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.

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.

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.

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.

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.

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.

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.

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.

Combined nighttime ultraviolet B irradiation and phytoseiid mite application provide optimal control of the spider mite Tetranychus urticae on greenhouse strawberry plants.

BACKGROUND: Tetranychus urticae is a hard-to-control pest of greenhouse strawberry production. Nighttime ultraviolet B (UV-B) radiation using light reflection sheets (LRS) has been applied as a physical method to control T. urticae through direct ovicidal effects (the UV method). However, because strawberry leaves grow more densely, UV-B radiation fails to reach the lower leaf surfaces inhabited by spider mites; therefore, a complementary method is required. We propose the supplemental application of phytoseiid mites in greenhouse strawberry production. We evaluated the control effects of UV-B irradiation, phytoseiid mite application and their combined use. The effects of UV-B irradiation on the degree of overlap relative to the independent distributions (ω) between predators and prey were also analyzed. RESULTS: The UV method alone maintained low T. urticae density levels from November to February; however, mite populations increased from March onward. Phytoseiid mite application in January and February without UV-B irradiation resulted in a temporary increase in spider mites in March and/or April. By contrast, combined application of the UV method and phytoseiid mites had a greater control effect during the strawberry growing season. The ω values were higher for the UV method compared with no UV-B irradiation, suggesting that UV-B irradiation increased phytoseiid mite foraging rates. CONCLUSION: The release of phytoseiid mites compensated for the shortcomings of the UV method, and UV-B irradiation promoted predation by phytoseiid mites by increasing the behavioral numerical response. Consequently, combined application of UV-B irradiation and phytoseiid mites is optimal for T. urticae control in greenhouse strawberry production. © 2023 Society of Chemical Industry.

Cross-resistance, inheritance and biochemical mechanism of abamectin resistance in a field-derived strain of the citrus red mite, Panonychus citri (Acari: Tetranychidae).

BACKGROUND: The citrus red mite, Panonychus citri (McGregor), a global pest of citrus, has developed different levels of resistance to various acaricides in the field. Abamectin is one of the most important insecticides/acaricides worldwide, targetting a wide number of insect and mite pests. The evolution of abamectin resistance in P. citri is threatening the sustainable use of abamectin for mite control. RESULTS: The abamectin resistant strain (NN-Aba), derived from a field strain NN by consistent selection with abamectin, showed 4279-fold resistance to abamectin compared to a relatively susceptible strain (SS) of P. citri. Cross-resistance of NN-Aba was observed between abamectin and emamectin benzoate, pyridaben, fenpropathrin and cyflumetofen. Inheritance analyses indicated that abamectin resistance in the NN-Aba strain was autosomal, incompletely recessive and polygenic. The synergy experiment showed that abamectin toxicity was synergized by piperonyl butoxide (PBO), diethyl maleate (DEM) and tributyl phosphorotrithiotate (TPP) in the NN-Aba strain, and synergy ratios were 2.72-, 2.48- and 2.13-fold, respectively. The glutathione-S-transferases activity in the NN-Aba strain were significantly increased by 2.08-fold compared with the SS strain. CONCLUSION: The abamectin resistance was autosomal, incompletely recessive and polygenic in P. citri. The NN-Aba strain showed cross-resistance to various acaricides with different modes of action. Metabolic detoxification mechanism participated in abamectin resistance in NN-Aba strain. These findings provide useful information for resistance management of P. citri in the field. © 2023 Society of Chemical Industry.

Enantioselective metabolism of fenpropathrin enantiomers by carboxyl/choline esterase 6 in Tetranychus cinnabarinus.

BACKGROUND: Tetranychus cinnabarinus is a polyphagous pest mite commonly found in agriculture. As an excellent acaricide, fenpropathrin (FEN) is frequently used to control T. cinnabarinus in agriculture. However, commercial FEN is a racemate with two enantiomers, R-FEN and S-FEN. Considering that investigations on the metabolism of FEN by T. cinnabarinus are based on racemate FEN, it is important to investigate the enantioselective metabolism of FEN in T. cinnabarinus. RESULTS: S-FEN was more toxic to T. cinnabarinus than R-FEN by more than 68.8-fold. Moreover, the synergist bioassay revealed that carboxylesterase and cytochrome P450 were the primary enzymes engaged in the detoxification of FEN in T. cinnabarinus, with carboxylesterase playing a leading role. Seven genes were substantially different after the induction of S-FEN and R-FEN. TcCCE06 was screened and selected as a key gene that related to FEN metabolism in T. cinnabarinus. The metabolic results showed that the recombinant TcCCE06 effectively metabolized 32.1% of the R-FEN and 13.8% of the S-FEN within 4 h of incubation. Moreover, R-FEN was demonstrated to have a higher affinity for the TcCCE06 protein than S-FEN based on molecular docking. CONCLUSION: Our results indicated that TcCCE06 mediates the enantioselective metabolism of FEN in T. cinnabarinus. Our findings will contribute to a more comprehensive understanding of the mechanisms underlying the differential toxicity of the FEN enantiomers against T. cinnabarinus. Furthermore, they also provide a new perspective for the development of enantiomer-enriched acaricides with higher activity and lower pesticide dosage and pollution risks. © 2023 Society of Chemical Industry.

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.

Functional analysis of a down-regulated transcription factor-SoxNeuroA gene involved in the acaricidal mechanism of scopoletin against spider mites.

BACKGROUND: Insight into the mode of action of plant-derived acaricides will help in the development of sustainable control strategies for mite pests. Scopoletin, a promising plant-derived bioactive compound, displays prominent acaricidal activity against Tetranychus cinnabarinus. The transcription factor SoxNeuroA plays a vital role in maintaining calcium ion (Ca2+ ) homeostasis. Down-regulation of SoxNeuroA gene expression occurs in scopoletin-exposed mites, but the functional role of this gene remains unknown. RESULTS: A SoxNeuroA gene from T. cinnabarinus (TcSoxNeuroA) was first cloned and identified. Reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qPCR), and Western blotting assays all confirmed that the gene expression and protein levels of TcSoxNeuroA were significantly reduced under scopoletin exposure. Furthermore, RNA interference silencing of the weakly expressed SoxNeuroA gene significantly enhanced the susceptibility of mites to scopoletin, suggesting that the acaricidal mechanism of scopoletin was mediated by the weakly expressed SoxNeuroA gene. Additionally, yeast one-hybrid (Y1H) and dual-luciferase reporter assays revealed that TcSoxNeuroA was a repressor of Orai1 Ca2+ channel gene transcription, and the key binding sequence was ATCAAAG (positions -361 to -368 of the Orai1 promoter). Importantly, site-directed mutagenesis and microscale thermophoresis assays further indicated that ASP185, ARG189, and LYS217, which were key predicted hydrogen-bonding sites in the molecular docking model, may be the vital binding sites for scopoletin in TcSoxNeuroA. CONCLUSION: These results demonstrate that the acaricidal mechanism of scopoletin involves inhibition of the transcription factor SoxNeuroA, thus inducing the activation of the Orai1 Ca2+ channel, eventually leading to Ca2+ overload and lethality. Elucidation of the transcription factor-targeted mechanism for this potent plant-derived acaricide has vital implications for the design of next-generation green acaricides with novel targets. © 2023 Society of Chemical Industry.