Browsing by Subject "Insektizidresistenz"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Publication
    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.
  • Thumbnail Image
    Publication
    Monitoring, mechanisms and management of insecticide resistance and insecticide mode of action in coleopteran pests of winter oilseed rape with special reference to neonicotinoid insecticides under laboratory and applied aspects
    (2014) Zimmer, Christoph Thomas; Zebitz, Claus P. W.
    Winter oilseed rape, Brassica napus L., has become a vital part of cereal-based crop rotations in Europe. It is attacked by numerous insect pests and their control relies on the intensive use of insecticides (compared to other broad acre crops). The exclusive and continuous use of pyrethroid insecticides for almost twenty years led to an enormous selection pressure and facilitated the development of resistance in oilseed rape pests in Europe. Unsurprising three out of the five major pests of the order Coleoptera are reported to be pyrethroid resistant at present: the pollen beetle, Meligethes aeneus F.; the cabbage stem flea beetle, Psylliodes chrysocephala L. and the cabbage seed weevil, Ceutorhynchus assimilis PAYK.. An adult vial bioassay, which is based on insecticide coated glass vials, was used to monitor the spread and strength of pyrethroid resistance and to determine cross-resistance pattern in pollen beetle and cabbage stem flea beetle. Furthermore, baseline susceptibility towards lambda-cyhalothrin (a widely used pyrethroid) was also established for the cabbage seed weevil. The vial bioassay methodology was adapted to thiacloprid, a neonicotinoid insecticide, to determine baseline susceptibility and to provide a methodology to allow long-term susceptibility monitoring of pollen beetle and cabbage seed weevil. Thiacloprid monitoring revealed that pollen beetle and cabbage seed weevil populations collected across Europe in 2009-2012 and 2012 respectively were highly susceptible to this insecticide class. Metabolism studies using native microsomal preparations as the enzyme source and deltamethrin as substrate revealed metabolism of deltamethrin with 4-OH-deltamethrin being the major metabolite. Metabolite formation in vitro was correlated with the observed pyrethroid resistance level in vivo and was suppressible by PBO. A degenerate PCR approach was used to identify partial P450 gene sequences from pollen beetle. qRT-PCR screening covering a range of pollen beetle populations differing in levels of pyrethroid resistance identified a single P450, CYP6BQ23, as significantly and highly overexpressed (up to ~900-fold) in resistant strains compared to susceptible strains. The expression of CYP6BQ23 was significantly correlated with both the level of resistance and with the rate of deltamethrin metabolism in microsomal preparations of these populations. Recombinant expression of this P450 in an insect cell line demonstrated that it is capable of hydroxylating deltamethrin and tau-fluvalinate. The turnover of these pyrethroids by CYP6BQ23 is in line with the observed moderate cross-resistant phenotype. Molecular modeling suggested a better fit of deltamethrin into the active site of CYP6BQ23 compared to tau-fluvalinate also supporting the biochemical results. The occurrence of target-site resistance was investigated by single nucleotide polymorphism (SNP) analysis of the para-locus encoding the voltage-gated sodium channel (VGSC) in insects. To achieve this goal a partial fragment (domain IIS4-6) encoding an important region of the pyrethroid binding site was PCR amplified and screened for non-synonymous SNPs. One SNP was identified causing a leucine to phenylalanine substitution at amino acid residue number 1014 (Musca domestica L. numbering), well known as knock down resistance (kdr) conferring an absolute cross-resistance to pyrethroids and DDT in various insect species. Sequencing of the very same gene region in the cabbage stem flea beetle also revealed the presence of the L1014F kdr mutation in pyrethroid resistant flea beetle populations, thus explaining the strong cross-resistance pattern observed in vitro. Most mechanistic studies of resistance have focused on elucidating the contribution of particular genes/gene families to pyrethroid resistance. To generate a comprehensive sequence resource and to elucidate global changes in gene regulation related to insecticide resistance in pollen beetle a de novo transcriptome was assembled from sequence pools generated by next-generation sequencing. RNA-sequencing of three pyrethroid resistant and one highly susceptible reference population allowed a global gene expression analysis by short read mapping against the generated transcriptome, as well as a SNP analysis. The implications of these results for resistance management in coleopteran pests in winter oilseed rape and opportunities for future work are discussed.