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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. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Simple Summary: Diaphorina citri is an important transmission vector of Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus Huanglongbing (HLB). The control of HLB mainly depends on the management of D. citri. The efficiency of D. citri nymphs in acquiring CLas bacteria is significantly higher than that of the adults, while the transmission efficiency of CLas bacteria by adults is significantly higher than that of nymphs. Therefore, it is of great significance to research the functions of key genes during the molting of fifth-instar nymphs. In this study, SMRT and Illumina sequencing were performed on D. citri fifth-instar nymphs and adults. SMRT-Seq-generated full-length transcripts provide valuable information for improving functional gene research in D. citri. Additionally, Illumina sequencing revealed that the Hippo pathway played an important role in regulating the transition of D. citri from fifth-instar nymphs to adults. Our findings provide useful reference information and lay a foundation for controlling D. citri. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid, Diaphorina citri is the principal vector of huanglongbing, which transmits Candidatus Liberibacter asiaticus. Trehalase is a key enzyme involved in trehalose hydrolysis and plays an important role in insect growth and development. The specific functions of this enzyme in D. citri have not been determined. In this study, three trehalase genes (DcTre1‐1, DcTre1‐2, and DcTre2) were identified based on the D. citri genome database. Bioinformatic analysis showed that DcTre1‐1 and DcTre1‐2 are related to soluble trehalase, whereas DcTre2 is associated with membrane‐bound trehalase. Spatiotemporal expression analysis indicated that DcTre1‐1 and DcTre1‐2 had the highest expression levels in the head and wing, respectively, and DcTre2 had high expression levels in the fat body. Furthermore, DcTre1‐1 and DcTre1‐2 expression levels were induced by 20‐hydroxyecdysone and juvenile hormone Ⅲ, but DcTre2 was unaffected. The expression levels of DcTre1‐1, DcTre1‐2, and DcTre2 were significantly upregulated, which resulted in high mortality after treatment with validamycin. Trehalase activities and glucose contents were downregulated, but the trehalose content increased after treatment with validamycin. In addition, the expression levels of chitin metabolism‐related genes significantly decreased at 24 and 48 h after treatment with validamycin. Furthermore, silencing of DcTre1‐1, DcTre1‐2, and DcTre2 reduced the expression levels of chitin metabolism‐related genes and led to a malformed phenotype of D. citri. These results indicate that D. citri trehalase plays an essential role in regulating chitin metabolism and provides a new target for control of D. citri. [ABSTRACT FROM AUTHOR]

Cathepsins belong to a group of mammalian papain-like cysteine proteases that play an important role in the insect immune response. In the present study, we identified two cathepsin genes from the Diaphorina citri genome database, cathepsin-L (DcCath-L) and cathepsin-O (DcCath-O). DcCath-L encodes a DcCath-L protein consisting of 348 amino acid residues, and DcCath-O encodes a DcCath-O protein consisting of 329 amino acid residues. DcCaths contain two conserved domains, the Inhibitor_I29 and Pept_C1 domains. Phylogenetic tree analysis revealed that DcCath-L and DcCath-O were divided into two different groups: Cathepsin-L and Cathepsin-O. Reverse transcription quantitative polymerase chain reaction analysis showed that both DcCath-O and DCCath-L were highly expressed in the midgut, while lower expression was observed in other tissues. Developmental stage expression analysis suggested that DcCath-O was mainly expressed in third instar nymph and adult, and DcCath-L was highly expressed in first and fourth instar nymph. Following exposure to two different heat-killed bacteria (Escherichia coli and Staphylococcus aureus) and Candidatus Liberibacter asiaticus, the expression of DcCath-O and DcCath-L was significantly increased and showed differential expression patterns at different time points. In addition, silencing of DcCath-L obvious affected the gene expression of members of the Toll pathway, while knock down of DcCath-L has no significantly influence. Overall, these data provide valuable information for further functional studies of D. citri cathepsins. Unlabelled Image • Expression profiles of four genes were analysed by RT-qPCR in different tissues and developmental stages. • DcCath-L and DcCath-O could be induced high expression by C Las. • DcCath-L and DcCath-O showed differential expression patterns after bacterial infection. • Silencing of DcCath-L obvious affected the gene expression of members of the Toll pathway. [ABSTRACT FROM AUTHOR]

• DcTm1-X2 showed high expression level in the leg of Diaphorina citri. • The expression level of DcTm1-X2 was down-regulated in C Las-infection groups. • DcTm1-X2 has interaction with CS and VAT. • The mortality rate of D. citri increased after knockdown of DcTm1-X2. • The infection rate of C Las was up-regulated after knockdown of DcTm1-X2. The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the transmitting vector of Candidatus Liberibacter asiaticus (C Las), which causes citrus disease Huanglongbing (HLB). In recent years, control of HLB has been achieved by reducing the vector population. In the present study, we identified an isoform of D. citri tropomyosin (herein designated as DcTm1-X1). DcTm1-X1 was down-regulated in C Las-infected ACPs compared with uninfected ACPs. Bioinformatics analysis revealed that the full-length DcTm1-X1 is 2955 bp and encodes a protein of 284 amino acids with a deduced molecular weight of 32.15 kDa. Phylogenetic tree analysis suggested that DcTm1-X1 shares a high amino acid identity with its homolog in Acyrthosiphon pisum. Higher DcTm1-X1 expression levels were found in the leg of the psyllid by reverse transcription quantitative PCR (RT-qPCR). According to Blue Native PAGE analysis and mass spectrometric analysis, DcTm1-X1 interacts with citrate synthase (CS) and V-type proton ATPase subunit B-like (VAT). In addition, knockdown of DcTm1-X1 by RNA interference (RNAi) significantly increased the mortality rate of nymphs and the infection rate of C Las at different time points. Taken together, our results show that DcTm1-X1 might play an important role in response to C Las, but also lay a foundation for further research on the functions of DcTm1-X1. [ABSTRACT FROM AUTHOR]

Simple Summary: Trehalose-6-phosphate synthase (TPS) is a key enzyme in regulating trehalose content in the insect hemolymph. The loss or dysfunction significantly affects the growth and development of insects. Diaphorina citri is a notorious phloem sap-sucking pest that can spread huanglongbing between the diseased tree and the healthy tree. The control of huanglongbing mainly depends the management of D. citri. So far, the management of D. citri populations has depended on using chemical pesticides, though pesticide abuse has caused serious problems. Therefore, it necessary to find new targets for D. citri control. In this paper, we identified a TPS gene from Diaphorina citri, and named it DcTPS1. Silencing of DcTPS1 induced an abnormal phenotype, and inhibited chitin metabolism and fatty acid metabolism. Moreover, the mortality and malformation rate significantly increased, and the molting rate decreased after inhibition of DcTPS1. KEGG analysis revealed that upregulated DEGs were mainly responsible for oxidative phosphorylation, whereas downregulated DEGs were mainly related to lysosome and ribosome. Overall, our data suggested that DcTPS1 might play a crucial role for the growth and 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. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important transmission vector of the citrus greening disease Candidatus Liberibacter asiaticus (CLas). The D. citri midgut exhibits an important tissue barrier against CLas infection. However, the molecular mechanism of the midgut response to CLas infection has not been comprehensively elucidated. In this study, we identified 778 differentially expressed genes (DEGs) in the midgut upon CLas infection, by comparative transcriptome analyses, including 499 upregulated DEGs and 279 downregulated DEGs. Functional annotation analysis showed that these DEGs were associated with ubiquitination, the immune response, the ribosome, endocytosis, the cytoskeleton and insecticide resistance. KEGG enrichment analysis revealed that most of the DEGs were primarily involved in endocytosis and the ribosome. A total of fourteen DEG functions were further validated by reverse transcription quantitative PCR (RT-qPCR). This study will contribute to our understanding of the molecular interaction between CLas and D. citri. [ABSTRACT FROM AUTHOR]

Chitin deacetylase (CDA) is a chitin degradation enzyme that strictly catalyzes the deacetylation of chitin to form chitosan, which plays an important role in regulating growth and development, as well as the immune response. In this study, a chitin deacetylase 3 gene (CDA3) was identified with a complete open reading frame (ORF) of 1362 bp from the genome database of Diaphorina citri, encoding a protein of 453 amino acids. Spatiotemporal expression analysis suggested that D. citriCDA3 (DcCDA3) had the highest expression level in the integument and third-instar nymph stage. Furthermore, DcCDA3 expression level can be induced by 20-hydroxyecdysone (20E). Injection of Escherichia coli and Staphylococcus aureus induced the upregulation of DcCDA3 in the midgut, while DcCDA3 was downregulated in the fat body. After silencing DcCDA3 by RNA interference, there was no influence on the D. citri phenotype. In addition, bactericidal tests showed that recombinant DcCDA3 inhibited gram-positive bacteria, including S. aureus and Bacillus subtilis (B. subtilis). In conclusion, our results suggest that DcCDA3 might play an important role in the immune response of D. citri. [ABSTRACT FROM AUTHOR]

Chitin synthase is a critical enzyme that catalyzes N-acetylglucosamine to form chitin, which plays an important role in the growth and development of insects. In this study, we identified a chitin synthase gene (CHS) with a complete open reading frame (ORF) of 3180 bp from the genome database of Diaphorina citri, encoding a protein of 1059 amino acid residues with the appropriate signature motifs (EDR and QRRRW). Reverse transcription-quantitative PCR (RT-qPCR) analysis suggested that D. citri CHS (DcCHS) was expressed throughout all developmental stages and all tissues. DcCHS had the highest expression level in the integument and fifth-instar nymph stage. Furthermore, the effects of diflubenzuron (DFB) on D. citri mortality and DcCHS expression level were investigated using fifth-instar nymph through leaf dip bioassay, and the results revealed that the nymph exposed to DFB had the highest mortality compared with control group (Triton-100). Silencing of DcCHS by RNA interference resulted in malformed phenotypes and increased mortality with decreased molting rate. In addition, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) also revealed corresponding ultrastructural defects. Our results suggest that DcCHS might play an important role in the development of D. citri and can be used as a potential target for psyllid control. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), also known as citrus greening disease, is caused by Candidatus Liberbacter asiaticus (C Las) and transmitted by Diaphorina citri. Previous studies reported that C Las infection significantly influences the structure of the D. citri cytoskeleton. However, the mechanisms through which C Las 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 C Las. Western blotting revealed a significant difference in ubiquitination levels between the C Las-free and C Las-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 C Las-infected groups compared with C Las-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 C Las bacterial content in D. citri. In summary, we provided the most comprehensive lysine ubiquitinome analysis of the D. citri response to C Las infection, thus furthering our understanding of the role of the ubiquitination of cytoskeleton proteins in C Las infection. [ABSTRACT FROM AUTHOR]