Mosquito Excito-Repellency: Effects on Behavior and the Development of Insecticide Resistance

Abstract

Mosquito’s resistance to avoiding insecticide-treated surfaces (“excito-repellency”) has two effects: irritation from direct contact with a treated area and repellency as an avoidance response to contact with treated surfaces. Nowadays, this behavior appears to reduce the success of mosquito control programs, particularly those based on insecticide-driven strategies. Different systems have been designed to assess the excito-repellency, evaluating numerous insecticides’ irritants, deterrents, and toxic properties at different concentrations. The information provides valuable insights regarding the patterns of mosquito behavior based on their physiological conditions, such as the age of the mosquitoes and the duration of the tests. However, the physiological processes resulting from chemical stimulus contact “chemoreception”) are still poorly explored and understood. This review provides an overview of insecticide effects on mosquito behavior and describes the mechanisms involved in chemical stimuli uptake, translation, and recognition.

1. Introduction

The worldwide spread and increasing transmission of diseases such as dengue, chikungunya, and Zika transmitted by Aedes aegypti have made this mosquito species a primary target for the vector control programs [1]. Ae. aegypti control still represents the primary strategy to reduce outbreaks of these diseases. Interventions include reducing or eliminating such breeding sites during their immature aquatic phase and insecticidal control using larvicides and adulticides [2]. The natural tendency of mosquitoes to avoid insecticide-treated surfaces appears to be a general behavioral phenomenon [3]. There are two types of behavioral responses to insecticides: irritation, which is defined as an insect leaving an insecticide-treated surface after tarsal contact with it, and repellency, or spatial repellency (deterrence or avoidance), which refers to the function of a compound to influence an avoidance response movement away from a chemical stimulus through actual physical contact with it, thus diverting insects from the treated surface [4, 5, 6]. However, this behavior and the changes in the vector’s resting habits and biting activity represent a challenge in the surveillance and vector control strategies [7].

Different authors have coined the term “excito-repellency” to refer to the combined effect of escape responses after the contact with the insecticide-treated area [5, 8, 9]. Likewise, studies based on the excito-repellency test system have reported that the nutritional status and physiological conditions (including age) and the duration of the tests can significantly influence the response behavior to insecticides. Therefore, chronological age, physiological status, and innate (circadian) activity patterns should be carefully considered for the proper selection of mosquitoes used in the tests [3].

The behavior of mosquitoes in response to insecticides continues to be poorly studied and understood. They do not refer to anything other than the locomotor response of mosquitoes after capturing a stimulus, either contact “irritation” or noncontact spatial repellency “deterrence” resulting from the capturing of chemical emanations “odors” by nature and/or evaporation of chemical substances [10, 11]. On the other hand, understanding mosquito behavior will enable the selection of insecticides, the operational planning of cost-effective and long-term interventions and the development of innovative surveillance and vector control tools and strategies [12, 13].

2. Vector control methods

Most of the interventions for Aedes mosquito control are based on adulticide and larvicide insecticides; however, this strategy alone is insufficient to reduce the mosquito populations and can induce different resistance mechanisms [2, 14]. In recent years, it has been necessary to further optimize current strategies within integrated approaches and advance the development of an alternative, an innovative design for the control [15, 16]. The integrated vector management (IVM) combines different methods for sustainable vector control; these methods are grouped into the following strategies: environmental control, mechanical/physical control, biological control, chemical control, and the use of new technologies such as the release of mosquitoes with endosymbiotic bacteria (Wolbachia), transgenic mosquitoes, or irradiated mosquitoes [2]. Additionally, WHO’s 2017–2030 Global Vector Control Response strategy points out that vector control can be improved by educating and empowering communities to identify and eliminate breeding sites around their homes, also improving the piped water supply system, adequate management of solid waste and screened housing to reduce densities of mosquitoes biting humans indoors and thus reduce human vector contact, and personal protection by repellents application, insecticide-impregnated net and curtains [1, 2, 17].

Environmental interventions (“Immature mosquito control”) are based on actions to eliminate breeding sites for these organisms, impacting adult populations. These interventions represent sustainable and safe methods as there are limited risks of environmental contamination and toxicity [18, 19]. Control of the immature stages of dengue vectors is generally conducted larval habitats using biological, chemical, environmental, or mechanical methods to maximize the reduction in vector population density [13]. The principal environmental methods are container covers with and without insecticides, waste management with and without direct garbage collection, elimination of breeding sites, drinking water supply, and urban planning [2, 19]. Similar to environmental interventions, mechanical and physical control, consists of cleaning breeding sites and physical barriers such as mosquito nets and curtains [15].

The biological control interventions to control mosquitos are based on pathogens and mosquito symbionts. Regarding successful experiences of biological control, some examples of interventions regarding the vertical or community-based introduction of Cyclopoid copepods (Mesocyclops longisetus) and one with fish of different species have been reported for the control of dengue vector populations [20, 21, 22]. However, their implementation and sustainability function depends on breeding sites and their ultimate productivity. Regarding biorational management, different formulations based on Bacillus thuringiensis var. israelensis (Bti) are efficient as bio-larvicides for malaria and dengue vectors [23, 24]. On the other hand, the use of innovative technologies based on interventions with “modified mosquitoes,” either genetically by dominant lethal genes (RISL) or immunity genes (RNAi), by irradiation as sterile insect technique (SIT) or biologically as incompatible insect technique IIT, the SIT and IIT strategies are not classified as genetically modified [25].

Chemical control is an essential element in vector control strategies worldwide and can be implemented under two approaches: 1) to reduce density/increase vector mortality by adulticides and larvicides application and 2) to reduce human vector contact by insecticide-treated materials (ITMs) such as long-lasting insecticide nets (LLINs), traditional nets, and personal repellents [2]. Vector control programs have favored insecticides to control adults, mainly based on ultralow-volume (ULV) space application for outdoors in open spaces due to the ease of covering large areas in the shortest possible time and thermal fog indoors [26, 27, 28]. Aerial ultralow-volume (AULV) applications are also being tested in México [29]. Targeted indoor residual spraying (IRS, TIRS) is another control method with evidence of efficacy in México [30].

The most common insecticide products to control malaria and any other mosquito vector transmission combine two different modes of action: 1) conventional insecticide activity that kills mosquitoes exposed to the insecticide, and 2) deterring mosquitoes away from humans [17]. Space spraying of insecticides is still considered to be a valuable tool to control the vectors of human diseases [31]. S-methoprene, pyriproxyfen, temephos, and Bti are among larvicidal and pupicidal agents recommended and approved by the World Health Organization (WHO) to treat Ae. aegypti larval environments. Temephos is the most extensively used larvicide for Ae. aegypti control [13]. Personal protection in the form of repellent application with DEET (N, N-diethyl-3-methylbenzamide) at a concentration of 25% can provide protection against Ae. aegypti [32].

Insecticide-treated materials (ITMs) can provide bite protection by killing or repelling vectors [6]. The use of nets impregnated with long-lasting insecticides used as pavilions or mosquito nets is currently one of the most promoted strategies to reduce the transmission of arboviral or parasitic diseases [17, 33]. Long-lasting insecticidal nets (LLINs) are materials pretreated with insecticides designed to prolong their useful life. Studies in several Latin American countries indicate that using LLINs fixed on doors and windows and insecticide-treated screening (ITS) are innovative approaches to control vector mosquito populations, and they may be promising in reducing the transmission of diseases such as dengue [34, 35, 36, 37]. The efficacy of these interventions is reflected in reduced vector-human interaction and sustained indoor adult vector densities as blood-fed and arbovirus-positive Ae. aegypti females [38, 39], either due to its effects of repellency and excito-repellency or due to its toxic action, causing the death of the vector, in addition to acting as a physical barrier [40, 41]. Furthermore, its use in the form of mosquito nets permanently installed on doors and windows could solve one of the main problems associated with interventions using LLINs: the decrease in coverage over time (due to disuse or misuse of the LLIN by the community), limiting its effectiveness [35].

Spatial repellents (SRs) are products containing volatile chemicals that disperse in the air under ambient conditions. Besides, the term “spatial repellency” is used here to refer to a range of insect behaviors induced by airborne chemicals that result in a reduction in human-vector contact and, therefore, personal protection [42, 43]. The behaviors can include movement away from a chemical stimulus, interference with host detection (attraction inhibition), and feeding response [43]. In clinical trials, SR products reduced malaria and Aedes-borne virus infection through mechanisms of reduced human-vector interaction, being and are effective against insecticide-susceptible and resistant mosquito vectors [44]. Lethal ovitraps are small to medium-sized plastic cups or buckets with an oviposition substrate (usually cloth or paper-based) treated with a residual insecticide or an adhesive that may be used indoors or outdoors to reduce Ae. aegypti populations [13].

3. Effect of insecticides on mosquito behavior

For example, indoor residual spraying against malaria vectors depends on whether mosquitoes rest indoors (i.e., endophilic behavior); likewise, the optimum effectiveness of insecticide-treated nets presumably depends on vectors biting at hours when most people are in bed. Prospects for genetic control by sterile males or genes rendering mosquitoes harmless to humans will depend on competitive mating behavior [45]. Some works have provided evidence for the existence of behavioral modification because of widespread IRS or ITN use [46]. The behavioral responses of mosquitoes to insecticide products must be determinate to understand the main mechanisms involved in their effectiveness; this task is vital not only for success in chemical control strategies used in vector control programs but also for the development of new insecticides and the design of innovative control strategies [6, 12, 47]. The devices developed to analyze the behavior of mosquitoes evaluate three main components of chemical action: 1) contact irritation (excito-repellency), 2) spatial repellency, and 3) toxicity [8, 47, 48, 49].

Malaithong et al. [11] criticize the subjective way of using terms such as avoidance, excitation, irritability, deterrence, and excito-repellency by different authors in a system based on “success-repellency” tests, to refer to locomotor response behavior caused by contact with insecticides. The experts point out that this test system distinguishes two main locomotor responses: 1) stimulation of the musculoskeletal apparatus “excitation,” “irritation” as a result of tarsal contact with a treated surface and 2) noncontact spatial repellency “deterrence” as a result of chemical emanations, that is, the capture of “odors” in the evaporation phase of chemical compounds at a distance, that is, the capture of “odors” in the evaporation phase of chemical compounds at a distance, without the need to make contact with the treated area and emphasizes the need for a clearer understanding of the behavior, stimuli, effects, and mechanisms involved in the response locomotive of mosquitoes against different xenobiotics.

Kongmee [50] reported no repellency effect without contact generated by insecticides in a study with anophelines based on excite-repellency tests with two pyrethroids; nevertheless, deltamethrin produced a high irritating effect, while bifenthrin exhibited low levels. At operational field doses of alpha-cypermethrin, low noncontact repellency was observed in three mosquito populations; thus, spatial repellency may play a minor role in escaping vectors from treated surfaces, with contact irritation being the most important main effect on Ae. aegypti populations [11]. Mongkalangoon et al. [51], in a study of irritability and repellency to synthetic pyrethroids, found a strong repellent effect of cyphenothrin in all doses evaluated compared with deltamethrin and d-tetramethrin; however, the irritant effect on contact was similar in the three insecticides evaluated. On the other hand, the presence of insecticide resistance by kdr and ace1 mutations can modify the mosquito response to DEET and natural repellents. These findings were validated for two resistant Anopheles gambie strains (KdrKis and AcerKis) in that the mutation can increase or decrease the effectiveness of DEET and natural compounds [52]. Cross-resistance has also been reported in the pyrethroid-resistant (PR) Puerto Rico strain of Ae. aegypti. All repellents tested in the study were less effective against the PR strain. Furthermore, the reduced susceptibility to these repellents may reflect a fitness cost caused by the PR strain’s kdr mutation. As a result, it is critical to understand the secondary effects of pesticide resistance evolution in mosquitos, as well as the importance of developing alternative resistance-control strategies [53].

4. Sensory receptor and stimulus uptake

The functions of insect chemoreceptors have primarily been studied using antennae (olfactory receptors) and mouthparts (gustatory receptors) through specialized structures called sensilla, which comprise neurons and non-neuronal support cells, extracellular lymph fluid, and a precisely shaped cuticle [54]. Other appendages with chemoreceptive sensilla include the leg tarsi and the anterior wing margin. Their specific roles in chemoreception and mosquito behavior remain largely unknown [55]. This review provides a brief description and illustration of the sensilla and its function in chemical stimulus, transduction, recognition, and understanding in insect behavior.

4.1 Sensilla

Modified cuticular structures are the basic sensory unit in insects; they are made up of four components: sensory neurons, a thermogenic cell (socket), a trichogenous cell that gives rise to the hair, and a thecogenous cell that surrounds and protects the axon terminal and provides it with ions and nutrients (Figure 1C), sensory neurons are bipolar, extending their dendrites from the cuticular portion and their axons toward the central nervous system (CNS). Due to their external morphology, they can be classified into trichoid, basiconic, placoid, styloconic, coeloconic, and bell-shaped [58].

Figure 1.

Sensory receptors in mosquitoes, the sensilla and the stimulus. A) Sensory receptors on appendages based on Sparks et al. [56], B) different types of sensilla ae. Aegypti adapted from Jonathan Bohbot’s lab (https://jonathanbohbot.weebly.com/the-mosquito-nose.html) [57], C) schematic organization of the trichoid sensilla (ORN: Olfactory receptor neuron), D) chemical stimulus, signal transduction, and recognition (OBP: Odorant-binding proteins) (A, C and D figures created with BioRender.com).

These structures, which are found on the surface of mosquito antennae and maxillary palps (olfactory sensilla), as well as mouthparts, tarsi, and wings (gustatory sensilla), play a major role in host detection and other sensory-mediated behaviors. (Figure 1A) [56, 59, 60]. In mosquitoes, the trichoid sensilla is the most common and abundant sensory structure. A single pore is typically observed at the distal end of a blunt tip. (Figure 1C) [60]. Bohbot et al. (2014) described the basiconica, chaetica, and campaniformia sensilla in Ae. aegypti and identified also in Culex pipiens with other classifications such as coeloconica, ampullacea, squamiformia, and styloconica. (Figure 1B) [59, 61]. Similar types of sensilla were observed on An. kochi antennae (ampullacea, chaetica, trichodea, basiconica, and coeloconica) [62]. Likewise, according to the type of stimulus they receive, these structures can be classified as: odorant receptors (chemoreceptors), gustatory receptors, ionotropic receptors, and olfactory receptors [56].

4.2 Chemoreceptors

Highly sensitive units can react to an external stimulus, mainly of “gustatory” character in the mouthparts, legs, and ovipositor, or olfactory character, mainly in the antennae and palps. Zwonitzer (1962) [63] within this classification includes those known as “general chemical sensitivity”, which in turn respond to volatile materials classified as “distance chemical receptors” that are moderately sensitive. The categorization also comprises the receptors that are stimulated by nonvolatile substances (“chemoreceptors senses”), and those that are relatively insensitive and lead to protective responses defined as “general chemical senses.” Chemoreception is the ability to perceive specific chemical stimuli. It is one of the most evolutionarily ancient forms of interaction between living organisms and their environment; this physiological process occurs because of the contact with a chemical stimulus and presents a broad spectrum of sensations [64, 65].

4.3 Stimulus reception

The chemical signals produced by semiochemicals and chemical substances reach the sensilla and penetrate the cuticle through pores; these substances are hydrophobic and cannot pass through the fluid. Lymphatic (aqueous medium) is achieved thanks to odorant-binding proteins (OBPs) that encapsulate them and direct them to the surface of the sensory cell where a structural change of charges in the membrane causes the stimulus to be expelled on the nerve receptor (Figure 1D) [58].

4.4 Chemical-sensory transduction

Once the union between the stimulus and the receptor has occurred (stimulus-receptor complex), the event must be communicated to other parts of the sensory cell to ensure the final message through the effect of action potentials transmitted to the brain. The amplification involves a series of membrane-bound intracellular molecules, usually calcium; at least one ion channel detects the increase in calcium and opens, thus triggering membrane depolarization [58].

4.5 Recognition and meaning in behavior

The sensory system acts as a filter allowing the insect to capture stimuli and to differentiate one stimulus from another. Therefore, receptor proteins and translation-associated molecules possess high specificity and sensitivity [66]. Several works have been published regarding the recognition by mosquitoes of emanations such as CO₂ and lactic acid on human skin; these works also show that high concentrations of these compounds can have a deterrent effect on mosquitoes [67, 68].

In female and male Ae. aegypti, chemoreceptors have been reported in the labella, tarsi, and at least in the first third of the legs; these receptors are curved setae present in the labella, and possibly they are those found in the tarsi. With these receptors, the mosquitoes can distinguish between acceptable and unacceptable solutions [69]. The presence of chemoreceptors in antennae has also been reported, it consists of sensilla classified as basiconic sensilla, and it is found in the ninth antennal segment [70]. However, multilayered molecular and cellular mechanisms determine the selectivity, sensitivity, and dynamic modulation of responses in insects [54]. Several studies on the development of attractants based on host-seeking behavior in mosquitoes have also provided important data to understand mosquito uptake of stimuli and different behavior patterns [71].

5. Effect of the physiological status of mosquitoes on the efficacy of insecticides

Different studies have compared the behavioral response of mosquitoes under different physiological conditions, including age. Experiments were carried out on Ae. aegyti females with four different physiological conditions: parous, without copulating, copulating (nulliparous), and fed to repletion. The results show that females without copulating and nulliparous females had higher responses of irritation and repellency than parid or newly fed females [3]. Polsomboon et al. [72] evaluated the relationship of the same physiological conditions in two pyrethroids; all the assessed females, regardless of their physiological state, were susceptible to deltamethrin and resistant to DDT; and two of the test groups from the same populations (without mating and nulliparous) showed higher mortality to DDT compared with parid females and recently fed females. Because blood can serve as an additional glycogen and protein storage, mosquitoes that were not mating or feeding showed reduced vigor in both insecticide tests.

Oliver & Brooke [73] were the first to demonstrate the expression of resistance to insecticides because of multiple blood feedings; they also point out the variability of the expression levels of detoxification enzymes as a function of age that presented a decrease in these due to aging. This could be because blood feeding can modify the expression of genes that affect the action of detoxifying enzymes. This expression is more evident during the first, second, and third days following a single blood feeding showing a dependence on sex [74].

This association seems to influence mosquito susceptibility or resistance to insecticides in terms of mosquito parasitism. According to Agnew et al. [75], parasitism can act as a source modifying the costs of resistance to organophosphate insecticides and as qualitatively different interactions (increasing or decreasing relative fitness in resistant individuals) that occur depending on the type of resistance involved.

In several insect groups and disease vectors across the world, “physiological” resistance, metabolic, and target site modifications to insecticides have been well documented [76], including highly physiologically insecticide-resistant mosquitoes. It also implies the application of chemical products at higher concentrations, which is neither practically feasible nor cost-effective [12].

6. Conclusions

Although the behavioral responses of mosquito vectors to insecticides differ based on the type of product and concentration, it is also true that the product’s properties (irritant-repellent) can help to reduce human-vector interaction. The behavioral response of avoiding the treated surface seeks to integrate products with such properties to reduce the transmission of pathogens because they reduce the opportunity for blood feeding. Understanding mosquito behavior, including oviposition site selection, dispersal behavior, and competitive mating, can allow the development of innovative mosquito surveillance and control strategies to control these important and deadly insects better. On the other hand, molecular and structural studies and the signaling pathways of these receptors must be studied better to understand their function and role in resistance development.

Conflict of interest

“The authors declare no conflict of interest.”