Incomplete reproductive barriers and genomic differentiation impact the spread of resistance mutations between green- and red-colour morphs of a cosmopolitan mite pest.

Pesticide resistance represents a clear and trackable case of adaptive evolution with a strong societal impact. Understanding the factors associated with the evolution and spread of resistance is imperative to develop sustainable crop management strategies. The two-spotted spider mite Tetranychus urticae, a major crop pest with worldwide distribution and a polyphagous lifestyle, has evolved resistance to most classes of pesticides. Tetranychus urticae exists as either a green- or a red-coloured morph. However, the extent of genetic divergence and reproductive compatibility vary across populations of these colour morphs, complicating their taxonomic resolution at the species level. Here, we studied patterns of genetic differentiation and barriers to gene flow within and between morphs of T. urticae in order to understand the factors that influence the spread of resistance mutations across its populations. We derived multiple iso-female lines from Tetranychus populations collected from agricultural crops. We generated genomic and morphological data, characterized their bacterial communities and performed controlled crosses. Despite morphological similarities, we found large genomic differentiation between the morphs. This pattern was reflected in the incomplete, but strong postzygotic incompatibility in crosses between colour morphs, while crosses within morphs from different geographical locations were largely compatible. In addition, our results suggest recent/on-going gene flow between green-coloured T. urticae and T. turkestani. By screening the sequences of 10 resistance genes, we found evidence for multiple independent origins and for single evolutionary origins of target-site resistance mutations. Our results indicate that target-site mutations mostly evolve independently in populations on different geographical locations, and that these mutations can spread due to incomplete barriers to gene flow within and between populations.

Imperfect diet choice reduces the performance of a predatory mite.

Two mutually unexclusive hypotheses prevail in the theory of nutritional ecology: the balanced diet hypothesis states that consumers feed on different food items because they have complementary nutrient and energy compositions. The toxin-dilution hypothesis poses that consumers feed on different food items to dilute the toxins present in each. Both predict that consumers should not feed on low-quality food when ample high-quality food forming a complete diet is present. We investigated the diet choice of Phytoseiulus persimilis, a predatory mite of web-producing spider mites. It can develop and reproduce on single prey species, for example the spider mite Tetranychus urticae. A closely related prey, T. evansi, is of notorious bad quality for P. persimilis and other predator species. We show that juvenile predators feeding on this prey have low survival and do not develop into adults. Adults stop reproducing and have increased mortality when feeding on it. Feeding on a mixed diet of the two prey decreases predator performance, but short-term effects of feeding on the low-quality prey can be partially reversed by subsequently feeding on the high-quality prey. Yet, predators consume low-quality prey in the presence of high-quality prey, which is in disagreement with both hypotheses. We suggest that it is perhaps not the instantaneous reproduction on single prey or mixtures of prey that matters for the fitness of predators, but that it is the overall reproduction by a female and her offspring on an ephemeral prey patch, which may be increased by including inferior prey in their diet.

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.

Point mutations in the voltage-gated sodium channel gene associated with pyrethroid resistance in Iranian populations of the European red mite Panonychus ulmi.

The European red mite Panonychus ulmi (Koch) is a major pest of apple trees worldwide and causes significant damage to apple orchards in Iran. Pyrethroid insecticides/acaricides, such as fenpropathrin and fenvalerate, are widely used to control P. ulmi, but their long-term use may lead to low efficacy. Earlier studies investigating pyrethroid resistance in closely related mites such as Tetranychus urticae revealed that pyrethroid resistance was associated with point mutations in the voltage-gated sodium channel gene (vgsc). The aim of this study was to investigate the biochemical and molecular mechanisms of fenpropathrin and fenvalerate resistance in Iranian populations of P. ulmi. Pyrethroid toxicity bioassays were carried out on different P. ulmi field populations. Marand (resistance ratio, RR = 149), Maraqeh (RR = 90) and Mianeh2 (RR = 71) populations exhibited high levels of resistance to fenpropathrin, compared to a susceptible field population (Shahin Dej). Resistance was also observed for fenvalerate with resistance ratio's ranging from 2- to 20-fold. Synergism experiments and enzyme activity assays predicted a minor role for classical detoxification enzymes. In contrast, two amino acid substitutions in the VGSC, L1024V and F1538I, that were previously shown to confer pyrethroid resistance, were detected in all three resistant P. ulmi populations and point towards target-site insensitivity as the most likely resistance mechanism. Furthermore, sequencing after cloning of vgsc fragments from single haploid males revealed the presence of multiple copies of vgsc in a highly resistant strain. The link between resistance mutations and vgsc copy number variation should be the subject of future study, as this might be used to develop molecular markers for monitoring pyrethroid resistance of P. ulmi in the field.

Comparative genome-wide transcriptome analysis of Vitis vinifera responses to adapted and non-adapted strains of two-spotted spider mite, Tetranyhus urticae.

BACKGROUND: The two-spotted spider mite, Tetranychus urticae, is an extreme generalist plant pest. Even though mites can feed on many plant species, local mite populations form host races that do not perform equally well on all potential hosts. An acquisition of the ability to evade plant defenses is fundamental for mite's ability to use a particular plant as a host. Thus, understanding the interactions between the plant and mites with different host adaptation status allows the identification of functional plant defenses and ways mites can evolve to avoid them. RESULTS: The grapevine genome-wide transcriptional responses to spider mite strains that are non-adapted and adapted to grapevine as a host were examined. Comparative transcriptome analysis of grapevine responses to these mite strains identified the existence of weak responses induced by the feeding of the non-adapted strain. In contrast, strong but ineffective induced defenses were triggered upon feeding of the adapted strain. A comparative meta-analysis of Arabidopsis, tomato and grapevine responses to mite feeding identified a core of 36 highly conserved genes involved in the perception, regulation and metabolism that were commonly induced in all three species by mite herbivory. CONCLUSIONS: This study describes the genome-wide grapevine transcriptional responses to herbivory of mite strains that differ in their ability to use grapevine as a host. It raises hypotheses whose testing will lead to our understanding of grapevine defenses and mite adaptations to them.

Transcriptome profiling of a spirodiclofen susceptible and resistant strain of the European red mite Panonychus ulmi using strand-specific RNA-seq.

BACKGROUND: The European red mite, Panonychus ulmi, is among the most important mite pests in fruit orchards, where it is controlled primarily by acaricide application. However, the species rapidly develops pesticide resistance, and the elucidation of resistance mechanisms for P. ulmi has not kept pace with insects or with the closely related spider mite Tetranychus urticae. The main reason for this lack of knowledge has been the absence of genomic resources needed to investigate the molecular biology of resistance mechanisms. RESULTS: Here, we provide a comprehensive strand-specific RNA-seq based transcriptome resource for P. ulmi derived from strains susceptible and resistant to the widely used acaricide spirodiclofen. From a de novo assembly of the P. ulmi transcriptome, we manually annotated detoxification enzyme families, target-sites of commonly used acaricides, and horizontally transferred genes implicated in plant-mite interactions and pesticide resistance. In a comparative analysis that incorporated sequences available for Panonychus citri, T. urticae, and insects, we identified radiations for detoxification gene families following the divergence of Panonychus and Tetranychus genera. Finally, we used the replicated RNA-seq data from the spirodiclofen susceptible and resistant strains to describe gene expression changes associated with resistance. A cytochrome P450 monooxygenase, as well as multiple carboxylcholinesterases, were differentially expressed between the susceptible and resistant strains, and provide a molecular entry point for understanding resistance to spirodiclofen, widely used to control P. ulmi populations. CONCLUSIONS: The new genomic resources and data that we present in this study for P. ulmi will substantially facilitate molecular studies of underlying mechanisms involved in acaricide resistance.

Adaptation of an arthropod predator to a challenging environment is associated with a loss of a genome-wide plastic transcriptional response.

BACKGROUND: Structural and chemical plant defence traits may reduce the efficacy of biological control agents in integrated pest management. Breeding programmes have shown arthropod predators' potential to acclimate to challenging host plants. However, whether and how these predators adapt to novel plant environments remain unclear. Using the predatory mite Phytoseiulus persimilis - herbivorous mite Tetranychus urticae system in an experimental evolution setup, we studied the adaptation mechanisms to tomato and cucumber, plants that possess a distinct repertoire of defensive traits. RESULTS: Experimental evolution experiments on whole plants revealed that allowing P. persimilis to adapt to tomatoes led to an ~100% larger population size. Independent feeding assays showed that tomato- and cucumber-adapted prey reduced predator fecundity. The deleterious effect of ingesting low-quality prey persisted after adaptation of the predator to both cucumber and tomato. We demonstrated that jasmonic acid (JA)-dependent defences reduce prey quality by evaluating predator performance on prey fed on JA defence-deficient tomato plants. Transcriptomic profiling of the replicated P. persimilis lines showed that long-term propagation on tomato and cucumber plants produces distinctive gene-expression levels. Predator adaptation to tomatoes results in the loss of a large transcriptional response, in which predicted cuticle-building rather than detoxification pathways are affected. CONCLUSION: We showed that the adaptation of predatory arthropods to a novel, challenging plant does not necessarily occur via the prey, but rather through the physical environment of the plant. We provided first insights into the underlying molecular mechanisms. © 2023 Society of Chemical Industry.

Selectivity and molecular stress responses to classical and botanical acaricides in the predatory mite Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae).

BACKGROUND: Acaricide application remains an integral component of integrated pest management (IPM) for the two-spotted spider mite Tetranychus urticae. Species and strains of phytoseiid predatory mites vary significantly in their response to acaricides. For the success of IPM, it is imperative to identify the determinants of selectivity and molecular stress responses of acaricides in predatory mites. RESULTS: The three classical acaricides bifenazate, cyflumetofen, and fenbutatin oxide did not affect the survival and fecundity of Phytoseiulus persimilis regardless of the route of exposure. Selectivity of the orange oil and terpenoid blend-based botanical acaricides was low via a combination of direct exposure, acaricide-laced diet, and residual exposure but improved when limiting exposure only to diet. To gain insights into the molecular stress responses, the transcriptome of P. persimilis was assembled. Subsequent gene expression analysis of predatory mites orally exposed to fenbutatin oxide and orange oil yielded only a limited xenobiotic stress response. In contrast, P. persimilis exhibited target-site resistance mutations, including I260M in SdhB, I1017M in CHS1, and kdr and super-kdr in VGSC. Extending the screen using available Phytoseiidae sequences uncovered I136T, S141F in cytb, G119S in AChE, and A2083V in ACC, well-known target-sites of acaricides. CONCLUSION: Selectivity of the tested botanical acaricides to P. persimilis was low but could be enhanced by restricting exposure to a single route. Differential gene expression analysis did not show a robust induced stress response after sublethal exposure. In contrast, this study uncovered target-site mutations that may help to explain the physiological selectivity of several classical acaricides to phytoseiid predators.

High-resolution QTL mapping in Tetranychus urticae reveals acaricide-specific responses and common target-site resistance after selection by different METI-I acaricides.

Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance. To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a non-canonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific.

Long-Term Population Studies Uncover the Genome Structure and Genetic Basis of Xenobiotic and Host Plant Adaptation in the Herbivore Tetranychus urticae.

Pesticide resistance arises rapidly in arthropod herbivores, as can host plant adaptation, and both are significant problems in agriculture. These traits have been challenging to study as both are often polygenic and many arthropods are genetically intractable. Here, we examined the genetic architecture of pesticide resistance and host plant adaptation in the two-spotted spider mite, Tetranychus urticae, a global agricultural pest. We show that the short generation time and high fecundity of T. urticae can be readily exploited in experimental evolution designs for high-resolution mapping of quantitative traits. As revealed by selection with spirodiclofen, an acetyl-CoA carboxylase inhibitor, in populations from a cross between a spirodiclofen-resistant and a spirodiclofen-susceptible strain, and which also differed in performance on tomato, we found that a limited number of loci could explain quantitative resistance to this compound. These were resolved to narrow genomic intervals, suggesting specific candidate genes, including acetyl-CoA carboxylase itself, clustered and copy variable cytochrome P450 genes, and NADPH cytochrome P450 reductase, which encodes a redox partner for cytochrome P450s. For performance on tomato, candidate genomic regions for response to selection were distinct from those responding to the synthetic compound and were consistent with a more polygenic architecture. In accomplishing this work, we exploited the continuous nature of allele frequency changes across experimental populations to resolve the existing fragmented T. urticae draft genome to pseudochromosomes. This improved assembly was indispensable for our analyses, as it will be for future research with this model herbivore that is exceptionally amenable to genetic studies.

Fitness costs of key point mutations that underlie acaricide target-site resistance in the two-spotted spider mite Tetranychus urticae.

The frequency of insecticide/acaricide target-site resistance is increasing in arthropod pest populations and is typically underpinned by single point mutations that affect the binding strength between the insecticide/acaricide and its target-site. Theory predicts that although resistance mutations clearly have advantageous effects under the selection pressure of the insecticide/acaricide, they might convey negative pleiotropic effects on other aspects of fitness. If such fitness costs are in place, target-site resistance is thus likely to disappear in the absence of insecticide/acaricide treatment, a process that would counteract the spread of resistance in agricultural crops. Hence, there is a great need to reliably quantify the various potential pleiotropic effects of target-site resistance point mutations on arthropod fitness. Here, we used near-isogenic lines of the spider mite pest Tetranychus urticae that carry well-characterized acaricide target-site resistance mutations to quantify potential fitness costs. Specifically, we analyzed P262T in the mitochondrial cytochrome b, the combined G314D and G326E substitutions in the glutamate-gated chloride channels, L1024V in the voltage-gated sodium channel, and I1017F in chitin synthase 1. Five fertility life table parameters and nine single-generation life-history traits were quantified and compared across a total of 15 mite lines. In addition, we monitored the temporal resistance level dynamics of populations with different starting frequency levels of the chitin synthase resistant allele to further support our findings. Three target-site resistance mutations, I1017F and the co-occurring G314D and G326E mutations, were shown to significantly and consistently alter certain fitness parameters in T. urticae. The other two mutations (P262T and L1024V) did not result in any consistent change in a fitness parameter analyzed in our study. Our findings are discussed in the context of the global spread of T. urticae pesticide resistance and integrated pest management.

The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae.

The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

A mutation in the PSST homologue of complex I (NADH:ubiquinone oxidoreductase) from Tetranychus urticae is associated with resistance to METI acaricides.

The acaricidal compounds pyridaben, tebufenpyrad and fenpyroximate are frequently used in the control of phytophagous mites such as Tetranychus urticae, and are referred to as Mitochondrial Electron Transport Inhibitors, acting at the quinone binding pocket of complex I (METI-I acaricides). Because of their very frequent use, resistance evolved fast more than 20 years ago, and is currently wide-spread. Increased activity of P450 monooxygenases has been often associated with resistance, but target-site based resistance mechanisms were never reported. Here, we report the discovery of a mutation (H92R) in the PSST homologue of complex I in METI-I resistant T. urticae strains. The position of the mutation was studied using the high-resolution crystal structure of Thermus thermophilus, and was located in a stretch of amino acids previously photo-affinity labeled by fenpyroximate. Selection experiments with a strain segregating for the mutant allele, together with marker-assisted back-crossing of the mutation in a susceptible background, confirmed the involvement of the mutation in METI-I resistance. Additionally, an independent genetic mapping approach; QTL analysis identified the genomic region of pyridaben resistance, which included the PSST gene. Last, we used CRISPR-Cas9 genome editing tools to introduce the mutation in the Drosophila PSST homologue.

Molecular analysis of cyenopyrafen resistance in the two-spotted spider mite Tetranychus urticae.

BACKGROUND: Cyenopyrafen is a recently developed acaricide with a new mode of action as a complex II inhibitor. However, it was recently shown that cross-resistance to cyenopyrafen can occur in resistant field strains of Tetranychus urticae, which might be linked to the previous use of classical METI acaricides. Here, we selected for cyenopyrafen resistance and studied the molecular mechanisms that underlie resistance. RESULTS: Selection for cyenopyrafen resistance confers cross-resistance to the complex II inhibitor cyflumetofen, but also to pyridaben, a frequently used complex I inhibitor. Cyenopyrafen resistance is highly synergised by piperonyl butoxide, and a 15-fold higher P450 activity was detected in the resistant strain. Target-site resistance was not detected. Genome-wide gene expression data, followed by a meta-analysis of previously obtained gene expression data, revealed the overexpression specifically of CYP392A11 and CYP392A12. CONCLUSIONS: Cyenopyrafen resistance is strongly linked to the overexpression of two P450s, which probably explains the observed cross-resistance. This information is highly valuable, as the novel complex II inhibitors cyenopyrafen and cyflumetofen are in the process of worldwide registration. The role of both CYP392A11 and CYP392A12 should be further supported by functional expression, but they are very promising candidates as molecular diagnostic markers for monitoring cyenopyrafen susceptibility in the field.

Complex Evolutionary Dynamics of Massively Expanded Chemosensory Receptor Families in an Extreme Generalist Chelicerate Herbivore.

While mechanisms to detoxify plant produced, anti-herbivore compounds have been associated with plant host use by herbivores, less is known about the role of chemosensory perception in their life histories. This is especially true for generalists, including chelicerate herbivores that evolved herbivory independently from the more studied insect lineages. To shed light on chemosensory perception in a generalist herbivore, we characterized the chemosensory receptors (CRs) of the chelicerate two-spotted spider mite, Tetranychus urticae, an extreme generalist. Strikingly, T. urticae has more CRs than reported in any other arthropod to date. Including pseudogenes, 689 gustatory receptors were identified, as were 136 degenerin/Epithelial Na+ Channels (ENaCs) that have also been implicated as CRs in insects. The genomic distribution of T. urticae gustatory receptors indicates recurring bursts of lineage-specific proliferations, with the extent of receptor clusters reminiscent of those observed in the CR-rich genomes of vertebrates or C. elegans Although pseudogenization of many gustatory receptors within clusters suggests relaxed selection, a subset of receptors is expressed. Consistent with functions as CRs, the genomic distribution and expression of ENaCs in lineage-specific T. urticae expansions mirrors that observed for gustatory receptors. The expansion of ENaCs in T. urticae to > 3-fold that reported in other animals was unexpected, raising the possibility that ENaCs in T. urticae have been co-opted to fulfill a major role performed by unrelated CRs in other animals. More broadly, our findings suggest an elaborate role for chemosensory perception in generalist herbivores that are of key ecological and agricultural importance.