Psyllid Gut Research Articles

Bacillus thuringiensis translocation in citrus scion/rootstock combinations and binding of cry toxins with the Asian citrus psyllid gut receptors

Bacillus thuringiensis (Bt) can be effective in controlling insect pests either in spray products or by their toxins expressed in transgenic crops. The discovery of Bt as endophytic in citrus opened new perspectives for the control of Diaphorina citri Kuwayama, the vector of the bacteria Candidatus Liberibacter spp. associated with Huanglongbing. In this study, the endophytic translocation of Bt in citrus seedlings (small and large plants) and in different scion-rootstock combinations were investigated. We also assessed the lethal concentration of D. citri nymphs caused by systemic Bt in citrus and, moreover, the binding of Cry toxins with the Asian citrus psyllid (ACP) gut receptors. Endophytic translocation of Bt in citrus seedlings and nursery trees was confirmed by colony isolation, PCR detection, and Btk-GFP microscopy analyses, which demonstrated that, after Bt inoculation into the substrate, the bacteria enter through lateral root emergence sites and are localized in stem xylem vessel elements. Mean lethal concentrations for D. citri third instar nymphs were 4.92 × 104 and 2.19 × 104 spores mL−1 for S1450 and S1302 strains, respectively. Nymphal mortality induced by the bacteria was higher in citrus seedlings than in citrus nursery trees. All Cry-toxins tested were able to bind to D. citri brush border membrane vesicles, suggesting the potential pathogenicity of Bt strains against psyllid. This study clearly demonstrates the penetration and Bt translocation in the xylem of small and large citrus plants and the potential of the bacteria to cause ACP nymphs mortality that fed on shoots from Bt-treated citrus.

RNA interference-mediated knockdown of genes involved in sugar transport and metabolism disrupts psyllid Bactericera cockerelli (Order: Hemiptera) gut physiology and results in high mortality.

The causal agent of zebra chip of potato and vein-greening diseases of tomato is "Candidatus Liberibacter solanacearum" (CLso), a fastidious bacterium transmitted by the potato psyllid. In the absence of disease-resistant cultivars, disease management has relied on minimizing vector population size to reduce CLso transmission, which requires frequent insecticide applications. There is growing interest in the use of RNA interference (RNAi) technology to supplant traditional insecticides with biopesticides. This requires knowledge of genes essential for insect livelihood whose knockdown leads to significant mortality or other phenotypes. Such candidate genes can be evaluated by reverse genetics approaches to further corroborate predicted gene function. Here, five potato psyllid genes involved in sugar homeostasis in the potato psyllid gut, α-glucosidase1 (AGLU1), aquaporin2 (AQP2), facilitated trehalose transporter1 (TRET1), Trehalase1 (TRE1), and Trehalase2 (TRE2), were investigated as candidates for effective gene silencing. Potato psyllid dsRNAs were designed to optimize knockdown of gene targets. Third instar PoP nymphs were given a 48-hr ingestion-access period (IAP) on individual or groups of dsRNA in 20% sucrose. Mortality was recorded 0, 3, 5, 7, and 9 days post-IAP. Gene knockdown was analyzed 9 days post-IAP by quantitative real-time reverse-transcriptase polymerase chain reaction amplification. The individual or stacked dsRNA combinations resulted in 20-60% and 20-40% knockdown, respectively, while subsequent psyllid mortality ranged from 20-40% to >60% for single and stacked dsRNA combinations, respectively. Reverse genetics analysis showed that simultaneous knockdown of the five selected candidate genes with predicted functions in pathways involved in sugar-homeostasis, metabolism, and -transport yielded the highest mortality, when compared with single or combinations of targets. Results confirmed the functions afforded by psyllid gut genes responsible for osmotic homeostasis and sugar metabolism/transport are essential for livelihood, identifying them as potentially lucrative RNAi biopesticide targets and highlighted the translational relevance of targeting multiple nodes in a physiological pathway simultaneously.

Potato psyllids mount distinct gut responses against two different 'Candidatus Liberibacter solanacearum' haplotypes.

'Candidatus Liberibacter solanacearum' (Lso) is a bacterial pathogen infecting several crops and causing damaging diseases. Several Lso haplotypes have been identified. Among the seven haplotypes present in North America, LsoA and LsoB are transmitted by the potato psyllid, Bactericera cockerelli (Šulc), in a circulative and persistent manner. The gut, which is the first organ pathogen encounters, could be a barrier for Lso transmission. However, the molecular interactions between Lso and the psyllid vector at the gut interface remain largely unknown. In this study, we investigated the global transcriptional responses of the adult psyllid gut upon infection with two Lso haplotypes (LsoA and LsoB) using Illumina sequencing. The results showed that each haplotype triggers a unique transcriptional response, with most of the distinct genes elicited by the highly virulent LsoB. The differentially expressed genes were mainly associated with digestion and metabolism, stress response, immunity, detoxification as well as cell proliferation and epithelium renewal. Importantly, distinct immune pathways were triggered by LsoA and LsoB in the gut of the potato psyllid. The information in this study will provide an understanding of the molecular basis of the interactions between the potato psyllid gut and Lso, which may lead to the discovery of novel molecular targets for the control of these pathogens.

Identifying the Gut Virome of Diaphorina citri from Florida Groves.

Asian citrus psyllid (Diaphorina citri) transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the putative causative agent of citrus Huanglongbing disease (HLB). Insect-specific viruses can act against insects as their natural enemies, and recently, several D. citri-associated viruses were discovered. The insect gut plays an important role as not only a pool for diverse microbes but also as a physical barrier to prevent the spread of pathogens such as CLas. However, there is little evidence of the presence of D. citri-associated viruses in the gut and of the interaction between them and CLas. Here, we dissected psyllid guts collected from five growing regions in Florida, and the gut virome was analyzed by high throughput sequencing. Four insect viruses, including D. citri-associated C virus (DcACV), D. citri densovirus (DcDV), D. citri reovirus (DcRV), and D. citri flavi-like virus (DcFLV), were identified, and their presence in the gut, including an additional D. citri cimodo-like virus (DcCLV), were confirmed with PCR-based assays. Microscopic analysis showed that DcFLV infection leads to morphological abnormalities in the nuclear structure in the infected psyllid gut cells. The complex and diverse composition of microbiota in the psyllid gut suggests a possible interaction and dynamics between CLas and the D. citri-associated viruses. Our study identified various D. citri-associated viruses that localized in the psyllid gut and provided more information that helps to evaluate the potential vectors for manipulating CLas in the psyllid gut.

Composition and abundance of midgut surface proteins in the Asian citrus psyllid, Diaphorina citri

The Asian citrus psyllid, Diaphorina citri, is the vector of Candidatus Liberibacter asiaticus (CLas), the presumed causative agent of citrus greening disease. For successful transmission, CLas must cross the gut barrier, requiring interaction with proteins on the midgut epithelium. We compared the relative abundance of gut surface proteins for both adult and nymph D. citri, as nymphs are particularly susceptible to CLas infection. To enrich for gut surface proteins, brush border membrane vesicles were prepared from dissected guts, and proteins identified from triplicate samples run on a timsTOF mass spectrometer. A total of 1516 and 1219 proteins were identified from D. citri adults and nymphs respectively. Based on bioinformatics analysis software and manual curation, 112 adult and 87 nymph proteins were predicted to localize to the surface of the microvilli and were further categorized into integral membrane and glycosylphosphatidylinositol (GPI)-anchored proteins. Proteins exploited by insect pathogens such as aminopeptidase, alkaline phosphatase, cadherin, ABC transporters, and carboxypeptidase were among the most abundant proteins on the gut surface. In addition to providing insights into hemipteran gut physiology, the D. citri gut surface proteome will inform novel approaches to interfere with CLas interaction with the psyllid gut to prevent the spread of citrus greening. Biological significanceThe Asian citrus psyllid (ACP), D. citri is one of the most serious pests of citrus worldwide. ACP transmits the pathogenic bacterium that causes citrus greening or huanglongbing (HLB), which has resulted in severe economic losses in global citriculture. The putative causative agent of this disease, the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), is vectored by the Asian citrus psyllid, D. citri, in a persistent and circulative manner. CLas must interact with gut surface proteins in order to enter midgut epithelial cells. However, the specific proteins exploited by CLas have yet to be identified. The characterization of the most abundant proteins on the surface of the D. citri gut provides insight into candidate receptors for CLas and other pathogens of D. citri. We hypothesize that pathogens of D. citri exploit the most abundant proteins on the surface of the gut for entry into the host insect. Importantly, the abundant gut surface proteins will provide the basis for novel approaches to disrupt CLas-D. citri interactions, with the goal of preventing further economic loss to the citrus industry.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

Identification of Autophagy-Related Genes in the Potato Psyllid, Bactericera cockerelli and Their Expression Profile in Response to 'Candidatus Liberibacter Solanacearum' in the Gut.

Simple SummaryIn North America, the bacterial plant pathogen ‘Candidatus Liberibacter solanacearum’ (Lso) infects solanaceous plants. Currently, Lso haplotypes, LsoA and LsoB are transmitted by potato psyllid, Bactericera cockerelli (Šulc). Because these bacteria are transmitted in a circulative and persistent manner, the gut of the psyllid is the first organ they encounter and could be a barrier to its transmission. Therefore, it is important to understand the molecular mechanisms involved in Lso acquisition and transmission. This study explored if an autophagic response was triggered in response to LsoA and/or LsoB in the gut of the adult potato psyllid. The results showed that Lso may induce the autophagic response in the adult psyllid gut since the majority of autophagy-related genes (ATGs) are sensitive and responsive to the exposure or infection of both LsoA and LsoB. Therefore, this study represents a stepping-stone towards understanding the molecular mechanisms involved in Lso transmission.Autophagy, also known as type II programmed cell death, is a cellular mechanism of “self-eating”. Autophagy plays an important role against pathogen infection in numerous organisms. Recently, it has been demonstrated that autophagy can be activated and even manipulated by plant viruses to facilitate their transmission within insect vectors. However, little is known about the role of autophagy in the interactions of insect vectors with plant bacterial pathogens. ‘Candidatus Liberibacter solanacearum’ (Lso) is a phloem-limited Gram-negative bacterium that infects crops worldwide. Two Lso haplotypes, LsoA and LsoB, are transmitted by the potato psyllid, Bactericera cockerelli and cause damaging diseases in solanaceous plants (e.g., zebra chip in potatoes). Both LsoA and LsoB are transmitted by the potato psyllid in a persistent circulative manner: they colonize and replicate within psyllid tissues. Following acquisition, the gut is the first organ Lso encounters and could be a barrier for transmission. In this study, we annotated autophagy-related genes (ATGs) from the potato psyllid transcriptome and evaluated their expression in response to Lso infection at the gut interface. In total, 19 ATGs belonging to 17 different families were identified. The comprehensive expression profile analysis revealed that the majority of the ATGs were regulated in the psyllid gut following the exposure or infection to each Lso haplotype, LsoA and LsoB, suggesting a potential role of autophagy in response to Lso at the psyllid gut interface.

Liberibacter, A Preemptive Bacterium: Apoptotic Response Repression in the Host Gut at the Early Infection to Facilitate Its Acquisition and Transmission.

“Candidatus Liberibacter solanacearum” (Lso) is a phloem-limited Gram-negative bacterium that infects crops worldwide. In North America, two haplotypes of Lso (LsoA and LsoB) are transmitted by the potato psyllid, Bactericera cockerelli (Šulc), in a circulative and persistent manner. Both haplotypes cause damaging plant diseases (e.g., zebra chip in potatoes). The psyllid gut is the first organ Lso encounters and could be a barrier for its transmission. However, little is known about the psyllid gut immune responses triggered upon Lso infection. In this study, we focused on the apoptotic response in the gut of adult potato psyllids at the early stage of Lso infection. We found that there was no evidence of apoptosis induced in the gut of the adult potato psyllids upon infection with either Lso haplotype based on microscopic observations. However, the expression of the inhibitor of apoptosis IAPP5.2 gene (survivin-like) was significantly upregulated during the period that Lso translocated into the gut cells. Interestingly, silencing of IAPP5.2 gene significantly upregulated the expression of two effector caspases and induced apoptosis in the psyllid gut cells. Moreover, RNA interference (RNAi) of IAPP5.2 significantly decreased the Lso titer in the gut of adult psyllids and reduced their transmission efficiency. Taken together, these observations suggest that Lso might repress the apoptotic response in the psyllid guts by inducing the anti-apoptotic gene IAPP5.2 at an early stage of the infection, which may favor Lso acquisition in the gut cells and facilitate its transmission by potato psyllid.

Acquisition and transmission of two \u2018Candidatus Liberibacter solanacearum\u2019 haplotypes by the tomato psyllid Bactericera cockerelli

‘Candidatus Liberibacter solanacearum’ (Lso) is a pathogen of solanaceous crops. Two haplotypes of Lso (LsoA and LsoB) are present in North America; both are transmitted by the tomato psyllid, Bactericera cockerelli (Šulc), in a circulative and propagative manner and cause damaging plant diseases (e.g. Zebra chip in potatoes). In this study, we investigated the acquisition and transmission of LsoA or LsoB by the tomato psyllid. We quantified the titer of Lso haplotype A and B in adult psyllid guts after several acquisition access periods (AAPs). We also performed sequential inoculation of tomato plants by adult psyllids following a 7-day AAP and compared the transmission of each Lso haplotype. The results indicated that LsoB population increased faster in the psyllid gut than LsoA. Further, LsoB population plateaued after 12 days, while LsoA population increased slowly during the 16 day-period evaluated. Additionally, LsoB had a shorter latent period and higher transmission rate than LsoA following a 7 day-AAP: LsoB was first transmitted by the adult psyllids between 17 and 21 days following the beginning of the AAP, while LsoA was first transmitted between 21 and 25 days after the beginning of the AAP. Overall, our data suggest that the two Lso haplotypes have distinct acquisition and transmission rates. The information provided in this study will improve our understanding of the biology of Lso acquisition and transmission as well as its relationship with the tomato psyllid at the gut interface.

Concanavalin A Toxicity Towards Potato Psyllid and Apoptosis Induction in Midgut Cells.

Concanavalin A (ConA), a legume lectin, has been drawing increasing attention in recent years concerning its toxicity against insects and its potential application in pest management. In an attempt to evaluate the effect of ConA on potato psyllid (Bactericera cockerelli), an economically important pest of solanaceous crops, the effect of ConA on potato psyllid survival, psyllid gut nuclear morphology, and expression of psyllid caspase genes were evaluated. Our results determined that artificial diet-feeding assays using ConA had deleterious effects on potato psyllids, resulting in significant psyllid mortality following ingestion. We also found that an apoptotic response was induced by ConA in psyllid midgut cells, which was demonstrated by the DNA fragmentation and abnormal nuclear architecture in the midgut cells. Following ConA ingestion, there was also upregulation of caspase genes in the psyllid midguts. Therefore, a key mechanism behind ConA toxicity towards potato psyllid probably involves the induction of apoptosis in midgut cells. This study could provide a better understanding of the mechanisms underlying ConA toxicity in insects and be a stepping stone towards the development of new psyllid control strategies based on plant lectins.

No Evidence of Apoptotic Response of the Potato Psyllid Bactericera cockerelli to "Candidatus Liberibacter solanacearum" at the Gut Interface.

"Candidatus Liberibacter solanacearum" is a pathogen transmitted by the potato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) in a persistent manner. In this study, we investigated the molecular interaction between "Ca. Liberibacter solanacearum" and the potato psyllid at the gut interface. Specifically, we focused on the apoptotic response of potato psyllids to the infection by two "Ca. Liberibacter solanacearum" haplotypes, LsoA and LsoB. To this end, we first quantified and localized "Ca. Liberibacter solanacearum" in the gut of adult psyllids. We then evaluated the existence of an apoptotic response in the insect gut using microscopy analyses to visualize the nuclei and the actin cytoskeleton of the gut cells and DNA fragmentation analyses by agarose gel electrophoresis. We also performed annexin V cell death assays to detect apoptosis. Finally, we annotated apoptosis-related genes from the potato psyllid transcriptome and evaluated their expression in response to "Ca. Liberibacter solanacearum" infection. The results showed no cellular markers of apoptosis despite the large amount of "Ca. Liberibacter solanacearum" present in the psyllid gut. In addition, only three genes potentially involved in apoptosis were regulated in the psyllid gut in response to "Ca. Liberibacter solanacearum": the apoptosis-inducing factor AIF3 was downregulated in LsoA-infected psyllids, while the inhibitor of apoptosis IAPP5 was downregulated and IAP6 was upregulated in LsoB-infected psyllids. Overall, no evidence of apoptosis was observed in the gut of potato psyllid adults in response to either "Ca. Liberibacter solanacearum" haplotype. This study represents a first step toward understanding the interactions between "Ca. Liberibacter solanacearum" and the potato psyllid, which is crucial to developing approaches to disrupt their transmission.

Combining 'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut.

Huanglongbing, or citrus greening disease, is an economically devastating bacterial disease of citrus. It is associated with infection by the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by Diaphorina citri, the Asian citrus psyllid (ACP). For insect transmission to occur, CLas must be ingested during feeding on infected phloem sap and cross the gut barrier to gain entry into the insect vector. To investigate the effects of CLas exposure at the gut-pathogen interface, we performed RNAseq and mass spectrometry-based proteomics to analyze the transcriptome and proteome, respectively, of ACP gut tissue. CLas exposure resulted in changes in pathways involving the TCA cycle, iron metabolism, insecticide resistance and the insect’s immune system. We identified 83 long non-coding RNAs that are responsive to CLas, two of which appear to be specific to the ACP. Proteomics analysis also enabled us to determine that Wolbachia, a symbiont of the ACP, undergoes proteome regulation when CLas is present. Fluorescent in situ hybridization (FISH) confirmed that Wolbachia and CLas inhabit the same ACP gut cells, but do not co-localize within those cells. Wolbachia cells are prevalent throughout the gut epithelial cell cytoplasm, and Wolbachia titer is more variable in the guts of CLas exposed insects. CLas is detected on the luminal membrane, in puncta within the gut epithelial cell cytoplasm, along actin filaments in the gut visceral muscles, and rarely, in association with gut cell nuclei. Our study provides a snapshot of how the psyllid gut copes with CLas exposure and provides information on pathways and proteins for targeted disruption of CLas-vector interactions at the gut interface.

Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli

The potato/tomato psyllid, Bactericerca cockerelli (B. cockerelli), and the Asian citrus psyllid, Diaphorina citri (D. citri), are very important plant pests, but they are also vectors of phloem-limited bacteria that are associated with two devastating plant diseases. B. cockerelli is the vector of Candidatus Liberibacter psyllaurous (solanacearum), which is associated with zebra chip disease of potatoes, and D. citri is the vector of Ca. Liberibacter asiaticus, which is associated with the Huanglongbing (citrus greening) disease that currently threatens the entire Florida citrus industry. Here we used EST sequence information from D. citri to identify potential targets for RNA interference in B. cockerelli. We targeted ubiquitously expressed and gut-abundant mRNAs via injection and oral acquisition of double-stranded RNAs and siRNAs and were able to induce mortality in recipient psyllids. We also showed knockdown of target mRNAs, and that oral acquisition resulted primarily in mRNA knockdown in the psyllid gut. Concurrent with gene knockdown was the accumulation of target specific ∼ 21 nucleotide siRNAs for an abundant mRNA for BC-Actin. These results showed that RNAi can be a powerful tool for gene function studies in psyllids, and give support for continued efforts for investigating RNAi approaches as possible tools for psyllid and plant disease control.