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1.  Infection of an Insect Vector with a Bacterial Plant Pathogen Increases Its Propensity for Dispersal 
PLoS ONE  2015;10(6):e0129373.
The spread of vector-transmitted pathogens relies on complex interactions between host, vector and pathogen. In sessile plant pathosystems, the spread of a pathogen highly depends on the movement and mobility of the vector. However, questions remain as to whether and how pathogen-induced vector manipulations may affect the spread of a plant pathogen. Here we report for the first time that infection with a bacterial plant pathogen increases the probability of vector dispersal, and that such movement of vectors is likely manipulated by a bacterial plant pathogen. We investigated how Candidatus Liberibacter asiaticus (CLas) affects dispersal behavior, flight capacity, and the sexual attraction of its vector, the Asian citrus psyllid (Diaphorina citri Kuwayama). CLas is the putative causal agent of huanglongbing (HLB), which is a disease that threatens the viability of commercial citrus production worldwide. When D. citri developed on CLas-infected plants, short distance dispersal of male D. citri was greater compared to counterparts reared on uninfected plants. Flight by CLas-infected D. citri was initiated earlier and long flight events were more common than by uninfected psyllids, as measured by a flight mill apparatus. Additionally, CLas titers were higher among psyllids that performed long flights than psyllid that performed short flights. Finally, attractiveness of female D. citri that developed on infected plants to male conspecifics increased proportionally with increasing CLas bacterial titers measured within female psyllids. Our study indicates that the phytopathogen, CLas, may manipulate movement and mate selection behavior of their vectors, which is a possible evolved mechanism to promote their own spread. These results have global implications for both current HLB models of disease spread and control strategies.
doi:10.1371/journal.pone.0129373
PMCID: PMC4471203  PMID: 26083763
2.  Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen 
PLoS Pathogens  2012;8(3):e1002610.
Transmission of plant pathogens by insect vectors is a complex biological process involving interactions between the plant, insect, and pathogen. Pathogen-induced plant responses can include changes in volatile and nonvolatile secondary metabolites as well as major plant nutrients. Experiments were conducted to understand how a plant pathogenic bacterium, Candidatus Liberibacter asiaticus (Las), affects host preference behavior of its psyllid (Diaphorina citri Kuwayama) vector. D. citri were attracted to volatiles from pathogen-infected plants more than to those from non-infected counterparts. Las-infected plants were more attractive to D. citri adults than non-infected plants initially; however after feeding, psyllids subsequently dispersed to non-infected rather than infected plants as their preferred settling point. Experiments with Las-infected and non-infected plants under complete darkness yielded similar results to those recorded under light. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen. Quantification of volatile release from non-infected and infected plants supported the hypothesis that odorants mediate psyllid preference. Significantly more methyl salicylate, yet less methyl anthranilate and D-limonene, was released by infected than non-infected plants. Methyl salicylate was attractive to psyllids, while methyl anthranilate did not affect their behavior. Feeding on citrus by D. citri adults also induced release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants. Infected plants were characterized by lower levels of nitrogen, phosphorus, sulfur, zinc, and iron, as well as, higher levels of potassium and boron than non-infected plants. Collectively, our results suggest that host selection behavior of D. citri may be modified by bacterial infection of plants, which alters release of specific headspace volatiles and plant nutritional contents. Furthermore, we show in a laboratory setting that this apparent pathogen-mediated manipulation of vector behavior may facilitate pathogen spread.
Author Summary
In this investigation, we experimentally demonstrate specific mechanisms through which a bacterial plant pathogen induces plant responses that modify behavior of its insect vector. Candidatus Liberibacter asiaticus, a fastidious, phloem-limited bacterium responsible for causing huanglongbing disease of citrus, induced release of a specific volatile chemical, methyl salicylate, which increased attractiveness of infected plants to its insect vector, Diaphorina citri, and caused vectors to initially prefer infected plants. However, the insect vectors subsequently dispersed to non-infected plants as their preferred location of prolonged settling because of likely sub-optimal nutritional content of infected plants. The duration of initial feeding on infected plants was sufficiently long for the vectors to acquire the pathogen before they dispersed to non-infected plants, suggesting that the bacterial pathogen manipulates behavior of its insect vector to promote its own proliferation. The behavior of psyllids in response to infected versus non-infected plants was not influenced by whether or not they were carriers of the pathogen and was similar under both light and dark conditions. Feeding on citrus by D. citri adults also induced the release of methyl salicylate, suggesting that it may be a cue revealing location of conspecifics on host plants.
doi:10.1371/journal.ppat.1002610
PMCID: PMC3310815  PMID: 22457628
3.  Inter-Population Variability of Endosymbiont Densities in the Asian Citrus Psyllid (Diaphorina citri Kuwayama) 
Microbial Ecology  2016;71:999-1007.
The Asian citrus psyllid (Diaphorina citri Kuwayama) is an insect pest capable of transmitting Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening in North America. D. citri also harbors three endosymbionts, Wolbachia, Candidatus Carsonella ruddii, and Candidatus Profftella armatura, which may influence D. citri physiology and fitness. Although genomic researches on these bacteria have been conducted, much remains unclear regarding their ecology and inter-population variability in D. citri. The present work examined the densities of each endosymbiont in adult D. citri sampled from different populations using quantitative PCR. Under field conditions, the densities of all three endosymbionts positively correlated with each other, and they are associated with D. citri gender and locality. In addition, the infection density of CLas also varied across populations. Although an analysis pooling D. citri from different populations showed that CLas-infected individuals tended to have lower endosymbiont densities compared to uninfected individuals, the difference was not significant when the population was included as a factor in the analysis, suggesting that other population-specific factors may have stronger effects on endosymbiont densities. To determine whether there is a genetic basis to the density differences, endosymbiont densities between aged CLas-negative females of two D. citri populations reared under standardized laboratory conditions were compared. Results suggested that inter-population variability in Wolbachia infection density is associated with the genotypes of the endosymbiont or the host. Findings from this work could facilitate understanding of D. citri-bacterial associations that may benefit the development of approaches for managing citrus greening, such as prevention of CLas transmission.
Electronic supplementary material
The online version of this article (doi:10.1007/s00248-016-0733-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s00248-016-0733-9
PMCID: PMC4944574  PMID: 26846216
Huanglongbing; Intracellular endosymbionts; Primary endosymbionts; Bacteriome
4.  Asian Citrus Psyllid Expression Profiles Suggest Candidatus Liberibacter Asiaticus-Mediated Alteration of Adult Nutrition and Metabolism, and of Nymphal Development and Immunity 
PLoS ONE  2015;10(6):e0130328.
The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the insect vector of the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease, or Huanglongbing (HLB). The widespread invasiveness of the psyllid vector and HLB in citrus trees worldwide has underscored the need for non-traditional approaches to manage the disease. One tenable solution is through the deployment of RNA interference technology to silence protein-protein interactions essential for ACP-mediated CLas invasion and transmission. To identify psyllid interactor-bacterial effector combinations associated with psyllid-CLas interactions, cDNA libraries were constructed from CLas-infected and CLas-free ACP adults and nymphs, and analyzed for differential expression. Library assemblies comprised 24,039,255 reads and yielded 45,976 consensus contigs. They were annotated (UniProt), classified using Gene Ontology, and subjected to in silico expression analyses using the Transcriptome Computational Workbench (TCW) (http://www.sohomoptera.org/ACPPoP/). Functional-biological pathway interpretations were carried out using the Kyoto Encyclopedia of Genes and Genomes databases. Differentially expressed contigs in adults and/or nymphs represented genes and/or metabolic/pathogenesis pathways involved in adhesion, biofilm formation, development-related, immunity, nutrition, stress, and virulence. Notably, contigs involved in gene silencing and transposon-related responses were documented in a psyllid for the first time. This is the first comparative transcriptomic analysis of ACP adults and nymphs infected and uninfected with CLas. The results provide key initial insights into host-parasite interactions involving CLas effectors that contribute to invasion-virulence, and to host nutritional exploitation and immune-related responses that appear to be essential for successful ACP-mediated circulative, propagative CLas transmission.
doi:10.1371/journal.pone.0130328
PMCID: PMC4474670  PMID: 26091106
5.  Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants 
Applied and Environmental Microbiology  2015;81(11):3728-3736.
Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 103 to 107 bacteria per insect. In spring, the infection densities were low in March, at ∼103 bacteria per insect, increasing up to 106 to 107 bacteria per insect in April and May, and decreasing to 105 to 106 bacteria per insect in late May, whereas the infection densities were constantly ∼106 to 107 bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼106 bacteria per insect) of “Ca. Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.
doi:10.1128/AEM.00707-15
PMCID: PMC4421049  PMID: 25819961
6.  Acquisition, Replication and Inoculation of Candidatus Liberibacter asiaticus following Various Acquisition Periods on Huanglongbing-Infected Citrus by Nymphs and Adults of the Asian Citrus Psyllid 
PLoS ONE  2016;11(7):e0159594.
The Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) implicated as causative agent of citrus huanglongbing (citrus greening), currently the most serious citrus disease worldwide. Las is transmitted by D. citri in a persistent-circulative manner, but the question of replication of this bacterium in its psyllid vector has not been resolved. Thus, we studied the effects of the acquisition access period (AAP) by nymphs and adults of D. citri on Las acquisition, multiplication and inoculation/transmission. D. citri nymphs or adults (previously non-exposed to Las) were caged on Las-infected citrus plants for an AAP of 1, 7 or 14 days. These ‘Las-exposed’ psyllids were then transferred weekly to healthy citrus or orange jasmine plants, and sampled via quantitative polymerase chain reaction (qPCR) analysis 1–42 days post-first access to diseased plants (padp); all tested nymphs became adults 7–14 days padp. Our results indicate that following 1 or 7 day AAP as nymphs 49–59% of Las-exposed psyllids became Las-infected (qPCR-positive), whereas only 8–29% of the psyllids were infected following 1–14 day AAP as adults. Q-PCR analysis also indicated that Las titer in the Las-exposed psyllids (relative to that of the psyllid S20 ribosomal protein gene) was: 1) significantly higher, and increasing at a faster rate, following Las acquisition as nymphs compared to that following Las acquisition as adults; 2) higher as post-acquisition time of psyllids on healthy plants increased reaching a peak at 14–28 days padp for nymphs and 21–35 days padp for adults, with Las titer decreasing or fluctuating after that; 3) higher with longer AAP on infected plants, especially with acquisition as adults. Our results strongly suggest that Las multiplies in both nymphs and adults of D. citri but attains much higher levels in a shorter period of time post-acquisition when acquired by nymphs than when acquired by adults, and that adults may require longer access to infected plants compared to nymphs for Las to reach higher levels in the vector. However, under the conditions of our experiments, only D. citri that had access to infected plants as nymphs were able to inoculate Las into healthy citrus seedlings or excised leaves. The higher probability of Las inoculation into citrus by psyllids when they have acquired this bacterium from infected plants during the nymphal rather than the adult stage, as reported by us and others, has significant implications in the epidemiology and control of this economically important citrus disease.
doi:10.1371/journal.pone.0159594
PMCID: PMC4956146  PMID: 27441694
7.  Annotation of the Asian Citrus Psyllid Genome Reveals a Reduced Innate Immune System 
Citrus production worldwide is currently facing significant losses due to citrus greening disease, also known as Huanglongbing. The citrus greening bacteria, Candidatus Liberibacter asiaticus (CLas), is a persistent propagative pathogen transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Hemipterans characterized to date lack a number of insect immune genes, including those associated with the Imd pathway targeting Gram-negative bacteria. The D. citri draft genome was used to characterize the immune defense genes present in D. citri. Predicted mRNAs identified by screening the published D. citri annotated draft genome were manually searched using a custom database of immune genes from previously annotated insect genomes. Toll and JAK/STAT pathways, general defense genes Dual oxidase, Nitric oxide synthase, prophenoloxidase, and cellular immune defense genes were present in D. citri. In contrast, D. citri lacked genes for the Imd pathway, most antimicrobial peptides, 1,3-β-glucan recognition proteins (GNBPs), and complete peptidoglycan recognition proteins. These data suggest that D. citri has a reduced immune capability similar to that observed in A. pisum, P. humanus, and R. prolixus. The absence of immune system genes from the D. citri genome may facilitate CLas infections, and is possibly compensated for by their relationship with their microbial endosymbionts.
doi:10.3389/fphys.2016.00570
PMCID: PMC5126049  PMID: 27965582
Diaphorina citri; immune; genes; Imd; liberibacter; antimicrobial peptide; virus
8.  Better Together: Association With ‘Candidatus Liberibacter Asiaticus’ Increases the Reproductive Fitness of Its Insect Vector, Diaphorina citri (Hemiptera: Liviidae) 
The duration of the evolutionary association between a pathogen and vector can be inferred based on the strength of their mutualistic interactions. A well-adapted pathogen is likely to confer some benefit or, at a minimum, exhibit low pathogenicity toward its host vector. Coevolution of the two toward a mutually beneficial association appears to have occurred between the citrus greening disease pathogen, Candidatus Liberibacter asiaticus (Las), and its insect vector, the Asian citrus psyllid, Diaphorina citri (Kuwayama). To better understand the dynamics facilitating transmission, we evaluated the effects of Las infection on the fitness of its vector. Diaphorina citri harboring Las were more fecund than their uninfected counterparts; however, their nymphal development rate and adult survival were comparatively reduced. The finite rate of population increase and net reproductive rate were both greater among Las-infected D. citri as compared with uninfected counterparts, indicating that overall population fitness of infected psyllids was improved given the greater number of offspring produced. Previous reports of transovarial transmission, in conjunction with increased fecundity and population growth rates of Las-positive D. citri found in the current investigation, suggest a long evolutionary relationship between pathogen and vector. The survival of Las-infected adult D. citri was lower compared with uninfected D. citri, which suggests that there may be a fitness trade-off in response to Las infection. A beneficial effect of a plant pathogen on vector fitness may indicate that the pathogen developed a relationship with the insect before secondarily moving to plants.
doi:10.1093/aesa/saw007
PMCID: PMC4874362  PMID: 27418697
Liberibacter; citrus greening; Huanglongbing; fitness
9.  Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama) 
FEBS Open Bio  2015;5:264-275.
Graphical abstract
Highlights
•A putative JH acid methyltransferase ortholog gene (jmtD) and its cognate cDNA were identified in D. citri.•This gene is expressed in all life stages in D. citri.•The purified enzyme expressed from cDNA in E. coli showed high preference for farnesoic acid (FA) and homoFA.•This suggests that this ortholog is a DcFA-o-methyl transferase gene (fmtD), and not jmtD.•First purified FA-o-MT from any insect order with preferred biological activity for FA and not JHA.
The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus ‘Liberibacter’ asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in JH biosynthesis in D. citri. A putative JH acid methyltransferase ortholog gene (jmtD) and its cognate cDNA were identified by searching D. citri genome database. Expression analysis shows expression in all life stages. In adults, it is expressed in the head-thorax, (containing the corpora allata), and the abdomen (containing ovaries and male accessory glands). A 3D protein model identified the catalytic groove with catalytically active amino acids and the S-adenosyl methionine (SAM)-binding loop. The cDNA was expressed in Escherichia coli cells and the purified enzyme showed high preference for farnesoic acid (FA) and homoFA (kcat of 0.752 × 10−3 and 0.217 × 10−3 s−1, respectively) as compared to JH acid I (JHA I) (cis/trans/cis; 2Z, 6E, 10cis), JHA III (2E, 6E, 10cis), and JHA I (trans/cis/cis; 2E, 2Z, 10cis) (kcat of 0.081 × 10−3, 0.013 × 10−3, and 0.003 × 10−3 s−1, respectively). This suggests that this ortholog is a DcFA-o-methyl transferase gene (fmtD), not a jmtD, and that JH biosynthesis in D. citri proceeds from FA to JH III through methyl farnesoate (MF). DcFA-o-MT does not require Ca2+, Mg2+ or Zn2+, however, Zn2+ (1 mM) completely inhibits the enzyme probably by binding H115 at the active groove. This represents the first purified FA-o-MT from Hemiptera with preferred biological activity for FA and not JHA.
doi:10.1016/j.fob.2015.03.012
PMCID: PMC4398755  PMID: 25893162
FA, farnesoic acid; FA-o-MT, farnesoic acid o-methyltransferase; fmtD, Diaphorina citri farnesoic acid o-methyltransferase gene; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; JH, juvenile hormone; JHA, juvenile hormone acid; JHAMT, juvenile hormone acid methyltransferase; jmtD, juvenile hormone acid methyl transferase ortholog gene of Diaphorina citri; MF, methyl farnesoate; MMLV, Moloney murine leukemia virus; RP, reversed phase; SAM, S-adenosyl methionine; SAM-MT, S-adenosyl methionine-dependent methyltransferase; Diaphorina citri; Juvenile hormone acid methyltransferase; Farnesoic acid methyltransferase; 3D modeling; Gene expression
10.  Comparison of Potato and Asian Citrus Psyllid Adult and Nymph Transcriptomes Identified Vector Transcripts with Potential Involvement in Circulative, Propagative Liberibacter Transmission 
Pathogens  2014;3(4):875-907.
The potato psyllid (PoP) Bactericera cockerelli (Sulc) and Asian citrus psyllid (ACP) Diaphorina citri Kuwayama are the insect vectors of the fastidious plant pathogen, Candidatus Liberibacter solanacearum (CLso) and Ca. L. asiaticus (CLas), respectively. CLso causes Zebra chip disease of potato and vein-greening in solanaceous species, whereas, CLas causes citrus greening disease. The reliance on insecticides for vector management to reduce pathogen transmission has increased interest in alternative approaches, including RNA interference to abate expression of genes essential for psyllid-mediated Ca. Liberibacter transmission. To identify genes with significantly altered expression at different life stages and conditions of CLso/CLas infection, cDNA libraries were constructed for CLso-infected and -uninfected PoP adults and nymphal instars. Illumina sequencing produced 199,081,451 reads that were assembled into 82,224 unique transcripts. PoP and the analogous transcripts from ACP adult and nymphs reported elsewhere were annotated, organized into functional gene groups using the Gene Ontology classification system, and analyzed for differential in silico expression. Expression profiles revealed vector life stage differences and differential gene expression associated with Liberibacter infection of the psyllid host, including invasion, immune system modulation, nutrition, and development.
doi:10.3390/pathogens3040875
PMCID: PMC4282890  PMID: 25436509
circulative-propagative transmission; fastidious plant bacteria; psyllid vector; transcriptome
11.  Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus 
Scientific Reports  2016;6:33418.
Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as Huanglongbing (HLB). CLas is transmitted by the Asian citrus psyllid (ACP) Diaphorina citri, in a circulative manner. Two major barriers to transmission within the insect are the midgut and the salivary glands. We performed a thorough microscopic analysis within the insect midgut following exposure to CLas-infected citrus trees. We observed changes in nuclear architecture, including pyknosis and karyorrhexis as well as changes to the actin cytoskeleton in CLas-exposed midgut cells. Further analyses showed that the changes are likely due to the activation of programmed cell death as assessed by Annexin V staining and DNA fragmentation assays. These results suggest that exposure to CLas-infected trees induces apoptotic responses in the psyllid midgut that should be further investigated. Understanding the adaptive significance of the apoptotic response has the potential to create new approaches for controlling HLB.
doi:10.1038/srep33418
PMCID: PMC5024303  PMID: 27630042
12.  Changes in Variable Number of Tandem Repeats in ‘Candidatus Liberibacter asiaticus’ through Insect Transmission 
PLoS ONE  2015;10(9):e0138699.
Citrus greening (huanglongbing) is the most destructive citrus disease worldwide. The disease is associated with three species of ‘Candidatus Liberibacter’ among which ‘Ca. Liberibacter asiaticus’ has the widest distribution. ‘Ca. L. asiaticus’ is commonly transmitted by a phloem-feeding insect vector, the Asian citrus psyllid Diaphorina citri. A previous study showed that isolates of ‘Ca. L. asiaticus’ were clearly differentiated by variable number of tandem repeat (VNTR) profiles at four loci in the genome. In this study, the VNTR analysis was further validated by assessing the stability of these repeats after multiplication of the pathogen upon host-to-host transmission using a ‘Ca. L. asiaticus’ strain from Japan. The results showed that some tandem repeats showed detectable changes after insect transmission. To our knowledge, this is the first report to demonstrate that the repeat numbers VNTR 002 and 077 of ‘Ca. L. asiaticus’ change through psyllid transmission. VNTRs in the recipient plant were apparently unrelated to the growing phase of the vector. In contrast, changes in the number of tandem repeats increased with longer acquisition and inoculation access periods, whereas changes were not observed through psyllid transmission after relatively short acquisition and inoculation access periods, up to 20 and 19 days, respectively.
doi:10.1371/journal.pone.0138699
PMCID: PMC4581716  PMID: 26402645
13.  The Phloem-Sap Feeding Mealybug (Ferrisia virgata) Carries ‘Candidatus Liberibacter asiaticus’ Populations That Do Not Cause Disease in Host Plants 
PLoS ONE  2014;9(1):e85503.
‘Candidatus Liberibacter asiaticus’ (Las) is the primary causal agent of huanglongbing (HLB), the most devastating disease of citrus worldwide. There are three known insect vectors of the HLB-associated bacteria, and all are members of the Hemiptera: Diaphorina citri (Psyllidae), Trioza erytreae (Triozidae), and Cacopsylla (Psylla) citrisuga (Psyllidae). In this study, we found that another hemipteran, the striped mealybug Ferrisia virgata (Cockerell) (Hemiptera: Pseudococcidae), was able to acquire and retain Las bacteria. The bacterial titers were positively correlated with the feeding acquisition time on Las-infected leaf discs, with a two-weeks feeding period resulting in Ct values ranging from 23.1 to 36.1 (8.24×107 to 1.07×104 Las cells per mealybug). We further discovered that the prophage/phage populations of Las in the mealybugs were different from those of Las in psyllids based on Las prophage-specific molecular markers: infected psyllids harbored the Las populations with prophage/phage FP1 and FP2, while infected mealybugs carried the Las populations with the iFP3 being the dominant prophage/phage. As in the psyllids, Las bacteria were shown to move through the insect gut wall to the salivary glands after being ingested by the mealybug based on a time-course quantitative polymerase chain reaction (qPCR) assay of the dissected digestive systems. However, Las populations transmitted by the mealybugs did not cause disease in host plants. This is the first evidence of genetic difference among Las populations harbored by different insect vectors and difference among Las populations with respect to whether or not they cause disease in host plants.
doi:10.1371/journal.pone.0085503
PMCID: PMC3896372  PMID: 24465578
14.  Stylet Morphometrics and Citrus Leaf Vein Structure in Relation to Feeding Behavior of the Asian Citrus Psyllid Diaphorina citri, Vector of Citrus Huanglongbing Bacterium 
PLoS ONE  2013;8(3):e59914.
The Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae), is the primary vector of the phloem-limited bacterium Candidatus Liberibacter asiaticus (LAS) associated with huanglongbing (HLB, citrus greening), considered the world’s most serious disease of citrus. Stylet morphometrics of ACP nymphs and adults were studied in relation to citrus vein structure and to their putative (histologically verified) feeding sites on Valencia orange leaves. ACP nymphs preferred to settle and feed on the lower (abaxial) side of young leaves either on secondary veins or on the sides of the midrib, whereas adults preferred to settle and feed on the upper (adaxial) or lower secondary veins of young or old leaves. Early instar nymphs can reach and probe the phloem probably because the distance to the phloem is considerably shorter in younger than in mature leaves, and is shorter from the sides of the midrib compared to that from the center. Additionally, the thick-walled ‘fibrous ring’ (sclerenchyma) around the phloem, which may act as a barrier to ACP stylet penetration into the phloem, is more prominent in older than in younger leaves and in the center than on the sides of the midrib. The majority (80–90%) of the salivary sheath termini produced by ACP nymphs and adults that reached a vascular bundle were associated with the phloem, whereas only 10–20% were associated with xylem vessels. Ultrastructural studies on ACP stylets and LAS-infected leaves suggested that the width of the maxillary food canal in first instar nymphs is wide enough for LAS bacteria to traverse during food ingestion (and LAS acquisition). However, the width of the maxillary salivary canal in these nymphs may not be wide enough to accommodate LAS bacteria during salivation (and LAS inoculation) into host plants. This may explain the inability of early instar nymphs to transmit LAS/HLB in earlier reports.
doi:10.1371/journal.pone.0059914
PMCID: PMC3608565  PMID: 23555830
15.  Horizontal Gene Acquisition of Liberibacter Plant Pathogens from a Bacteriome-Confined Endosymbiont of Their Psyllid Vector 
PLoS ONE  2013;8(12):e82612.
he Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits the phloem-inhabiting alphaproteobacterial ‘Candidatus Liberibacter asiaticus’ and allied plant pathogens, which cause the devastating citrus disease called Huanglongbing or greening disease. D. citri harbors two distinct bacterial mutualists in the symbiotic organ called bacteriome: the betaproteobacterium ‘Candidatus Profftella armatura’ in the syncytial cytoplasm at the center of the bacteriome, and the gammaproteobacterium ‘Candidatus Carsonella ruddii’ in uninucleate bacteriocytes. Here we report that a putative amino acid transporter LysE of Profftella forms a highly supported clade with proteins of L. asiaticus, L. americanus, and L. solanacearum. L. crescens, the most basal Liberibacter lineage currently known, lacked the corresponding gene. The Profftella-Liberibacter subclade of LysE formed a clade with proteins from betaproteobacteria of the order Burkholderiales, to which Profftella belongs. This phylogenetic pattern favors the hypothesis that the Liberibacter lineage acquired the gene from the Profftella lineage via horizontal gene transfer (HGT) after L. crescens diverged from other Liberibacter lineages. KA/KS analyses further supported the hypothesis that the genes encoded in the Liberibacter genomes are functional. These findings highlight the possible evolutionary importance of HGT between plant pathogens and their insect vector’s symbionts that are confined in the symbiotic organ and seemingly sequestered from external microbial populations.
doi:10.1371/journal.pone.0082612
PMCID: PMC3857777  PMID: 24349319
16.  Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing 
PLoS ONE  2015;10(12):e0145132.
Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.
doi:10.1371/journal.pone.0145132
PMCID: PMC4696824  PMID: 26717484
17.  Metabolic Interplay between the Asian Citrus Psyllid and Its Profftella Symbiont: An Achilles’ Heel of the Citrus Greening Insect Vector 
PLoS ONE  2015;10(11):e0140826.
‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including ‘Candidatus Profftella armatura’, are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.
doi:10.1371/journal.pone.0140826
PMCID: PMC4651294  PMID: 26580079
18.  Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick 
BMC Microbiology  2014;14:86.
Background
Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions.
Results
A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids.
Conclusions
Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors.
doi:10.1186/1471-2180-14-86
PMCID: PMC4021466  PMID: 24708539
Huanglongbing; Candidatus Liberibacter asiaticus; Diaphorina citri
19.  Survey of Endosymbionts in the Diaphorina citri Metagenome and Assembly of a Wolbachia wDi Draft Genome 
PLoS ONE  2012;7(11):e50067.
Diaphorina citri (Hemiptera: Psyllidae), the Asian citrus psyllid, is the insect vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening disease. Sequencing of the D. citri metagenome has been initiated to gain better understanding of the biology of this organism and the potential roles of its bacterial endosymbionts. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the D. citri metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Genome alignment with the complete sequences reveals membership of Wolbachia wDi in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida D. citri isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese D. citri isolates, supporting the hypothesis that the D. citri introduced into Florida did not originate from China.
doi:10.1371/journal.pone.0050067
PMCID: PMC3500351  PMID: 23166822
20.  Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats ▿ †  
Applied and Environmental Microbiology  2011;77(18):6663-6673.
“Candidatus Liberibacter asiaticus” is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of “Ca. Liberibacter” associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes (hyvI and hyvII) were identified in the prophage regions of the Psy62 “Ca. Liberibacter asiaticus” genome. Sequence analyses of the hyvI and hyvII genes in 35 “Ca. Liberibacter asiaticus” DNA isolates collected globally revealed that the hyvI gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyvII contains up to 2 NITRs and 4 partial repeats and shares homology with hyvI. Frequent deletions or insertions of these repeats within the hyvI and hyvII genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of “Ca. Liberibacter asiaticus” bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single “Ca. Liberibacter asiaticus”-infected sample. This is the first evidence of different “Ca. Liberibacter asiaticus” populations coexisting in a single HLB-affected sample. The Florida “Ca. Liberibacter asiaticus” isolates contain both hyvI and hyvII, while all other global “Ca. Liberibacter asiaticus” isolates contain either one or the other. Interclade assignments of the putative HyvI and HyvII proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple “Ca. Liberibacter asiaticus” populations in the world and a multisource introduction of the “Ca. Liberibacter asiaticus” bacterium into Florida.
doi:10.1128/AEM.05111-11
PMCID: PMC3187138  PMID: 21784907
21.  Oral Delivery of Double-Stranded RNAs and siRNAs Induces RNAi Effects in the Potato/Tomato Psyllid, Bactericerca cockerelli 
PLoS ONE  2011;6(11):e27736.
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.
doi:10.1371/journal.pone.0027736
PMCID: PMC3218023  PMID: 22110747
22.  Candidatus Liberibacter americanus induces significant reprogramming of the transcriptome of the susceptible citrus genotype 
BMC Genomics  2013;14:247.
Background
Citrus huanglongbing (HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria named Candidatus Liberibacter africanus (CaLaf), Ca. L. asiaticus (CaLas), and Ca. L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector, Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam.
Results
The microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes from Arabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas.
Conclusions
Many gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB.
doi:10.1186/1471-2164-14-247
PMCID: PMC3635983  PMID: 23586643
Gene expression; Sweet orange; huanglongbing; Plant-pathogen interaction
23.  Temperature Studies with the Asian Citrus Psyllid, Diaphorina citri: Cold Hardiness and Temperature Thresholds for Oviposition 
This study was conducted to obtain information on the cold hardiness of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in Florida and to assess upper and lower temperature thresholds for oviposition. The psyllid is an important pest in citrus because it transmits the bacterial pathogens responsible for citrus greening disease, Huanglongbing, considered the most serious citrus disease worldwide. D. citri was first found in Florida during 1998, and the disease was discovered during 2005. Little was known regarding cold hardiness of D. citri, but Florida citrus is occasionally subjected to notable freeze events. Temperature and duration were each significant sources of variation in percent mortality of D. citri subjected to freeze events. Relatively large percentages of adults and nymphs survived after being exposed for several hours to temperatures as low as -5 to -6° C. Relatively large percentages of eggs hatched after being exposed for several hours to temperatures as low as -8° C. Research results indicated that adult D. citri become cold acclimated during the winter through exposure to cooler winter temperatures. There was no evidence that eggs became cold acclimated during winter. Cold acclimation in nymphs was not investigated. Research with adult D. citri from laboratory and greenhouse colonies revealed that mild to moderate freeze events were usually nonlethal to the D. citri irrespective of whether they were cold acclimated or not. Upper and lower temperature thresholds for oviposition were investigated because such information may be valuable in explaining the geographic distribution and potential spread of the pest from Florida as well as how cooler winter temperatures might limit population growth. The estimated lower and upper thresholds for oviposition were 16.0 and 41.6° C, respectively; the estimated temperature of peak oviposition over a 48 h period was 29.6° C.
doi:10.1673/031.011.8301
PMCID: PMC3281434  PMID: 21870969
insecticide; cold acclimation; Candidatus Liberibacter asiaticus; Huanglongbing
24.  Characterization of the Relative Abundance of the Citrus Pathogen Ca. Liberibacter Asiaticus in the Microbiome of Its Insect Vector, Diaphorina citri, using High Throughput 16S rRNA Sequencing 
The relationship between the causal agent of Huanglongbing (HLB), Ca. Liberibacter asiaticus(Las), and the naturally occurring endosymbiotic community of its insect vector, the Asian citrus psyllid (ACP), Diaphorina citri, was studied. Variation was observed in the titer of Las within an ACP population feeding on the same material. The cause of this disparity is unknown, and has implications for Las transmission and the spread of HLB. This study utilizes culture independent methods to establish the relationship between the ACP’s microbial community and Las acquisition. DNA from 21 psyllids was amplified using universal 16S rRNA primers with Illumina adaptor regions and a sample-specific 7- base identifier. These amplicons were then batch-sequenced on the Illumina platform. The resulting sequences were separated by the identifier, and compared to known sequences in a 16S rRNA database. The microbial communities of each psyllid were compared to determine whether a correlation exists between the ACP’s endosymbionts and level of Las acquisition.
ACPs were dominated by the same four bacterialgenera regardless of the abundance of Ca.Liberibacter. A combination of qPCR and Illumina sequencing was used to establish an infection gradient among the sampled ACPs. The Ca. Liberibacter titer within the insect was found to have a strong negative relationship with an endosymbiont residing in the syncytium of the mycetocyte and a positive relationship with Wolbachia. These correlations have implications in the acquisition of Las by the ACP as well as the activities of Las within this vector.
doi:10.2174/1874285801206010029
PMCID: PMC3330398  PMID: 22529882
Bacteriocyte; Huanglongbing; Illumina; multiplex; psyllid; syncytium endosymbiont.
25.  Repellent Activity of Botanical Oils against Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae) 
Insects  2016;7(3):35.
The Asian citrus psyllid, Diaphorina citri Kuwayama, is the insect vector of the pathogen causing huanglongbing. We selected three botanical oils to evaluate behavioral activity against D. citri. In laboratory olfactometer assays, fir oil was repellent to D. citri females, while litsea and citronella oils elicited no response from D. citri females. In choice settling experiments, D. citri settled almost completely on control plants rather than on plants treated with fir oil at a 9.5 mg/day release rate. Therefore, we conducted field trials to determine if fir oil reduced D. citri densities in citrus groves. We found no repellency of D. citri from sweet orange resets that were treated with fir oil dispensers releasing 10.4 g/day/tree as compared with control plots. However, we found a two-week decrease in populations of D. citri as compared with controls when the deployment rate of these dispensers was doubled. Our results suggest that treatment of citrus with fir oil may have limited activity as a stand-alone management tool for D. citri and would require integration with other management practices.
doi:10.3390/insects7030035
PMCID: PMC5039548  PMID: 27429006
citrus greening disease; essential oil; repellent; push-pull; integrated pest management

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