While bioassays are useful to monitor the susceptibility of mosquitoes to insecticides, such behavioural assays may miss the presence of insecticide resistance alleles in the mosquito population at low frequencies, particularly if they are recessive.

Genes and alleles

Some genes can have more than one form, which can encode for different characteristics in an organism. Of particular interest to us are the different forms of genes that provide different degrees of insecticide susceptibility. Such specific forms of a given gene are called ‘alleles’. Those that allow an insect to survive exposure to an insecticide are called ‘resistant alleles’. Whereas insects that succumb to the insecticide likely express a ‘susceptible allele’, or form of the gene.

Mosquitoes have two copies of every gene; the copies, or alleles, may be different, potentially expressing different characteristics. Generally, only one allele is expressed. Some alleles may be dominant and are always expressed, whereas others may be recessive, and are only expressed when two copies of the recessive allele are present. Specific alleles are known that confer resistance to certain mode of action classes of insecticide.

Mutations of a gene may very occasionally give rise to an allele that offers a selective advantage to an organism, such as insecticide resistance. As a result, individuals with these mutants, or new alleles, may have more offspring, and so the allele becomes more frequent in the population.

If you can use molecular techniques to identify the presence of a ‘resistant allele’ in a mosquito, or mosquito population, it can act as a marker, allowing you to predict whether it will be susceptible to a given insecticide class.

Molecular tools

If a mosquito carries only one recessive copy of the resistance allele, it will still die in the behavioural assay. However, its offspring may express resistance if they inherit a copy with the mutation from both parents.

Molecular diagnostics can track the spread of resistance alleles and help detect mutations at an early stage, before resistance becomes widespread. Moreover, identifying the molecular mechanisms of resistance present in a mosquito population helps in making an informed choice of insecticide in a control programme, allowing you to identify mode of action classes that are less likely to be resisted.

There are several Polymerase Chain Reaction (PCR) diagnostics based on molecular DNA markers for target-site resistance mutations, such as knockdown resistance (kdr), against pyrethroids and DDT.

However, very few diagnostic DNA markers have been identified so far that are associated with the increased production of enzymes that break down insecticides – so-called metabolic resistance. Therefore, current molecular approaches may miss detection of insecticide resistance, particularly metabolic resistance, where we have limited diagnostics available.

Advantages of molecular approaches

An advantage of the molecular diagnostic approach is that it doesn’t need to use living mosquitoes. Dead mosquitoes, or even parts of a mosquito, such as a leg, can be collected in the field and returned to the laboratory at a later date for molecular analysis. This approach can also provide further useful information about the mosquito, including its exact species.

Where markers for metabolic insecticide resistance are not available, an alternative is to measure the expression levels of genes implicated in conferring metabolic resistance and compare them to susceptible mosquito reference colonies. Current techniques include, for example, quantitative PCR, DNA microarray platforms or RNA sequencing. However, these techniques require access to specialised laboratory equipment and trained personnel, and are costly.

Biochemical approaches

An alternative to gene expression analysis are biochemical assays. Biochemical assays measure more directly the activity and quantity of detoxification enzymes in field populations and compare them to susceptible reference colonies. However, these assays require a cold chain and measure the expression of enzyme families rather than specific enzymes, limiting their sensitivity and specificity. Nevertheless, the use of studies with synergists may be still useful. Synergists are molecules that limit enzyme activity, including that of insecticide-metabolising enzymes. The synergists are used in combination with the behavioural insecticide susceptibility assays.

Mosquitoes are first exposed to the synergist before they are exposed to the insecticide of interest. If the synergist restores the activity of the insecticide, we have an indication of metabolic resistance. An example is PBO, a synergist that blocks the activity of pyrethroid-detoxifying enzymes. ‘Next generation’ LLINs containing the synergist PBO have been developed which can increase their effectiveness against metabolic-resistant mosquitoes. Synergist assays with PBO would help to identify whether such nets could mitigate metabolic resistance mechanisms in a given mosquito population.

Looking forward

Research efforts to discover resistance alleles, or markers, are ongoing, and accelerating, as genome sequencing of mosquito field populations becomes more feasible. At the same time, with the introduction of novel insecticides, prospective analysis is required to anticipate the development of novel insecticide resistance mechanisms and so to have appropriate markers already available.

Author: Pie Müller