Biochemical mechanisms of resistance in Daphnia magna exposed to the insecticide fenitrothion

Resistance to fenitrothion and enzyme activities associated with the toxicity and metabolism of organophosphorus insecticides were measured in three genetically unique Daphnia magna clones collected from rice fields of Delta del Ebro (NE Spain) during the growing season and a lab sensitive clone. The studied clones showed up to sixfold differences in resistance to fenitrothion. The lack of correlation between in vitro sensitivity of acetylcholinesterase (AChE) to fenitrooxon and resistance to fenitrothion indicated that insensitivity of AChE to the most active oxon metabolite was not involved in the observed differences in resistance. Inhibition of mixed- function oxidases (MFOs) by piperonyl butoxide (PBO) increased the tolerance to fenitrothion by almost 20-fold in all clones without altering their relative ranking of resistance. Conversely, when exposed to fenitrooxon, the studied clones showed similar levels of tolerance, thus indicating that clonal differences in the conversion of fenitrothion to fenitrooxon by MFOs were involved in the observed resistance patterns. Despite that resistant clones showed over 1.5 higher activities of carboxilesterase (CbE) than sensitive ones, toxicity tests with 2-(O-cresyl)-4H-1,3,2-benzodioxaphosphorin-2 oxide, which is a specific inhibitor of these enzymes, evidenced that this system only contributed marginally to the observed clonal differences in tolerance. Glutathione-S-transferases activity (GST) varied across clones but not under exposure to fenitrothion, and was only related with tolerance levels in the field clones. In summary, our results indicate that MFO mediated differences on the bio-activation of the phosphorotionate OP pesticide to its active oxon metabolite contributed mostly in explaining the observed moderate levels of resistance, whereas the activities of CbE and GST had only a marginal role.