**7. Insecticide resistance mechanisms**

In mosquitoes, resistance is mediated through complex mechanisms, including behavioural and/or physiological changes resulting in insecticide avoidance (reduced contact with insecticide), reduced cuticular penetration (of the insecticide through the cuticle), increased sequestration (i.e., stored in the body where it is not harmful), target site insensitivity (i.e., the target site is altered and not affected by the insecticide), or increased bio-degradation (so that it is detoxified before it reaches the target site) [84], and possible increased excretion. The molecular basis of insecticide resistance has been attributed to the existence of mutations in target site genes or metabolic alterations at the level of the activity of the detoxification proteins [56, 85]. Insecticide resistance mechanisms have a biochemical basis and target-site resistance and detoxification enzyme-based resistance remain the two major forms of biochemical resistance [52, 56]. Alone or in combination, target site resistance, which results from the inability of the insecticide to bind to its target, and metabolic resistance resulting from failure of the insecticide to reach its site of action due to enhanced levels of modified activities of detoxification enzymes, confer various levels of resistance to all classes of insecticides [84]. Though long- and well-recognised, the importance of behavioural and cuticular resistance in malaria vectors has been largely overlooked. While resistance arises through Darwinian selection in a population, it is often a combination of factors that results in the overall expres‐ sion of the phenomenon [15]. Thus, gene over-expression, amplification, and structural mutations have been linked to insecticide resistance mechanisms in some insects [84]. Cogni‐ zant of detailed elaborations given elsewhere [86], a brief description of the key mechanisms is outlined below:

measure AChE insensitivity using a carbamate or an oxon analogue of a phosphorothioate insecticide exist [77], the applicability of biochemical assays for the GSTs and P450s are not

*Molecular Assays:* Molecular techniques can be used to detect some well-characterised resist‐ ance mechanisms. Most techniques employ the method of polymerase chain reaction (PCR). Mutations in the insecticides' target site lend themselves to detection through simple PCR assays, which can readily be used in many field settings. Allele-specific PCR assays have been developed for three major target sites, the GABA receptors [78, 79], the sodium channels (*kdr*) [80, 81], and AChE. The challenge is to adapt these assays for high-throughput field applica‐ tions as they have the advantage of detecting heterozygous-resistant individuals that may be missed by other assays [29]. Recent advances in genomics have allowed a much more rapid identification of genes that are up or down regulated in insecticide resistant insects using microarray technology [82]. The detoxification microarray chip, developed for *An. gambiae,* contains all potential insecticide resistance genes. The detoxification chips have been devel‐

Bioassay data as measured by either the CDC or WHO technique have a common limitation of inability to accurately determine gene frequency or predict the epidemiological impact of resistance [83]. Resistance management requires that resistance is detected at very low frequencies, but the two approaches are not sensitive enough to achieve this. While contem‐ porary techniques diverge in their sophistication and ease of use, there is still a need for approaches to enable measurement of the frequency of different mechanisms of resistance in

In mosquitoes, resistance is mediated through complex mechanisms, including behavioural and/or physiological changes resulting in insecticide avoidance (reduced contact with insecticide), reduced cuticular penetration (of the insecticide through the cuticle), increased sequestration (i.e., stored in the body where it is not harmful), target site insensitivity (i.e., the target site is altered and not affected by the insecticide), or increased bio-degradation (so that it is detoxified before it reaches the target site) [84], and possible increased excretion. The molecular basis of insecticide resistance has been attributed to the existence of mutations in target site genes or metabolic alterations at the level of the activity of the detoxification proteins [56, 85]. Insecticide resistance mechanisms have a biochemical basis and target-site resistance and detoxification enzyme-based resistance remain the two major forms of biochemical resistance [52, 56]. Alone or in combination, target site resistance, which results from the inability of the insecticide to bind to its target, and metabolic resistance resulting from failure of the insecticide to reach its site of action due to enhanced levels of modified activities of detoxification enzymes, confer various levels of resistance to all classes of insecticides [84]. Though long- and well-recognised, the importance of behavioural and cuticular resistance in malaria vectors has been largely overlooked. While resistance arises through Darwinian

oped for *An. stephensi,* pyrethroid resistant *An. funestus*, and *A. aegypti* [29].

easily amenable in the field [29].

162 Insecticides Resistance

mosquito populations [29].

**7. Insecticide resistance mechanisms**

*Target site resistance* (also called phenotypic resistance) is based on alterations of amino acids in the site of action where the insecticide is supposed to bind, rendering them less sensitive to the active ingredient [58]. Majority of insecticides used in vector control are nerve poisons and target: the acetylcholinesterase (AChE) that hydrolyses the neurotransmitter acetylcholine in the synapses particularly in carbamates and organophosphates; the sodium channels respon‐ sible for raising the action potential in the neurons during the nerve impulses involved in the resistance in organochlorines and pyrethroids; or the c-aminobutyric acid (GABA) receptors responsible for chloride-ion neurotransmission in the nervous system, specific for cyclodienes [87, 88]. Mutations have been observed in neuronal enzymes and receptors, leading to welldefined target site alteration and resistance to chemical insecticides [89]. Knock down resist‐ ance (Kdr) occurs due to a single or multiple substitutions/mutations in the para-gated sodium channel gene [29]. In *An. gambiae*, two mutations (Leu-Phe [90] and Leu-Ser [91]) have been identified at the same codon. As this is the target site of DDT and pyrethroids, this mechanism produces cross-resistance to the two insecticide classes. Organophosphate and carbamate insecticides inhibit acetylcholinesterase (AChE). Many insect vectors have developed resist‐ ance through structural alterations of this target site [58]. These point mutations may act individually or in combination.

*Metabolic resistance*, on the other hand, usually involves over-expression/over-production of a complex array of specific enzymes capable of detoxifying insecticides or modifications in the amino acid sequences that cause alterations in the levels and activity of detoxifying proteins [58, 90]. The mechanism of increased detoxification contributes to a decrease in the effective dose of insecticides available at the target site [67]. The overproduction of these endogenous detoxifying enzymes may be achieved via two nonexclusive mechanisms: 1) increase in the gene's copy number of available molecules (by gene amplification or expression activation); or 2) mutation in the enzyme coding portion of the gene, so that its product metabolises the insecticide more efficiently [92], preventing it from reaching its target in the nervous system. Metabolic resistance occurs through increased biodegradation of the insecticide, usually through overproduction of detoxification enzymes such as P450s, GST, and carboxylesterases (EST). The P450 cytochromes primarily metabolises pyrethoids and to a lesser extent, of carbamates and organophosphates, carboxylesterases largely detoxify organophosphate and carbamate and to a lesser extent in pyrethroid resistance [93]. GSTs are involved in the detoxification of a wide range of xenobiotics, including the organochloride insecticide DDT [94]. In *A. gambiae*, metabolic resistance to insecticides can be conferred by elevation in the activity of these three classes of detoxifying enzymes. In contrast, there are few examples in literature regarding insect behavioural changes and tegument alterations.

*Behavioural resistance* involves behaviour changes in response to prolonged exposure to an insecticide resulting in avoidance and reduced contact with lethal doses of an insecticide [95]. Behavioural resistance does not have the same "importance" as physiological resistance but may be considered to be a contributing factor [96, 97]. It remains unclear whether adaptation of malaria vectors species to insecticidal-based vector control interventions may result from a phenotypic plasticity or from selected behavioural traits [98]. Notably, behavioural resistance is characteristically difficult to quantify [99].

*Penetration resistance*: Reduced penetration involves changes that decrease the rate of penetra‐ tion or absorption of insecticide through the insect cuticle or digestive tract linings and confers low levels of resistance [68, 100]. This resistance mechanism is not specific and can affect a broad range of insecticides. Reduced uptake of insecticide, often referred to as cuticular resistance, is frequently described as a minor resistance mechanism. More effort is required to identify the significance of cuticular resistance in phenotypic resistance [86].

The understanding of the development of resistance and the design of novel strategies to manage it and to effectively control disease vectors is greatly owed to the characterisation of genes and the molecular mechanisms involved in insecticide resistance [101]. However, the mechanisms of insecticide resistance are generally far less well-understood. Particularly, the contribution these enzymes make towards pyrethroid resistance and their biochemical relationships with P450-mediated resistance is still unclear [84].
