**4.2.2 Behavioral resistance**

150 Pesticides in the Modern World - Risks and Benefits

The mechanisms of resistance are behavioral, reduced penetration, metabolism of toxicant to inactive product and target site insensitivity. These mechanisms can be detected using biochemical assay techniques (spectrophotometric and fluorometric methods) and molecular assays (base on DNA diagnostic) in one individual or small number of insect. Identification of resistance mechanisms is critical for determining of the cross resistance

"Molecular methods and traditional assays (ie. bioassay) used for distinguish heterozygotes (SR), homozygous susceptible (SS) and homozygous resistant (RR) genotypes" (Scott, 1995). The environmental conditions such as temperatures, humidity, pH and light increase errors in biochemical and bioassay results but these conditions can not affect the results of molecular methods (Scott, 1995). Now, PCR-based techniques have been designed for field detection of modified acetylcholinesterase (AChE) and *knock down (Kdr)* in individual *Myzus persicae* (Field et al., 1996). "The amplified E4 or FE4 genes can be identified by restriction enzyme analysis or polymerase chain reaction (PCR)-based methods" (Field et al., 1996).

Biochemical and molecular basis of resistance mechanisms to pesticides in insects, acari, fungi, bacteria, weeds and vertebrate pests are similar. An exhaustive knowledge on biochemical and molecular resistance mechanisms in pests are useful for designing insecticide resistance management (IRM) strategies. Also, identification of resistance mechanisms is necessary for developing discriminating techniques for detecting and monitoring resistance genes and cross resistance spectrum in the field populations of pests (Hammock and Soderlund, 1986). The factors affecting pesticides effectiveness were distinguished in two classes: The first class decreases the amount of pesticide dose in action site including behavioural resistance, reduced penetration or adsorption, sequestration and detoxification. The second class is decreased target site sensitivity to pesticides that reduce the affinity of target protein toward activated pesticide (van leeuwen, et al., 2009). "In practice, probably more than 90% of all resistance cases in insects and mites are caused by a less sensitive target site and/or an enhanced pesticide detoxification" (Roush and Tabashnik, 1990 as cited in van leeuwen, et al., 2009). The relative importance of these

Genetic mechanisms of pesticide resistance involve some point mutations in genes and their

Devonshire and Moores, 1982 showed that the gene amplification of one of two closely related carboxylesterases (E4 and FE4) in *M. persicae* were associated with resistance to OP, carbamates and pyrethroids. Carboxylesterases sequester or degrade carbamate and OP insecticides before they reach to AChE in the nervous system. E4 and EF4 overproduction in resistant strains of *M. persicae* is due to amplification of structural genes encoding these

The research showed that cytochrome P450 enzyme were over expressed in some resistance strain of *M. domestica* through the increase of gene transcription by up-regulated

mechanisms depends on pest species and history of chemical application.

over expression. These mechanisms were elucidated as follow:

spectrum (Brogdon and McAllister, 1998).

**4.2 Mechanisms of resistance to pesticides** 

**4.2.1 Genetic mechanisms** 

**4.2.1.1 Gene amplification** 

enzymes (Field et al., 1988).

**4.2.1.2 Up- and down-regulation** 

"Behavioral mechanisms, defined as evolved behaviors that reduce an insect's exposure to toxic compounds or that allow an insect to survive in what would otherwise be a toxic and fatal environment" (Sparks et al.,1989). There is a little literature on behavioural resistance mechanisms in insect due to difficulties in detection (as cited in Jensen, 2000). It seems the significance of this mechanism for resistance is less than other resistance mechanisms.

## **4.2.3 Reduced penetration**

Reduced penetration of insecticide as a resistance mechanism has been studied in few insect species such as *Leptinotarsa decemlineata*. Reduced insecticide penetration via cuticle led to decrease the amount of dose in action site. The resistance ratio by this mechanism was lower than 3-fold (Scott, 1990), but because several different mechanisms are responsible for resistance to an insecticide and multiple resistance mechanisms may co‐exist in an insect and act either additively or synergistically.

Patil & Guthrie, 1979 compared the composition of the cuticular lipids of two resistant strains of *M. domestica* and their results showed that "total lipids, monoglycerides, diglycerides and sterol esters, sterols, fatty acids and phospholipid phosphorus were higher in resistant strains than in the susceptible strain".

Three methods for detecting of this mechanism include: Wash-off, diffusion cell and disk technique. In wash-off radiolabelled insecticide was topically applied to the insects and then, at fixed times after application, un-penetrated insecticide was washed off with an appropriate solvent and quantified (as cited in Jensen, 2000).
