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Browsing by Subject "Resistance mechansims"

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    Characterization of mechanisms of resistance to common insecticides in noctuid pest species and resistance risk assessment for the new lepidopteran specific compound flubendiamide
    (2009) Konanz, Stefanie; Zebitz, Claus P. W.
    Noctuid species, such as the beet armyworm Spodoptera exigua, the cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens, are well-known pests in many agricultural cropping systems worldwide. The extensive and widespread use of insecticides against these species has led to the development of resistance against almost all commercially used compounds. The focus of this thesis was on the novel lepidopteran specific compound flubendiamde, to get a detailed overview about the efficacy using different kinds of bioassays for a resistance risk assessment. On the other hand, the underlying possible resistance mechanisms of a S. exigua strain from southern Spain was investigated on the toxicological, biochemical, pharmacokinetic and molecular level. The basic of all further experiments was two bioassay techniques, a leaf-disc spray application and an artificial diet bioassay, on 2nd instar larvae. Flubendiamide and 11 competitors with different mode of actions were tested on three susceptible laboratory strains (HELI-AR, HELI-VI, SPOD-EX S) and one field strain from Spain (SPOD-EX E-98). The aim was to get baseline susceptibilities of the three noctuid species and additionally the resistance profile of strain SPOD-EX E-98 against the different insecticides. The results of the two bioassay techniques were comparable with each other, emamectin shown the highest efficacy followed by low EC50-values for flubendiamide, deltamethrin and indoxacarb. The robust artificial diet bioassay was used for a worldwide resistance monitoring for flubendiamide, and resulting in a mean efficacy of 93% in 18 test populations. An interesting cross-resistance pattern against insecticides with different mode of actions demonstrated the S. exigua strain SPOD-EX E-98, and can be called as ?multi-resistant?. High resistance to three classes of insecticides, pyrethroids, benzoylphenyl ureas and carbamates/organophosphates, was detected in this strain, also moderate resistance levels to endosulfan and indoxacarb. The in vivo application study with the synergist PBO (monooxygenase inhibitor) has shown an additionally participation of this enzyme system towards the deltamethrin resistance in this strain. Additionally, PBO and two esterase inhibitors had no effect to the resistance level of triflumuron (benzoylphenyl urea) and so target-site resistance is likely in this case. Using molecular biological methods, a point mutation (kdr) in the voltage-gated sodium channel of the resistant strain SPOD-EX E-98 was detected, this channel is the target of the pyrethroids. This target-site resistance is particularly responsible for the extremely high resistance factor (~900) of the pyrethroid deltamethrin detected in the bioassays. In order to investigate the pharmacokinetic profiles of deltamethrin, triflumuron and flubendiamide in S. exigua larvae radiolabelled compounds were used. No differences in penetration or excretion of the compounds were observed between the two strains. This suggests that physiological changes have not influence as a possible mechanism of deltamethrin and triflumuron resistance. Four biochemical markers, carboxylesterases (CEs), cytochrome P450-dependent monooxygenases (monooxygenases), glutathione S-transferases (GSTs) and acetylcholinesterases (AChEs), were investigated in both S. exigua strains. These enzyme systems are known to be linked with metabolic detoxification/resistance to diverse insecticides. With the exception of GST, in the multi-resistant SPOD-EX E-98 strain was found significantly higher enzyme activities to the other three marker enzymes. The higher CE activity in the multi-resistant strain SPOD-EX E-98 was further investigated, using an nPAGE to obtaining the iso-enzyme banding patterns of both strains. In comparison, the strain SPOD-EX E-98 exhibited an additional thick band and it was not possible to inhibiting this band in vitro through the esterase inhibitor DEF. It is possible that this result is involved in resistance, but only further investigations could be clarify the exact role of the additional band in this resistant strain. The AChE of both S. exigua strains was used for in vitro inhibition studies. The resistant strain SPOD-EX E-98 was insensitive against several organophosphates and carbamates compared with strain SPOD-EX S. Only one exception was detected, the AChE of the resistant strain was more sensible to carbofuran than the enzyme from strain SPOD-EX S. This phenomenon of ?negative cross-insensitivity? is generally correlated with an altered substrate binding site of the AChE. Further investigations of kinetic parameters exhibited a higher turnover of the substrate in strain SPOD-EX E-98. The bioassays confirmed these results and so the resistance mechanism of the two chemical classes is an altered AChE in the resistant strain.