Genome-Wide Identification of the PIP5K Gene Family in Camellia sinensis and Their Roles in Metabolic Regulation.

Spider mite infestation has a severe impact on tea growth and quality. In this study, we conducted a deep exploration of the functions and regulations of the CsPIP5K gene family using chromosomal localization and collinearity analysis. Additionally, we carefully examined the cis elements within these genes. To fully understand the metabolic response of CsPIP5K under spider mite infection, we integrated previously published metabolomic and transcriptomic data. Our analysis revealed that multiple CsPIP5K genes are associated with phospholipid metabolism, with CsPIP5K06 showing the strongest correlation. Therefore, we employed qPCR and subcellular localization techniques to determine the expression pattern of this gene and its functional location within the cell. Overall, this study not only comprehensively elucidated the characteristics, structure, and evolution of the CsPIP5K gene family but also identified several candidate CsPIP5K genes related to phospholipid biosynthesis and associated with spider mites based on previously published data. This research makes a significant contribution to enhancing the resistance of tea to spider mite and maintaining optimal tea quality.

Chromosome-level genome assembly of the two-spotted spider mite Tetranychus urticae.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a notorious pest in agriculture that has developed resistance to almost all chemical types used for its control. Here, we assembled a chromosome-level genome for the TSSM using Illumina, Nanopore, and Hi-C sequencing technologies. The assembled contigs had a total length of 103.94 Mb with an N50 of 3.46 Mb, with 87.7 Mb of 34 contigs anchored to three chromosomes. The chromosome-level genome assembly had a BUSCO completeness of 94.8%. We identified 15,604 protein-coding genes, with 11,435 genes that could be functionally annotated. The high-quality genome provides invaluable resources for the genetic and evolutionary study of TSSM.

Overexpression of a nuclear receptor HR96 contributes to spirodiclofen susceptibility in Panonychus citri (McGregor).

The citrus red mite, Panonychus citri, is one of the most notorious and devastating citrus pests around the world that has developed resistance to multiple chemical acaricides. In previous research, we found that spirodiclofen-resistant is related to overexpression of P450, CCE, and ABC transporter genes in P. citri. However, the regulatory mechanisms of these detoxification genes are still elusive. This study identified all hormone receptor 96 genes of P. citri. 8 PcHR96 genes contained highly conserved domains. The expression profiles showed that PcHR96h was significantly upregulated in spirodiclofen resistant strain and after exposure to spirodiclofen. RNA interference of PcHR96h decreased expression of detoxification genes and increased spirodiclofen susceptibility in P. citri. Furthermore, molecular docking, heterologous expression, and drug affinity responsive target stability demonstrated that PcHR96h can interact with spirodiclofen in vitro. Our research results indicate that PcHR96h plays an important role in regulating spirodiclofen susceptibility and provides theoretical support for the resistance management of P. citri.

Metabolomic profiling of virulent and non-virulent Beauveria bassiana strains: insights into the pathogenicity of Tetranychus truncatus.

In this study, the pathogenicity of local Beauveria bassiana strains was elucidated using molecular and metabolomics methodologies. Molecular verification of the B. bassiana-specific chitinase gene was achieved via phylogenetic analysis of the Bbchit1 region. Subsequent metabolomic analyses employing UPLC-Q-TOF-MS revealed a different number of non-volatile metabolite profiles among the six B. bassiana strains. Bb6 produced the most non-volatile compounds (17) out of a total of 18, followed by Bb15 (16) and Bb12 (15). Similarly, Bb5, Bb8, and Bb21, three non-virulent B. bassiana strains, produced 13, 14, and 14 metabolites, respectively. But unique secondary metabolites like bassianolide and beauvericin, pivotal for virulence and mite management, were exclusively found in the virulent strains (Bb6, Bb12, and Bb15) of B. bassiana. The distinctive non-volatile metabolomic profiles of these strains underscore their pathogenicity against Tetranychus truncatus, suggesting their promise in bio-control applications.

Unveiling fenpropathrin resistance levels in field populations of Tetranychus cinnabarinus (Boisduval): Insights, risks, and RNAi strategy.

Indoor cases of Tetranychus cinnabarinus displaying resistance have been documented, but the resistance level in field populations remains unexplored in China. This study delves into the resistance dynamics of T. cinnabarinus to fenpropathrin in various field populations across China, a pressing concern in contemporary agricultural pest control. The conventional bioassay and amplicon sequencing reveal a notable absence of significant fenpropathrin resistance in field populations, contrasting with known resistance in indoor cases. Current study highlights the limitations of traditional bioassays in detecting early-stage resistance and underscores the nuanced capabilities and constraints of amplicon sequencing in resistance gene frequency analysis. By employing an integrated approach, we combined dose-response bioassays, amplicon sequencing, and statistical modeling to assess resistance levels and investigate underlying genetic factors. The model with empirical data indicates that a 5% mutation frequency represents the threshold before resistance emerges. However, the detection of the kdr mutation in certain populations ranging from 0 to 1.2%, signals an early looming threat of future resistance emergence. Additionally, we further assessed a specific dsRNA targeting VGSC genes at two concentrations (10 ng/µL and 100 ng/µL), both inducing substantial mortality by silencing target genes effectively. The exploration of RNA interference (RNAi) as a novel, more environmentally friendly pest control measure opens new avenues, despite the ongoing challenge of resistance evolution. Overall, this study underscores the necessity for evolving pest management strategies, integrating advanced biotechnological approaches with traditional methods, to effectively counter pesticide resistance and ensure sustainable agricultural productivity.

A novel spider venom peptide from the predatory mite Neoseiulus barkeri shows lethal effect on phytophagous pests.

The long-term use of pesticides in the field, and the high fertility and adaptability of phytophagous mites have led to resistance problems; consequently, novel safe and efficient active substances are necessary to broaden the tools of pest mite control. Natural enemies of arthropods typically secrete substances with paralytic or lethal effects on their prey, and those substances are a resource for future biopesticides. In this study, two putative venom peptide genes were identified in a parasitic mite Neoseiulus barkeri transcriptome. Recombinant venom NbSP2 peptide injected into Tetranychus cinnabarinus mites was significantly more lethal than recombinant NBSP1. NbSP2 was also lethal to Spodoptera litura when injected but not when fed to third instar larvae. The interaction proteins of NbSP2 in T. cinnabarinus and S. litura were identified by affinity chromatography. Among these proteins, ATP synthase subunit ß (ATP SSß) was deduced as a potential target. Four binding sites were predicted between NBSP2 and ATP SSß of T. cinnabarinus and S. litura. In conclusion, we identified a venom peptide with activity against T. cinnabarinus and S. litura. This study provides a novel component for development of a new biological pesticide.

QTL analysis on a lemon population provides novel insights on the genetic regulation of the tolerance to the two-spotted spider mite attack.

BACKGROUND: Among the Citrus species, lemon (Citrus limon Burm f.) is one of the most affected by the two-spotted spider mite (Tetranychus urticae Koch). Moreover, chemical control is hampered by the mite's ability to develop genetic resistance against acaricides. In this context, the identification of the genetic basis of the host resistance could represent a sustainable strategy for spider mite control. In the present study, a marker-trait association analysis was performed on a lemon population employing an association mapping approach. An inter-specific full-sib population composed of 109 accessions was phenotyped through a detached-leaf assays performed in modified Huffaker cells. Those individuals, complemented with two inter-specific segregating populations, were genotyped using a target-sequencing approach called SPET (Single Primer Enrichment Technology), the resulting SNPs were employed for the generation of an integrated genetic map. RESULTS: The percentage of damaged area in the full-sib population showed a quantitative distribution with values ranging from 0.36 to 9.67%. A total of 47,298 SNPs were selected for an association mapping study and a significant marker linked with resistance to spider mite was detected on linkage group 5. In silico gene annotation of the QTL interval enabled the detection of 13 genes involved in immune response to biotic and abiotic stress. Gene expression analysis showed an over expression of the gene encoding for the ethylene-responsive transcription factor ERF098-like, already characterized in Arabidopsis and in rice for its involvement in defense response. CONCLUSION: The identification of a molecular marker linked to the resistance to spider mite attack can pave the way for the development of marker-assisted breeding plan for the development of novel selection coupling favorable agronomical traits (e.g. fruit quality, yield) with a higher resistance toward the mite.

The chitinase genes TuCht4 and TuCht10 are indispensable for molting and survival of Tetranychus urticae.

Insect chitinases (Chts) play a crucial role in the molting process, enabling continuous growth through sequential developmental stages. Based on their high homology to insect Chts, TuCht1 (group II), TuCht4 (group I) and TuCht10 (group IV) were identified, and their roles during molting process were investigated. TuCht1 was mainly expressed in the deutonymphal stage, while TuCht4 was mainly expressed in the nymphal stage and the highest expression level of TuCht10 was observed in the larvae. Feeding RNAi assays have shown that group I TuCht4 and group Ⅳ TuCht10 are involved in mite molting. Suppression of TuCht4 or TuCht10 resulted in high mortality, molting abnormalities and the absence of distinct electron dense layers of chitinous horizontal laminae in the cuticle, as demonstrated by scanning electron microscopy and transmission electron microscopy. The nanocarrier mediated RNAi had significantly higher RNAi efficiency and caused higher mortality. The results of the present study suggest that chitinase genes TuCht4 and TuCht10 are potential targets for dietary RNAi, and demonstrates a nanocarrier-mediated delivery system to enhance the bioactivity of dsRNA, providing a potential technology for green pest management.

UGT201H1 overexpression confers cyflumetofen resistance in Tetranychus cinnabarinus (Boisduval).

BACKGROUND: Tetranychus cinnabarinus is one of the most common polyphagous arthropod herbivores, and is primarily controlled by the application of acaricides. The heavy use of acaricides has led to high levels of resistance to acaricides such as cyflumetofen, which poses a threat to global resistance management programs. Cyflumetofen resistance is caused by an increase in metabolic detoxification; however, the role of uridine diphosphate (UDP)-glycosyltransferase (UGT) genes in cyflumetofen resistance remains to be determined. RESULTS: Synergist 5-nitrouracil (5-Nul) significantly enhanced cyflumetofen toxicity in T. cinnabarinus, which indicated that UGTs are involved in the development of cyflumetofen resistance. Transcriptomic analysis and quantitative (q)PCR assays demonstrated that the UGT genes, especially UGT201H1, were highly expressed in the YN-CyR strain, compared to those of the YN-S strain. The RNA interference (RNAi)-mediated knockdown of UGT201H1 expression diminished the levels of cyflumetofen resistance in YN-CyR mites. The findings additionally revealed that the recombinant UGT201H1 protein plays a role in metabolizing cyflumetofen. Our results also suggested that the aromatic hydrocarbon receptor (AhR) probably regulates the overexpression of the UGT201H1 detoxification gene. CONCLUSION: UGT201H1 is involved in cyflumetofen resistance, and AhR may regulates the overexpression of UGT201H1. These findings provide deeper insights into the molecular mechanisms underlying UGT-mediated metabolic resistance to chemical insecticides. © 2024 Society of Chemical Industry.

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.

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.

A novel target-site mutation (H146Q) outside the ubiquinone binding site of succinate dehydrogenase confers high levels of resistance to cyflumetofen and pyflubumide in Tetranychus urticae.

Mitochondrial electron transfer inhibitors at complex II (METI-II), also referred to as succinate dehydrogenase inhibitors (SDHI), represent a recently developed class of acaricides encompassing cyflumetofen, cyenopyrafen, pyflubumide and cyetpyrafen. Despite their novelty, resistance has already developed in the target pest, Tetranychus urticae. In this study a new mutation, H146Q in a highly conserved region of subunit B of complex II, was identified in a T. urticae population resistant to all METI-IIs. In contrast to previously described mutations, H146Q is located outside the ubiquinone binding site of complex II. Marker-assisted backcrossing of this mutation in a susceptible genetic background validated its association with resistance to cyflumetofen and pyflubumide, but not cyenopyrafen or cyetpyrafen. Biochemical assays and the construction of inhibition curves with isolated mitochondria corroborated this selectivity. In addition, phenotypic effects of H146Q, together with the previously described H258L, were further examined via CRISPR/Cas9 gene editing. Although both mutations were successfully introduced into a susceptible T. urticae population, the H146Q gene editing event was only recovered in individuals already harboring the I260V mutation, known to confer resistance towards cyflumetofen. The combination of H146Q + I260V conferred high resistance levels to all METI-II acaricides with LC50 values over 5000 mg a.i./L for cyflumetofen and pyflubumide. Similarly, the introduction of H258L via gene editing resulted in high resistance levels to all tested acaricides, with extreme LC50 values (>5000 mg a.i./L) for cyenopyrafen and cyetpyrafen, but lower resistance levels for pyflubumide and cyflumetofen. Together, these findings indicate that different mutations result in a different cross-resistance spectrum, probably also reflecting subtle differences in the binding mode of complex II acaricides.

Prey-mediated effects of Mpp51Aa2-producing cotton on longevity and reproduction of Orius majusculus.

Genetically engineered (GE) cotton event MON 88702, producing Mpp51Aa2 (previously mCry51Aa2) from Bacillus thuringiensis (Bt), controls sucking pests, such as Lygus spp. (Hemiptera: Miridae) and thrips (Thysanoptera). Ingesting high doses of the insecticidal protein resulted in adverse effects on life table parameters of beneficial, predatory Orius spp. (Hemiptera: Anthocoridae). This triggered laboratory studies with more realistic food treatments, including different combinations of prey types with and without Bt protein to further characterize risks to this important group of non-target organisms. In this work, exclusive feeding of frozen spider mites (Tetranychus urticae, Acari: Tetranychidae) from Bt cotton confirmed adverse effects on longevity and fecundity of O. majusculus adults. Alternate feeding of Bt protein-containing spider mites and Bt-free Ephestia kuehniella (Lepidoptera: Pyralidae) eggs mitigated effects on longevity, but not on fecundity. When living larvae of Spodoptera littoralis (Lepidoptera: Noctuidae) from Bt cotton were fed to the predators, however, no effects on longevity and reproduction of female O. majusculus were observed, despite the fact that Bt protein concentrations in larvae were almost as high as concentrations in spider mites. When a diverse mix of prey species with various Bt protein concentrations is consumed in the field, it is unlikely that exposure of Orius spp. to Mpp51Aa2 is high enough to exert adverse effects on predator populations. MON 88702 cotton may thus be a valuable tool for integrated management of sucking pests.

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 novel mutation in mitochondrial cytochrome b conferring resistance to bifenazate in two-spotted spider mite Tetranychus urticae Koch (Acarina: Tetranychidae).

BACKGROUND: The two-spotted spider mite Tetranychus urticae causes significant damage to ornamental, cotton, sugarcane and horticultural crops in Australia. It has a long history of developing resistance to many acaricides including bifenazate. A mutation in the conserved cd1- and ef-helices of the Qo pocket of cytochrome b is recognized as the primary mechanism of bifenazate resistance. To investigate the resistance mechanisms against bifenazate in Australian two-spotted spider mite, we sequenced the complete mitochondrion genome of five mite strains including a susceptible and bifenazate-resistant strain. RESULTS: We identified a novel mutation D252N in the G126S background at cytochrome b being the cause of bifenazate resistance in a bifenazate-resistant strain, Bram. We validated the role of this mutation combination by reciprocal crosses between a bifenazate resistant and susceptible strain. By doing these crosses we confirmed the pattern of inheritance was maternal. Additionally, mitochondrial heteroplasmy was not observed by single mite genotyping of the mutations in cytb in a known bifenazate-resistant strain Bram. The phylogenetic analysis with the complete mitochondrion genome sequences revealed that Australian two-spotted spider mite strains are closely related to the green form of T. urticae found in China. CONCLUSIONS: The novel mutation D252N found in the cytochrome b in the G126S background was revealed to be the main cause of bifenazate resistance in the Australian T. urticae strain Bram. © 2024 Society of Chemical Industry.

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.

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.

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.