Candidatus Liberibacter asiaticus influences the emergence of the Asian citrus psyllid Diaphorina citri by regulating key cuticular proteins.

The Asian citrus psyllid, Diaphorina citri, is the primary vector of the HLB pathogen, Candidatus Liberibacter asiaticus (CLas). The acquisition of CLas shortens the developmental period of nymphs, accelerating the emergence into adulthood and thereby facilitating the spread of CLas. Cuticular proteins (CPs) are involved in insect emergence. In this study, we investigated the molecular mechanisms underlying CLas-promoted emergence in D. citri via CP mediation. Here, a total of 159 CP genes were first identified in the D. citri genome. Chromosomal location analysis revealed an uneven distribution of these CP genes across the 13 D. citri chromosomes. Proteomic analysis identified 54 differentially expressed CPs during D. citri emergence, with 14 CPs exhibiting significant differential expression after CLas acquisition. Five key genes, Dc18aa-1, Dc18aa-2, DcCPR-24, DcCPR-38 and DcCPR-58, were screened from the proteome and CLas acquisition. The silencing of these 5 genes through a modified feeding method significantly reduced the emergence rate and caused various abnormal phenotypes, indicating the crucial role that these genes play in D. citri emergence. This study provides a comprehensive overview of the role of CPs in D. citri and reveals that CLas can influence the emergence process of D. citri by regulating the expression of CPs. These key CPs may serve as potential targets for future research on controlling huanglongbing (HLB) transmission.

RNA interference of Argonaute-1 delays ovarian development in the oriental fruit fly, Bactrocera dorsalis (Hendel).

BACKGROUND: With the development of rapid resistance, new modes of action for pesticides are needed for insect control, such as RNAi-based biopesticides targeting essential genes. To explore the function of Argonaute-1 (Ago-1) and potential miRNAs in ovarian development of Bactrocera dorsalis, an important agricultural pest, and to develop a novel control strategy for the pest, BdAgo-1 was first identified in B. dorsalis. RESULTS: Spatiotemporal expression analysis indicated that BdAgo-1 had a relatively high transcriptional level in the ovarian tissues of adult female B. dorsalis during the sexual maturation period. RNA interference (RNAi) experiment showed that BdAgo-1 knockdown significantly decreased the expression levels of ovarian development-related genes and delayed ovarian development. Although RNAi-mediated silencing of Ago-1 led to a reduced ovary surface area, a subsequent oviposition assay revealed that the influence was minimal over a longer time period. Small RNA libraries were constructed and sequenced from different ovarian developmental stages of B. dorsalis adults. Among 161 identified miRNAs, 84 miRNAs were differentially expressed during the three developmental stages of the B. dorsalis ovary. BdAgo-1 silencing caused significant down-regulation of seven differentially expressed miRNAs (DEMs) showing relatively high expression levels (>1000 TPM (Transcripts per kilobase of exon model per million mapped reads)). The expression patterns of these seven core DEMs and their putative target genes were analyzed in the ovaries of B. dorsalis. CONCLUSION: The results indicate that Ago-1 and Ago-1-dependent miRNAs are indispensable for normal ovarian development in B. dorsalis and help identify miRNA targets useful for control of this pest.

An odorant-binding protein of Asian citrus psyllid, Diaphorina citri, participates in the response of host plant volatiles.

BACKGROUND: Odorant-binding proteins (OBPs) in insects contribute to the sensitivity of the olfactory system and connect external odorants to olfactory receptor neurons. Determination of the chemosensory functions in Diaphorina citri, a vector of the citrus Huanglongbing pathogen, may help in developing a potential target for pest management. RESULTS: Diaphorina citri showed dose-dependent electroantennogram recording (EAG) responses to 12 host plant volatiles. A two-choice behavioral trap experiment showed that four compounds (methyl salicylate, linalool, citral and R-(+)-limonene) that elicited high EAG responses also had significant attraction to adults. The expression profiles induced by these compounds were detected in nine OBP genes, DcitOBP1-9. DcitOBP3, DcitOBP6 and DcitOBP7 commonly showed significant upregulation or downregulation compared with the control. Microscale thermophoresis (MST) showed that the recombinant protein DcitOBP7 had high in vitro binding affinities (Kd < 10 µm) to methyl salicylate, linalool and R-(+)-limonene, and moderate binding affinity to citral with a Kd value of 15.95 µm. Furthermore, RNA interference (RNAi)-suppressed messenger RNA (mRNA) expression of DcitOBP7 resulted in a significant reduction in EAG activity and in adult D. citri behavioral responses to tested volatiles and the preferred host, Murraya paniculata. The hydrophilic residue Arg107 of DcitOBP7 may have a key role in binding odorants via formation of hydrogen bonds. CONCLUSION: These results show that DcitOBP7 plays an important role in the olfactory response. This finding may provide new insight into the functions of OBP families in D. citri and aid in the development of safe strategies for managing D. citri populations. © 2021 Society of Chemical Industry.

A Transcriptomic and Proteomic Analysis of the Diaphorina citri Salivary Glands Reveals Genes Responding to Candidatus Liberibacter asiaticus.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is the principal vector of the Candidatus Liberibacter asiaticus (CLas) bacterium that causes Huanglongbing (HLB) disease. The D. citri salivary glands (SG) is an important barrier to the transmission of CLas. Despite its importance, the transcriptome and proteome of SG defense against CLas are unstudied in D. citri. In the present study, we generated a comparative transcriptome dataset of the SG in infected and uninfected D. citri using an Illumina RNA-Seq technology. We obtained 407 differentially expressed genes (DEGs), including 159 upregulated DEGs and 248 downregulated DEGs. Functional categories showed that many DEGs were associated with the ribosome, the insecticide resistance, the immune response and the digestion in comparison with CLas-infected SG and CLas-free SG. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases confirmed that metabolism and immunity were important functions in the SG. Among the DEGs, 68 genes (35 upregulated and 33 downregulated) encoding putative-secreted proteins were obtained with a signal peptide, suggesting that these genes may play important roles in CLas infection. A total of 673 SG proteins were identified in uninfected D. citri by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, and 30 DEGs (15 upregulated and 15 downregulated) were found using the local tBLASTP programs. Among the 30 DEGs, many DEGs mainly involved in the metabolism and cellular processes pathways. This study provides basic transcriptome and proteome information for the SG in D. citri, and helps illuminate the molecular interactions between CLas and D. citri.

Odorant binding protein 2 reduces imidacloprid susceptibility of Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is the principal vector of Huanglongbing pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe economic losses to citrus industry worldwide. Use of broad-spectrum insecticides to control D. citri has resulted in considerable resistance development. Specific chemosensory proteins such as odorant binding proteins (OBPs) are potentially involved in reduced insecticide susceptibility. However, functional data on the contribution of OBPs to reduced susceptibility of D. citri are unavailable. We found that DcitOBP2 was stably expressed in different developmental stages and highly expressed in the legs, head and cuticle of D. citri. Expression of DcitOBP2 was significantly induced by 12 to 48 h of imidacloprid exposure and ranged from a 1.34- to 2.44-fold increase. RNAi of DcitOBP2 increased the susceptibility of D. citri adults to imidacloprid. The purified recombinant protein of DcitOBP2 expressed in Escherichia coli showed strong in vitro binding activity (Kd = 62.39 nM) to imidacloprid using microscale thermophoresis technology (MST). DcitOBP2 also had strong binding ability to thiamethoxam and dinotefuran but it had no response to abamectin, fenpropathrin and chlorpyrifos. The results showed that DcitOBP2 can interact with several neonicotinoid insecticides. This suggests that DcitOBP2 is involved in the decreased susceptibility of D. citri to imidacloprid. Our data reveal a new function of insect OBPs as a buffering protein that helps insects survive insecticide exposure. Our investigation may also aid in the development of new methods for resistance management of D. citri.

Topical dsRNA delivery induces gene silencing and mortality in the pea aphid.

BACKGROUND: With the growing number of available aphid genomes and transcriptomes, an efficient and easy-to-adapt tool for gene function study is urgently required. RNA interference (RNAi), as a post-transcriptional gene silencing mechanism, is important as a research tool for determining gene functions and has potential as a novel insect control strategy. However, these applications have been hampered by the lack of effective dsRNA delivery approaches in aphids. RESULTS: Here, we developed a convenient and efficient dsRNA delivery method, topical RNAi, in aphids. An investigation of its dose and time-dependent RNAi efficiencies revealed that with as little as 60 ng dsRNA per adult pea aphid (Acyrthosiphon pisum), the indicator gene, Aphunchback, could be significantly silenced within 2 h of exposure. The method was further validated by successfully silencing other different genes, and it was also efficient toward two other aphid species, Aphis citricidus and Myzus persicae. Furthermore, a noticeable mortality was also observed in pea aphids using topical RNAi-mediated gene silencing, within 4 days post-dsRNA application for four out of seven tested genes. CONCLUSION: Compared with the currently used dsRNA delivery methods in aphids, microinjection and ingestion, topical RNAi is time- and cost-effective, which could greatly influence RNAi-based gene functional studies and potential candidate gene selection for developing RNAi-based aphid control strategies in the future. © 2019 Society of Chemical Industry.