| Abstract |
Tetranychus urticae (Koch) is one of the most destructive agricultural pests. Its control is based mainly on the use of insecticides / acaricides. Due to the extensive use of chemical compounds and the life history traits (short cycle, high proliferation high fecundity and arrhenotokous parthenogenesis) they develop resistance quickly, showing striking phenotypes in some cases. Among them, several studies report resistance to abamectin, METIs and pyrethroids.
Both target site resistance and metabolic resistance have been associated with abamectin, METIs and pyrethroids resistance. Gene expression data on abamectin and METI resistant populations compared to susceptible populations indicated that many detoxification genes and particularly cytochrome P450s are over - expressed in the resistant strains and they are associated with the phenotype.
In this study (Chapter 2) a functional link between cytochrome P450 metabolism and multi-insecticide resistance was investigated by expressing several P450s (CYP392A16, CYP392A11, CYP392A12, CYP392D8 and CYP392D10) in an E. coli model system. I showed that the cytochrome P450 CYP392A16, which is strongly associated with abamectin resistance at the gene expression level, is capable to metabolise abamectin to a less toxic compound (24- or 26- hydroxymethyl metabolite) as confirmed by bioassays with the purified metabolite. An antibody was developed against CYP392A16, and successfully tested on resistant and susceptible spider mite homogenates showing high specificity and sensitivity in detecting the elevated levels of the 55 kDa CYP392A16 protein. Also, CYP392A11, a cytochrome P450 strongly associated with METI resistance at the gene expression level, is capable to metabolise two METI acaricides, fenpyroximate and cyenopyrafen, a novel METI recently introduced in the market, but never used against the mite strains analysed in this study. It was shown that fenpyroximate metabolism produces a non toxic compound (“metabolite M5”), while cyenopyrafen was metabolized to a hydroxylated compound.
In Chapter 3 I employed the GAL4/UAS system for ectopic co-expression of T. urticae cytochrome P450s (CYP392A16 and CYP392A11) and CPR in Drosophila, in order to validate their role in resistance to abamectin and METIs, in vivo. The transgenic lines co-expressing CYP392A16; TuCPR under GAL4 driver were successfully generated, and toxicity bioassays showed that they were resistant to abamectin in comparison to the control line (CYP32A16; TuCPR x w1118). Also, TuCPR; CYP392A11 x GAL4 line was tested against fenpyroximate showing 2.6 folds resistance to the specific insecticide / acaricide compared to the control line (TuCPR; CYP392A11 x w1118).
In the last chapter (Chapter 4), I examined the relative contribution of known avermectin and pyrethroid target-site mutations (G314D and G326E in glutamate gated chloride channels and L1024V, F1538I and A1215D in voltage gated sodium channel) in the resistance phenotype of T. urticae by undertaking a genetic approach. I introduced G314D, G326E on glutamate gated chloride channels and L1024V and F1538I on sodium channel in a susceptible T. urticae genetic background through multiple genetic crosses, in order to obtain homozygous lines that carry these mutations, alone or in combinations. Their contribution to the phenotype of resistance was examined by toxicity assays. It was shown that 314D and 326E alone have minor effect to abamectin and milbemectin resistance. On the other hand, their combination (G314D; G326E) provides higher levels of resistance to abamectin and milbemectin, without reaching the resistance levels of the parental resistant strain though. The results indicate possible involvement of additional mechanisms in the phenotype and cross -resistance to milbemectin. Also, the relative contribution of sodium channel mutations (L1024V and F1538I) in pyrethroid resistance was examined, by conducting toxicity assays with bifenthrin, fluvalinate and fenpropathrin. The results indicate that homozygous lines for either the 1024V or the 1538I show high resistance levels to bifenthrin, fluvalinate and fenpropathrin.
The findings, their impact on insecticide resistance research and Insecticide Resistance Management (IRM) strategies, and some future research directions are discussed in the last session (general discussion).
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