate that through microencapsulation and nanoemulsion formulation technologies, effective and longer usage of essential oils on cotton fabrics or ITNs can be achieved.

Neem-based insecticides can also be effectively used for the control of mosquitoes. They are considered more eco-friendly than synthetic insecticides and are less prone to induce resistance because of their multiple modes of action on insects. Another advantage of neem oil formulations is that it causes mortality at relatively low concentrations making them potential alternatives to synthetic insecticides in the control of malaria vectors. Microencapsulation of neem seed oil and karanja oil has been used for the control of larvae of *Ae. aegypti* [211]. The major drawback of using neem oil is that its dosage should be considered when applied in the field because neem can cause risks to non-target organisms at higher doses.

Natural pyrethrins are now considered as a potential alternative to synthetic DTT and can overcome hazardous effects of pyrethroids. However, they have major drawbacks which include their high instability and quick degradation in the presence of sunlight. Stability concerns and short duration of their knockdown effect cause inadequate field applications against mosquito populations during the day [212]. However, the application of pyrethrin-based insecticides after sunset against *Culex* and *Anopheles* has shown a decrease in mosquito populations and protection against non-target insects [213]. Pyrethrins are also more effective when used with a synergist. They can be easily degraded before having an impact on mosquitoes, thus should be applied with a synergist of non-synthetic origin. Since pyrethrin-based chemicals are detected via mosquito olfactory organs and processed through olfactory signal transduction mechanisms, pyrethrin-based repellent molecules should be developed and implemented in order to interfere with the host-seeking behavior of mosquitoes for an effective reduction in disease transmission.

Despite our increasing knowledge on plant-based bioactive compounds and their multiple mode of actions on insects, a few of them, such as essential oil-based and neem-based insecticides, have been commercially available for pest managemen<sup>t</sup> [205]. One of the reasons that causes their limited usage in the field is the formulation problem to overcome phytotoxic effects. The chemical composition of each compound should be formulated in such a way that it should be bioactive to target insects and non-toxic to non-target organisms. In addition, formulation of plant-based bioinsecticides should ensure that it can be produced in large quantities through biomass production of plants and administered in recommended dosages to minimize toxic effects, and biological activity can be maintained for longer shelf-life. As discussed above, microencapsulation and nanoemulsion technologies have benefits in solving formulation issues of phytochemicals. A new formulation in the form of tablets containing a lectin preparation showed mosquitocidal activity against different developmental stages of *Ae. aegypti* mosquitoes, and this formulation method

is proposed as a new control strategy for *Ae. aegypti* populations [214]. Phytochemicals break down rapidly and this causes a need for continuous and more frequent applications in the field for a satisfactory impact on mosquito control. Further studies are needed with the implementation of new methods for the development of effective bioinsecticides from other plant-based bioactive compounds.
