Arthropod P450 Dokuwiki

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====== P450 and pesticide resistance ======

For a recent and general review of the role of P450s in pesticide resistance, see [[https://doi.org/10.1146/annurev-ento-070621-
061328|Nauen et al., 2022]]

A reminder of "resistance" definitions: 

The classical definitions are from the WHO (1957): Resistance is the development of an ability in a strain of insects to tolerate a dose of toxicants which would prove lethal to the majority of individuals in a normal population of the same species; and from the FAO: resistance is the decreased response of a population of animal or plant species to a pesticide or control agent as a result of their application.

A Forum by [[https://doi.org/10.1603/EC13458 |Tabashnik et al. 2014]] discusses critically the many ways "resistance" is defined, and proposes a general definition of resistance as a "genetically based //decrease// in susceptibility to a pesticide". (//italics// are used here to point out that not all genetically based variation in susceptibility would qualify as resistance).

The fact that //resistance// is heritable and that it results at the population level from selection by a toxic agent is implicit in many definitions, yet of the essence of resistance. While many authors avoid or dislike the term //tolerance//, the definition of Finney (1971) is very useful: the highest concentration of a particular pesticide that an individual can withstand without being killed. It follows that natural or experimental variation in the toxicity of a pesticide is a measure of tolerance, not necessarily of resistance.

===== Phenotype, genotype and causal relationships =====

There are at least two ways of looking at resistance: 

One way is to look at the phenotype: a biochemical or physiological change that causes resistance. 

A second way is to look at the molecular genetic modification that leads to this phenotype. 

The first way is the classical way whereby pesticide resistance is achieved in a selected strain or population (i) by an alteration of the target site (toxicodynamic change), (ii) by an alteration of the effective dose of insecticide that reaches the target (toxicokinetic change) or (iii) by a combination of the two. The resistance phenotypes have long been analyzed according to these useful biochemical and physiological criteria. 

The second way documents at the molecular genetic level the classes of mutations that can account for these phenotypes (Taylor and Feyereisen, 1996; [[http://dx.doi.org/10.1016/j.pestbp.2015.01.004|Feyereisen et al., 2015]]). These **two dimensions of resistance** are illustrated in this table, with checkmarks for cases documented by 2015. For P450-mediated resistance, focus is on toxicokinetics (middle column).

{{ ::blinking_lights.jpg?400 |}} 

Different classes of mutations affecting a P450 gene and thus leading to resistance can be distinguished. They fall broadly in three categories as shown in the first column of the table above. The first are mutations affecting the coding sequence of the gene and thereby structurally alter the gene product. The second are mutations causing an increase in gene dosage or expression. The third are mutations causing a decrease in gene dosage or expression. The latter two classes can be further distinguished in mutations affecting the whole gene (such as duplication and amplification, or disruption and loss) or just the //cis// regulation or the //trans// regulatory elements of the gene.

Examples of all these precise molecular mutations responsible for P450-mediated resistance are known. The //cis//-mutation (Accord element insertion) in the 5‘UTR of the //[[cyp6g1_case_study|Cyp6g1]]// gene in //Drosophila// causing up-regulation of expression ([[http://doi.org/10.1126/science.1074170|Daborn et al., 2002]]) is one of the most detailed account. 

Traditionally, the first line of evidence for a role of a P450 enzyme in resistance has been the use of an insecticide [[P450 inhibition|synergist]] (e.g. piperonyl butoxide, PBO)(Feyereisen, 2015). A suppression or decrease in the level of resistance by treatment with the synergist being diagnostic. In cases too many to list here, this initial and indirect evidence is probably correct, however there are cases where piperonyl butoxide synergism has not been explained by increased detoxification (Kennaugh et al., 1993). Piperonyl butoxide may also be a poor inhibitor of the P450(s) responsible for resistance, or may inhibit some esterase activity so that the use of a second synergist, structurally unrelated to PBO may be warranted (Brown et al., 1996, Zhang et al., 1997). In addition, the synergist as P450 inhibitor can decrease the activation of a proinsecticide, so that lack of resistance suppression can be misleading. An independent and additional line of evidence is the measurement of total P450 levels or metabolism of selected model substrates. An increase in either or both being viewed as diagnostic. Such evidence is indirect, and the absence of change is not informative. 

An increase in the metabolism of the insecticide itself in the resistant strain is more conclusive and has been demonstrated in many studies. For instance, permethrin metabolism to 4'-hydroxypermethrin was higher in a microsomes from //Culex quinquefasciatus// larvae that are highly resistant to permethrin (Kasai et al., 1998b) than in their susceptible counterparts. Total P450 and cytochrome b5 levels were 2.5 times higher in the resistant strain. Both permethrin toxicity and metabolism were inhibited by two unrelated synergists, TCPPE and piperonyl butoxide. A similarly convincing approach was taken to show P450 involvement in the resistance of house fly larvae of the YPPF strain to pyriproxifen. Gut and fat body microsomes were shown to metabolize the IGR to 4'-OH-pyriproxyfen and 5"-OH-pyriproxyfen at higher rates than microsomes of the susceptible strains and this metabolism was synergist-suppressible (Zhang et al., 1998). The major, dominant resistance factor was linked to chromosome 2 in that strain (Zhang et al., 1997). 

Increased levels of transcripts for one or more P450 genes in insecticide resistant strains has now been reported in many cases. These transcriptomic studies suggest that overexpression of one or more P450 genes is a very common phenomenon of metabolic resistance but they do not by themselves establish a causal relationship with resistance. Instead, they merely provide lists of candidate genes. 

Genetic linkage between increased mRNA or protein levels for a particular P450 and resistance has been obtained to the chromosome level or closer to marker genes (Feyereisen, 2005; Bogwitz et al., 2005; Wee et al., 2008; Hardstone et al., 2010). Linkage is just the first step in establishing a causal link between a P450 gene and resistance. QTL mapping of resistance in mosquitoes has also provided short lists of candidate genes for further functional studies.
 
[[in_vitro_activities|Functional expression]] of the P450 enzymes in heterologous systems (in vitro activity of the enzyme) is required to establish the contribution to resistance of a CYP gene. However, while //necessary//, this criterion is not //sufficient//. Indeed, several P450s can contribute to the metabolism of a chemical in susceptible strains, while perhaps just one of them (through expression or sequence change or both) is in fact responsible for resistance.

[[transgenic_expression|Transgenic expression]] in Drosophila or other insects followed by in vivo toxicity assays can provide convincing evidence, but the interpretation of such experiments is not always straightforward. For instance, while Bogwitz et al. (2005) demonstrated that Cyp12a4 overexpression in the midgut and Malpighian tubules confers lufenuron resistance in Drosophila, they also showed that high level ectopic expression of the gene was embryonic lethal (for an unknown reason).

[[null_mutants_or_crispr_cas_knockouts|CRISPR/Cas gene editing]], such as gene knockouts or even knockout of gene clusters is an increasingly accessible and very effective technique to study the contribution of one or more P450 genes to xenobiotic metabolism.

[[effects_of_p450_knockdown_by_rnai|RNAi suppression of CYP gene expression]] is very informative, but this technique is not equally effective in all insect species. Furthermore, the effects observed are often marginal. 

Ultimately, a combination of genetic and biochemical evidence is needed to make a compelling case for the involvement of one or more P450s to resistance.

Pages below under construction. Contributions are welcome [[arthropodP450@gmail.com]]

===== P450 overexpression in cis =====

===== P450 overexpression in trans =====

===== P450 underexpression =====

===== P450 duplications and amplifications =====

===== Structural changes in P450 proteins =====

===== Resistance case studies =====

A brief discussion of //selected cases// of P450 genes associated with insecticide resistance is provided below. 

[[CYP6G1 case study|CYP6G1 in Drosophila]]

[[CYP6A1 case study|CYP6A1 in the house fly]] 

[[CYP6D1 case study|CYP6D1 in the house fly]]