Host Plant Adaptation Drives Changes in Diaphorina citri Proteome Regulation, Proteoform Expression, and Transmission of 'Candidatus Liberibacter asiaticus', the Citrus Greening Pathogen.

The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, 'Candidatus Liberibacter asiaticus' (CLas), which is associated with citrus greening disease. The citrus relative Murraya paniculata (orange jasmine) is a host plant of D. citri but is more resistant to CLas compared with all tested Citrus genotypes. The effect of host switching of D. citri between Citrus medica (citron) and M. paniculata plants on the acquisition and transmission of CLas was investigated. The psyllid CLas titer and the proportion of CLas-infected psyllids decreased in the generations after transfer from CLas-infected citron to healthy M. paniculata plants. Furthermore, after several generations of feeding on M. paniculata, pathogen acquisition (20 to 40% reduction) and transmission rates (15 to 20% reduction) in psyllids transferred to CLas-infected citron were reduced compared with psyllids continually maintained on infected citron. Top-down (difference gel electrophoresis) and bottom-up (shotgun MS/MS) proteomics methods were used to identify changes in D. citri protein expression resulting from host plant switching between Citrus macrophylla and M. paniculata. Changes in expression of insect metabolism, immunity, and cytoskeleton proteins were associated with host plant switching. Both transient and sustained feeding on M. paniculata induced distinct patterns of protein expression in D. citri compared with psyllids reared on C. macrophylla. The results point to complex interactions that affect vector competence and may lead to strategies to control the spread of citrus greening disease.

Diaphorina citri Nymphs Are Resistant to Morphological Changes Induced by "Candidatus Liberibacter asiaticus" in Midgut Epithelial Cells.

"Candidatus Liberibacter asiaticus" is the causative bacterium associated with citrus greening disease. "Ca Liberibacter asiaticus" is transmitted by Diaphorina citri more efficiently when it is acquired by nymphs rather than adults. Why this occurs is not known. We compared midguts of D. citri insects reared on healthy or "Ca Liberibacter asiaticus"-infected citrus trees using quantitative PCR, confocal microscopy, and mitochondrial superoxide staining for evidence of oxidative stress. Consistent with its classification as propagative, "Ca Liberibacter asiaticus" titers were higher in adults than in nymphs. Our previous work showed that adult D. citri insects have basal levels of karyorrhexis (fragmentation of the nucleus) in midgut epithelial cells, which is increased in severity and frequency in response to "Ca Liberibacter asiaticus." Here, we show that nymphs exhibit lower levels of early-stage karyorrhexis than adults and are refractory to the induction of advanced karyorrhexis by "Ca Liberibacter asiaticus" in the midgut epithelium. MitoSox Red staining showed that guts of infected adults, particularly males, experienced oxidative stress in response to "Ca Liberibacter asiaticus." A positive correlation between the titers of "Ca Liberibacter asiaticus" and the Wolbachia endosymbiont was observed in adult and nymph midguts, suggesting an interplay between these bacteria during development. We hypothesize that the resistance of the nymph midgut to late-stage karyorrhexis through as yet unknown molecular mechanisms benefits "Ca Liberibacter asiaticus" for efficient invasion of midgut epithelial cells, which may be a factor explaining the developmental dependency of "Ca Liberibacter asiaticus" acquisition by the vector.

Effect of Cyantraniliprole, a Novel Insecticide, on the Inoculation of Candidatus Liberibacter Asiaticus Associated with Citrus Huanglongbing by the Asian Citrus Psyllid (Hemiptera: Liviidae).

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the principal vector of 'Candidatus Liberibacter asiaticus' (CLas) associated with huanglongbing (HLB), the most serious citrus disease worldwide. New control measures including pesticides are urgently needed to combat HLB, especially to protect young or newly planted citrus trees from CLas-inoculation by vector psyllids. Here, we tested CLas-inoculation by D. citri adults (CLas-exposed, reared on infected plants) by feeding them for 7 d on excised healthy citrus leaves with dry residues of cyantraniliprole (Exirel), a novel insecticide, in comparison with fenpropathrin (Danitol 2.4EC), an insecticide commonly used against D. citri. Fewer adults settled (putatively feeding or probing) on leaves treated with cyantraniliprole than those treated with fenpropathrin or water controls. Also, psyllid adults died at a slower rate on leaves treated with cyantraniliprole than those treated with fenpropathrin, although the final cumulative mortality did not differ between the two treatments. In quantitative real-time polymerase chain reaction tests, 59.0-65.3% of the CLas-exposed psyllid adults were proven to be CLas-positive. Inoculation rates of CLas (using 10 adults per leaf) into untreated healthy citrus leaves (47.5-85%) were significantly higher than rates into leaves treated with cyantraniliprole or fenpropathrin (2.5-12.5%). Reduced inoculation rates to leaves treated with cyantraniliprole probably occurred as a result of reduced feeding or probing by D. citri. The excised leaf assay method, which took only a few weeks compared with up to a year or longer using whole plants, can be an effective tool for testing the effect of new pesticides or other treatments in reducing CLas inoculation or transmission by psyllid vectors.

New excised-leaf assay method to test inoculativity of Asian citrus psyllid (Hemiptera: Psyllidae) with Candidatus Liberibacter asiaticus associated with citrus huanglongbing disease.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) associated with huanglongbing, or citrus greening, the most devastating citrus (Citrus spp.) disease worldwide. Here, we developed a new "excised-leaf assay" that can speed up Las-inoculativity tests on Asian citrus psyllid from the current 3-12 mo (when using whole citrus seedlings for inoculation) to only 2-3 wk. Young adults of Asian citrus psyllid that had been reared on Las-infected plants were caged on excised healthy sweet orange [Citrus sinensis (L.) Osbeck] leaves for a 1-2-wk inoculation access periods (IAP), and then both psyllids and leaves were tested later by quantitative polymerase chain reaction (PCR). When single adults were tested per leaf, percentages of Las-positive leaves averaged 2-6% by using HLBaspr primers and 10-20% by using the more sensitive LJ900 primers. Higher proportions of Las-positive leaves were obtained with 1) higher densities of inoculating psyllids (5-10 adults per leaf), 2) longer IAPs, and 3) incubation of leaves for 1 wk postinoculation before PCR. Logistic regression analysis indicated a positive correlation between Las titer in Asian citrus psyllid adults tested singly and the probability of detecting Las in the inoculated leaves, correlations that can be very useful in epidemiological studies. Comparison between excised leaves and whole seedlings, inoculated consecutively for 1 wk each by one or a group of psyllids, indicated no significant difference between Las detection in excised leaves or whole plants. This new excised-leaf assay method saves considerable time, materials, and greenhouse space, and it may enhance vector relation and epidemiological studies on Las and potentially other Liberibacter spp. associated with huanglongbing disease.

The bacterium Pantoea stewartii uses two different type III secretion systems to colonize its plant host and insect vector.

Plant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (herein referred to as P. stewartii), the causative agent of Stewart's bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis, P. stewartii has a second T3SS (Pantoea secretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector, Chaetocnema pulicaria (Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence of P. stewartii in flea beetle guts and the beetle's ability to transmit P. stewartii to maize. Ectopic expression of the psaN gene complements these phenotypes. In addition, the PSI-2 psaN gene is not required for P. stewartii pathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle of P. stewartii as it alternates between its insect vector and plant host.

Spiroplasma-like organisms closely associated with the gut in five leafhopper species (Hemiptera: Cicadellidae).

Spiroplasmas are bacteria in the Class Mollicutes that are frequently associated with insects and/or plants. Here, we describe the ultrastructure, localization, and occurrence of apparent commensal/symbiotic spiroplasma-like organisms (SLOs) in the midgut and hindgut of five leafhopper species from laboratory-reared colonies. Those found in Dalbulus elimatus, Endria inimica, and Macrosteles quadrilineatus were long and tubular shaped, whereas those in Dalbulus maidis and Graminella nigrifrons were shorter and mostly rod-shaped in their host organisms. These SLOs were found in great numbers in the gut lumen frequently associated with the gut microvilli, but unlike the plant-pathogenic mollicutes, they did not seem to invade the gut epithelium or other tissues in any of these five leafhopper species. Large accumulations of these gut-associated organisms were more commonly found by confocal laser scanning microscopy in males than in females and in crowded than in singly reared leafhoppers. Ultrastructural evidence suggests that these SLOs may be horizontally transmitted between leafhoppers by contamination of the mouth parts with leafhopper excretions.

Plant host range and leafhopper transmission of maize fine streak virus.

Maize fine streak virus (MFSV), an emerging Rhabdovirus sp. in the genus Nucleorhabdovirus, is persistently transmitted by the black-faced leafhopper, Graminella nigrifrons (Forbes). MFSV was transmitted to maize, wheat, oat, rye, barley, foxtail, annual ryegrass, and quackgrass by G. nigrifrons. Parameters affecting efficiency of MFSV acquisition (infection) and transmission (inoculation) to maize were evaluated using single-leafhopper inoculations and enzyme-linked immunosorbent assay. MFSV was detected in ≈20% of leafhoppers that fed on infected plants but <10% of insects transmitted the virus. Nymphs became infected earlier and supported higher viral titers than adults but developmental stage at aquisition did not affect the rate of MFSV transmission. Viral titer and transmission also increased with longer post-first access to diseased periods (PADPs) (the sum of the intervals from the beginning of the acquisition access period to the end of the inoculation access period). Length of the acquisition access period was more important for virus accumulation in adults, whereas length of the interval between acquisition access and inoculation access was more important in nymphs. A threshold viral titer was needed for transmission but no transmission occurred, irrespective of titer, with a PADP of <4 weeks. MFSV was first detected by immunofluorescence confocal laser scanning microscopy at 2-week PADPs in midgut cells, hemocytes, and neural tissues; 3-week PADPs in tracheal cells; and 4-week PADPs in salivary glands, coinciding with the time of transmission to plants.

Insect vector interactions with persistently transmitted viruses.

The majority of described plant viruses are transmitted by insects of the Hemipteroid assemblage that includes aphids, whiteflies, leafhoppers, planthoppers, and thrips. In this review we highlight progress made in research on vector interactions of the more than 200 plant viruses that are transmitted by hemipteroid insects beginning a few hours or days after acquisition and for up to the life of the insect, i.e., in a persistent-circulative or persistent-propagative mode. These plant viruses move through the insect vector, from the gut lumen into the hemolymph or other tissues and finally into the salivary glands, from which these viruses are introduced back into the plant host during insect feeding. The movement and/or replication of the viruses in the insect vectors require specific interactions between virus and vector components. Recent investigations have resulted in a better understanding of the replication sites and tissue tropism of several plant viruses that propagate in insect vectors. Furthermore, virus and insect proteins involved in overcoming transmission barriers in the vector have been identified for some virus-vector combinations.

Asian citrus psyllid adults inoculate huanglongbing bacterium more efficiently than nymphs when this bacterium is acquired by early instar nymphs.

The Asian citrus psyllid (Diaphorina citri) transmits the bacterium 'Candidatus Liberibacter asiaticus' (CLas), which causes huanglongbing (citrus greening) disease, in a circulative-propagative manner. We compared CLas inoculation efficiency of D. citri nymphs and adults into healthy (uninfected) citron leaves when both vector stages were reared from eggs on infected plants. The proportion of CLas-positive leaves was 2.5% for nymphs and 36.3% for adults. CLas acquisition by early instar nymphs followed by dissections of adults and 4th instar nymphs revealed that CLas bacterium had moved into the head-thorax section (containing the salivary glands) in 26.7-30.0% of nymphs and 37-45% of adults. Mean Ct values in these sections were 31.6-32.9 and 26.8-27.0 for nymphs and adults, respectively. Therefore, CLas incidence and titer were higher in the head-thorax of adults than in nymphs. Our results suggest that following acquisition of CLas by early instar D. citri nymphs, emerging adults inoculate the bacteria into citrus more efficiently than nymphs because adults are afforded a longer latent period necessary for multiplication and/or translocation of CLas into the salivary glands of the vector. We propose that CLas uses D. citri nymphs mainly for pathogen acquisition and multiplication, and their adults mainly for pathogen inoculation and spread.

Feeding Behavior of Asian Citrus Psyllid [Diaphorina citri (Hemiptera: Liviidae)] Nymphs and Adults on Common Weeds Occurring in Cultivated Citrus Described Using Electrical Penetration Graph Recordings.

Asian citrus psyllid, Diaphorina citri, transmits Candidatus Liberibacter asiaticus (CLas), the putative causal agent of Huanglongbing disease. Although they primarily feed on the phloem of Citrus and related plants, when grove or host conditions are unfavorable, D. citri may be able to use weed species as alternate food sources for survival. To explore this possibility, electrical penetration graph (EPG) recordings (18 h) were performed to investigate the feeding behavior of psyllid adults and nymphs on three common south Florida weeds (Bidens alba, Eupatorium capillifolium, and Ludwigia octovalvis). EPG recordings revealed that the proportion of time spent by D. citri feeding on xylem was similar on all tested weed species (19%-22%) and on the positive control (20%), the preferred host, Citrus macrophylla. Very little to no phloem feeding was observed on weed species by either nymphs or adults. Histological studies using epifluorescence microscopy showed that salivary sheaths were branched and extended into xylem of weed species, whereas they ended in phloem on citrus plants. No choice behavioral assays showed that adults can obtain some nutrition by feeding on weed species (xylem feeding) and they may be able to survive on them for short intervals, when host conditions are unfavorable.

AY-WB phytoplasma secretes a protein that targets plant cell nuclei.

The fully sequenced genome of aster yellows phytoplasma strain witches' broom (AY-WB; Candidatus Phytoplasma asteris) was mined for the presence of genes encoding secreted proteins based on the presence of N-terminal signal peptides (SP). We identified 56 secreted AY-WB proteins (SAP). These SAP are candidate effector proteins potentially involved in interaction with plant and insect cell components. One of these SAP, SAP11, contains an N-terminal SP sequence and a eukaryotic bipartite nuclear localization signal (NLS). Transcripts for SAP11 were detected in AY-WB-infected plants. Yellow fluorescence protein (YFP)-tagged SAP11 accumulated in Nicotiana benthamiana cell nuclei, whereas the nuclear targeting of YFP-tagged SAP11 mutants with disrupted NLS was inhibited. The nuclear transport of YFP-SAP11 was also inhibited in N. benthamiana plants in which the expression of importin alpha was knocked down using virus-induced gene silencing (VIGS). Furthermore, SAP11 was detected by immunocytology in nuclei of young sink tissues of China aster plants infected with AY-WB. In summary, this work shows that AY-WB phytoplasma produces a protein that targets the nuclei of plant host cells; this protein is a potential phytoplasma effector that may alter plant cell physiology.

Large accumulations of maize streak virus in the filter chamber and midgut cells of the leafhopper vector Cicadulina mbila.

Maize streak virus (MSV, Mastrevirus, Geminiviridae) is persistently transmitted by Cicadulina mbila, apparently without propagation in its leafhopper vector. MSV was shown earlier by quantitative PCR to accumulate in the alimentary canal of C. mbila. We examined the alimentary canals of C. mbila leafhoppers that acquired MSV from diseased plants for various acquisition access periods (AAP) by immunofluorescence confocal laser scanning microscopy (iCLSM) and by immunogold labelling transmission electron microscopy (iTEM). Following a 7-day AAP and a 7-day inoculation period (IP) on healthy seedlings, MSV was detected by iCLSM mainly in the filter chamber and anterior midgut. Using iTEM, large accumulations of MSV particles, usually enclosed in membranous vesicles, were detected only in cells of the midgut, inside and outside the filter chamber, following 14- or 30-day AAPs, and also following 7-day AAP and 7-day IP on healthy plants. No virus was detected in the control non-vector species C. chinaï. Coated pits or vesicles, typical of clathrin-mediated endocytosis, were not observed. We discuss an alternative endocytosis pathway and suggest that the MSV accumulations are stored in endosomes in the midgut epithelial cells.

Immuno-Ultrastructural Localization and Putative Multiplication Sites of Huanglongbing Bacterium in Asian Citrus Psyllid Diaphorina citri.

Huanglongbing, the most destructive citrus disease worldwide, is caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas) and is vectored by the Asian citrus psyllid (ACP). Very little is known about the form and distribution of CLas in infected psyllids, especially at the ultrastructural level. Here, we examined these aspects by transmission electron microscopy, combined with immunogold labeling. In CLas-exposed ACP adults, the CLas bacterial cells were found to be pleomorphic taking tubular, spherical, or flask-shaped forms, some of which seemed to divide further. Small or large aggregates of CLas were found in vacuolated cytoplasmic pockets of most ACP organs and tissues examined, including the midgut, filter chamber, hindgut, Malpighian tubules, and secretory cells of the salivary glands, in addition to fat tissues, epidermis, muscle, hemocytes, neural tissues, bacteriome, and walls of the female spermatheca and oviduct. Large aggregates of CLas were found outside the midgut within the filter chamber and between the sublayers of the basal lamina of the hindgut and Malpighian tubules. Novel intracytoplasmic structures that we hypothesized as 'putative CLas multiplication sites' were found in the cells of the midgut, salivary glands, and other tissues in CLas-exposed ACP. These structures, characterized by containing a granular matrix and closely packed bacterial cells, were unbound by membranes and were frequently associated with rough endoplasmic reticulum. Our results point to the close association between CLas and its psyllid vector, and provide support for a circulative-propagative mode of transmission.

A neurotropic route for Maize mosaic virus (Rhabdoviridae) in its planthopper vector Peregrinus maidis.

To investigate the dissemination route of Maize mosaic virus (MMV, Rhabdoviridae) in its planthopper vector Peregrinus maidis (Delphacidae, Hemiptera), temporal and spatial distribution of MMV was studied by immunofluorescence confocal laser scanning microscopy following 1-week acquisition feeding of planthoppers on infected plants. MMV was detected 1-week post first access to diseased plants (padp) in the midgut and anterior diverticulum, 2-week padp in the esophagus, nerves, nerve ganglia and visceral muscles, and 3-week padp in hemocytes, tracheae, salivary glands and other tissues. MMV is neurotropic in P. maidis; infection was more extensive in the nervous system compared to other tissues. A significantly higher proportion of planthoppers had infected midguts (28.1%) compared to those with infected salivary glands (20.4%) or to those that transmitted MMV (15.7%), suggesting the occurrence of midgut and salivary gland barriers to MMV transmission in P. maidis. In this planthopper, the esophagus and anterior diverticulum are located between the compound ganglionic mass and the salivary glands. We postulate that MMV may overcome transmission barriers in P. maidis by proceeding from the midgut to the anterior diverticulum and esophagus, and from these to the salivary glands via the nervous system: a neurotropic route similar to that of some vertebrate-infecting rhabdoviruses.

Salivary Sheaths of the Asian Citrus Psyllid Show Signs of Degradation 3-4 Weeks Following Their Deposition into Citrus Leaves by the Feeding Psyllids.

BACKGROUND: Salivary sheaths, also known as stylet sheaths or stylet tracks, are essential features of the piercing-sucking feeding mechanism of plant-feeding hemipteran insects, many of which are vectors of economically important plant viral and bacterial pathogens. Although knowledge of their structure and function is incomplete, these salivary sheaths are frequently used by researchers to study hemipteran's feeding behavior, host preference, or host resistance, because these sheaths remain in the plant tissues after the insect withdraws its stylets following its feeding or probing on these tissues. However, in most cases, it is not known how long these salivary sheaths may last in plant tissues after their deposition by the feeding insects. An earlier report suggested that the salivary sheaths of the Asian citrus psyllid, Diaphorina citri (Hemiptera, Liviidae), vector of the devastating huanglongbing (citrus greening) disease bacterium, start to dissipate 1 week after their deposition in citrus leaves. METHODS AND RESULTS: Here, using epifluorescence microscopy of cross sections in citron leaves, we found that D. citri salivary sheaths show signs of degradation in 3-4 weeks and become mostly degraded by 5-6 weeks, following their deposition by the psyllids into citrus tissues. Degradation of the salivary sheath starts at or near the "flange" area close to the leaf surface and continues gradually inward through the intercellular part of the sheath, within the mesophyll tissue, but apparently does not extend to the deeper or intracellular parts of the sheath in or near the phloem. Staining citron leaf sections with the fluorescent stain calcofluor white, which stains fungi, or propidium iodide (DNA/RNA stain) suggested that the degraded parts of the older salivary sheaths are not associated with fungi or bacterial accumulations. CONCLUSION: We speculate that degradation of the salivary sheaths may be due to enzymatic activities in the host plant, especially in the extracellular matrix of the mesophyll tissue.

Laser surgery reveals the biomechanical and chemical signaling functions of aphid siphunculi (cornicles).

Aphids are an attractive food source to many predators and parasitoids because of their small size, soft bodies and slow movement. To combat predation, aphids evolved both behavioral and chemical defensive mechanisms that are operated via siphunculi (cornicles), differently developed structures that more or less extend from their abdomen. Although both direct and indirect linkages between siphunculi and their defensive mechanisms have been explored, their ultimate effects on aphid fitness are still broadly debated. To explicitly test the influence of siphunculi on brown citrus aphid, Aphis (Toxoptera) citricida (Kirkaldy), fitness, we razor-cut and laser-sealed the siphunculi. Siphunculi removal resulted in two distinct behavior modifications, (false aggregation and increased drop-off rates) that led to decreased survival and the loss of the ability to right themselves from an inverted position. These results together indicate that siphunculi play an important role in survival, and removal of these organs will have negative effect on aphid fitness. Furthermore, results suggested that released alarm pheromone may play an important role in communication among aphid clone-mate, and omitting it results in miscommunication and competition among clonemates. These findings will help in better understanding the aphid biology.

Epifluorescence and Stereomicroscopy of Trichomes Associated with Resistant and Susceptible Host Plant Genotypes of the Asian Citrus Psyllid (Hemiptera: Liviidae), Vector of Citrus Greening Disease Bacterium.

BACKGROUND AND SIGNIFICANCE: Foliar trichomes (tiny hair-like structures) are part of the plant defense mechanisms that may confer resistance to some herbivore pests. Trifoliate orange, Poncirus trifoliata, is a genotype resistant to infestations by the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), vector of the economically important citrus greening (huanglongbing) disease bacterium. We discovered that dense trichomes are associated with young leaves of trifoliate orange plants and hypothesized that these might be responsible for reduced infestation by this psyllid. MATERIALS AND METHODS: Epifluorescence and stereomicroscopy were used to study the density and structure of trichomes associated with young flush leaves and stems of trifoliate orange and of five other plant genotypes that are highly susceptible to colonization by the psyllid: lemon, grapefruit, sweet orange, curry leaf, and orange jasmine. RESULTS: Simple unicellular trichomes were observed at moderate-to-large densities on young leaves and stems of each genotype except lemon and sweet orange, which had considerably fewer trichomes. Trichomes were generally abundant on young leaves of curry leaf and orange jasmine, two genotypes that are often heavily colonized by the psyllid. Although we did not quantify oviposition rates on these genotypes, we observed that psyllid females deposited eggs on young leaves, buds, and stems regardless of the density of trichomes present, sometimes directly within or close to a dense bed of trichomes. CONCLUSIONS: While trichomes were moderately abundant on young leaves of trifoliate orange, our results strongly suggest that these trichomes may play little or no role in reduced colonization by the psyllid on this genotype.

The quest for a non-vector psyllid: Natural variation in acquisition and transmission of the huanglongbing pathogen 'Candidatus Liberibacter asiaticus' by Asian citrus psyllid isofemale lines.

Genetic variability in insect vectors is valuable to study vector competence determinants and to select non-vector populations that may help reduce the spread of vector-borne pathogens. We collected and tested vector competency of 15 isofemale lines of Asian citrus psyllid, Diaphorina citri, vector of 'Candidatus Liberibacter asiaticus' (CLas). CLas is associated with huanglongbing (citrus greening), the most serious citrus disease worldwide. D. citri adults were collected from orange jasmine (Murraya paniculata) hedges in Florida, and individual pairs (females and males) were caged on healthy Murraya plants for egg laying. The progeny from each pair that tested CLas-negative by qPCR were maintained on Murraya plants and considered an isofemale line. Six acquisition tests on D. citri adults that were reared as nymphs on CLas-infected citrus, from various generations of each line, were conducted to assess their acquisition rates (percentage of qPCR-positive adults). Three lines with mean acquisition rates of 28 to 32%, were classified as 'good' acquirers and three other lines were classified as 'poor' acquirers, with only 5 to 8% acquisition rates. All lines were further tested for their ability to inoculate CLas by confining CLas-exposed psyllids for one week onto healthy citrus leaves (6-10 adults/leaf/week), and testing the leaves for CLas by qPCR. Mean inoculation rates were 19 to 28% for the three good acquirer lines and 0 to 3% for the three poor acquirer lines. Statistical analyses indicated positive correlations between CLas acquisition and inoculation rates, as well as between CLas titer in the psyllids and CLas acquisition or inoculation rates. Phenotypic and molecular characterization of one of the good and one of the poor acquirer lines revealed differences between them in color morphs and hemocyanin expression, but not the composition of bacterial endosymbionts. Understanding the genetic architecture of CLas transmission will enable the development of new tools for combating this devastating citrus disease.

Prolonged phloem ingestion by Diaphorina citri nymphs compared to adults is correlated with increased acquisition of citrus greening pathogen.

Citrus greening disease (huanglongbing), currently the most destructive citrus disease worldwide, is putatively caused by Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium transmitted by the Asian citrus psyllid Diaphorina citri. Electrical penetration graph (EPG) recordings over 42 h were performed to compare the feeding behavior of D. citri adults and 4th or 5th instar nymphs feeding on CLas-infected or healthy citron plants. Nymphs performed more individual bouts of phloem ingestion (E2) and recorded longer phloem ingestion total time compared with adults, whereas adults performed more bouts of xylem ingestion (G) and recorded greater total time of xylem ingestion compared with nymphs. Quantitative polymerase chain reaction tests indicated that 58% of nymphs and 6% of adults acquired CLas during the 42 h EPG-recorded feeding on infected plants. In a histological study, a greater proportion of salivary sheaths produced by nymphs were branched compared to those of the adults. Our results strongly suggest that more bouts and longer feeding time in the phloem by nymphs may explain their more efficient CLas acquisition from infected plants compared to adults. This is the first EPG study comparing nymphs and adults of D. citri on healthy and infected citrus plants in relation to CLas acquisition.

Ultrastructure of the salivary glands, alimentary canal and bacteria-like organisms in the Asian citrus psyllid, vector of citrus huanglongbing disease bacteria.

The Asian citrus psyllid (ACP, Diaphorina citri, Hemiptera: Liviidae) is the principal vector of Candidatus Liberibacter asiaticus (Las), the putative bacterial agent of citrus greening/huanglongbing (HLB); currently the most serious citrus disease worldwide. Las is transmitted in a persistent-propagative manner by ACP, and the salivary glands and midgut have been suggested as transmission barriers that can impede translocation of Las within the vector. However, no detailed ultrastructural studies have been reported on these organs in this or other psyllid species, although some bacterium-like structures have been described in them and assumed to be the causal agents of HLB. In this study, we describe the ultrastructure of the salivary glands, filter chamber, other parts of the alimentary canal, and other organs and tissues of ACP including the compound ganglionic mass (in the thorax) and the bacteriome (in the abdomen). Furthermore, in addition to two ultrastructurally apparently different symbiotic bacteria found in the bacteriome, other morphological types of bacteria were found in the gut epithelial cells and salivary glands of both Las-infected (quantitative polymerase chain reaction positive) and noninfected (quantitative polymerase chain reaction negative) ACP. These results show the importance of immunolabeling, fluorescence in situ hybridization, or other labeling techniques that must be used before identifying any bacterium-like structures in ACP or other vectors as Las or other possible agents of HLB. This ultrastructural investigation should help future work on the cellular and subcellular aspects of pathogen-psyllid relationships, including the study of receptors, binding sites, and transmission barriers of Las and other pathogens within their psyllid vectors.