Huanglongbing (HLB), a devastating citrus disease with no known cure, necessitates early on-site detection for effective disease management. However, current diagnostic methods remain limited by suboptimal detection rates, biosafety concerns, and lack of portability. This study presents the HLB-OSD system, a field-ready, portable heating and fluorometric device for the rapid, on-site identification of Candidatus Liberibacter asiaticus (CLas), the causative agent of HLB. The system integrates cross-priming amplification with highly specific molecular beacon probes in a single-tube, isothermal (63 °C) assay, enabling the direct detection of CLas from crude citrus leaf DNA extracts within 25-40 min without complex instrumentation. With 8.3 copy/μL detection limit and no cross-reactivity, the assay showed 94.24% concordance with qPCR (180/191). Field validation in Yongxiu county, a key HLB containment buffer zone in northern Jiangxi Province, confirmed a 9.0% (44/487) infection rate. Our on-site system provides lab-accurate detection, enabling timely monitoring of HLB for citrus protection.

The diamondback moth (Plutella xylostella), a globally destructive pest, has Brassicaceae as its long-term co-evolved host and can also utilize Fabaceae as an alternative field host. The primary differential factor between these plant families is glucosinolates (GLs). Conventional transcriptome data revealed high midgut expression of glucosinolate sulfatases (GSSs) in response to glucosinolates. However, due to the inability of conventional transcriptomics to resolve cellular heterogeneity, it remained unclear which specific cell types express GSSs and whether exposure to different field hosts triggers distinct differentiation fates in these cells. To address these questions, we constructed a high-resolution atlas of 28,451 midgut cells from larvae reared on radish and pea leaves. Marker gene-based clustering identified 16 distinct cell types, including enterocytes (ECs), enteroendocrine cells (EEs), and intestinal stem cells (ISCs). ScRNA-seq and qRT-PCR analyses revealed host-dependent differential expression patterns of three GSS genes (GSS1-GSS3) among ECs, EEs, and ISCs. Notably, elevated GSS3 protein levels in radish-fed larvae were confirmed by Western blot (WB) and Immunohistochemistry (IHC), while GSS1 and GSS2 exhibited more variable expression patterns. Pseudo-time trajectory analysis further demonstrated that all three GSSs followed differentiation pathways from ISCs into EEs or ECs, but dietary conditions led to distinct differentiation trajectories. This study elucidates the diet-dependent regulatory landscape of GSSs in P. xylostella and delineates the differentiation trajectory of GSS-expressing cells.

The Asian citrus psyllid (ACP), Diaphorina citri, serves as the primary vector for Candidatus Liberibacter asiaticus (CLas), the pathogen responsible for citrus Huanglongbing (HLB). D. citri modulates the expression of its key proteins in response to CLas infection. Previous research has revealed that CLas infection significantly alters the expression levels of E3 ubiquitin ligases in D. citri; however, the specific functions of these E3 ligases remain largely uncharacterized. In this study, a total of 11 E3 ubiquitin ligases were identified from the proteomics database of D. citri, among which E3 ubiquitin ligase RNF115 was significantly upregulated following CLas infection. RING finger protein 115 (RNF115) consists of 156 amino acids and contains a RING finger domain at its N-terminus. Silencing RNF115 via RNA interference (RNAi) and injecting the inhibitor disulfiram, which targets RNF115, significantly increased CLas bacterial content in D. citri. In contrast, injection of recombinant RNF115 protein markedly inhibited CLas bacterial proliferation. Furthermore, interaction between RNF115 and D. citri histone H1 was confirmed using yeast 2-hybrid assay, pull-down experiments and molecular docking analysis. Knockdown of histone H1 via RNAi significantly reduced CLas bacterial content, whereas injection of recombinant histone H1 protein led to an increase in CLas content within D. citri. These findings suggest that CLas infection may induce an upregulation of RNF115 expression in D. citri, leading to subsequent interactions with histone H1 that facilitate the ubiquitination of histone H1, ultimately resulting in reduced expression levels and inhibiting CLas proliferation within D. citri.

The Asian citrus psyllid (Diaphorina citri) serves as the primary vector of the bacterium Candidatus Liberibacter asiaticus (CLas), which is responsible for causing citrus Huanglongbing. Neonicotinoid pesticides such as thiamethoxam are frequently utilized for D. citri management worldwide, but their use have resulted in considerable development of insecticide resistance within D. citri populations. Recent reports have revealed that chemosensory proteins (CSPs)-mediated resistance represents a novel insecticide resistance mechanism in insects. In this finding, we found that exposure to thiamethoxam at different concentrations resulted in significant upregulation of transcriptional levels of DcitCSP8 by 2.41, 2.24 and 1.74-fold respectively. The expression profiles among different tissues showed that DcitCSP8 highly occurred in the legs and wings, and its expression being significantly induced by thiamethoxam with 2.40-fold increase specifically in the legs. The purified recombinant protein DcitCSP8, derived from Escherichia coli expression, present strong in vitro binding affinity (Ki = 4.74 mu M) to thiamethoxam. Furthermore, the nanocarrier star polycation (SPc) was used to enhance silencing efficiency, resulting in a significant reduction of DcitCSP8 by 85.37% and 91.80% at 24 and 48 h post application of SPc + ds DcitCSP8. Knockdown of DcitCSP8 transcription significantly increased the susceptibility of D. citri adults in response to thiamethoxam, while reinjection of DcitCSP8 protein could restore the resistance in DcitCSP8silenced individuals. Our findings highlight the association between DcitCSP8 and thiamethoxam resistance via high binding affinity that mitigates toxicity in D. citri, shedding light on CSP-mediated insecticide resistance and potentially contributing to the development of novel strategies targeting CSPs for managing resistance in D. citri.

BACKGROUND:The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), a notorious pest accountable for citrus Huanglongbing (HLB), has developed high levels of resistance to a variety of commercial insecticides. RNA interference (RNAi) is a promising and effective approach for pest management. The first step for effective pest control by RNAi is the development of effective and reliable target genes. Tyrosine hydroxylase (TH) plays important roles in immunity, cuticle tanning and pigmentation. RESULTS:In this study, we focused on the functions of TH during the molting of D. citri, and whether TH could be employed as a target for the pest control. The D. citri tyrosine hydroxylase (DcTH) complementary DNA (cDNA) sequence was identified from the genome database of D. citri and cloned. Its open reading frame (ORF) was 1782 bp, encoding a protein consisting of 593 amino acids. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that DcTH was highly expressed in the wing, and its expression level was significantly higher in the early stages of each instar compared with the late stages. Additionally, the expression level of DcTH was higher in the early stage of molting than in the late stage, while TH activity presented the opposite trend. The 3-iodo-tyrosine (3-IT) treatment or double-stranded RNA (dsRNA) injection significantly decreased the expression level of DcTH and TH activity, and augmented the mortality of D. citri. The silencing of DcTH by RNAi significantly disrupted the metamorphosis to the adult stage and reduced the content of 3,4-dihydroxyphenylalanine (DOPA) in D. citri. Transmission electron microscopy (TEM) analysis indicated that the synthesis of the exocuticle was suppressed following the inhibition of DcTH. Furthermore, the down-regulation of DcTH significantly inhibited the expression levels of three cuticle tanning pathway genes. Additionally, a drop RNAi method targeting DcTH based on star polycation (SPc) nanoparticles-wrapped dsRNA significantly increased the mortality of D. citri. CONCLUSIONS:These results imply that DcTH plays a crucial role in exocuticle tanning in D. citri, and it could be an ideal target for RNAi-mediated control of D. citri. © 2025 Society of Chemical Industry.

The root-associated microbiome significantly influences plant health and pest resistance, yet the temporal dynamics of its compositional and functional change in response to Ectropis grisescens Warren (Lepidoptera: Geometridae) infestation remain largely unexplored. The study took samples of leaves, roots, and rhizosphere soil at different times after the plants were attacked by E. grisescens. These samples were analyzed using transcriptomic and high-throughput sequencing of 16S rRNA techniques. The goal was to understand how the plant’s defense mechanisms and the microbial community around the roots changed after the attack. Additionally, bacterial feedback assays were conducted to evaluate the effects of selected microbial strains on plant growth and pest defense responses. By conducting 16S rRNA sequencing on the collected soil samples, we found significant shifts in bacterial communities by the seventh day, suggesting a lag in community adaptation. Transcriptomic analysis revealed that E. grisescens attack induced reprogramming of the tea root transcriptome, upregulating genes related to defensive pathways such as phenylpropanoid and flavonoid biosynthesis. Metagenomic data indicated functional changes in the rhizosphere microbiome, with enrichment in genes linked to metabolic pathways and nitrogen cycling. Network analysis showed a reorganization of core microbial members, favoring nitrogen-fixing bacteria like Burkholderia species. Bacterial feedback assays confirmed that selected strains, notably Burkholderia cepacia strain ABC4 (T1) and a nine-strain consortium (T5), enhanced plant growth and defense responses, including elevated levels of flavonoids, polyphenols, caffeine, jasmonic acid, and increased peroxidase (POD) and superoxide dismutase (SOD) activities. This study emphasizes the potential of utilizing root-associated microbial communities for sustainable pest management in tea cultivation, thereby enhancing resilience in tea crops while maintaining ecosystem balance.

Rubisco activase (RCA) is an ATP-dependent enzyme that plays a crucial role in plant stress responses by regulating the catalytic activity of Rubisco. However, the alternative splicing and functional characteristics of the RCA gene exhibit notable species-specific diversity. The variable splice forms and functions of the RCA gene in mangrove plants remain poorly understood. Herein, we cloned the RCA cDNA in the leaves of mangrove plant Kandelia candel (L.) in response to combined flooding and salinity stress, and performed systematic expression analysis and functional validation. Our results demonstrated that the RCA gene undergoes alternative splicing to produce two isoforms, designated as KcRCAl (GenBank accession: MG492021) and KcRCAs (GenBank accession: MG492022), respectively. The KcRCAl encodes a 440-amino acid protein (42.49 kDa) belonging to the β-isoforms, while KcRCAs encodes a 474-amino acid protein (46.10 kDa) classified as the α-isoforms. Moreover, protein structure analysis revealed that both isoforms contain phosphorylation and lysine acetylation modification sites. Phylogenetic analysis indicated that KcRCA shares the closest evolutionary relationship with RCA from Cicer arietinum (chickpea) and Durio zibethinus (durian). Furthermore, RT-qPCR analysis revealed that the expression levels of KcRCAl and KcRCAs were significantly upregulated in K. Candel leaves under the combined stress condition. The following functional validation studies in transgenic Arabidopsis demonstrated that overexpression of the KcRCA cDNA enhances the plant’s tolerance to resist flooding and salinity stress while improving antioxidant capacity and increasing RCA and Rubisco activity, thereby maintaining photosynthetic efficiency under combined flooding and salinity stress. Our study not only provides new experimental evidence for understanding the molecular mechanisms of plant flooding and salinity stress, but also offers theoretical foundations for breeding flooding- and salinity-tolerant crops.

Plants perceive proteins from insect-derived oral secretion (OS) and regulate the classical endogenous hormone jasmonic acid to resist insects, but the role of abscisic acid (ABA) in this process is poorly understood. In this study, we used the specialist herbivorous caterpillar Plutella xylostella and cruciferous plants as a model to investigate how the ABA hormone responds to the OS and its core peptide from the insect. Through proteomics and Western blotting analysis, glucosinolate sulfatase 1 (GSS1) was identified in OS. Yeast library screening revealed that GSS1 and its 28-amino-acid core peptide (GSS1-P1) interact with ABA biosynthetic enzyme ABA1. Arabidopsis overexpressing GSS1 and plants treated with synthetic GSS1-P1 showed elevated ABA levels. Transcriptome analysis and RT-qPCR confirmed that GSS1-P1 upregulates WRKY18 and ABA1 expression, modulating ABA production. Both GSS1-P1 application and optimal ABA concentrations enhanced plant resistance to herbivory. Our study shows that GSS1 and its peptide stimulate ABA production, boosting plant-insect resistance and highlighting ABA's potential role in pest-stress response.

Short-chain fatty acids (SCFAs) are the primary energy source of colonic epithelial cells, but oral SCFAs are digested, absorbed, or degraded before reaching the colon. The acylated starch with SCFAs can be fermented and release specific SCFAs under the action of colonic intestinal microbiota. This review first introduces the preparation method, reaction mechanism, and substitution factors. Second, the structure, physical and chemical properties, in vitro function, and mechanism of acylated starch were expounded. Finally, the application of acylated starch in foods is introduced, and its safety is evaluated, providing a basis for the further development of acylated starch-based foods. The acylated starch obtained by different acylation types and preparation methods is different in particle, molecular, and crystal structures, leading to changes in the function and physicochemical properties. Meanwhile, acylated starch has the functional potential of targeted delivery of SCFAs to the colon, which can increase SCFAs in feces and intestine, selectively regulate the intestinal microbiota, and produce a prebiotic effect conducive to host health. The safety of acetylated starch has been supported by relevant studies, which have been widely used in various food fields and have great potential in the food industry.

Hydroxyl radical (·OH) scavenging capacity (HOSC) estimation is essential for evaluating antioxidants, natural extracts, or drugs against clinical diseases. While nanozymes offer advantages in related applications, they still face limitations in activity and selectivity. In response, this work showcases the fabrication of laminarin-modulated osmium (laminarin-Os) nanoclusters (1.45 ± 0.05 nm), functioning as peroxidase-like nanozymes within a colorimetric assay tailored for rational HOSC estimation. This study validates both the characterization and remarkable stability of laminarin-Os. By leveraging the abundant surface negative charges of laminarin-Os and the surface hydroxyls of laminarin, oxidation reactions are facilitated, augmenting laminarin-Os’s affinity for 3,3′,5,5′-tetramethylbenzidine (TMB) (KM = 0.04 mM). This enables the laminarin-Os-based colorimetric assay to respond to ·OH more effectively than citrate-, albumin-, or other polysaccharides-based Os. In addition, experimental results also validate the selective peroxidase-like behavior of laminarin-Os under acidic conditions. Antioxidants like ascorbic acid, glutathione, tannic acid, and cysteine inhibit absorbance at 652 nm in the colorimetric platform using laminarin-Os’s peroxidase-like activity. Compared with commercial kits, this assay demonstrates superior sensitivity (e.g., responds to ascorbic acid 0.01–0.075 mM, glutathione 1–15 µg/mL, tannic acid 0.5–5 µM, and monoammonium glycyrrhizinate cysteine 1.06–10.63 µM) and HOSC testing for glutathione, tannic acid, and monoammonium glycyrrhizinate cysteine. Overall, this study introduces a novel Os nanozyme with exceptional TMB affinity and ·OH selectivity, paving the way for HOSC estimation in biomedical research, pharmaceutical analysis, drug quality control, and beyond.

α-Glucosidase (α-Glu) is implicated in the progression and pathogenesis of type II diabetes (T2D). In this study, we developed a rapid colorimetric technique using platinum nanoparticles stabilized by chitosan (Ch-PtNPs) to detect α-Glu activity and its inhibitor. The Ch-PtNPs facilitate the conversion of 3,3′,5,5′-tetramethylbenzidine (TMB) into oxidized TMB (oxTMB) in the presence of dissolved O2. The catalytic hydrolysis of 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) by α-Glu produces ascorbic acid (AA), which reduces oxTMB to TMB, leading to the fading of the blue color. However, the presence of α-Glu inhibitors (AGIs) hinders the generation of AA, allowing Ch-PtNPs to re-oxidize colorless TMB back to blue oxTMB. This unique phenomenon enables the colorimetric detection of α-Glu activity and AGIs. The linear range for α-Glu was found to be 0.1–1.0 U mL−1 and the detection limit was 0.026 U mL−1. Additionally, the half-maximal inhibition value (IC50) for acarbose, an α-Glu inhibitor, was calculated to be 0.4769 mM. Excitingly, this sensing platform successfully detected α-Glu activity in human serum samples and effectively screened AGIs. These promising findings highlight the potential application of the proposed strategy in clinical diabetes diagnosis and drug discovery.

In this study, four types of maize starch with different amylose contents (3 %, 25 %, 40 %, and 70 %) were used to prepare butyrylated starches. Based on amylose contents, the influence of butyryl group distribution on the structure, thermal and digestive properties of butyrylated maize starch was investigated. The butyrylation reaction mainly substituted butyryl groups on amylose, and the butyryl groups were most easily substituted for the hydroxyl group at the C6 position. The degree of substitution of butyrylated starch reached its maximum when the amylose content was 40 %, and the degree of substitution did not correlate linearly with the amylose content. The butyrylation reaction increased the surface roughness, decreased the crystallinity, enthalpy value and molecular weight of native starch granules, resulting in a decrease in the degree of internal order of the starch and inducing the rearrangement of the amylose molecular chains in the amorphous region of the starch. The combination of the amylose content and the substitution of butyryl groups on amylose affected the digestibility of starch and ultimately increased its resistance. The Pearson correlation coefficient further confirmed the correlation between the distribution of butyryl groups and the structure and properties of butyrylated starch.

BACKGROUND: The insect cuticle consists of chitin fibers and a protein matrix, which plays an important role in protecting the body from invasion of various pathogens and prevents water loss. Periodic synthesis and degradation of the cuticle is required for the growth and development of insects. Key genes involved in cuticle formation have long been considered a potential target for pest control. RESULTS: In this study, a member of the RR-2 subfamily of cuticular protein 8 (DcCP8) was identified from the Diaphorina citri genome database. Immunofluorescence analysis suggested that DcCP8 was mainly located in the Diaphorina citri exocuticle and can be induced to up-regulate 12 h following 20-hydroxyecdysone (20E) treatment. Silencing of DcCP8 by RNA interference (RNAi) significantly disrupted the metamorphosis to the adult stage, and improved the permeability of the cuticle. Transmission electron microscopy (TEM) analysis revealed that the synthesis of the exocuticle was impressed after silencing of DcCP8. Furthermore, the recombinant DcCP8 protein exhibited chitin-binding properties in vitro, down-regulation of DcCP8 significantly inhibited expression levels of chitin metabolism-related genes. Additionally, a sprayable RNAi method targeting DcCP8 based on star polycation (SPc) nanoparticles-wrapped double-stranded RNA (dsRNA) significantly increased Diaphorina citri mortality. Transcriptome sequencing further confirmed that genes associated with the endocytic pathway and immune response were up-regulated in Diaphorina citri after SPc treatment. CONCLUSIONS: The current study indicated that DcCP8 is critical for the formation of Diaphorina citri exocuticles, and lays a foundation for Diaphorina citri control based on large-scale dsRNA nanoparticles. (c) 2024 Society of Chemical Industry.

Flupyrimin (FLP) is a novel class of insecticide acting on insect nicotinic acetylcholine receptor (nAChR) and shows robust insecticidal activity. However, the toxicological effects of FLP on Spodoptera litura have not been revealed. In this study, the results showed that the larval survival rate decreased significantly with increasing concentration of FLP. The hematoxylin-eosin (HE) staining showed that FLP exposure damages the structure of the larval midgut. Additionally, FLP treatments significantly increased the activities of detoxification (GST and CarE) and digestive (alpha-Amylase and Trypsin) enzymes and reduced lipase activity. Transcriptome sequencing identified 855, 1493 and 735 differentially expressed genes (DEGs) at 12 h, 24 h and 48 h after exposure to 3 mM FLP, respectively. Gene function enrichment analysis revealed that DEGs were mainly related to fatty acid metabolic, protein processing in the endoplasmic reticulum and drug metabolism-cytochrome P450. The DEGs associated with food digestion and detoxification was validated by reverse-transcription quantitative PCR (RTqPCR). Furthermore, a total of fifteen energy-related metabolites were identified, among which thirteen metabolisms were significantly influenced after FLP treatment based on 1 H NMR-based metabolome analysis, including tyrosine, glucose, trehalose, malate, threonine, proline, glycine, lysine, citrate, alanine, lactate, valine, and leucine. Taken together, these results provide useful information for revealing the toxicological effect of FLP against S. litura. .

Oral cancer is a disease with high morbidity and mortality worldwide and greatly impacts the quality of life, especially in patients with advanced stages. Photodynamic therapy (PDT) is one of the most effective clinical treatments for oral cancers. However, most clinically applied photosensitizers have several deficiencies, including oxygen dependence, poor aqueous solubility, and a lack of tumor-targeting ability. Herein, the carrier-free multifunctional Sorafenib (Sor), chlorin e6 (Ce6), and Fe3+ self-assembly co-delivery nanoparticles (Sor-Ce6 NPs) were constructed via combining a ferroptosis inducer Sor and a photosensitizer Ce6 for synergetic therapy. The as-synthesized Sor-Ce6 NPs presented excellent colloidal stability and water dispersity with good in vivo tumor-targeting ability. More significantly, the low dose of Sor-Ce6 NPs had little dark toxicity but produced significantly enhanced ROS and supplied O2 sustainably to increase phototoxicity through ferroptosis pathway. Notably, the Sor-Ce6 NPs showed significantly higher in vitro and in vivo anti-tumor efficacy than the Sor/Ce6 mixture due to the improvement of cellular uptake and the incorporation of foreign Fe ions in the system, which also confer the T1 magnetic resonance-guided imaging ability to the formed Sor-Ce6 NPs. Our study demonstrates a promising self-assembled strategy for overcoming hypoxia-related PDT resistance for oral cancer treatment.

The abundance of molecules on early Earth likely enabled a wide range of prebiotic chemistry, with peptides playing a key role in the development of early life forms and the evolution of metabolic pathways. Among peptides, those with enzyme-like activities occupy a unique position between peptides and enzymes, combining both structural flexibility and catalytic functionality. However, their full potential remains largely untapped. Further exploration of these enzyme-like peptides at the nanoscale could provide valuable insights into modern nanotechnology, biomedicine, and even the origins of life. Hence, this review introduces the groundbreaking concept of “peptide nanozymes (PepNzymes)”, which includes single peptides exhibiting enzyme-like activities, peptide-based nanostructures with enzyme-like activities, and peptide-based nanozymes, thus enabling the investigation of biological phenomena at nanoscale dimensions. Through the rational design of enzyme-like peptides or their assembly with nanostructures and nanozymes, researchers have found or created PepNzymes capable of catalyzing a wide range of reactions. By scrutinizing the interactions between the structures and enzyme-like activities of PepNzymes, we have gained valuable insights into the underlying mechanisms governing enzyme-like activities. Generally, PepNzymes play a crucial role in biological processes by facilitating small-scale enzyme-like reactions, speeding up molecular oxidation-reduction, cleavage, and synthesis reactions, leveraging the functional properties of peptides, and creating a stable microenvironment, among other functions. These discoveries make PepNzymes useful for diagnostics, cellular imaging, antimicrobial therapy, tissue engineering, anti-tumor treatments, and more while pointing out opportunities. Overall, this research provides a significant journey of PepNzymes’ potential in various biomedical applications, pushing them towards new advancements.

Developing sensor arrays capturing comprehensive fluorescence (FL) spectra from a single probe is crucial for understanding sugar structures with very high similarity in biofluids. Therefore, the analysis of highly similar sugar' structures in biofluids based on the entire FL of a single nanozyme probe needs more concern, which makes the development of novel alternative approaches highly wanted for biomedical and other applications. Herein, a well-designed deep learning model with intrinsic information of 3D FL of CuO nanoparticles (NPs)' oxidase-like activity was developed to classify and predict the concentration of a group of sugars with very similar chemical structures in different media. The findings presented that the overall accuracy of the developed model in classifying the nine selected sugars was (99-100 %), which prompted us to transfer the developed model to predict the concentration of the selected sugars at a concentration range of (1-100 mu M). The transferred model also gave excellent results (R2 = 97-100 %). Therefore, the model was extended to other more complex applications, namely the identification of mixtures of sugars in serum and the detection of polysaccharides in different media such as serum and lake water. Notably, LOD for fructose was determined at 4.23 nM, marking a 120-fold decrease compared to previous studies. Our developed model was also compared with other deep learning-based models, and the results have demonstrated remarkable progress. Moreover, the identification of other possible coexisting interference substances in lake water samples was considered. This work marks a significant advancement, opening avenues for the widespread application of sensor arrays integrating nanozymes and deep learning techniques in biomedical and other diverse fields.

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing. In the current study, we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella, in addition to its known functions in the ovarian development in female adults and egg hatching. Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein (GFP) gene into autosomal Pxfl(2)d for labelling mutants could be achieved. However, this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity. We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9. The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences. We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein (CFP) expression cassettes on a Z-linked region in P. xylostella, which can also be used for early sex detection. By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain, the gene editing efficiency reached 29.73%, confirming the success of expressing a functional Cas9 gene. Taken together, we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P. xylostella, while the establishment of a heritable strain required the positioning of appropriate sites. This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

The ongoing interplay among plants, insects, and bacteria underscores the intricate balance of defense mechanisms in ecosystems. Regurgitant bacteria directly/indirectly impact plant immune responses, but the underlying mechanism is unclear. Here, we focus on the interaction between regurgitant bacteria, diamondback moth (DBM), and plant. Six culturable bacteria were isolated from DBM gut regurgitant, including three Enterobacter strains (RB1-3), Micrococcus sp. (RB4), Staphylococcus haemolyticus (RB5), and Bacillus cereus (RB6). These RB strains suppressed genes related to jasmonic acid and glucosinolate signaling pathways but had little effect on salicylic acid signaling pathway genes in Arabidopsis thaliana wounds. RB1 and RB5 inhibited DBM development on A. thaliana but not on an artificial diet. RB1 and RB5 significantly suppressed GOX genes and proteins in DBMs. However, the Pxgox2 insect mutant strain inoculated with RB1 or RB5 did not significantly affect DBM feeding on A. thaliana compared to the wild type. Six RB have been functionally identified, with RB1 and RB5 negatively regulating GOX-mediated host adaptability. The deliberate addition of RB1 and RB5 can negatively affect DBM herbivory and fitness. Our study provides a molecular basis for the further application of RB for insect pest management by modulating insect–plant interactions.

The use of conventional antibiotic therapies is in question owing to the emergence of drug-resistant pathogenic bacteria. Therefore, novel, highly efficient antibacterial agents to effectively overcome resistant bacteria are urgently needed. Accordingly, in this work, we described a novel class luminogen of 6-Aza-2-thiothymine-decorated gold nanoclusters (ATT-AuNCs) with aggregation-induced emission property that possessed potent antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). Scanning electron microscopy was performed to investigate the interactions between ATT-AuNCs and MRSA. In addition, ATT-AuNCs exhibited excellent ROS generation efficiency and could effectively ablate MRSA via their internalization to the cells. Finally, tandem mass tag-labeling proteome analysis was carried out to investigate the differential expression proteins in MRSA strains. The results suggested that ATT-AuNCs killed MRSA cells through altering the expression of multiple target proteins involved in DNA replication, aminoacyl-tRNA synthesis, peptidoglycan and arginine biosynthesis metabolism. Parallel reaction monitoring technique was further used for the validation of these proteome results. ATT-AuNCs could also be served as a wound-healing agent and accelerate the healing process. Overall, we proposed ATT-AuNCs could serve as a robust antimicrobial aggregation-induced emission luminogen (AIEgen) that shows the ability to alter the activities of multiple targets for the elimination of drug-resistant bacteria.