CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

Polygenic adaptation of a cosmopolitan pest to a novel thermal environment.

The fluctuation in temperature poses a significant challenge for poikilothermic organisms, notably insects, particularly in the context of changing climatic conditions. In insects, temperature adaptation has been driven by polygenes. In addition to genes that directly affect traits (core genes), other genes (peripheral genes) may also play a role in insect temperature adaptation. This study focuses on two peripheral genes, the GRIP and coiled-coil domain containing 2 (GCC2) and karyopherin subunit beta 1 (KPNB1). These genes are differentially expressed at different temperatures in the cosmopolitan pest, Plutella xylostella. GCC2 and KPNB1 in P. xylostella were cloned, and their relative expression patterns were identified. Reduced capacity for thermal adaptation (development, reproduction and response to temperature extremes) in the GCC2-deficient and KPNB1-deficient P. xylostella strains, which were constructed by CRISPR/Cas9 technique. Deletion of the PxGCC2 or PxKPNB1 genes in P. xylostella also had a differential effect on gene expression for many traits including stress resistance, resistance to pesticides, involved in immunity, trehalose metabolism, fatty acid metabolism and so forth. The ability of the moth to adapt to temperature via different pathways is likely to be key to its ability to remain an important pest species under predicted climate change conditions.

Thermal acclimation uncovers a simple genetic basis of adaptation to high temperature in a cosmopolitan pest.

Understanding a population's fitness heterogeneity and genetic basis of thermal adaptation is essential for predicting the responses to global warming. We examined the thermotolerance and genetic adaptation of Plutella xylostella to exposure to hot temperatures. The population fitness parameters of the hot-acclimated DBM strains varied in the thermal environments. Using genome scanning and transcription profiling, we find a number of genes potentially involved in thermal adaptation of DBM. Editing two ABCG transporter genes, PxWhite and PxABCG, confirmed their role in altering cuticle permeability and influencing thermal responses. Our results demonstrate that SNP mutations in genes and changes in gene expression can allow DBM to rapidly adapt to thermal environment. ABCG transporter genes play an important role in thermal adaptation of DBM. This work improves our understanding of genetic adaptation mechanisms of insects to thermal stress and our capacity to predict the effects of rising global temperatures on ectotherms.

Deep learning-based quantification and transcriptomic profiling reveal a methyl jasmonate-mediated glandular trichome formation pathway in Cannabis sativa.

Cannabis glandular trichomes (GTs) are economically and biotechnologically important structures that have a remarkable morphology and capacity to produce, store, and secrete diverse classes of secondary metabolites. However, our understanding of the developmental changes and the underlying molecular processes involved in cannabis GT development is limited. In this study, we developed Cannabis Glandular Trichome Detection Model (CGTDM), a deep learning-based model capable of differentiating and quantifying three types of cannabis GTs with a high degree of efficiency and accuracy. By profiling at eight different time points, we captured dynamic changes in gene expression, phenotypes, and metabolic processes associated with GT development. By integrating weighted gene co-expression network analysis with CGTDM measurements, we established correlations between phenotypic variations in GT traits and the global transcriptome profiles across the developmental gradient. Notably, we identified a module containing methyl jasmonate (MeJA)-responsive genes that significantly correlated with stalked GT density and cannabinoid content during development, suggesting the existence of a MeJA-mediated GT formation pathway. Our findings were further supported by the successful promotion of GT development in cannabis through exogenous MeJA treatment. Importantly, we have identified CsMYC4 as a key transcription factor that positively regulates GT formation via MeJA signaling in cannabis. These findings provide novel tools for GT detection and counting, as well as valuable information for understanding the molecular regulatory mechanism of GT formation, which has the potential to facilitate the molecular breeding, targeted engineering, informed harvest timing, and manipulation of cannabinoid production.

Developmental Shifts in the Microbiome of a Cosmopolitan Pest: Unraveling the Role of Wolbachia and Dominant Bacteria.

Wolbachia bacteria (phylum Proteobacteria) are ubiquitous intracellular parasites of diverse invertebrates. In insects, coevolution has forged mutualistic associations with Wolbachia species, influencing reproduction, immunity, development, pathogen resistance, and overall fitness. However, the impact of Wolbachia on other microbial associates within the insect microbiome, which are crucial for host fitness, remains less explored. The diamondback moth (Plutella xylostella), a major pest of cruciferous vegetables worldwide, harbors the dominant Wolbachia strain plutWB1, known to distort its sex ratio. This study investigated the bacterial community diversity and dynamics across different developmental life stages and Wolbachia infection states in P. xylostella using high-throughput 16S rDNA amplicon sequencing. Proteobacteria and Firmicutes dominated the P. xylostella microbiome regardless of life stage or Wolbachia infection. However, the relative abundance of dominant genera, including an unclassified genus of Enterobacteriaceae, Wolbachia, Carnobacterium, and Delftia tsuruhatensis, displayed significant stage-specific variations. While significant differences in bacterial diversity and composition were observed across life stages, Wolbachia infection had no substantial impact on overall diversity. Nonetheless, relative abundances of specific genera differed between infection states. Notably, Wolbachia exhibited a stable, high relative abundance across all stages and negatively correlated with an unclassified genus of Enterobacteriaceae, Delftia tsuruhatensis, and Carnobacterium. Our findings provide a foundational understanding of the complex interplay between the host, Wolbachia, and the associated microbiome in P. xylostella, paving the way for a deeper understanding of their complex interactions and potential implications for pest control strategies.

Identification of floral volatiles from Fagopyrum esculentum that attract Cotesia vestalis with potentially better biocontrol efficacy against Plutella xylostella.

BACKGROUND: Nectar plants provide extra nourishment for parasitoids, which can utilize floral volatiles to locate nectar-rich flowers. A promising strategy is to screen potential floral species based on the wasps' olfactory preferences for nectar sources, and to ensure their suitability for both natural enemies and targeted pests. Cotesia vestalis (Haliday) is a dominant parasitoid of the oligophagous pest Plutella xylostella, which poses a significant threat to cruciferous vegetables globally. However, the chemical cues in plant-parasitoid complexes mediating Cotesia vestalis to locate nectar food resources and the positive effect of nectar plants on the Cotesia vestalis population are poorly understood. RESULTS: The results showed that Fagopyrum esculentum was the most attractive plant that attracted Cotesia vestalis, not Plutella xylostella in 44 flowering plants from 19 families. 1,2-Diethyl benzene and 1,4-diethyl benzene, identified from the floral volatiles from F. esculentum in full bloom, were found to elicit dose-dependent electrophysiological responses and attract Cotesia vestalis adults, demonstrating their potential as semiochemicals. Moreover, the age-stage, two-sex life table revealed that feeding on nectar food increased the efficacy of Cotesia vestalis adults against Plutella xylostella. CONCLUSION: In summary, the findings provide insights into the chemical ecology of plant-parasitoid complexes and support the potential use of F. esculentum as insectary plants in habitat manipulation against Plutella xylostella by supplying natural nectar food for the Cotesia vestalis population. Our results suggest an attract and reward strategy based on an attractant for Cotesia vestalis to control Plutella xylostella, or the development of volatile-based artificial food for Cotesia vestalis. © 2023 Society of Chemical Industry.

Comparative Metabolomic Analysis Reveals the Differences in Nonvolatile and Volatile Metabolites and Their Quality Characteristics in Beauty Tea with Different Extents of Punctured Leaves by Tea Green Leafhopper.

The fresh leaves were processed into beauty tea from the Camellia sinensis "Jinxuan" cultivar, which were punctured by tea green leafhoppers to different extents. Low-puncturing dry tea (LPDT) exhibited a superior quality. Altogether, 101 and 129 differential metabolites, including tea polyphenols, lipids, and saccharides, were identified from the fresh leaves and dry beauty tea, respectively. Most metabolite levels increased in the fresh leaves punctured by leafhoppers, but the opposite was observed for the dry beauty tea. According to relative odor activity values (rOAVs) and partial least-squares discriminant analysis (PLS-DA), four characteristic volatiles, including linalool, geraniol, benzeneacetaldehyde, and dihydrolinalool, were selected. Mechanical injury to leaves caused by leafhoppers, watery saliva secreted by the leafhopper, and different water contents of the fresh leaves in different puncturing degrees are the possible reasons for the difference in the quality of the beauty tea with different levels of puncturing. Overall, this study identified a wide range of chemicals that are affected by the degrees of leafhopper puncturing.

Effects of Antibiotic Treatment on the Development and Bacterial Community of the Wolbachia-Infected Diamondback Moth.

Based on the important role of antibiotic treatment in the research of the interaction between Wolbachia and insect hosts, this study aimed to identify the most suitable antibiotic and concentration for Wolbachia elimination in the P. xylostella, and to investigate the effect of Wolbachia and antibiotic treatment on the bacterial community of P. xylostella. Our results showed that the Wolbachia-infected strain was plutWB1 of supergroup B in the P. xylostella population collected in Nepal in this study; 1 mg/mL rifampicin could remove Wolbachia infection in P. xylostella after 1 generation of feeding treatment and the toxic effect was relatively low; among the 29 samples of adult P. xylostella in our study (10 WU samples, 10 WA samples, and 9 WI samples), 52.5% of the sequences were of Firmicutes and 47.5% were of Proteobacteria, with the dominant genera being mainly Carnobacterium (46.2%), Enterobacter (10.1%), and Enterococcus (6.2%); Moreover, antibiotic removal of Wolbachia infection in P. xylostella and transfer to normal conditions for 10 generations no longer significantly affected the bacterial community of P. xylostella. This study provides a theoretical basis for the elimination method of Wolbachia in the P. xylostella, as well as a reference for the elimination method of Wolbachia in other Wolbachia-infected insect species, and a basis for the study of the extent and duration of the effect of antibiotic treatment on the bacterial community of the P. xylostella.

Chemical and Quality Analysis of Beauty Tea Processed from Fresh Leaves of Tieguanyin Variety with Different Puncturing Degrees.

Beauty tea with special flavor can be affected by the degree of leafhopper puncturing. The present research adopted widely targeted metabolomics to analyze the characteristic metabolites of fresh tea leaves and beauty tea with different degrees of leafhopper puncturing. Low-puncturing beauty tea (LPBT) exhibited a superior quality. Altogether, 95 and 65 differential metabolites, including tea polyphenols, saccharides, and lipids, were identified from fresh leaves and beauty tea, respectively. The partial least squares regression (PLSR) analysis results showed that isomaltulose, theaflavic acid, and ellagic acid, may be the characteristic metabolites that form the different taste outlines of beauty tea. Based on odor activity values (OAVs) and partial least squares discriminant analysis (PLS-DA), dihydrolinalool and cis-linalool oxide were identified as characteristic volatile components, which may be essential for the formation of the different aroma characteristic of beauty tea. The results provide a theoretical basis for selecting raw materials, performing quality research, and developing beauty tea industrially.

Development of an efficient insecticide substrate and inhibitor screening system of insect P450s using fission yeast.

Metabolic resistance is one of the most frequent mechanisms of insecticide resistance, characterized by an increased expression of several important enzymes and transporters, especially cytochrome P450s (CYPs). Due to the large number of P450s in pests, determining the precise relationship between these enzymes and the insecticide substrates is a challenge. Herein, we developed a luminescence-based screening system for efficient identification of insecticide substrates and insect P450 inhibitors. We recombinantly expressed Bemisia tabaci CYP6CM1vQ (Bt CYP6CM1vQ) in the fission yeast Schizosaccharomyces pombe and subsequently permeabilized the yeast cells to convert them into "enzyme bags". We exploited these enzyme bags to screen the activity of twelve luciferin substrates and identified Luciferin-FEE as the optimal competing probe that was further used to characterize the metabolism of eight candidate commercial insecticides. Among them, Bt CYP6CM1vQ exhibited notable activity against pymetrozine and imidacloprid. Their binding modes were predicted by homology modeling and molecular docking, revealing the mechanisms of the metabolism. We also tested the inhibitory effect of eight known P450 inhibitors using our system and identified letrozole and 1-benzylimidazole as showing significant activity against Bt CYP6CM1vQ, with IC50 values of 23.74 µM and 1.30 µM, respectively. Their potential to be developed as an insecticide synergist was further proven by an in vitro toxicity assay using imidacloprid-resistant Bemisia tabaci. Overall, our luciferin-based enzyme bag method is capable of providing a robust and efficient screening of insect P450 substrates and, more importantly, inhibitors to overcome the resistance.

Gut microbiota assemblages of generalist predators are driven by local- and landscape-scale factors.

The gut microbiomes of arthropods have significant impact on key physiological functions such as nutrition, reproduction, behavior, and health. Spiders are diverse and numerically dominant predators in crop fields where they are potentially important regulators of pests. Harnessing spiders to control agricultural pests is likely to be supported by an understanding of their gut microbiomes, and the environmental drivers shaping microbiome assemblages. This study aimed to deciphering the gut microbiome assembly of these invertebrate predators and elucidating potential implications of key environmental constraints in this process. Here, we used high-throughput sequencing to examine for the first time how the assemblages of bacteria in the gut of spiders are shaped by environmental variables. Local drivers of microbiome composition were globally-relevant input use system (organic production vs. conventional practice), and crop identity (Chinese cabbage vs. cauliflower). Landscape-scale factors, proportion of forest and grassland, compositional diversity, and habitat edge density, also strongly affected gut microbiota. Specific bacterial taxa were enriched in gut of spiders sampled from different settings and seasons. These findings provide a comprehensive insight into composition and plasticity of spider gut microbiota. Understanding the temporal responses of specific microbiota could lead to innovative strategies development for boosting biological control services of predators.

Clip-SP1 cleavage activates downstream prophenoloxidase activating protease (PAP) in Plutella xylostella.

Melanization is a component of the humoral immune defense of insects and is induced by serine protease-mediated phenoloxidase (PO) catalysis. Prophenoloxidase (PPO) in the midgut of Plutella xylostella is activated by the CLIP domain serine protease (clip-SP) in response to Bacillus thuringiensis (Bt) infection, but the detailed signaling cascade following this activation is unknown. Here, we report that activation of clip-SP enhances PO activity in the P. xylostella midgut by cleaving three downstream PPO-activating proteases (PAPs). First, the expression level of clip-SP1 was increased in the midgut after Bt8010 infection of P. xylostella. Then, purified recombinant clip-SP1 was able to activate three PAPs - PAPa, PAPb and PAP3 - which in turn enhanced their PO activity in the hemolymph. Furthermore, clip-SP1 showed a dominant effect on PO activity compared to the individual PAPs. Our results indicate that Bt infection induces the expression of clip-SP1, which is upstream of a signaling cascade, to efficiently activate PO catalysis and mediate melanization in the midgut of P. xylostella. And it provides a basis for studying the complex PPO regulatory system in the midgut during Bt infection.

A very long-chain fatty acid enzyme gene, PxHacd2 affects the temperature adaptability of a cosmopolitan insect by altering epidermal permeability.

Temperature fluctuations pose challenges to poikilotherms, such as insects, especially under climate change conditions. Very long-chain fatty acids (VLCFAs) form important structural components of membranes and epidermal surfaces, so play important roles in adaptation to temperature stress in plants. It has been unclear whether VLCFAs are involved in epidermis formation and thermal resistance in insects. In this study, we focused on the 3-hydroxy acyl-CoA dehydratase 2 (Hacd2), an important enzyme in the synthesis pathway of VLCFAs, in a cosmopolitan pest, the diamondback moth, Plutella xylostella. Hacd2 was cloned from P. xylostella and the relative expression pattern was identified. Epidermal permeability increased with the decreased VLCFAs in the Hacd2-deficient P. xylostella strain, which was constructed by using the CRISPR/Cas9 system. Survival and fecundity of the Hacd2-deficient strain was significantly lower than that of the wildtype strain when subject to desiccating environmental stress. Hacd2 mediates thermal adaptability in P. xylostella by changing epidermal permeability so is likely to be key to its remaining a major pest species under predicted climate change conditions.

Enhanced resistance to Bacillus thuringiensis Cry1Ac toxin mediated by the activation of prophenoloxidase in a cosmopolitan pest.

Plutella xylostella has evolved resistance to Bacillus thuringiensis Cry1Ac toxin over a long evolutionary period. Enhanced immune response is an important factor in insect resistance to a variety of insecticides, and whether phenoloxidase (PO), an immune protein, is involved in resistance to Cry1Ac toxin in P. xylostella remains unclear. Here, spatial and temporal expression patterns showed that prophenoloxidase (PxPPO1 and PxPPO2) in the Cry1S1000-resistant strain was more highly expressed in eggs, 4th instar, head, and hemolymph than those in G88-susceptible strain. The results of PO activity analysis showed that after treatment with Cry1Ac toxin PO activity was about 3 times higher than that before treatment. Furthermore, knockout of PxPPO1 and PxPPO2 significantly increased the susceptibility to Cry1Ac toxin. These findings were further supported by the knockdown of Clip-SPH2, a negative regulator of PO, which resulted in increased PxPPO1 and PxPPO2 expression and Cry1Ac susceptibility in the Cry1S1000-resistant strain. Finally, the synergistic effect of quercetin showed that larval survival decreased from 100 % to <20 % compared to the control group. This study will provide a theoretical basis for the analysis of immune-related genes (PO) genes involved in the resistance mechanism and pest control of P. xylostella.

Analysis of the Effect of Plutella xylostella Polycalin and ABCC2 Transporter on Cry1Ac Susceptibility by CRISPR/Cas9-Mediated Knockout.

Many insects, including the Plutella xylostella (L.), have developed varying degrees of resistance to many insecticides, including Bacillus thuringiensis (Bt) toxins, the bioinsecticides derived from Bt. The polycalin protein is one of the potential receptors for Bt toxins, and previous studies have confirmed that the Cry1Ac toxin can bind to the polycalin protein of P. xylostella, but whether polycalin is associated with the resistance of Bt toxins remains controversial. In this study, we compared the midgut of larvae from Cry1Ac-susceptible and -resistant strains, and found that the expression of the Pxpolycalin gene was largely reduced in the midgut of the resistant strains. Moreover, the spatial and temporal expression patterns of Pxpolycalin showed that it was mainly expressed in the larval stage and midgut tissue. However, genetic linkage experiments showed that the Pxpolycalin gene and its transcript level were not linked to Cry1Ac resistance, whereas both the PxABCC2 gene and its transcript levels were linked to Cry1Ac resistance. The larvae fed on a diet containing the Cry1Ac toxin showed no significant change in the expression of the Pxpolycalin gene in a short term. Furthermore, the knockout of polycalin and ATP-binding cassette transporter subfamily C2 (ABCC2) genes separately by CRISPR/Cas9 technology resulted in resistance to decreased susceptibility to Cry1Ac toxin. Our results provide new insights into the potential role of polycalin and ABCC2 proteins in Cry1Ac resistance and the mechanism underlying the resistance of insects to Bt toxins.

Expression and purification of snustorr snarlik protein from Plutella xylostella.

Snustorr snarlik (Snsl) is a type of extracellular protein essential for insect cuticle formation and insect survival, but is absent in mammals, making it a potential selective target for pest control. Here, we successfully expressed and purified the Snsl protein of Plutella xylostella in Escherichia coli. Two truncated forms of Snsl protein, Snsl 16-119 and Snsl 16-159, were expressed as a maltose-binding protein (MBP) fusion protein and purified to a purity above 90% after a five-step purification protocol. Snsl 16-119, forming stable monomer in solution, was crystallized, and the crystal was diffracted to a resolution of ∼10 Å. Snsl 16-159, forming an equilibrium between monomer and octamer in solution, was shown to form rod-shaped particles on negative staining electron-microscopy images. Our results lay a foundation for the determination of the structure of Snsl, which would improve our understanding of the molecular mechanism of cuticle formation and related pesticide resistance and provide a template for structure-based insecticide design.

Diversity of Wolbachia infection and its influence on mitochondrial DNA variation in the diamondback moth, Plutella xylostella.

Plutella xylostella is a pest that severely damages cruciferous vegetables worldwide and has been shown to be infected with the maternally inherited bacteria Wolbachia, with the main infected strain was plutWB1. In this study, we performed a large-scale global sampling of P. xylostella and amplified 3 mtDNA genes of P. xylostella and 6 Wolbachia genes to analyze the infection status, diversity of Wolbachia in P. xylostella, and its effect on mtDNA variation in P. xylostella. This study provides a conservative estimate of Wolbachia infection rates in P. xylostella, which was found to be 7% (104/1440). The ST 108 (plutWB1) was shared among butterfly species and the moth species P. xylostella, revealing that Wolbachia strain plutWB1 acquisition in P. xylostella may be through horizontal transmission. The Parafit analyses indicated a significant association between Wolbachia and Wolbachia-infected P. xylostella individuals, and individuals infected with plutWB1 tended to cluster in the basal positions of the phylogenetic tree based on the mtDNA data. Additionally, Wolbachia infections were associated with increased mtDNA polymorphism in the infected P. xylostella population. These data suggest that Wolbachia endosymbionts may have a potential effect on mtDNA variation of P. xylostella.

Oxygen stress on age-stage, two-sex life tables and transcriptomic response of diamondback moth (Plutella xylostella).

Elucidating the genetic basis of local adaption is one of the important tasks in evolutionary biology. The Qinghai-Tibet Plateau has the highest biodiversity for an extreme environment worldwide, and provides an ideal natural laboratory to study adaptive evolution. The diamondback moth (DBM), Plutella xylostella, is one of the most devastating pests of the global Brassica industry. A highly heterozygous genome of this pest has facilitated its adaptation to a variety of complex environments, and so provides an ideal model to study fast adaptation. We conducted a pilot study combining RNA-seq with an age-stage, two-sex life table to study the effects of oxygen deprivation on DBM. The developmental periods of all instars were significantly shorter in the hypoxic environment. We compared the transcriptomes of DBM from Fuzhou, Fujian (low-altitude) and Lhasa, Tibet (high-altitude) under hypoxia treatment in a hypoxic chamber. Some DEGs are enriched in pathways associated with DNA replication, such as DNA repair, nucleotide excision repair, base excision repair, mismatch repair and homologous recombination. The pathways with significant changes were associated with metabolism process and cell development. Thus, we assumed that insects could adapt to different environments by regulating their metabolism. Our findings indicated that although adaptive mechanisms to hypoxia in different DBM strains could be similar, DBM individuals from Tibet had superior tolerance to hypoxia compared with those of Fuzhou. Local adaptation of the Tibetan colony was assumed to be responsible for this difference. Our research suggests novel mechanisms of insect responses to hypoxia stress.

The Potential Role of the Methionine Aminopeptidase Gene PxMetAP1 in a Cosmopolitan Pest for Bacillus thuringiensis Toxin Tolerance.

Methionine aminopeptidases (MetAPs) catalyze the cleavage of the N-terminal initiator methionine (iMet) in new peptide chains and arylamides, which is essential for protein and peptide synthesis. MetAP is differentially expressed in two diamondback moth (DBM; Plutella xylostella) strains: the G88 susceptible strain and the Cry1S1000 strain, which are resistant to the Bt toxin Cry1Ac, implicating that MetAP expression might be associated with Bt resistance. In this study, we identified and cloned a MetAP gene from DBMs, named PxMetAP1, which has a CDS of 1140 bp and encodes a 379 amino acid protein. The relative expression of PxMetAP1 was found to be ~2.2-fold lower in the Cry1S1000 strain compared to that in the G88 strain. PxMetAP1 presents a stage- and tissue-specific expression pattern, with higher levels in the eggs, adults, integument, and fatbody of DBMs. The linkage between PxMetAP1 and Cry1Ac resistance is verified by genetic linkage analysis. The knockout of PxMetAP1 in G88 by CRISPR/Cas9 leads to a ~5.6-fold decrease in sensitivity to the Cry1Ac toxin, further supporting the association between the PxMetAP1 gene and Bt tolerance. Our research sheds light on the role of MetAP genes in the development of Bt tolerance in P. xylostella and enriches the knowledge for the management of such a cosmopolitan pest.

Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth.

The CRISPR/Cas9 system is an efficient tool for reverse genetics validation, and the application of this system in the cell lines provides a new perspective on target gene analysis for the development of biotechnology tools. However, in the cell lines of diamondback moth, Plutella xylostella, the integrity of the CRISPR/Cas9 system and the utilization of this cell lines still need to be improved to ensure the application of the system. Here, we stabilize the transfection efficiency of the P. xylostella cell lines at different passages at about 60% by trying different transfection reagents and adjusting the transfection method. For Cas9 expression in the CRIPSPR/Cas9 system, we identified a strong endogenous promoter: the 217-2 promoter. The dual-luciferase and EGFP reporter assay demonstrated that it has a driving efficiency close to that of the IE1 promoter. We constructed pB-Cas9-Neo plasmid and pU6-sgRNA plasmid for CRISPR/Cas9 system and subsequent cell screening. The feasibility of the CRISPR/Cas9 system in P. xylostella cell lines was verified by knocking out endogenous and exogenous genes. Finally, we generated a transgenic Cas9 cell line of P. xylostella that would benefit future exploitation, such as knock-in and multi-threaded editing. Our works provides the validity of the CRISPR/Cas9 system in the P. xylostella cell lines and lays the foundation for further genetic and molecular studies on insects, particularly favoring gene function analysis.