Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

Behavioral Plasticity in Probing by <Emphasis Type="Italic">Diaphorina citri</Emphasis> (Hemiptera,Liviidae): Ingestion from Phloem Versus Xylem is Influenced by Leaf Age and Surface

Diaphorina citri is a major pest of citrus because it transmits Candidatus Liberibacter asiaticus, a phloem-limited bacterium that putatively causes Huanglongbing (HLB). The disease moves slowly through a tree, and the vector facilitates further within-tree movement via transmission of the pathogen. However, this only happens when D. citri stylets contact the phloem, to inoculate bacteria during phloem salivation and acquire bacteria during phloem sap ingestion. Behavioral changes in D. citri associated with different plant parts would affect how long it takes to reach phloem and how long the psyllids stays in phloem to ingest, thereby influencing the risk of disease spread. D. citri feeding was recorded on the abaxial and adaxial surfaces of mature and immature citrus leaves. Adults in the field can be found on these surfaces at all times of year. On abaxial surface of immature leaves, phloem salivation would occur after 11 h on average, but rarely as soon as 0.56 h. The corresponding values on mature leaves were 16 and 2.7. In general, psyllids spent more time ingesting phloem sap on immature leaves than on mature leaves. Psyllids on abaxial surfaces spent more time ingesting from phloem, though the strength of this effect was less than for immature versus mature leaves. In contrast, xylem ingestion increased on mature leaves compared with young. The biological differences that could produce this outcome are discussed. The results discussed herein are of relevance to further studies on the efficacy of an insecticide to act quickly enough to prevent pathogen transmission.     

Characterization of the electrical penetration graphs of the psyllid Bactericera trigonica on carrots

The psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae) is a carrot and celery pest recently described as a vector of the plant pathogenic bacterium Candidatus Liberibacter solanacearum (Lso) on Apiaceae. Detailed information on vector stylet penetration activities is essential in the study of Lso transmission. In this study we used the electrical penetration graph (EPG) technique, characterized waveforms produced during the various stylet penetration activities in carrot leaves, and correlated them with stylet tracks and salivary sheath termini on plant tissues as well as with Lso inoculation. In addition, the effect of Lso in B. trigonica on the stylet penetration activities was tested. The EPG waveforms identified were: waveforms C1 and C2 detected in the mesophyll, waveforms D, E1, and E2 near or in the phloem sieve elements, and waveform G in the xylem vessels. A waveform pattern not previously reported for psyllids was the ‘pseudo‐potential drop’ (pseudo‐pd), characterized by sudden voltage dips similar to potential drops. However, the lowered voltage appeared to be inverted when the plant voltage is negative, indicating that it is caused by an increased resistance period and not due to a cell puncture. A direct correlation is shown between the waveform E1 and salivation into phloem sieve elements by B. trigonica as the inoculation of Lso occurred in a period as short as 30 s of E1; Lso transmission occurred in 17 of 35 plants (48%). Stylet activities during waveforms C or D had no consequences on the inoculation of Lso. In conclusion, Lso infection directly affects the probing behaviour of B. trigonica by increasing the total duration of C and D waveforms, but not variables related to phloem salivation (Lso inoculation) or ingestion (Lso acquisition). The reported information here is fundamental for identifying the psyllid vector traits of behaviour associated with transmission of Lso to Apiaceae.     

Feeding disruption of potato psyllid,Bactericera cockerelli,by imidacloprid as measured by electrical penetration graphs

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a serious pest of potatoes that can cause yield loss by direct feeding and by transmitting a bacterial pathogen, Candidatus Liberibacter psyllaurous (also known as Candidatus L. solanacearum), which is associated with zebra chip disease of this crop. Current pest management practices rely on the use of insecticides for control of potato psyllid to lower disease incidences and increase yields. Imidacloprid is typically applied at potato planting, and it remains unknown if imidacloprid has any effect on potato psyllid feeding behavior. Thus, our specific objectives of this study were to determine and characterize the effects of imidacloprid treatment (0.11 ml l^?1) to potato plants on adult potato psyllid feeding behavior 1, 2, and 4 weeks post‐application. Electrical penetration graph (EPG) recordings of potato psyllid feeding revealed six EPG waveforms, which include non‐probing (NP), intercellular stylet penetration (C), initial contact with phloem tissue (D), salivation into phloem sieve elements (E1), phloem sap ingestion (E2), and ingestion of xylem sap (G). The number of NP events and the duration of individual NP events significantly increased on plants treated with imidacloprid compared with untreated controls. Potato psyllids exhibited significant decreases in the number of phloem salivation events on plants treated with imidacloprid. Waveform durations and waveform durations per event for E2 and G were significantly decreased for psyllids on plants treated with imidacloprid compared with untreated controls. These data suggest that the effective use of imidacloprid to reduce transmission of Ca. Liberibacter psyllaurous is related to the negative effects of imidacloprid on psyllid feeding.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

Suitability of native North American Rutaceae to serve as host plants for the Asian citrus psyllid (Hemiptera: Liviidae)

The Asian citrus psyllid, Diaphorina citri Kuwayama, a vector of citrus huanglongbing, is now present in all citrus‐producing states in the USA and Mexico. In addition to citrus, the insect can reproduce on several other plant species in the Rutaceae family; orange jasmine (Murraya spp.) and curry leaf (Bergera koenigii) are among its preferred hosts. There are several indigenous Rutaceae species in North America, and some are popular ornamentals. A study was therefore initiated to determine the suitability of some of these plants for feeding and development of the psyllid in choice and no‐choice experiments. D. citri was found to reproduce successfully on Choisya ternata, C. arizonica and Helietta parvifolia in no‐choice tests, but preferentially selected orange jasmine and curry leaf for feeding and reproduction, in choice tests. On Amyris madrensis, A. texana and Zanthoxylum fagara, adult psyllids laid eggs which hatched, but no successful nymphal development was recorded beyond the first instars. No oviposition was recorded on Esenbeckia berlandieri, Ptelea trifoliata and Casimiroa tetrameria, although adult psyllids were able to survive on these species for several days. Results showed that C. ternata, C. arizonica and H. parvifolia can serve host plants of D. citri and this constitutes the first report of these plants serving as host for D. citri. The findings of the present study suggest that native rutaceous host plants can serve as host plants and thus affect D. citri population dynamics and the epidemiology of Huanglongbing, the deadly citrus greening disease whose pathogen is vectored by D. citri. Thus, area‐wide management of this pest also should target these riparian habitats where these host plants are present with D. citri biological control agents for sustainable management of this pest.     

Incidence of “Candidatus Liberibacter asiaticus” in a Florida population of Asian citrus psyllid

The incidence of a bacterium “Candidatus Liberibacter asiaticus” was assessed in a Florida population of Asian citrus psyllid, Diaphorina citri. The bacterium is the presumed causal agent of Asiatic huanglongbing, a serious citrus disease. Adult D. citri were periodically collected between May 2010 and September 2012 in a block of diseased trees located in east‐central Florida. The psyllids were individually subjected to molecular analyses (quantitative polymerase chain reaction assays using HLBaspr primers) to determine whether the bacterium was present and, if so, the population level of the pathogen based on qPCR cycle threshold (CT) values. Significantly greater percentages of females tested positive for the pathogen than males, but there were no significant differences between females and males with respect to population levels of the pathogen within the psyllids. No significant differences were found among the three D. citri colour morphs with respect to percentages of adults testing positive for the pathogen. Among 47 sample dates, a mean of 17.5% of adults per sample date tested positive (CT < 36) for the pathogen with a mean CT value of 31.1. The incidence of the pathogen was generally higher during late fall or early winter and often lower during mid‐ to late summer. There was a significant negative correlation between percentages of D. citri testing positive and air temperature. Increases in the incidence of the pathogen may not necessarily correspond to increases in transmission, as a number of factors both internal and external to D. citri can influence transmission. Transmission rates may be highest during periods when D. citri infestation levels are large, a high percentage of adults carry a high population of the pathogen in their salivary glands, and citrus flush is abundant.     

Silencing of V-ATPase-E gene causes midgut apoptosis of Diaphorina citri and affects its acquisition of Huanglongbing pathogen

The Asian citrus psyllid, Diaphorina citri Kuwayama, is among the most important pests of citrus. It is the main vector of the Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe losses in citrus crops. Control of D. citri is therefore of paramount importance to reduce the spread of HLB. In this regard, using RNA interference (RNAi) to silence target genes is a useful strategy to control psyllids. In this study, using RNAi, we examined the biological functions of the V-ATPase subunit E (V-ATP-E) gene of D. citri, including its effect on acquisition of CLas. The amino acid sequence of V-ATP-E from D. citri had high homology with proteins from other insects. V-ATP-E was expressed at all D. citri life stages analyzed, and the expression level in mature adults was higher than that of teneral adults. Silencing of V-ATP-E resulted in a significant increase in mortality, reduced body weight, and induced cell apoptosis of the D. citri midgut. The reduced expression of V-ATP-E was indicated to inhibit CLas passing through the midgut and into the hemolymph, leading to a majority of CLas being confined to the midgut. In addition, double-stranded RNA of D. citri V-ATP-E was safe to non-target parasitic wasps. These results suggest that V-ATP-E is an effective RNAi target that can be used in D. citri control to block CLas infection.     

Proteomic maps of subcellular protein fractions of the Asian citrus psyllid Diaphorina citri,the vector of citrus huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect vector that transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) associated with the destructive citrus disease, citrus huanglongbing (HLB). Currently, D. citri is the major target in HLB management, although insecticidal control and disruption of the D. citri–CLas interactions both face numerous challenges. The present study reports the subcellular proteomic profiles of D. citri, encompassing the three main subcellular protein fractions: cytosol, mitochondria and microsomes. After optimization, subcellular proteins of both high and low abundance are obtained by two‐dimensional gel electrophoresis (2‐DE). A total of 1170 spots are detected in the 2‐DE gels of the three subcellular fractions. One hundred and sixty‐four differentially expressed proteins are successfully identified using liquid chromatography‐dual mass spectroscopy. An efficient protocol for subcellular protein fractionation from D. citri is established and a clear protein separation is achieved with the chosen protein fractionation protocol. The identified cytosolic proteins are mainly metabolic enzymes, whereas a large portion of the identified proteins in the mitochondrial and microsomal fractions are involved in ATP biosynthesis and protein metabolism, respectively. Protein–protein interaction networks are predicted for some identified proteins known to be implicated in pathogen–vector interactions, such as actin, tubulin and ATP synthase, as well as insecticide resistance, such as the cytochrome P450 superfamily. The findings should provide useful information to help identify the mechanism responsible for the CLas–D. citri interactions and eventually contribute to D. citri control.     

Plant volatiles and density-dependent conspecific female odors are used by Asian citrus psyllid to evaluate host suitability on a spatial scale

We investigated how chemical cues derived from female Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae) and their host plants affect host acceptance choices by conspecifics. In four-choice cage and two-choice olfactometer assays, female psyllids avoided conspecific female cues in a density-dependent manner. However, odors from citrus plants actively damaged by psyllid feeding were attractive to conspecific females. When odors from feeding-damaged plants were presented simultaneously with odors from female D. citri, attraction of female conspecifics was no longer observed as compared with a clean air control in olfactometer assays. In subsequent experiments, D. citri females were released within arenas that contained actively feeding-damaged or non-damaged (control) citrus plants, each with previously psyllid-infested and uninfested young leaves. D. citri development is linked to the presence of these newly emerging leaves which is the only site of nymphal development. Female D. citri were initially attracted by the actively damaged plants as compared with non-damaged controls. After acceptance of plants that were actively damaged by feeding, D. citri females preferentially chose and settled on uninfested young leaves as compared with previously infested young leaves. A herbivore-induced plant volatile attractant and a female-specific odor repellent appear to be complementary foraging cues providing psyllids with information at two spatial scales: (1) the whole plant level for choosing a plant potentially harboring male conspecifics for mating, and (2) the within plant level to reduce intra-conspecific competition by identifying previously exploited resources.     

Feeding behavior of Diaphorina citri and its transmission of ‘Candidatus Liberibacter asiaticus’ to citrus

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive disease of citrus, responsible for huge economic losses in the world's major citrus production areas. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits ‘Candidatus Liberibacter asiaticus’ (Clas), the pathogen responsible to cause HLB. Understanding of vector, pathogen, and host plant interactions is important for the management of this vector‐disease complex. We used the direct‐current electrical penetration graph (DC‐EPG) system to evaluate feeding behavior of Clas‐infected D. citri adults, and their potential to transmit the pathogen to healthy citrus, Citrus reticulata Blanco cv. Sunki (Rutaceae), following a 24‐h inoculation access period. Plants were tested for the presence of Clas by qPCR 6 months after inoculation. Findings suggest that inoculation was associated with salivation into the phloem sieve elements (waveform E1). The minimum feeding time for successful transmission by a single adult was 88.8 min, with a minimum E1 duration of 5.1 min. Regression analysis indicated a significant relationship between E1 duration and transmission efficiency. The adults successful in transmitting Clas to healthy citrus were able to penetrate and feed in the phloem much earlier than those which did not transmit. The minimum duration of E1 for a female was shorter than that of a male, but transmission was higher. However, durations of other EPG parameters were not significantly different between male and female. Feeding by single Clas‐infected D. citri adults on 6‐month‐old plants (Sunki) resulted in 23% HLB‐positive plants 6 months after inoculation. Multiple nymphs or adults could transmit the pathogen more efficiently than individual adults in the field, and further enhance the severity of the disease. Effective tactics are warranted to control D. citri and disrupt transmission of Clas.     

Secondary hosts of the Asian citrus psyllid,Diaphorina citri Kuwayama: Survivorship and preference

The Asian citrus psyllid, Diaphorina citri Kuwayama, is the most economically important and difficult to manage citrus pest in Florida due to its role as vector of huanglongbing or citrus greening disease. Spread of the disease is a function of dispersal patterns and thus influenced by availability of suitable hosts. Young leaves of citrus or related plants are required for reproduction, but in their absence, secondary hosts may provide needed resources to enhance survival. Therefore, survivorship on and preference for three abundant weed species was investigated. The suitability of potential secondary host plants Bidens alba (L.), DC, Ludwigia octovalvis (Jacqu.) P. H. Raven, and Eupatorium capillifolium (Lam.) Small was compared to a reproductive host, Murraya paniculata (L.) Jack, in no‐choice tests by assessing survivorship of D. citri adults confined to these plants in cages. Preference was evaluated by choice tests where D. citri adults were released into cages containing all three secondary hosts alone or with M. paniculata. Both B. alba and E. capillifolium increased D. citri survivorship by twofold compared to starvation conditions with only water available. Choice trials revealed no difference in initial selection between true and secondary hosts; however, the true host was favoured over time. This result suggested that hosts were selected initially by sight, and only later by taste and/or smell. While secondary hosts are unable to support reproduction or long‐term survival, these findings establish the ability of D. citri to use secondary hosts that are ubiquitous in Florida citrus groves as temporary reservoirs for food and moisture when ideal host conditions are scarce or absent.     

Infection of an Insect Vector with a Bacterial Plant Pathogen Increases Its Propensity for Dispersal

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.     

Induced Release of a Plant-Defense Volatile ‘Deceptively’ Attracts Insect Vectors to Plants Infected with a Bacterial Pathogen

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.     

Effect of plant barriers and citrus leaf age on dispersal of Diaphorina citri (Hemiptera: Liviidae)

Studies designed to measure dispersal capacity of Diaphorina citri Kuwayama (Hemiptera: Liviidae) are needed to provide the epidemiological knowledge necessary to improve management of citrus huanglongbing. In this study, a mark–release–recapture technique was used to investigate whether 1) host or non‐host plants of D. citri can act as barriers for dispersing insects and 2) presence or absence of young citrus leaves influence movement of D. citri towards citrus plants. The experimental field consisted of four circular and adjacent areas containing citrus trees, Citrus sinensis (L.) Osbeck cv. ‘Hamlin’, planted in concentric circles at 18, 24 and 30 m from the release centre. Insect activity was monitored by recapturing at each distance using yellow stick traps. Dense plantings of tall non‐host plants of D. citri such as corn had no effect on insect dispersal towards citrus plants when compared to a shorter cover crop such as grass. In contrast, suitable host plants acted as traps decreasing movement of D. citri. Diaphorina citri dispersed at greater speeds in the absence of young leaves reaching 140 m within 6 hours after release, whereas in the presence of young leaves, individuals reached at most 60 m at 1 day after release. Results suggest that D. citri control measures may be more efficient during periods of highest vegetative activity when insects are less active. Moreover, the use of suitable host plants for D. citri as trap plants may be a potential tactic to prevent movement of insects into the crop.     

Metabolomic analyses of the haemolymph of the Asian citrus psyllid Diaphorina citri,the vector of huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama is currently threatening the citrus industry by transmitting the causative agent Candidatus Liberibacter asiaticus (CLas) of huanglongbing. Multiplication of CLas in haemolymph of D. citri indicates that it contains the necessary nutrients for CLas. Although many studies examine D. citri, the haemolymph composition of this dangerous pest remains to be investigated. In the present study, the haemolymph of D. citri is collected using a nanolitre syringe after the removal of one of its forelegs. The haemolymph is either derivatized with methyl chloroformate (MCF) or trimethylsilyl (TMS) derivatizing reagent and analyzed with gas chromatography–mass spectrometry. Nineteen amino acids, two organic acids and seven fatty acids are detected in the haemolymph after MCF derivatization. More metabolites are detected after TMS derivatization. Sugars are the most abundant metabolites in the haemolymph. Glucose and fructose are the main monosaccharides. Trehalose and sucrose are the major disaccharides. Furthermore, three inositol isomers (myo‐inositol, scyllo‐inositol and chiro‐inositol) are detected in the haemolymph. Organic acids are found in low amounts, whereas phosphoric acid is found at a higher concentration. Twenty‐four nucleotides and sugar nucleotides, including ATP, ADP and AMP, are detected using high‐performance anion‐exchange chromatography. Adenine nucleotides are the most abundant nucleotides followed by uridine and guanosine. The adenylate energy charge for the haemolymph is 0.77. Our results show that many metabolites found in the citrus phloem sap are also found in the haemolymph of D. citri.     

Diurnal Patterns of Flight Activity and Effects of Light on Host Finding Behavior of the Asian Citrus Psyllid

The Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae), is an invasive pest of citrus in the United States. The psyllid feeds and reproduces primarily on new flush growth of citrus and other rutaceous plants. Because it vectors the bacterial causal agents of the deadly citrus greening disease, D. citri is potentially a pest of economic importance in all citrus growing areas where it occurs together with the disease. We investigated the diurnal patterns of its flight activity in the field and the effects of light on its host selection and egg laying behaviors. The numbers of adult psyllids caught on yellow sticky traps were 3 to 4-fold higher during daytime than nighttime. Daytime flight activity of D. citri adults also varied with time of the day with peak catches occurring at midday from 1200 to 1500 h. Illumination of the traps at night increased their attractiveness to adult psyllids by 5-fold. Similarly, light significantly increased plant colonization by adults and female egg deposition on potted plants in the laboratory. These results showed that the flight activity and host selection behavior of adult psyllids are regulated by light and circadian rhythms. Thus, adult psyllids utilize light as visual cues in their host-plant selection process.     

Genetic variation and potential coinfection of Wolbachia among widespread Asian citrus psyllid (Diaphorina citri Kuwayama) populations

Wolbachia can profoundly influence the survival, reproduction, and defenses of insect hosts. These interactions could potentially be harnessed for managing pests or insecttransmitted diseases. Diaphorina citri Kuwayama is a phloem-feeding pest capable of transmitting the putative causal agent of citrus greening, Candidatus Liberibacter asiaticus (CLas). Like many insects, D. citri is also infected with Wolbachia (wDi). Recent studies indicate that the relative abundance of wDi could be associated with the abundance of CLas, and that wDi may contribute to regulating expression of phage lytic cycle genes in CLas, suggesting the need for better understanding of wDi biology in general. This study investigated the genetic diversity of wDi among D. citri in populations spanning eleven countries and two U.S. territories. Six Wolbachia genes, wsp, coxA.fbpA.ftsZ, gatB, and hep A, were sequenced and compared across samples. Two prevalent wDi strains were identified across the samples, and screening of clone libraries revealed possible coinfection of wDi strains in specific populations. D. citri mitochondrial cytochrome oxidase subunit I gene (mtCOI) were more divergent between D. citri populations that were infected with different wDi strains or had different infection statuses (single infection vs. coinfection). While we could not eliminate the possibility that maternal transmission may contribute to such patterns, it is also possible that wDi may induce cytoplasmic incompatibility in their host. These fin dings should contribute to the understanding of wDi population ecology, which may facilitate manipulation of this endosymbiont for management of citrus greening disease worldwide.     

Transmission parameters for Candidatus liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae)

The purpose of this investigation was to evaluate acquisition and inoculation (together, transmission) efficiency of Candidatus Liberibacter asiaticus (Las), the pathogen associated with citrus huanglongbing (HLB) by the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae). In laboratory studies, nymphs reared on Las infected plants were more likely to acquire the bacterium than adults. Acquisition by nymphs ranged from 60 to 100%, whereas acquisition by adults only reached 40% after 5 wk of feeding on Las-infected plants. Similar rates of pathogen acquisition by psyllids after nymphal and adult feeding were observed in the field. Transmission of Las from parent to offspring (transovarial) occurred at a rate of 2-6%. One year after psyllid inoculations, successful transmission by individual D. citri ranged from 4 to 10%, whereas groups of 100 or more D. citri transmitted the pathogen at a rate of approximately 88%. In addition, the proportion of Las-positive adult psyllids, determined using quantitative real-time polymerase chain reaction, decreased over time when held on healthy plants. Due to the low rate of pathogen acquisition and long time period required for successful inoculation by adult D. citri, experiments designed to determine the latent period required for replication and successful inoculation of Las by D. citri did not result in Las-infected plants after >1 yr of incubation after inoculation. Collectively, these results indicate that adult D. citri which acquire the HLB pathogen as adults are poor vectors of the pathogen compared with adults that acquired the pathogen as nymphs.     

Bacteria causing important diseases of citrus utilise distinct modes of pathogenesis to attack a common host

In this review, we summarise the current knowledge on three pathogens that exhibit distinct tissue specificity and modes of pathogenesis in citrus plants. Xanthomonas axonopodis pv. citri causes canker disease and invades the host leaf mesophyll tissue through natural openings and can also survive as an epiphyte. Xylella fastidiosa and Candidatus Liberibacter are vectored by insects and proliferate in the vascular system of the host, either in the phloem (Candidatus Liberibacter) or xylem (X. fastidiosa) causing variegated chlorosis and huanglongbing diseases, respectively. Candidatus Liberibacter can be found within host cells and is thus unique as an intracellular phytopathogenic bacterium. Genome sequence comparisons have identified groups of species-specific genes that may be associated with the particular lifestyle, mode of transmission or symptoms produced by each phytopathogen. In addition, components that are conserved amongst bacteria may have diverse regulatory actions underpinning the different bacterial lifestyles; one example is the divergent role of the Rpf/DSF cell–cell signalling system in X. citri and X. fastidiosa. Biofilm plays a key role in epiphytic fitness and canker development in X. citri and in the symptoms produced by X. fastidiosa. Bacterial aggregation may be associated with vascular occlusion of the xylem vessels and symptomatology of variegated chlorosis.