Automated imaging coupled with AI-powered analysis accelerates the assessment of plant resistance to Tetranychus urticae.

The two-spotted spider mite (TSSM), Tetranychus urticae, is among the most destructive piercing-sucking herbivores, infesting more than 1100 plant species, including numerous greenhouse and open-field crops of significant economic importance. Its prolific fecundity and short life cycle contribute to the development of resistance to pesticides. However, effective resistance loci in plants are still unknown. To advance research on plant-mite interactions and identify genes contributing to plant immunity against TSSM, efficient methods are required to screen large, genetically diverse populations. In this study, we propose an analytical pipeline utilizing high-resolution imaging of infested leaves and an artificial intelligence-based computer program, MITESPOTTER, for the precise analysis of plant susceptibility. Our system accurately identifies and quantifies eggs, feces and damaged areas on leaves without expert intervention. Evaluation of 14 TSSM-infested Arabidopsis thaliana ecotypes originating from diverse global locations revealed significant variations in symptom quantity and distribution across leaf surfaces. This analytical pipeline can be adapted to various pest and host species, facilitating diverse experiments with large specimen numbers, including screening mutagenized plant populations or phenotyping polymorphic plant populations for genetic association studies. We anticipate that such methods will expedite the identification of loci crucial for breeding TSSM-resistant plants.

A chromosome-level genome assembly of the spider mite Tetranychus piercei McGregor.

Despite the rapid advances in sequencing technology, limited genomic resources are currently available for phytophagous spider mites, which include many important agricultural pests. One of these pests is Tetranychus piercei (McGregor), a serious banana pest in East Asia exhibiting remarkable tolerance to high temperature. In this study, we assembled a high-quality genome of T. piercei using a combination of PacBio long reads and Illumina short reads sequencing. With the assistance of chromatin conformation capture technology, 99.9% of the contigs were anchored into three pseudochromosomes with a total size of 86.02 Mb. Repetitive elements, accounting for 14.16% of this genome (12.20 Mb), are predominantly composed of long-terminal repeats (30.7%). By combining evidence of ab initio prediction, transcripts, and homologous proteins, we annotated 11,881 protein-coding genes. Both the genome and proteins have high BUSCO completeness scores (>94%). This high-quality genome, along with reliable annotation, provides a valuable resource for investigating the high-temperature tolerance of this species and exploring the genomic basis that underlies the host range evolution of spider mites.

Identification and Functional Characterization of an Omega-Class Glutathione S-Transferase Gene PcGSTO1 Associated with Cyetpyrafen Resistance in Panonychus citri (McGregor).

Cyetpyrafen is a recently developed acaricide. The citrus red mite, Panonychus citri (McGregor), has developed significant resistance to cyetpyrafen. However, the molecular mechanism underlying the cyetpyrafen resistance in P. citri remains unclear. Glutathione S-transferases (GSTs) play a critical role in arthropod pesticide resistance. This study showed that GSTs were potentially related to the resistance of P. citri to cyetpyrafen through synergistic experiments and enzyme activity analysis. An omega-family GST gene, PcGSTO1, was significantly up-regulated in the egg, nymph, and adult stages of the cyetpyrafen-resistant strain. Additionally, silencing of PcGSTO1 significantly increased the mortality of P. citri to cyetpyrafen and recombinant PcGSTO1 demonstrated the ability to metabolize cyetpyrafen. Our results indicated that the overexpression of PcGSTO1 is associated with cyetpyrafen resistance in P. citri, and they also provided valuable information for managing resistance in P. citri.

A multiplex direct PCR method for the rapid and accurate discrimination of three species of spider mites (Acari: Tetranychidae) in fruit orchards in Beijing.

Spider mites (Acari: Tetranychidae) are polyphagous pests of economic importance in agriculture, among which the two-spotted spider mite Tetranychus urticae Koch has spread widely worldwide as an invasive species, posing a serious threat to fruit tree production in China, including Beijing. The hawthorn spider mite, Amphitetranychus viennensis Zacher, is also a worldwide pest of fruit trees and woody ornamental plants. The cassava mite, Tetranychus truncatus Ehara, is mainly found in Asian countries, including China, Korea and Japan, and mainly affects fruit trees and agricultural crops. These three species of spider mites are widespread and serious fruit tree pests in Beijing. Rapid and accurate identification of spider mites is essential for effective pest and plant quarantine in Beijing orchard fields. The identification of spider mite species is difficult due to their limited morphological characteristics. Although the identification of insect and mite species based on PCR and real-time polymerase chain reaction TaqMan is becoming increasingly common, DNA extraction is difficult, expensive and time-consuming due to the minute size of spider mites. Therefore, the objective of this study was to establish a direct multiplex PCR method for the simultaneous identification of three common species of spider mites in orchards, A. viennensis, T. truncatus and T. urticae, to provide technical support for the differentiation of spider mite species and phytosanitary measures in orchards in Beijing. Based on the mitochondrial cytochrome c oxidase subunit I (COI) of the two-spotted spider mite and the cassava mite and the 18S gene sequence of the hawthorn spider mite as the amplification target, three pairs of specific primers were designed, and the primer concentrations were optimized to establish a direct multiplex PCR system for the rapid and accurate discrimination of the three spider mites without the need for DNA extraction and purification. The method showed a high sensitivity of 0.047 ng for T. truncatus and T. urticae DNA and 0.0002 ng for A. viennensis. This method eliminates the DNA extraction and sequencing procedures of spider mite samples, offers a possibility for rapid monitoring of multiple spider mites in an integrated microarray laboratory system, reducing the time and cost of leaf mite identification and quarantine monitoring in the field.

SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods.

The genome editing technique CRISPR/Cas9 has led to major advancements in many research fields and this state-of-the-art tool has proven its use in genetic studies for various arthropods. However, most transformation protocols rely on microinjection of CRISPR/Cas9 components into embryos, a method which is challenging for many species. Alternatively, injections can be performed on adult females, but transformation efficiencies can be very low as was shown for the two-spotted spider mite, Tetranychus urticae, a minute but important chelicerate pest on many crops. In this study, we explored different CRISPR/Cas9 formulations to optimize a maternal injection protocol for T. urticae. We observed a strong synergy between branched amphipathic peptide capsules and saponins, resulting in a significant increase of CRISPR/Cas9 knock-out efficiency, exceeding 20%. This CRISPR/Cas9 formulation, termed SYNCAS, was used to knock-out different T. urticae genes - phytoene desaturase, CYP384A1 and Antennapedia - but also allowed to develop a co-CRISPR strategy and facilitated the generation of T. urticae knock-in mutants. In addition, SYNCAS was successfully applied to knock-out white and white-like genes in the western flower thrips, Frankliniella occidentalis. The SYNCAS method allows routine genome editing in these species and can be a game changer for genetic research in other hard to transform arthropods.

Knockdown of the ABCG23 Gene Disrupts the Development and Lipid Accumulation of Panonychus citri (Acari/Tetranychidae).

Panonychus citri is a worldwide citrus pest that is currently controlled through the use of insecticides. However, alternative strategies are required to manage P. citri. Recent studies suggest that the ATP-binding cassette (ABC) transporter G subfamily plays a crucial role in transporting cuticular lipids, which are essential for the insect's barrier function against microbial penetration. Therefore, investigating the potential of the ABC transporter G subfamily as a control measure for P. citri could be a promising approach. Based on the genome database, the gene was cloned, and the transcriptional response of ABCG23 for the different developmental stages of P. citri and under spirobudiclofen stress was investigated. Our results showed that the expression level of ABCG23 was significantly lower in adult females exposed to treatment compared to the control and was higher in females than males. The knockdown of ABCG23 using RNAi led to a decrease in the survival rate, fecundity, and TG contents of P. citri. Additionally, a lethal phenotype was characterized by body wrinkling and darkening. These results indicate that ABCG23 may be involved in cuticular lipid transportation and have adverse effects on the development and reproduction of P. citri, providing insight into the discovery of new targets for pest management based on the insect cuticle's penetration barrier function.

Fatty Acid Elongase Gene PcELO7 is Essential for Lipid Accumulation and Fecundity of Panonychus citri (Acari: Tetranychidae).

RNA interference (RNAi) has been proposed as a promising strategy for sustainable and ecofriendly pest control. The insect cuticle lipids were deposited on the body surface and functioned as a defense against chemical xenobiotics. They consisted of aliphatic compounds, including free fatty acids (FFAs). However, elongase of very long chain fatty acids (ELOs) is essential for FFA biosynthesis; the function of ELO is still unknown in many arthropods, including Panonychus citri (P. citri). In this study, three ELOs were cloned. Developmental-specific mRNA expression results revealed that three PcELOs were highly expressed in egg and adult females. Whereas PcELO7 was dominantly expressed in adult females. Under spirobudiclofen stress, ELOs mRNA expression had different changes, and PcELO7 was down-regulated. The silencing of PcELO7 resulted in a dramatic reduction of oviposition and hatchability. Significant reduction of FFA contents was also examined within PcELO7-repressed P. citri. In addition, we found that PcELO7 mRNA levels were related to fecundity and could affect triacylglycerol (TG) contents. The findings demonstrated that the introduction of dsPcELO7 via oral feeding induced the RNA interference-mediated silencing of a special target gene and could result in mortality and reproduction. In conclusion, PcELO7 is a special RNAi target for P. citri control, and its lethal mechanism might be disturbing lipids biosynthesis.

Contrasting roles of cytochrome P450s in amitraz and chlorfenapyr resistance in the crop pest Tetranychus urticae.

The molecular mechanisms of amitraz and chlorfenapyr resistance remain only poorly understood for major agricultural pests and vectors of human diseases. This study focusses on a multi-resistant field strain of the crop pest Tetranychus urticae, which could be readily selected in the laboratory to high levels of amitraz and chlorfenapyr resistance. Toxicity experiments using tralopyril, the active toxophore of chlorfenapyr, suggested decreased activation as a likely mechanism underlying resistance. Starting from the same parental strain, transcriptome profiling revealed that a cluster of detoxifying genes was upregulated after amitraz selection, but unexpectedly downregulated after chlorfenapyr selection. Further functional validation associated the upregulation of CYP392A16 with amitraz metabolism and the downregulation of CYP392D8 with reduced activation of chlorfenapyr to tralopyril. Genetic mapping (QTL analysis by BSA) was conducted in an attempt to unravel the genetic mechanisms of expression variation and resistance. This revealed that chlorfenapyr resistance was associated with a single QTL, while 3 QTLs were uncovered for amitraz resistance. Together with the observed contrasting gene expression patterns, we argue that transcriptional regulators most likely underly the distinct expression profiles associated with resistance, but these await further functional validation.

The nuclear receptor gene E75 plays a key role in regulating the molting process of the spider mite, Tetranychus urticae.

The nuclear receptor gene Ecdysone-induced protein 75 (E75), as the component of ecdysone response genes in the ecdysone signaling pathway, has important regulatory function for insect molting. However, the regulatory function of E75 during the molting process of spider mites is not yet clear. In this study, the expression pattern of E75 in the molting process of the spider mite Tetranychus urticae was analyzed. The results showed that there was a peak at 8 h post-molting, followed by a decline 8 h after entering each respective quiescent stage across various developmental stages. During the deutonymph stage, the expression dynamics of E75, observed at 4-h intervals, indicated that the transcript levels of TuE75 peaked at 24 h, coinciding with the onset of molting in the mites. To investigate the function of TuE75 during the molting process, silencing TuE75 through dsRNA injection into deutonymph mites at the age of 8 h yielded a notable outcome: 78% of the deutonymph mites were unable to progress to the adult stage. Among these phenotypic mites, 37% were incapable of transitioning into the quiescent state and eventually succumbed after a certain period. An additional 41% of the mites successfully entered the quiescent state but encountered difficulties in shedding the old epidermis, leading to eventual mortality. In summary, these results suggested that TuE75 plays a key role in the molting process of T. urticae.

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.

Osmotin1 is involved in rice resistance to Schizotetranychus oryzae (Acari: Tetranychidae) infestation.

BACKGROUND: Rice is one of the most consumed cereals in the world. Productivity losses are caused by different biotic stresses. One of the most common is the phytophagous mite Schizotetranychus oryzae Rossi de Simons (Acari: Tetranychidae), which inhibits plant development and seed production. The identification of plant defense proteins is important for a better understanding of the mite-plant interaction. We previously detected a high expression of Osmotin1 protein in mite-resistant rice cultivars, under infested conditions, suggesting it could be involved in plant defense against mite attack. We therefore aimed to evaluate the responses of three rice lines overexpressing Osmotin1 (OSM1-OE) and three lines lacking the Osmotin1 gene (osm1-ko) to mite attack. RESULTS: The numbers of individuals (adults, immature stages, and eggs) were significantly lower in OSM1-OE lines than those in wild-type (WT) plants. On the other hand, the osm1-ko lines showed larger numbers of mites per leaf than WT plants. When plants reached the full maturity stage, two out of the three infested OSM1-OE lines presented lower plant height than WT, while the three osm1-ko lines (infested or not) presented higher plant height than WT. The reduction in seed number caused by mite infestation was lower in OSM1-OE lines (12-19%) than in WT plants (34%), while osm1-ko lines presented higher reduction (24-54%) in seed number than WT plants (13%). CONCLUSION: These data suggest that Osmotin1 is involved in rice resistance to S. oryzae infestation. This is the first work showing increased plant resistance to herbivory overexpressing an Osmotin gene. © 2023 Society of Chemical Industry.

CYP4CL2 Confers Metabolic Resistance to Pyridaben in the Citrus Pest Mite Panonychus citri.

The citrus red mite Panonychus citri has developed strong resistance to acaricides. Cytochrome P450 monooxygenases (P450s) can detoxify pesticides and are involved in pesticide resistance in many insects. Here, a pyridaben-resistant P. citri strain showed cross-resistance to cyenopyrafen, bifenazate, fenpyroximate, and tolfenpyrad. Piperonyl butoxide, a P450 inhibitor, significantly increased the toxicity of pyridaben to resistant (Pyr_Rs) and susceptible (Pyr_Control) P. citri strains. P450 activity was significantly higher in Pyr_Rs than in Pyr_Control. Analyses of RNA-Seq data identified a P450 gene (CYP4CL2) that is potentially involved in pyridaben resistance. Consistently, it was up-regulated in two field-derived resistant populations (CQ_WZ and CQ_TN). RNA interference-mediated knockdown of CYP4CL2 significantly decreased the pyridaben resistance in P. citri. Transgenic Drosophila melanogaster expressing CYP4CL2 showed increased pyridaben resistance. Molecular docking analysis showed that pyridaben could bind to several amino acids at substrate recognition sites in CYP4CL2. These findings shed light on P450-mediated pyridaben resistance in pest mites.

Transcriptomic and metabolomic analyses reveals keys genes and metabolic pathways in tea (Camellia sinensis) against six-spotted spider mite (Eotetranychus Sexmaculatus).

BACKGROUND: Six-spotted spider mite (Eotetranychus sexmaculatus) is one of the most damaging pests of tea (Camellia sinensis). E. sexmaculatus causes great economic loss and affects tea quality adversely. In response to pests, such as spider mites, tea plants have evolved resistance mechanisms, such as expression of defense-related genes and defense-related metabolites. RESULTS: To evaluate the biochemical and molecular mechanisms of resistance in C. sinensis against spider mites, "Tianfu-5" (resistant to E. sexmaculatus) and "Fuding Dabai" (susceptible to E. sexmaculatus) were inoculated with spider mites. Transcriptomics and metabolomics based on RNA-Seq and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technology were used to analyze changes in gene expression and metabolite content, respectively. RNA-Seq data analysis revealed that 246 to 3,986 differentially expressed genes (DEGs) were identified in multiple compared groups, and these DEGs were significantly enriched in various pathways, such as phenylpropanoid and flavonoid biosynthesis, plant-pathogen interactions, MAPK signaling, and plant hormone signaling. Additionally, the metabolome data detected 2,220 metabolites, with 194 to 260 differentially abundant metabolites (DAMs) identified in multiple compared groups, including phenylalanine, lignin, salicylic acid, and jasmonic acid. The combined analysis of RNA-Seq and metabolomic data indicated that phenylpropanoid and flavonoid biosynthesis, MAPK signaling, and Ca2+-mediated PR-1 signaling pathways may contribute to spider mite resistance. CONCLUSIONS: Our findings provide insights for identifying insect-induced genes and metabolites and form a basis for studies on mechanisms of host defense against spider mites in C. sinensis. The candidate genes and metabolites identified will be a valuable resource for tea breeding in response to biotic stress.

The Capsicum terpenoid biosynthetic module is affected by spider-mite herbivory.

In response to herbivory, Capsicum annuum leaves adapt their specialized metabolome that may protect the plant against herbivore feeding either directly or indirectly through volatile metabolites acting as cues for natural enemies of the herbivore. The volatile blend of spider-mite infested leaves differs from non-challenged leaves predominantly by a higher contribution of mono- and sesquiterpenes. In addition to these terpenoids released into the headspace, the terpenoid composition of the leaves alters upon herbivory. All this suggests an important role for terpenoids and their biosynthetic machinery in the defence against herbivory. Here, we show that the C. annuum genome contains a terpene synthase (TPS) gene family of 103 putative members of which structural analysis revealed that 27 encode functional enzymes. Transcriptome analysis showed that several TPS loci were differentially expressed upon herbivory in leaves of two C. annuum genotypes, that differ in susceptibility towards spider mites. The relative expression of upstream biosynthetic genes from the mevalonate and the methylerythritol phosphate pathway also altered upon herbivory, revealing a shift in the metabolic flux through the terpene biosynthetic module. The expression of multiple genes potentially acting downstream of the TPSs, including cytochrome P450 monooxygenases, UDP-glucosyl transferases, and transcription factors strongly correlated with the herbivory-induced TPS genes. A selection of herbivory-induced TPS genes was functionally characterized through heterologous expression and the products that these enzymes catalysed matched with the volatile and non-volatile terpenoids induced in response to herbivory.

The host plant strongly modulates acaricide resistance levels to mitochondrial complex II inhibitors in a multi-resistant field population of Tetranychus urticae.

The two-spotted spider mite Tetranychus urticae is a polyphagous pest with an extraordinary ability to develop acaricide resistance. Here, we characterize the resistance mechanisms in a T. urticae population (VR-BE) collected from a Belgian tomato greenhouse, where the grower was unsuccessful in chemically controlling the mite population resulting in crop loss. Upon arrival in the laboratory, the VR-BE population was established both on bean and tomato plants as hosts. Toxicity bioassays on both populations confirmed that the population was highly multi-resistant, recording resistance to 12 out of 13 compounds tested from various mode of action groups. DNA sequencing revealed the presence of multiple target-site resistance mutations, but these could not explain resistance to all compounds. In addition, striking differences in toxicity for six acaricides were observed between the populations on bean and tomato. The highest difference was recorded for the complex II inhibitors cyenopyrafen and cyflumetofen, which were 4.4 and 3.3-fold less toxic for VR-BE mites on tomato versus bean. PBO synergism bioassays suggested increased P450 based detoxification contribute to the host-dependent toxicity. Given the involvement of increased detoxification, we subsequently determined genome-wide gene expression levels of VR-BE on both hosts, in comparison to a reference susceptible population, revealing overexpression of a large set of detoxification genes in VR-BE on both hosts compared to the reference. In addition, a number of mainly detoxification genes with higher expression in VR-BE on tomato compared to bean was identified, including several cytochrome P450s. Together, our work suggests that multi-resistant field populations can accumulate a striking number of target-site resistance mutations. We also show that the host plant can have a profound effect on the P450-associated resistance levels to cyenopyrafen and cyflumetofen.

A link between energy metabolism and plant host adaptation states in the two-spotted spider mite, Tetranychus urticae (Koch).

Energy metabolism is a highly conserved process that balances generation of cellular energy and maintenance of redox homeostasis. It consists of five interconnected pathways: glycolysis, tricarboxylic acid cycle, pentose phosphate, trans-sulfuration, and NAD+ biosynthesis pathways. Environmental stress rewires cellular energy metabolism. Type-2 diabetes is a well-studied energy metabolism rewiring state in human pancreatic ß-cells where glucose metabolism is uncoupled from insulin secretion. The two-spotted spider mite, Tetranychus urticae (Koch), exhibits a remarkable ability to adapt to environmental stress. Upon transfer to unfavourable plant hosts, mites experience extreme xenobiotic stress that dramatically affects their survivorship and fecundity. However, within 25 generations, mites adapt to the xenobiotic stress and restore their fitness. Mites' ability to withstand long-term xenobiotic stress raises a question of their energy metabolism states during host adaptation. Here, we compared the transcriptional responses of five energy metabolism pathways between host-adapted and non-adapted mites while using responses in human pancreatic islet donors to model these pathways under stress. We found that non-adapted mites and human pancreatic ß-cells responded in a similar manner to host plant transfer and diabetogenic stress respectively, where redox homeostasis maintenance was favoured over energy generation. Remarkably, we found that upon host-adaptation, mite energy metabolic states were restored to normal. These findings suggest that genes involved in energy metabolism can serve as molecular markers for mite host-adaptation.

Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage. UGTs are well-known for their role in phase II metabolism; however, their contribution to host adaptation and acaricide resistance in arthropods, such as T. urticae, is not yet resolved. TuUGT202A2 (Tetur22g00270) has been linked to the ability of this pest to adapt to tomato plants. Moreover, it was shown that this enzyme can glycosylate a wide range of flavonoids. To understand this relationship at the molecular level, structural, functional, and computational studies were performed. Structural studies provided specific snapshots of the enzyme in different catalytically relevant stages. The crystal structure of TuUGT202A2 in complex with UDP-glucose was obtained and site-directed mutagenesis paired with molecular dynamic simulations revealed a novel lid-like mechanism involved in the binding of the activated sugar donor. Two additional TuUGT202A2 crystal complexes, UDP-(S)-naringenin and UDP-naringin, demonstrated that this enzyme has a highly plastic and open-ended acceptor-binding site. Overall, this work reveals the molecular basis of substrate promiscuity of TuUGT202A2 and provides novel insights into the structural mechanism of UGTs catalysis.

The Arabidopsis thioredoxin TRXh5regulates the S-nitrosylation pattern of the TIRK receptor being both proteins essential in the modulation of defences to Tetranychus urticae.

The interaction between plants and phytophagous arthropods encompasses a complex network of molecules, signals, and pathways to overcome defences generated by each interacting organism. Although most of the elements and modulators involved in this interplay are still unidentified, plant redox homeostasis and signalling are essential for the establishment of defence responses. Here, focusing on the response of Arabidopsis thaliana to the spider mite Tetranychus urticae, we demonstrate the involvement in plant defence of the thioredoxin TRXh5, a small redox protein whose expression is induced by mite infestation. TRXh5 is localized in the cell membrane system and cytoplasm and is associated with alterations in the content of reactive oxygen and nitrogen species. Protein S-nitrosylation signal in TRXh5 over-expression lines is decreased and alteration in TRXh5 level produces changes in the JA/SA hormonal crosstalk of infested plants. Moreover, TRXh5 interacts and likely regulates the redox state of an uncharacterized receptor-like kinase, named THIOREDOXIN INTERACTING RECEPTOR KINASE (TIRK), also induced by mite herbivory. Feeding bioassays performed withTRXh5 over-expression plants result in lower leaf damage and reduced egg accumulation after T. urticae infestation than in wild-type (WT) plants. In contrast, mites cause a more severe injury in trxh5 mutant lines where a greater number of eggs accumulates. Likewise, analysis of TIRK-gain and -loss-of-function lines demonstrate the defence role of this receptor in Arabidopsis against T. urticae. Altogether, our findings demonstrate the interaction between TRXh5 and TIRK and highlight the importance of TRXh5 and TIRK in the establishment of effective Arabidopsis defences against spider mite herbivory.

Cardinium symbionts are pervasive in Iranian populations of the spider mite Panonychus ulmi despite inducing an infection cost and no demonstrable reproductive phenotypes when Wolbachia is a symbiotic partner.

Maternally transmitted symbionts such as Cardinium and Wolbachia are widespread in arthropods. Both Cardinium and Wolbachia can cause cytoplasmic incompatibility, a reproductive phenotype that interferes with the development of uninfected eggs that are fertilized by infected sperm. In haplodiploid hosts, these symbionts can also distort sex allocation to facilitate their spread through host populations. Without other fitness effects, symbionts that induce strong reproductive phenotypes tend to spread to high and stable infection frequencies, whereas variants that induce weak reproductive phenotypes are typically associated with intermediate and variable frequencies. To study the spread of Cardinium in a haplodiploid host, we sampled Iranian populations of the economically important spider mite Panonychus ulmi in apple orchards. Within several field populations, we also studied the Wolbachia infection frequencies. All P. ulmi field populations carried a Cardinium infection and exhibited high infection frequencies. In contrast, Wolbachia frequency ranged between ca. 10% and ca. 70% and was only found in co-infected mites. To test whether Cardinium induce reproductive phenotypes in P. ulmi, a Cardinium-cured derived line was generated by antibiotic treatment from a co-infected field population. Genetic crosses indicated that Cardinium do not induce demonstrable levels of cytoplasmic incompatibility and sex allocation distortion in co-infected P. ulmi. However, Cardinium infection was associated with a longer developmental time and reduced total fecundity for co-infected females. We hypothesize that Cardinium spread through P. ulmi populations via uncharacterized fitness effects and that co-infection with Wolbachia might impact these drive mechanisms.

Assessment of the role of an ABCC transporter TuMRP1 in the toxicity of abamectin to Tetranychus urticae.

The rapid evolution of pest resistance threatens the sustainable utilization of bioinsecticides such as abamectin, and so deciphering the molecular mechanisms affecting toxicity and resistance is essential for their long-term application. Historical studies of abamectin resistance in arthropods have mainly focused on mechanisms involving the glutamate-gated chloride channel (GluCl) targets, with the role of metabolic processes less clear. The two-spotted spider mite, Tetranychus urticae, is a generalist herbivore notorious for rapidly developing resistance to pesticides worldwide, and abamectin has been widely used for its control in the field. After reanalyzing previous transcriptome and RNA-seq data, we here identified an ABC transporter subfamily C gene in T. urticae named multidrug resistance-associated protein 1 (TuMRP1), whose expression differed between susceptible and resistant populations. Synergism bioassays with the inhibitor MK-571, the existence of a genetic association between TuMRP1 expression and susceptibility to abamectin, and the effect of RNA interference mediated silencing of TuMRP1 were all consistent with a direct role of this transporter protein in the toxicity of abamectin. Although ABC transporters are often involved in removing insecticidal compounds from cells, our data suggest either an alternative role for these proteins in the mechanism of action of abamectin or highlight an indirect association between their expression and abamectin toxicity.