The complex II resistance mutation H258Y in succinate dehydrogenase subunit B causes fitness penalties associated with mitochondrial respiratory deficiency.

BACKGROUND: The acaricides cyflumetofen, cyenopyrafen and pyflubumide inhibit the mitochondrial electron transport chain at complex II [succinate dehydrogenase (SDH) complex]. A target site mutation H258Y was recently discovered in a resistant strain of the spider mite pest Tetranychus urticae. H258Y causes strong cross-resistance between cyenopyrafen and pyflubumide, but not cyflumetofen. In fungal pests, fitness costs associated with substitutions at the corresponding H258 position that confer resistance to fungicidal SDH inhibitors have not been uncovered. Here, we used H258 and Y258 near-isogenic lines of T. urticae to quantify potential pleiotropic fitness effects on mite physiology. RESULTS: The H258Y mutation was not associated with consistent significant changes of single generation life history traits and fertility life table parameters. In contrast, proportional Sanger sequencing and droplet digital polymerase chain reaction showed that the frequency of the resistant Y258 allele decreased when replicated 50:50 Y258:H258 experimentally evolving populations were maintained in an acaricide-free environment for approximately 12 generations. Using in vitro assays with mitochondrial extracts from resistant (Y258) and susceptible (H258) lines, we identified a significantly reduced SDH activity (48% lower activity) and a slightly enhanced combined complex I and III activity (18% higher activity) in the Y258 lines. CONCLUSION: Our findings suggest that the H258Y mutation is associated with a high fitness cost in the spider mite T. urticae. Importantly, while it is the most common approach, it is clear that only comparing life history traits and life table fecundity does not allow to reliably estimate fitness costs of target site mutations in natural pest populations. © 2023 Society of Chemical Industry.

Long-term survey and characterization of cyflumetofen resistance in Tetranychus urticae populations from Turkey.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is the most economically important mite pest in agricultural areas and chemical acaricides are widely used to control T. urticae populations. Cyflumetofen is a recently introduced acaricide that inhibits the mitochondrial electron transport chain at complex II (succinate dehydrogenase, SDH), which represents the most recently developed mode of action for mite control worldwide. In the present study, started upon the launch of cyflumetofen in Turkey, a five-year survey was performed to monitor cyflumetofen susceptibility in 28 T. urticae populations collected from agricultural fields across the country. The first resistance case that might cause control failure in practical field conditions was uncovered in 2019, three years after the registration of cyflumetofen. In addition, an extremely resistant population (1722-fold resistance) was also detected towards the end of 2019. Cyflumetofen resistance did not decrease in the laboratory after relaxation of selection pressure for over one year in field-collected populations, suggesting the absence of a fitness cost associated with resistance in these populations. Next to phenotypic resistance, metabolic and physiological mechanisms underlying the decreased susceptibility were also investigated. Synergism assays showed the involvement of P450 monooxygenases in cyflumetofen resistance. Downregulation of carboxylesterases as resistance mechanism, is underpinned by the fact that pre-treatment with esterase inhibitor DEF decreased cyflumetofen toxicity in field-collected strains. Furthermore, a novel H258L substitution in the subunit B of complex II was uncovered in a field population. In silico modeling of the new mutation suggested that the mutation might indeed influence toxicity to complex II inhibitors cyenopyrafen and pyflubumide, but most likely not cyflumetofen. However, further studies are needed to uncover the exact role of this mutation in resistance to this new class of complex II inhibitors.

A H258Y mutation in subunit B of the succinate dehydrogenase complex of the spider mite Tetranychus urticae confers resistance to cyenopyrafen and pyflubumide, but likely reinforces cyflumetofen binding and toxicity.

Succinate dehydrogenase (SDH) inhibitors such as cyflumetofen, cyenopyrafen and pyflubumide, are selective acaricides that control plant-feeding spider mite pests. Resistance development to SDH inhibitors has been investigated in a limited number of populations of the spider mite Tetranychus urticae and is associated with cytochrome P450 based detoxification and target-site mutations such as I260 T/V in subunit B and S56L in subunit C of SDH. Here, we report the discovery of a H258Y substitution in subunit B of SDH in a highly pyflubumide resistant population of T. urticae. As this highly conserved residue corresponds to one of the ubiquinone binding residues in fungi and bacteria, we hypothesized that H258Y could have a strong impact on SDH inhibitors toxicity. Marker assisted introgression and toxicity bioassays revealed that H258Y caused high cross resistance between cyenopyrafen and pyflubumide, but increased cyflumetofen toxicity. Resistance associated with H258Y was determined as dominant for cyenopyrafen, but recessive for pyflubumide. In vitro SDH assays with extracted H258 mitochondria showed that cyenopyrafen and the active metabolites of pyflubumide and cyflumetofen, interacted strongly with complex II. However, a clear shift in IC50s was observed for cyenopyrafen and the metabolite of pyflubumide when Y258 mitochondria were investigated. In contrast, the mutation slightly increased affinity of the cyflumetofen metabolite, likely explaining its increased toxicity for the mite lines carrying the substitution. Homology modeling and ligand docking further revealed that, although the three acaricides share a common binding motif in the Q-site of SDH, H258Y eliminated an important hydrogen bond required for cyenopyrafen and pyflubumide binding. In addition, the hydrogen bond between cyenopyrafen and Y117 in subunit D was also lost upon mutation. In contrast, cyflumetofen affinity was enhanced due to an additional hydrogen bond to W215 and hydrophobic interactions with the introduced Y258 in subunit B. Altogether, our findings not only highlight the importance of the highly conserved histidine residue in the binding of SDH inhibitors, but also reveal that a resistance mutation can provide both positive and negative cross-resistance within the same acaricide mode of action group.

QTL mapping suggests that both cytochrome P450-mediated detoxification and target-site resistance are involved in fenbutatin oxide resistance in Tetranychus urticae.

The organotin acaricide fenbutatin oxide (FBO) - an inhibitor of mitochondrial ATP-synthase - has been one of the most extensively used acaricides for the control of spider mites, and is still in use today. Resistance against FBO has evolved in many regions around the world but only few studies have investigated the molecular and genetic mechanisms of resistance to organotin acaricides. Here, we found that FBO resistance is polygenic in two genetically distant, highly resistant strains of the spider mite Tetranychus urticae, MAR-AB and MR-VL. To identify the loci underlying FBO resistance, two independent bulked segregant analysis (BSA) based QTL mapping experiments, BSA MAR-AB and BSA MR-VL, were performed. Two QTLs on chromosome 1 were associated with FBO resistance in each mapping experiment. At the second QTL of BSA MAR-AB, several cytochrome P450 monooxygenase (CYP) genes were located, including CYP392E4, CYP392E6 and CYP392E11, the latter being overexpressed in MAR-AB. Synergism tests further implied a role for CYPs in FBO resistance. Subunit c of mitochondrial ATP-synthase was located near the first QTL of both mapping experiments and harbored a unique V89A mutation enriched in the resistant parents and selected BSA populations. Marker-assisted introgression into a susceptible strain demonstrated a moderate but significant effect of the V89A mutation on toxicity of organotin acaricides. The impact of the mutation on organotin inhibition of ATP synthase was also functionally confirmed by ATPase assays on mitochondrial preparations. To conclude, our findings suggest that FBO resistance in the spider mite T. urticae is a complex interplay between CYP-mediated detoxification and target-site resistance.

Molecular and genetic analysis of resistance to METI-I acaricides in Iranian populations of the citrus red mite Panonychus citri.

The citrus red mite, Panonychus citri, is a major pest on citrus all around the world. Mitochondrial Electron Transport Inhibitors of complex I (METI-I) acaricides such as fenpyroximate have been used extensively to control P. citri populations, which resulted in multiple reports of METI-I resistant populations in the field. In this study, biochemical and molecular mechanisms of fenpyroximate resistance were investigated in P. citri. Seven populations were collected from Northern provinces of Iran. Resistance ratios were determined and reached up to 75-fold in comparison to a fenpyroximate susceptible population. Cross-resistance to two additional METI-I acaricides, pyridaben and tebufenpyrad, was detected. PBO synergism experiments, in vivo enzyme assays and gene expression analysis suggest a minor involvement of cytochrome P450 monooxygenases in fenpyroximate resistance, which is in contrast with many reported cases for the closely related Tetranychus urticae. Next, we determined the frequency of a well-known mutation in the target-site of METI-Is, the PSST subunit, associated with METI-I resistance. Indeed, the H92R substitution was detected in a highly fenpyroximate resistant P. citri population. Additionally, a new amino acid substitution at a conserved site in the PSST subunit was detected, A94V, with higher allele frequencies in a moderately resistant population. Marker-assisted back-crossing in a susceptible background confirmed the potential involvement of the newly discovered A94V mutation in fenpyroximate resistance. However, introduction of the A94V mutation in the PSST homologue of D. melanogaster using CRISPR-Cas9 did not result in fenpyroximate resistant flies. In addition, differences in binding curves between METI-Is and complex I measured directly, in isolated transgenic and wildtype mitochondria preparations, could not be found.