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.

Citrus Huanglongbing (HLB), a severe and destructive plant disease caused by the Gram-negative, phloem-limited bacterium “ Candidatus Liberibacter asiaticus ( C Las)” and transmitted by Diaphorina citri , has been extensively studied. Previous studies have reported that protein post-translational modifications play a crucial role in D. citri response to C Las infection. However, comprehensive phosphoproteomic profiling of D. citri induced by C Las remains underexplored. In this study, a total of 144 differentially expressed proteins (DEPs) and 997 differentially phosphorylated proteins (DPPs) were identified by 4D label-free quantitative proteomics and phosphoproteomics. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that DEPs were mainly associated with molecular binding, structural constituent of cuticle and cytochrome P450, whereas DPPs were predominately involved in acting and calcium binding. A total of thirteen proteins were selected for parallel reaction monitoring (PRM) analysis to validate the reliability of proteomics. Integrated proteomic and phosphoproteomic analyses identified seven co-expressed proteins: vitellogenin-A1 (Vg-A1), alpha-crystallin A chain (αA- crystallin), facilitated trehalose transporter Tret1 (Tret1), LOC103509854, zinc finger protein 319 (ZFP319), LOC113471498 and Protein argonaute-2 (Ago-2). Furthermore, RNA interference (RNAi)-mediated knockdown of vitellogenin-A1 and alpha-crystallin A chain significantly reduced C Las content in D. citri . In conclusion, this study provides the most comprehensive phosphorylation profiles of D. citri in response to C Las infection and identifies two potential targets implicated in C Las infection.

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.

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.

Huanglongbing (HLB) is a systemic plant disease caused by ‘Candidatus Liberibacter asiaticus (CLas)’ and transmitted by Diaphorina citri. D. citri acquires the CLas bacteria in the nymph stage and transmits it in the adult stage, indicating that molting from the nymph to adult stages is crucial for HLB transmission. However, the available D. citri reference genomes are incomplete, and gene function studies have been limited to date. In the current research, PacBio single-molecule real-time (SMRT) and Illumina sequencing were performed to investigate the transcriptome of D. citri nymphs and adults. In total, 10,641 full-length, non-redundant transcripts (FLNRTs), 594 alternative splicing (AS) events, 4522 simple sequence repeats (SSRs), 1086 long-coding RNAs (lncRNAs), 281 transcription factors (TFs), and 4459 APA sites were identified. Furthermore, 3746 differentially expressed genes (DEGs) between nymphs and adults were identified, among which 30 DEGs involved in the Hippo signaling pathway were found. Reverse transcription–quantitative PCR (RT-qPCR) further validated the expression levels of 12 DEGs and showed a positive correlation with transcriptome data. Finally, the spatiotemporal expression pattern of genes involved in the Hippo signaling pathway exhibited high expression in the D. citri testis, ovary, and egg. Silencing of the D. citri transcriptional co-activator (DcYki) gene significantly increased D. citri mortality and decreased the cumulative molting. Our results provide useful information and a reliable data resource for gene function research of D. citri.

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. .

Coumarin and its derivatives are plant-derived compounds that exhibit potent insecticidal properties. In this study, we found that natural coumarin significantly inhibited the growth and development of Spodoptera litura larvae through toxicological assay. By transcriptomic sequencing, 80 and 45 differentially expressed genes (DEGs) related to detoxification were identified from 0 to 24 h and 24 to 48 h in S. litura after coumarin treatment, respectively. Enzyme activity analysis showed that CYP450 and acetylcholinesterase (AChE) activities significantly decreased at 48 h after coumarin treatment, while glutathione S-transferases (GST) activity increased at 24 h. Silencing of SlCYP324A16 gene by RNA interference significantly increased S. litura larval mortality and decreased individual weight after treatment with coumarin. Additionally, the expression levels of DEGs involved in glycolysis and tricarboxylic acid (TCA) cycle were inhibited at 24 h after coumarin treatment, while their expression levels were upregulated at 48 h. Furthermore, metabonomics analysis identified 391 differential metabolites involved in purine metabolism, amino acid metabolism, and TCA cycle from 0 to 24 h after treated with coumarin and 352 differential metabolites associated with ATP-binding cassette (ABC) transporters and amino acid metabolism. These results provide an in-depth understanding of the toxicological mechanism of coumarin on S. litura.

Brassica plants have glucosinolate (GLs)-myrosinase defense mechanisms to deter herbivores. However, Plutella xylostella specifically feeds on Brassica vegetables. The larvae possess three glucosinolate sulfatases (PxGSS1-3) that compete with plant myrosinase for shared GLs substrates and produce nontoxic desulfo-GLs (deGLs). Although PxGSSs are considered potential targets for pest control, the lack of a comprehensive review has hindered the development of PxGSSs-targeted pest control methods. Recent advances in integrative multi-omics analysis, substrate-enzyme kinetics, and molecular biological techniques have elucidated the evolutionary origin and functional diversity of these three PxGSSs. This review summarizes research progress on PxGSSs over the past 20 years, covering sequence properties, evolution, protein modification, enzyme activity, structural variation, substrate specificity, and interaction scenarios based on functional diversity. Finally, we discussed the potential applications of PxGSSs-targeted pest control technologies driven by artificial intelligence, including CRISPR/Cas9-mediated gene drive, transgenic plant-mediated RNAi, small-molecule inhibitors, and peptide inhibitors. These technologies have the potential to overcome current management challenges and promote the development and field application of PxGSSs-targeted pest control.

Chitin plays an important role in the development and molting of insects. The key genes involved in chitin metabolism were considered promising targets for pest control. In this study, two splice variants of chitin deacetylase 2 (CDA2) from Diaphorina citri were identified, including DcCDA2a and DcCDA2b. Bioinformatics analysis revealed that DcCDA2a and DcCDA2b encoded 550 and 544 amino acid residues with a signal peptide, respectively. Spatio-temporal expression patterns analysis showed that DcCDA2a and DcCDA2b were highly expressed in D. citri wing and nymph stages. Moreover, DcCDA2a and DcCDA2b expression levels were induced by 20-hydroxyecdysone (20E). Silencing DcCDA2a by RNA interference (RNAi) significantly disrupted the D. citri molting and increased D. citri mortality and malformation rate, whereas inhibition of DcCDA2b resulted in a semimolting phenotype. Furthermore, silencing DcCDA2a and DcCDA2b significantly suppressed D. citri chitin and fatty acid metabolism. Our results indicated that DcCDA2 might play crucial roles in regulating D. citri chitin and fatty acid metabolism, and it could be used as a potential target for controlling D. citri.

Insect ScienceEarly View LETTER TO THE EDITOR Clathrin heavy chain is involved in infection of Candidatus Liberibacter asiaticus in the host vector Diaphorina citri Lianjie Xie, Lianjie Xie National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China These authors contributed equally to this work.Search for more papers by this authorXiangdong Zeng, Xiangdong Zeng National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China These authors contributed equally to this work.Search for more papers by this authorMuhammad Bilal Amir, Muhammad Bilal Amir National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, ChinaSearch for more papers by this authorXiaojin Zou, Xiaojin Zou National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, ChinaSearch for more papers by this authorJingfu Yuan, Jingfu Yuan Agriculture and Rural Affairs Office of Datangbu Town, Ganzhou, Jiangxi Province, ChinaSearch for more papers by this authorHaizhong Yu, Corresponding Author Haizhong Yu [email protected] National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China Correspondence: Haizhong Yu, Zhanjun Lu, and Wei Chen, National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China. Email: [email protected]; [email protected]; and [email protected]Search for more papers by this authorZhanjun Lu, Corresponding Author Zhanjun Lu [email protected] orcid.org/0000-0001-8926-6312 National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China Correspondence: Haizhong Yu, Zhanjun Lu, and Wei Chen, National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China. Email: [email protected]; [email protected]; and [email protected]Search for more papers by this authorWei Chen, Corresponding Author Wei Chen [email protected] orcid.org/0000-0001-9570-3662 National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China Correspondence: Haizhong Yu, Zhanjun Lu, and Wei Chen, National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China. Email: [email protected]; [email protected]; and [email protected]Search for more papers by this author Lianjie Xie, Lianjie Xie National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China These authors contributed equally to this work.Search for more papers by this authorXiangdong Zeng, Xiangdong Zeng National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China These authors contributed equally to this work.Search for more papers by this authorMuhammad Bilal Amir, Muhammad Bilal Amir National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, ChinaSearch for more papers by this authorXiaojin Zou, Xiaojin Zou National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, ChinaSearch for more papers by this authorJingfu Yuan, Jingfu Yuan Agriculture and Rural Affairs Office of Datangbu Town, Ganzhou, Jiangxi Province, ChinaSearch for more papers by this authorHaizhong Yu, Corresponding Author Haizhong Yu [email protected] National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China Correspondence: Haizhong Yu, Zhanjun Lu, and Wei Chen, National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China. Email: [email protected]; [email protected]; and [email protected]Search for more papers by this authorZhanjun Lu, Corresponding Author Zhanjun Lu [email protected] orcid.org/0000-0001-8926-6312 National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China Correspondence: Haizhong Yu, Zhanjun Lu, and Wei Chen, National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China. Email: [email protected]; [email protected]; and [email protected]Search for more papers by this authorWei Chen, Corresponding Author Wei Chen [email protected] orcid.org/0000-0001-9570-3662 National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou, Jiangxi Province, China Correspondence: Haizhong Yu, Zhanjun Lu, and Wei Chen, National Navel Orange Engineering Research Center, School of Life Sciences, Gannan Normal University, Ganzhou 341000, Jiangxi Province, China. Email: [email protected]; [email protected]; and [email protected]Search for more papers by this author First published: 13 December 2023 https://doi.org/10.1111/1744-7917.13305 Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Supporting Information Filename Description ins13305-sup-0001-SuppMat.docx3.2 MB Table S1 Primers used in this study. Table S2 Relative expression level of DcChc in D. citri in the field. Table S3 Relative expression level of DcChc in lab. Table S4 CLas copy number in different samples of D. citri after dsRNA of DcChc treatment for 24 and 48 hours. Fig. S1. Comparison of Chc protein sequence in different species. Fig. S2. Analysis of DcChc gene expression between male and female adults. Student's t-test was performed to analyze the expression of DcChc. ns: P > 0.05. Fig. S3. Upper: gel electrophoresis image for navel orange of CLas detection. Bottom: Ct value for navel orange qPCR of CLas detection. NC: negative control; PC: positive control. Fig. S4. Left: analysis of DcChc gene expression for RNA interference after 24 hours; Right: survival rate of D citri after RNA interference.**: P < 0.01. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. References Abbasi, R., Heschuk, D., Kim, B. and Whyard, S. 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The relationship of DNA methylation and sex-biased gene expression is of high interest, it allows research into mechanisms of sexual dimorphism and the development of potential novel strategies for insect pest control. The Asian citrus psyllid, Diaphorina citri Kuwayama, is a major vector for the causative agents of Huanglongbing (HLB), which presents an unparalleled challenge to citrus production worldwide. Here, we identify the X chromosome of D. citri and investigate differences in the transcription and DNA methylation landscapes between adult virgin males and females. We find a large number of male-biased genes on the autosomes and a depletion of such on the X chromosome. We have also characterized the methylome of D. citri, finding low genome-wide levels, which is unusual for an hemipteran species, as well as evidence for both promoter and TE methylation. Overall, DNA methylation profiles are similar between the sexes but with a small number of differentially methylated genes found to be involved in sex differentiation. There also appears to be no direct relationship between differential DNA methylation and differential gene expression. Our findings lay the groundwork for the development of novel epigenetic-based pest control methods, and given the similarity of the D. citri methylome to some other insect species, these methods could be applicable across agricultural insect pests.

Huanglongbing (HLB), also known as citrus greening disease, is caused by Candidatus Liberbacter asiaticus (CLas) and transmitted by Diaphorina citri. Previous studies reported that CLas infection significantly influences the structure of the D. citri cytoskeleton. However, the mechanisms through which CLas manipulates cytoskeleton-related proteins remain unclear. In this study, we performed quantitative ubiquitylome crosstalk with the proteome to reveal the roles of cytoskeleton-related proteins during the infection of D. citri by CLas. Western blotting revealed a significant difference in ubiquitination levels between the CLas-free and CLas-infected groups. According to ubiquitylome and 4D label-free proteome analysis, 343 quantified lysine ubiquitination (Kub) sites and 666 differentially expressed proteins (DEPs) were identified in CLas-infected groups compared with CLas-free groups. A total of 53 sites in 51 DEPs were upregulated, while 290 sites in 192 DEPs were downregulated. Furthermore, functional enrichment analysis indicated that 18 DEPs and 21 lysine ubiquitinated proteins were associated with the cytoskeleton, showing an obvious interaction. Ubiquitination of D. citri tropomyosin was confirmed by immunoprecipitation, Western blotting, and LC-MS/MS. RNAi-mediated knockdown of tropomyosin significantly increased CLas bacterial content in D. citri. In summary, we provided the most comprehensive lysine ubiquitinome analysis of the D. citri response to CLas infection, thus furthering our understanding of the role of the ubiquitination of cytoskeleton proteins in CLas infection.

Glycogen is a predominant carbohydrate reserve in various organisms, which provides energy for different life activities. Glycogen synthase kinase 3 (GSK3) is a central player that catalyzes glucose and converts it into glycogen. In this study, a GSK3 gene was identified from the D. citri genome database and named DcGSK3. A reverse transcription quantitative PCR (RT-qPCR) analysis showed that DcGSK3 was expressed at a high level in the head and egg. The silencing of DcGSK3 by RNA interference (RNAi) led to a loss-of-function phenotype. In addition, DcGSK3 knockdown decreased trehalase activity, glycogen, trehalose, glucose and free fatty acid content. Moreover, the expression levels of the genes associated with chitin and fatty acid synthesis were significantly downregulated after the silencing of DcGSK3. According to a comparative transcriptomics analysis, 991 differentially expressed genes (DEGs) were identified in dsDcGSK3 groups compared with dsGFP groups. A KEGG enrichment analysis suggested that these DEGs were primarily involved in carbon and fatty acid metabolism. The clustering analysis of DEGs further confirmed that chitin and fatty acid metabolism-related DEGs were upregulated at 24 h and were downregulated at 48 h. Our results suggest that DcGSK3 plays an important role in regulating the chitin and fatty acid metabolism of D. citri.

为掌握近年来赣南柑桔木虱发生动态及其携带黄龙病菌变化情况,2016-2020年定点、定时监测了赣南10个县(区)20个正常管理果园的柑桔木虱成虫发生动态变化,并于2018年和2020年利用实时荧光定量PCR检测了 8个县(市)正常管理果园和失管果园柑桔木虱成虫带菌情况.结果表明,赣南地区柑桔木虱成虫发生高峰集中在5月下旬至10月上旬;2016-2019年发生数量呈逐年下降趋势,但2020年发生量较前几年有所增加,平均虫量最高峰达93头/百梢.2020年正常管理果园和失管果园柑桔木虱带菌率均低于2018年,失管果园柑桔木虱带菌率明显高于正常管理果园.经相关回归分析,柑桔木虱带菌率与果园黄龙病发病株率呈极显著正相关.

Ferritin heavy-chain homolog (FerHCH), an iron-binding protein, plays an important role in the host defense against oxidative stress and pathogen infections. In our previous research, Bombyx mori native ferritin had an interaction with B. mori nucleopolyhedrovirus (BmNPV). However, the underlying molecular mechanism of single ferritin homolog responses to BmNPV infection remains unclear. In this study, we found that BmNPV titer and B. mori FerHCH (BmFerHCH) expression were positively correlated with the ferric iron concentration. We performed RNA interference (RNAi) and overexpression experiments to investigate the effects of BmFerHCH on BmNPV proliferation. BmFerHCH knockdown suppressed BmNPV proliferation in vivo and in vitro, whereas BmFerHCH overexpression facilitated BmNPV proliferation. In addition, the oxidative stress level was increased significantly in BmN cells after budded virus infection, while BmFerHCH could neutralize the increased ROS production induced by BmNPV. Of note, we found that ROS was involved in BmNPV-induced apoptosis. Through inhibiting ROS, apoptosis was suppressed by BmFerHCH, whereas BmFerHCH knockdown facilitated apoptosis. Therefore, we hypothesize that BmFerHCH-mediated inhibition of virus-induced apoptosis depends on suppressing ROS accumulation and, thereby, facilitates virus replication. These results suggest that BmFerHCH plays an important role in facilitating BmNPV proliferation and modulating BmFerHCH is potential strategy for studying host-pathogen interactions.

Chitin is a major component of the arthropod exoskeleton, always working together with chitin-binding proteins to maintain the functions of extracellular structures. In the present study, we identified a cuticle protein 64 from Diaphorina citri using a chitin-binding assay. Bioinformatics analysis revealed that DcCP64 contained eight conserved PYPV motifs but lacked a Rebers–Riddiford (R–R) consensus and other chitin-binding domains. RT-qPCR analysis suggested that DcCP64 had the highest expression level in the wing and fifth-instar nymph stage. Knockdown of DcCP64 by RNA interference (RNAi) resulted in a malformed-wing phenotype, higher mortality and decreased molting rate. Furthermore, transcriptomics analysis revealed that 1244 differentially expressed genes (DEGs) were up-regulated and 580 DEGs were down-regulated, compared with dsDcCP64 groups and dsGFP groups. KEGG enrichment analysis revealed that up-regulated DEGs were mainly related to oxidative phosphorylation, whereas down-regulated DEGs were mainly involved in the MAPK and FoxO signaling pathways. Moreover, inhibition of DcCP64 significantly affected the cuticle surface, and increased the permeability of the abdomen and wings. Further chitin- and cellulose-binding assay confirmed the chitin-binding properties of recombinant DcCP64 in vitro. These results indicate that DcCP64 might play an important role in the cuticle and wing development of D. citri.

Trehalose-6-phosphate synthase (TPS) plays an important role in the synthesis of trehalose. In the current study, a TPS gene was obtained from Diaphorina citri, and named as DcTPS1 which encoded a protein of 833 amino acid residues. Real-time quantitative PCR (qPCR) analysis revealed that DcTPS1 had the highest expression level in the midgut and fifth-instar nymph stage. Knockdown of DcTPS1 by RNA interference (RNAi) induced an abnormal phenotype and increased mortality and malformation rate with a decreased molting rate. In addition, silencing of DcTPS1 significantly inhibited D. citri chitin metabolism and fatty acid metabolism, while the expression levels of fatty acid decomposition-related genes were downregulated. Furthermore, comparative transcriptomics analysis revealed that 791 differentially expressed genes (DEGs) were upregulated and 678 DEGs were downregulated when comparing dsDcTPS1 groups with dsGFP groups. Bioinformatics analysis showed that upregulated DEGs were mainly involved in oxidative phosphorylation, whereas downregulated DEGs were mainly attributed to the lysosome and ribosome. These results indicated that DcTPS1 played an important role in the growth and development of D. citri.

烟碱型乙酰胆碱受体(nACh凡)是脊椎动物和无脊椎动物中的主要兴奋性神经递质受体.在昆虫中,nAChRs是杀虫剂的重要分子靶点,如新烟碱类和多杀菌素.在此研究中,根据稻纵卷叶螟(Cnaphalocrocis medinalis)转录组数据库和生物信息学方法鉴定两个稻纵卷叶螟nAChRs基因(Cmα8和Cmα9).Cmα8和Cmα9的开放阅读框(ORF)分别由1,650和999个核苷酸组成,这两种基因都具有nAChR家族成员的典型特征.系统发育树分析显示,Cmα8与丽蝇蛹集金小蜂(Nasonia vitripennis)的Nvα8同源性最高,Cmα9与家蚕Bombyx mori α9(Bmα9)的同源性最高.组织表达模式分析表明两个基因在中肠和脂肪体中高度表达,在蛹和成虫阶段,Cmα8和Cmα9的表达量最高,最低的表达量则位于幼虫阶段.此外,吡虫啉在中肠中显著诱导Cmα8和Cmα9的表达,而在脂肪体中则没有明显变化.综上所述,本文将为理解稻纵卷叶螟(C.medinalis)中nA ChRs的功能提供有用的信息.