**6. Methods for detecting insecticide resistance**

Information on insecticide resistance is important to inform effective vector control policy formulation. As such, detection and monitoring of insecticide resistance in malaria vectors is crucial and has to be conducted together with other entomological surveys [29]. Insecticide resistance can be detected and investigated at many levels; from dose-related phenotypical observations and genotypic approaches ranging from molecular characterisation of genes conferring resistance and their biochemical products, to the role these gene products play in overcoming the toxic effects of insecticides. In order to detect resistance, one needs to be continually looking for it. Measuring phenotypic resistance using bioassays is the recom‐ mended initial step in establishing resistance levels before genotyping for target-site and metabolic resistance and biochemical assays [59, 60]. Establishing an effective resistance management plan requires simple assays to monitor and evaluate insecticide resistance and its underlying mechanisms. To this end, different biochemical and molecular approaches have been devised, some of which are amenable for field operations. The contemporary applicable methods for resistance monitoring of field populations of mosquitoes are outlined below:

*WHO Diagnostic Assays:* A bioassay is used to determine the relationship between a physio‐ logically active agent and the effect that it produces in a living organism [61, 62]. Bioassays with the dosage or the exposure time as the variable are carried out to test the resistance status of insect populations. The WHO diagnostic assay is a useful and handy approach to detect resistance. Insecticide impregnated filter paper is used as a contact surface for exposed mosquitoes. The assay uses insecticide discriminating dosages twice the LD99 that kills 100% of non-blood-fed, adult-susceptible Anopheline mosquitoes of known age [59, 63, 64]. The demerits associated with the assays include: the assay is only able to detect high levels of resistance, i.e., if more than 5% of insects survive the exposure, with the potential exception of dieldrin bioassays, they cannot monitor resistance gene frequencies accurately; cannot give an indication of the underlying mechanisms of resistance; and cannot be used to predict crossresistance between insecticides [29]. Therefore, the resistance status detected using bioassays, can then be further studied by looking at the mechanisms responsible for resistance using biochemical and molecular assays.

The level of resistance in insect populations is dependent on the amount and frequency of insecticides used, and the inherent characteristics of the insect species selected. Mosquitoes, for instance, are endowed with all attributes suited for rapid resistance development including high reproductive potentials and short life cycles producing several generations per season with abundant progeny [27, 52]. Mostly resistance in a particular species is considered to occur throughout the control area, but in reality, insecticide resistance can be focal in nature and is very heterogeneous even over very small distances. It often develops within a small part of the population of one species of *Anopheles* and assumes different patterns depending on the type of selection pressure [27]. In Guatemala, sampling sites for *Anopheles albimanus* only a few kilometres apart varied not only by presence or absence of resistance, but also by level of resistance and the mix of mechanisms responsible for resistance [56]. The WHO Global Technical Strategy for Malaria 2016–2030 highlights insecticide resistance as a major obstacle to achieving malaria control targets [57]. The current major emphasis in research into vector resistance is double pronged. The first approach strives towards understanding the molecular mechanisms underlying resistance with the view of developing novel vector-control methods that avoid or minimise resistance problems. The second approach to research involves rational resistance management, which is developing and implementing control methods that mini‐ mise the likelihood that vectors will evolve strong resistance to important insecticides [58].

Information on insecticide resistance is important to inform effective vector control policy formulation. As such, detection and monitoring of insecticide resistance in malaria vectors is crucial and has to be conducted together with other entomological surveys [29]. Insecticide resistance can be detected and investigated at many levels; from dose-related phenotypical observations and genotypic approaches ranging from molecular characterisation of genes conferring resistance and their biochemical products, to the role these gene products play in overcoming the toxic effects of insecticides. In order to detect resistance, one needs to be continually looking for it. Measuring phenotypic resistance using bioassays is the recom‐ mended initial step in establishing resistance levels before genotyping for target-site and metabolic resistance and biochemical assays [59, 60]. Establishing an effective resistance management plan requires simple assays to monitor and evaluate insecticide resistance and its underlying mechanisms. To this end, different biochemical and molecular approaches have been devised, some of which are amenable for field operations. The contemporary applicable methods for resistance monitoring of field populations of mosquitoes are outlined below:

*WHO Diagnostic Assays:* A bioassay is used to determine the relationship between a physio‐ logically active agent and the effect that it produces in a living organism [61, 62]. Bioassays with the dosage or the exposure time as the variable are carried out to test the resistance status of insect populations. The WHO diagnostic assay is a useful and handy approach to detect resistance. Insecticide impregnated filter paper is used as a contact surface for exposed mosquitoes. The assay uses insecticide discriminating dosages twice the LD99 that kills 100% of non-blood-fed, adult-susceptible Anopheline mosquitoes of known age [59, 63, 64]. The

**6. Methods for detecting insecticide resistance**

160 Insecticides Resistance

*CDC Bottle Assay:* These are similar to the WHO discriminating dose assays. However, the CDC bottle assay relies on time mortality data, which are measures of the time it takes an insecticide to penetrate a vector, traverse its intervening tissues, get to the target site, and act on that site. Mosquitoes are exposed to glass bottle surfaces coated with an acetone- or alcohol-based formulation of insecticides. The advantage of this assay is that the rate of insecticide knock down can easily be scored during the course of the exposure period. It is able to give predictive kdr-type resistance mechanism with rapid acting insecticides, such as pyrethroids. However, caution should be exercised, as metabolic resistance mechanisms are capable of eliciting reduced knock-down phenotype without any concomitant change in sensitivity at the sodium channel target site [65].

*Synergists:* Synergists are enzyme inhibitors of insecticide detoxification enzymes. The synergists, piperonyl butoxide (PBO) and S, S, S- tributylphosphorotrithioate (DEF) are inhibitors of monooxygenases and esterases respectively [64, 66]. Glutathione s-transferase activity is inhibited by Ethacrynic acid (EA), diethyl maleate (DM), and chlorfenethol (CF). By inhibiting specific detoxification enzymes, insecticide synergists can reduce or eliminate the selective advantage of individuals possessing over-expressed or mutated enzymes [67]. Therefore, they are used to suggest the type of metabolic resistance mechanisms present in insect populations [68]. For example, they are used in bioassays to counteract or inhibit the enzymes responsible for resistance to the insecticide. Some are used in control to reduce the dose or rate of application [64]. For example, piperonyl butoxide is commonly added to pyrethroid-based aerosol formulations to decrease the time to knock down and increase the time to recover from the insecticide.

*Biochemical Assays:* There are two ways that metabolic enzymes can produce resistance [69]; overproduction of the enzyme, which leads to either increased metabolism or sequestration of the insecticide and an alteration in the catalytic centre activity of the enzyme, which increases the rate of insecticide metabolism by the enzyme. Sequestration occurs when the overproduced enzyme rapidly binds and slowly metabolises the insecticide, therefore preventing it from reaching the target site within the insecticide [70]. With sequestration, the resistance level is proportional to the increase in the quantity of the enzyme produced because of the slow insecticide turn-over rate [70]. Biochemical assays are used to give a first indication of the enzyme system involved in resistance [69]. A number of simple biochemical assays [71] are available to detect increased activity of three enzyme systems, esterases [72, 73], glutathione-S-transferases (GST) [74, 75], and cytochrome P450-dependent monoxygenases (P450s) [76] involved in insecticide metabolism. Many of these assays detect increased enzymatic activity against model substrates in resistant individuals. While simple microtitre plate assays to 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 easily amenable in the field [29].

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

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 mosquito populations [29].
