Detection and monitoring of insecticide resistance and its underlying mechanisms is of primary importance for effective vector and pest control program. Detection of resistance when it first appears is crucial for a timely resistance management actions. Several methods have been developed over the years to monitor, and detect insecticide resistance. In some cases even the mechanism involved can be ascertained. Traditional methods includes bioassay tests to measure the resistance, while biochemical assays and molecular biology and genetics approaches provide a more insight as for the mechanisms involved
3.1. Bioassay test:
This method has traditionally been used to detect insecticide resistance in the field and is principally designed to measure the level of a population resistance to a given insecticide in relation to a susceptible genotype. It is a dose-response approach that consists to collect insects in the field and rear them in the laboratory through several generation without insecticide exposure. Larvae or adults are tested for resistance by assessing their mortality after exposure to a range of doses of the insecticide. For susceptible and field populations, LD50 or LC50 values were calculated by using probit analysis. The results are compared with those from standard susceptible populations and conclusion is drawn. Resistance detection could also be done on individual insect to a range of concentration of insecticide, and conclusion is drawn based on dose-response relationship. The disadvantage of this technique result from the fact that it can only detect resistance when it is already established in a population.
3.2. Biochemichal assays
Biochemichal techniques use enzymes assay to detect enzymes activity in insect homogenates using model structure, as a way to determine resistance. The assays are conducted on a set of enzymes, which are known to metabolize a given insecticide, and a model substrate is used to conduct the assay. The most important assays used involve esterases and acetylcholinesterase For instance, naphtyl acetate has been used as substrate to detect total esterase activity in Myzus persicae (sawicki et al.1980), and in various mosquito species (Pasteur and Georgiou,1989). Similarly acetylthiocholine iode has been use as substrate to monitor acetylcholinesterase activity in housefly (Devonshire and Moores 1984). Biochemical assays offer the advantage of detecting the initial stages of resistance in a population and the mechanism of resistance involved.
3.3. Molecular biology and genetics approaches
The advancements in molecular biology and genetics compounded over the last few decades has equipped entomologists with novel approaches to unravel the underlying mechanism of insecticide resistance. The sequencing of multiple insect species provides a great opportunity to compare detoxification gene families. DNA sequencing is used in various insect species to establish the presence of mutation in a specific insecticide receptor proteins. PCR assays was also used to detect two knock down resistance (kdr)-like mutations (L1014S and L1014F) in the sodium channel of Anopheles stephensi (Singh et al., 2011). In this study PCR assays followed by DNA sequencing were developed to reveal the presence of these mutations.
In Colorado potato beetle, Malekmohammadi et al. (2012) used a combination of polymerase chain reaction and restriction fragment length polymorphism to monitor the S291G resistance mutation of the AchE gene in azinphosmethyl-resistant field populations from various locations. From this study 25% of collected samples were found to carry the resistance mutation
In the cat flea, Ctenocephalides felis, resistance to cyclodiene insecticides is caused by amino acid substitutions at a single residue (A302) within the M2 transmembrane region of the gamma-aminobutyric acid (GABA) receptor sub-unit termed Rdl (resistance to dieldrin). These mutations (A302S and A302G) have also been shown to confer varying levels of cross-resistance to fipronil, a phenylpyrazole insecticide with a similar mode of action to cyclodienes. To investigate the possible occurrence of these mutations in the cat flea, Ctenocephalides felis a 176-bp fragment of the cat flea Rdl gene, encompassing the mutation site, was PCR amplified and sequenced from nine laboratory flea strains. The A302S mutation was found in eight of the nine strains analysed. Only one strain (R6) was found to be homozygous for the S302 allele in all the tested individuals. A PCR-based diagnostic assay, was developed to screen individual fleas for this mutation. The A302S mutation was present at a high frequency in these domestic pet populations (Bass et al., 2004).