The development of an egg-soaking method for delivering dsRNAs into spider mites.

Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.

Design, synthesis, and evaluation of novel arecoline-linked amino acid derivatives for insecticidal and antifungal activities.

A series of arecoline derivatives with amino acid moieties were designed and synthesised using an acylamide condensation strategy, taking arecoline as the foundational structure. The insecticidal efficacy of these compounds against Aphis craccivora and Tetranychus cinnabarinus was evaluated. Notably, derivatives 3h and 3i demonstrated superior insecticidal activity compared with arecoline. Additionally, 3h and 3i showed good fungicidal effectiveness against two types of plant fungi. Moreover, molecular docking analyses suggested that 3h and 3i could affect the nervous systems of A. craccivora and T. cinnabarinus by binding to neuronal nicotinic acetylcholine receptors. These findings suggest that compounds 3h and 3i represent promising leads for further development in insecticide and fungicide research.

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.

Influence of abiotic factors on diapause termination and temperature requirements for postdiapause development in the European red mite, Panonychus ulmi (Acari: Tetranychidae).

The European red mite Panonychus ulmi (Koch) is widely distributed and it can severely affect pome fruit crops, particularly apple. Pest outbreaks are related to an overuse of non-selective pesticide treatments that lead to the development of resistance and the absence of natural enemies in the orchard. A key aspect to optimize the use of pesticide treatments in the context of IPM is to increase the knowledge on the biology and ecology of the pest to better predict population dynamics and outbreaks. For the European red mite, knowledge on the conditions that lead to diapause breaking by winter eggs is essential to model population dynamics. To increase this knowledge, winter eggs were collected during field surveys in northen Spain during three years and egg hatching was monitored under controlled temperature and photoperiod conditions in the laboratory. The "number of days exposed to cold temperatures" was the most significant factor that positively affected hatching of overwintering eggs. The time required for 50% of the egg population to hatch (T50%) was also negatively modulated by the duration of exposure to cold temperature. The temperature threshold for postdiapause eggs development collected from the field was estimated between 5 and 6 ºC in 2005 and 2007, respectively. Moreover, the degree-days required for post diapause development were estimated between 263.2 and 270.3, depending on the year of collection. Collectively, we provide additional information on the diapause termination and postdiapause development of the European red mite that may effectively contribute to optimize pest population models.

Novel Potential of Rose Essential Oil as a Powerful Plant Defense Potentiator.

Terpenoids, natural compounds released by plants, function to enhance plant defense. The aim of this study was to investigate the effects of terpenoid-enriched essential oils (EOs) on tomato plants. From the application of a highly diluted solution of 11 different EOs to potted tomato soil, our study showed that rose essential oil (REO), rich in ß-citronellol, played a crucial role in activating defense genes in tomato leaves. As a result, leaf damage caused by herbivores, such as Spodoptera litura and Tetranychus urticae, was significantly reduced. In addition, our results were validated in field trials, providing evidence that REO is an effective biostimulant for enhancing plant defense against pests. Notably, the REO solution also had the added benefit of attracting herbivore predators, such as Phytoseiulus persimilis. Our findings suggest a practical approach to promote organic tomato production that encourages environmentally friendly and sustainable practices.

Prey stage preference of Amblyseius paraaerialis (Acari: Phytoseiidae) on varied life stages of the spider mites Tetranychus urticae, Tetranychus macfarlanei and Oligonychus biharensis (Acari: Tetranychidae) and exploring the mass rearing possibilities of this predatory mite on alternative diets.

Spider mites Tetranychus urticae, Tetranychus macfarlanei and Oligonychus biharensis are considered to be highly polyphagous in nature and causes severe damage to a wide range of plants around the world. Amblyseius paraaerialis is an efficient phytoseiid predator of spider mites with a potential to survive on both natural and alternative diets. Evaluation of predatory potential and prey stage preference provides valuable information on the efficacy of the predatory species in controlling mite population. Feeding experiments were conducted on mulberry leaf discs under the laboratory conditions of 30 ± 2 °C and 70 ± 5% relative humidity (RH). After 24 h of feeding experiment, the adult female predator exhibited a significant preference in feeding towards the eggs of T. macfarlanei (42.6%) and the larval stages of T. urticae (46%) and O. biharensis (25.3%). The mass rearing possibilities of A. paraaerialis was tested by tracking and comparing the developmental duration of individual life stages on varied food sources like, honey, castor (Ricinus communis) pollen, honey-pollen mixture and mixed life stages of T. urticae. The predator was failed to complete its development on honey and pollen when supplied separately. However it was successfully developed on honey-pollen mixture and mixed life stages of T. urticae. The developmental studies unravelled a shortest developmental duration and an extended adult longevity and lifespan of A. paraaerialis when reared on the alternative diet, thus opened up the mass rearing possibility of the predatory species under laboratory conditions.

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.

Construction and Single-Crystal Structures of N-Isoxazolin-5-ylcarbonylindole Derivatives, and Their Pesticidal Activities and Toxicology Study.

To explore natural product-based pesticide candidates, a series of indole derivatives containing the isoxazoline skeleton at the N-1 position were synthesized by 1,3-dipolar [2 + 3] cycloaddition reaction. Their structures were characterized by melting points (mp), infrared (IR) spectra, proton nuclear magnetic resonance spectra (1H NMR), carbon-13 nuclear magnetic resonance spectra (13C NMR), and high resolution mass spectrometry (HRMS). The single-crystal structures of five compounds were presented. Against Tetranychus cinnabarinus Boisduval, compound 3b showed greater than 3.8-fold acaricidal activity of indole and good control effects under glasshouse conditions. Against Aphis citricola Van der Goot, compounds 3b and 3q exhibited 48.3- and 36.8-fold aphicidal activity of indole and 6-methylindole, respectively. Particularly, compound 3b showed good bioactivities against T. cinnabarinus and A. citricola. Against Eriosoma lanigerum Hausmann, compound 3h and 3i showed 2.1 and 1.9 times higher aphicidal activity compared to indole. Furthermore, the construction of the epidermal cuticle layer of 3b-treated carmine spider mites was distinctly damaged, which ultimately led to their death.

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.

An exploratory analysis of the structure of tetranychid and phytoseiid assemblages in walnut orchards in California.

Spider mites were considered secondary pests of walnut production in California, under the control of phytoseiid predators. Due to increased importance as walnut pests in recent decades there is renewed interest in the structure and function of the associated phytoseiid assemblage. In this study we report the results from a 3 year survey of the tetranychid and phytoseiid assemblages in walnut orchards in the Central Valley of California. The survey was conducted to determine the range and dominance of web-spinning Tetranychus species present, to investigate the species richness and dominance of the phytoseiid species present, and to explore whether patterns of variation in the relative abundance of phytoseiid species could be explained by one or more explanatory variables. Tetranychus urticae was the dominant spider mite in all growing regions and years with T. pacificus and T. turkestani also present in orchards in the southern San Joaquin Valley. Phytoseiid species richness declined with latitude among the three walnut growing regions and of the 13 species found Amblyseius similoides, Euseius stipulatus, Galendromus occidentalis and Typhlodromus caudiglans were the most abundant and widespread species present. Mean proportional abundance significantly increased from early (mid May-July) to late (August-mid October) season and from southern to northern growing regions for Type II and IV predators, but significantly decreased from early to late season and from southern to northern growing regions for Type III predators. The mean proportional abundance of Type II predators, particularly G. occidentalis, significantly increased and that of Type III predators significantly decreased with mean Tetranychus density in individual orchards. The current survey provides a more in-depth analysis of mite assemblages in walnut orchards in California and can be used to better inform adaptive management strategies for integrated mite management in the future.

Herbivore-induced plant volatiles emitted by citrus in response to spider mite infestation can attract predatory mites.

Understanding the nutritional interplay among plants, pests, and natural enemies is essential for sustainable pest management. Enhancing the efficiency of natural enemies, such as Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) is critical, and exploiting herbivore-induced plant volatiles (HIPVs) offers a promising approach. However, N. californicus has rarely been reported to utilize HIPVs to improve their biological control capabilities. Our research revealed a significant difference in the diversity of volatile compounds detected in clean Citrus reticulata Blanco leaves compared to those in C. reticulata leaves infested with Panonychus citri (McGregor) (Acari: Tetranychidae), regardless of mite presence. This suggests that P. citri infestation induces a wide array of HIPVs in C. reticulata leaves. We conducted olfactory behavioral assays to evaluate the response of N. californicus to synthetic HIPVs. Results revealed that linalool (1.00 mg/mL), 2,2,4-trimethylpentane (10.0 mg/mL), undecylcyclohexane (1.00 mg/mL), and (+)-dibenzoyl-L-tartaric anhydride (10.0 mg/mL) significantly attracted N. californicus while pentadecanal (1.00 mg/mL) significantly deterred it. A 3-component blend of linalool, undecylcyclohexane, and (+)-dibenzoyl-L-tartaric anhydride was better than other combinations in attracting N. californicus. This combination provided the basis for developing an attractant for N. californicus, facilitating the rate of its dispersal to enhance its biological control of pests. Consequently, this research offers vital insights into improving the sustainable pest control potential of predatory mites.

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.

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.

The influence of high-temperature frequency variation on the life-history traits of pyridaben-sensitive and -resistant strains of Tetranychus truncatus.

With a generally warming global climate, the number of Tetranychus truncatus specimens in the Hexi region in China has been increasing. As ectotherms, the growth and development of T. truncatus are greatly affected by changes in environmental temperature. The effect of heatwaves on organisms depends on a delicate balance between damage and repair periods. Therefore, we simulated nine patterns of periodically recurring changes in the frequency of high-temperature days using an intraday gradual temperature change model to study and compare the effects on the development and reproduction of pyridaben-sensitive and -resistant strains of T. truncatus. The results showed that the influence of the frequency of high-temperature days on developmental stages, longevity and fecundity was different between the two strains. The egg and immature stages of the sensitive strain were all affected by hot days, whereas the adult stage was less affected by the frequency. The egg stage of the resistant strain was less affected; it was mainly affected in the immature and adult stages. Under the moderate condition of increasing the proportion of days at normal temperature, the longevity of the resistant strain gradually increased and reached a maximum at a 1:3 frequency, and then it decreased with the increase in high-temperature days. The longevity of the sensitive strain was less affected by frequency, and there was no significant difference between most treatment and control groups. In addition, both sensitive and resistant strains were able to complete growth and development under all nine frequencies of high-temperature days, but the reproductive rate was lower than it was at normal temperatures, indicating that both strains of T. truncatus adapted to high temperatures at the expense of reduced reproduction rates. This lays a key theoretical foundation for predicting the occurrence of agricultural pest populations under the background of climate warming and developing appropriate control strategies.

Sublethal and transgenerational effects of lufenuron on the biological traits of Panonychus citri (McGregor) (Acari: Tetranychidae).

The citrus red mite, Panonychus citri (McGregor), is a globally important pest that has developed severe resistance to various pesticides. Lufenuron has been widely used in the control of the related pests in citrus orchard ecosystem. In this study, the susceptibilities of egg, larva, deutonymph and female adult of P. citri to lufenuron was determined, and the LC50 values were 161.354 mg/L, 49.595 mg/L, 81.580 mg/L, and 147.006 mg/L, respectively. Life-table analysis indicated that the fecundities were significantly increased by 11.86% and 26.84% after the mites were treated with LC20 concentrations of lufenuron at the egg or deutonymph stages, respectively. After eggs were treated with lufenuron, the immature stage and longevity were also affected, and resulted in a significant increase in r, R0 and λ. After exposure of female adults to LC20 of lufenuron, the fecundity and longevity of F0 generation significantly decreased by 31.99% and 10.94%, respectively. Furthermore, the expression level of EcR and Vg was significantly inhibited upon mites was treated with lufenuron. However, lufenuron exposure has a positive effect on fecundity and R0 in F1 generation, the expression of all reproduction-related genes was significantly up-regulated. In conclusion, there was a stimulating effect on the offspring population. Our results will contribute to the assessment of the resurgence of P. citri in the field after the application of lufenuron and the development of integrated pest control strategies in citrus orchards.

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.

Assessing the impact of piercing-sucking pests on greenhouse-grown industrial hemp (Cannabis sativa L.).

Cannabis sativa or hemp, defined as <0.3% total tetrahydrocannabinol (THC), is a specialty crop in the United States, of particular interest among growers in the southeastern United States to replace tobacco production. Tetranychus urticae (twospotted spider mite), Aculops cannabicola (hemp russet mite), Polyphagotarsonemus latus (broad mites), and Phorodon cannabis (cannabis aphids) are considered the most significant pests in greenhouse grown hemp. Mite and aphid injury can cause cupping and yellowing of leaves, resulting in leaf drop, and reduced flower and resin production. We sought to understand the effects of feeding by T. urticae and Myzus persicae (green peach aphid), as a proxy for P. cannabis, on the concentration of economically significant cannabinoids through a series of experiments on greenhouse grown plants. First, we compared the variability of chemical concentrations in samples collected from individual plants versus pooled samples from 5 plants, and found that chemical concentrations in single plants were similar to those in pooled plant samples. Next, we compared chemical concentrations prior to arthropod infestation and post infestation. When evaluating the mite feeding damage in 2020, cannabinoids in plants infested with high densities of T. urticae increased more slowly than in uninfested control plants or plants infested with low T. urticae densities. In 2021, the concentration of tetrahydrocannabinol did not differ significantly between treatments. Cannabidiol increased more slowly in plants with low T. urticae densities when compared to uninfested controls but did not differ from the high T. urticae densities 14 days after infestation.

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.

A new spider mite elicitor triggers plant defence and promotes resistance to herbivores.

Herbivore-associated elicitors (HAEs) are active molecules produced by herbivorous insects. Recognition of HAEs by plants induces defence that resist herbivore attacks. We previously demonstrated that the tomato red spider mite Tetranychus evansi triggered defence in Nicotiana benthamiana. However, our knowledge of HAEs from T. evansi remains limited. Here, we characterize a novel HAE, Te16, from T. evansi and dissect its function in mite-plant interactions. We investigate the effects of Te16 on spider mites and plants by heterologous expression, virus-induced gene silencing assay, and RNA interference. Te16 induces cell death, reactive oxygen species (ROS) accumulation, callose deposition, and jasmonate (JA)-related responses in N. benthamiana leaves. Te16-mediated cell death requires a calcium signalling pathway, cytoplasmic localization, the plant co-receptor BAK1, and the signalling components SGT1 and HSP90. The active region of Te16-induced cell death is located at amino acids 114-293. Moreover, silencing Te16 gene in T. evansi reduces spider mite survival and hatchability, but expressing Te16 in N. benthamiana leaves enhances plant resistance to herbivores. Finally, Te16 gene is specific to Tetranychidae species and is highly conserved in activating plant immunity. Our findings reveal a novel salivary protein produced by spider mites that elicits plant defence and resistance to insects, providing valuable clues for pest management.