Diaphorina Citri (Hemiptera: Liviidae) Vector Competence for the Citrus Greening Pathogen ‘Candidatus Liberibacter Asiaticus’

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Journal of Economic Entomology Advance Access published March 21, 2015

FORUM Diaphorina citri (Hemiptera: Liviidae) Vector Competence for the Citrus Greening Pathogen ‘Candidatus Liberibacter Asiaticus’

WALTER J. TABACHNICK1

Florida Medical Entomology Laboratory, University of Florida – IFAS, 200 9th St., SE, Vero Beach, FL 32962.

J. Econ. Entomol. 1–10 (2015); DOI: 10.1093/jee/tov038 ABSTRACT Characterizing the vector competence of Diaphorina citri Kuwayama for ‘Candidatus Liberibacter asiaticus,’ the pathogen causing citrus greening, is essential for understanding the epidemiology of this disease that is threatening the U.S. citrus industry. Vector competence studies have been difficult because of the biology of D. citri, the inability to culture the pathogen, and the available diagnostic methods used to detect the bacteria in plant and insect tissues. The methods employed in many studies of D. citri vector competence may have overestimated amounts of live ‘Ca. L. asiaticus’ in both plant and insect tissues, and it is possible that the amounts of phloem ingested by psyllids may not contain sufficient detectable pathogen using current diagnostic methods. As a result of the difficulty in characterizing D. citri vector competence, the several daunting challenges for providing D. citri that are unable to inoculate ‘Ca. L. asiaticus’, as a novel method to control greening are discussed. Suggestions to overcome some of these challenges are provided.

KEY WORDS citrus greening disease, vector competence, psyllid

Citrus greening is a disease of citrus caused by insect- reported in Florida in 1998, and HLB was first associated phloem-limited bacteria Candidatus Liberi- reported in Florida in 2005. HLB and D. citri have bacter. There are three types of Ca. Liberibacter: been detected in California, Texas, South Carolina, ‘Ca. L. asiaticus,’ ‘Ca. L. americanus,’ and ‘Ca. L. Louisiana, and Georgia. africanus.’ The most prevalent is ‘Ca. L. asiaticus’ The effects of greening on the Florida citrus indus- which causes Asian huanglongbing (HLB) or citrus try have been devastating. The Florida citrus industry, greening. Dispersal of this plant pathogen is through representing ca. 65% of the total of U.S. citrus pro- transmission by the Asian citrus psyllid, Diaphorina duction, with estimated annual revenue of ca. US$9 citri Kuwayama. Once transmitted to susceptible citrus billion, lost ca. US$4.5 billion between 2006–2011 the bacteria reside in the phloem where it can be due to greening (Hodges and Spreen 2012). Citrus transmitted to phloem-ingesting D. citri that can subse- greening has recently arrived in California. The pre- quently inoculate the bacteria when they feed on citrus dicted consequences are dire should citrus greening hosts. All commercial citrus varieties are susceptible to spread throughout California, with ca. 35% of total HLB. The pathogen ultimately causes a decline in the U.S. citrus production over a 20-yr period if fruit production and fruit quality of the infected tree. unchecked. Infected trees decline until they die in a process that could take as few as five years. Reviews of citrus Reducing the Impact of Greening on U.S. greening are available elsewhere (i.e., da Grac¸a and Citrus. Many different approaches are being pursued Kirsten 2004, Bove´2006, Gottwald 2010, Grafton- to reduce the effects of greening on U.S. citrus. These Cardwell et al. 2013, Wang and Trivedi, 2013, Hall include developing citrus varieties that are resistant to et al. 2013). HLB, show fewer symptoms upon infection, or both; Interest in citrus greening has increased in the past reducing greening symptoms in citrus by using 20 yr, as the pathogen and D. citri have spread from improved citrus nutrition and management; and using Asia to the world’s major citrus-producing countries. novel compounds to kill ‘Ca. L. asiaticus’ in infected D. citri has been present in Brazil for ca. 60 yr, and it citrus trees. Strategies are being sought that reduce was found in many countries starting in the 1990s. psyllid populations and their ability to transmit ‘Ca. L. HLB has been reported in Mexico, Belize, Jamaica, asiaticus’ to citrus. This includes development of pesti- Puerto Rico, Cuba, and the United States. It was first cides against psyllids and biological control strategies such as using a predator wasp that attacks and kills psyllids. RNAi and drugs are being sought to reduce ‘Ca. L. asiaticus’ in citrus or in the psyllid. Currently no approach has provided a sustainable solution that con- 1 Corresponding author, e-mail: [email protected]. trols greening. Irey et al. (2008) estimated the proposed

VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 2JOURNAL OF ECONOMIC ENTOMOLOGY greening management programs might increase citrus during arthropod juvenile stages like juvenile density production costs by 40%. and nutrition, influence vector competence. Further, Several of the approaches to control the spread of influential factors interact with one another in largely greening require an understanding of the ability of nonlinear unpredictable ways that greatly add to the D. citri to transmit ‘Ca. L. asiaticus.’ The goals of this complexity of the controlling factors and to the diffi- paper are to review current knowledge about D. citri culty in conducting and interpreting observations. As a vector competence for ‘Ca. L. asiaticus’ with focus on result, studies of vector competence must be attentive areas that need further research. Although progress has to a host of environmental conditions to be able to been made in understanding D. citri vector compe- assess the causes of variation in competence and to be tence for ‘Ca. L. asiaticus,’ much remains unknown. able to compare observations between individual vec- The purpose of this paper is to evaluate the methods tors and between different studies. that have been used to characterize D. citri vector com- Though the presence of ‘Ca. L. asiaticus’ in D. citri petence, with attention to their effects on understand- salivary glands is consistent with expulsion of the patho- ing vector competence, and suggestions are provided to gen in saliva during ingestion (Xu et al. 1988, Ammar meet identified challenges. et al 2011b), there have been few studies on other Vector Competence. Vector competence encom- potential routes of psyllid acquisition, retention, and passes all the intrinsic factors that allow individual inoculation. Psyllid biology further complicates vector arthropods to transmit a specific pathogen (Hardy et al. competence studies. For example, mosquitoes acquire 1983, Gray and Banergee 1999, Tabachnick 2013). pathogens through a single bloodmeal of known size. Primarily vector competence includes the ability of the The amount (titer) of the pathogen that the mosquito arthropod to become infected with the pathogen upon receives can be quantified by measuring the pathogen feeding or some other form of acquiring the pathogen in the mosquito immediately after blood feeding. In and subsequent ability to transmit or inoculate the comparison, psyllids ingest plant phloem more or less pathogen. In circulative propagative pathogen systems, continuously, contacting increasing quantities of susceptibility to infection entails ingesting the pathogen phloem as they feed, while exuding some portion as and it subsequently escaping from the arthropod’s sugar-laden honeydew excreted from their anal open- digestive tissues to other insect tissues where the ing. Therefore it is difficult to measure the amounts of pathogen propagates. Ultimately pathogens gain access phloem being ingested by different psyllids and the to the arthropod feeding mechanism. In circulative titer of ingested ‘Ca. L. asiaticus.’ This information is systems this involves invading the salivary glands where vital to ensure that tested psyllids had ingested similar the pathogens escape the vector upon subsequent sali- titers of the pathogen. vation into a host during feeding or probing a host in Both sexes of D. citri ingest phloem and are capable preparation to feeding. of acquiring and inoculating ‘Ca. L. asiaticus.’ There There are other modes of transmission that do not have been no studies to determine if the sex of the psy- involve propagation and salivary glands. The pathogen llid influences its vector competence. As a result, in may be associated with the arthropod’s mouthparts dur- this review, potential differences in vector competence ing feeding, remain on the mouthparts, and be inocu- between the sexes will not be considered. lated into another host without multiplying in the Methods to Assess D. citri Vector Competence arthropod or spreading to other tissues. Such mechani- for ‘Ca. L. asiaticus’. There have been many studies cal transmission does not involve the biological proc- describing D. citri vector competence for ‘Ca. L. asiat- esses encompassed in vector competence. Transmission icus’ (Xu et al. 1988; Inoue et al. 2009; Pelz-Stelinski may occur if the pathogen is excreted as part of the 2010; Ammar et al. 2011a,b; Coletta-Filho et al. 2014). waste products. The pathogen may be regurgitated Studies of D. citri competence for ‘Ca. L. asiaticus’ from the stomach that could infect the host. Pathogens have been difficult because ‘Ca. L. asiaticus’ is not could be passed to uninfected offspring vertically from amenable to growth in cell culture. Hence, most stud- infected mothers through the egg, or through venereal ies have not provided the titer of ‘Ca. L. asiaticus’ in transmission where infected males or females transfer the inoculum actually ingested by the psyllids. Typi- and infect the other sex during mating. The vector may cally, D. citri ingest ‘Ca. L. asiaticus’ by feeding on be infected through cofeeding next to other vectors or plants that vary in pathogen titers. In addition, the feeding at a site in proximity to where an infected vec- inability to culture ‘Ca. L. asiaticus’ has resulted in reli- tor had recently fed on the host where they may ingest ance on quantitative polymerase chain reaction (qPCR) the pathogen from these other infected arthropods techniques to characterize levels of infection in both prior to the host being infected (Jones et al. 1987, Lord plant and insect tissues (Jagoueix et al. 1996; Hung and Tabachnick 2002). et al. 2004; Teixeira et al. 2005; Benyon et al. 2008; Arthropod vector competence is influenced by Manjunath et al. 2008; Li et al 2009, 2006; Lin et al. an array of factors (Tabachnick 2013). Variation in 2010; Coy et al 2014). vector competence is due to genetic variation in the Several studies have reported difficulties in using vector and genetic variation in the pathogen. A variety qPCR to detect low titers of ‘Ca. L. asiaticus’ (Lin et al. of environmental factors, i.e., the temperature during 2010, Coy et al. 2014). As a result more sensitive qPCR pathogen development in the arthropod, the dose of methods have been developed in an attempt to the pathogen, genetic variants of the pathogen, the age overcome this problem (Benyon et al. 2008, Lin et al. of the vector, its size, and even environmental factors 2010, Coy et al. 2014). In addition, qPCR does not 2015 TABACHNICK:PSYLLID VECTOR COMPETENCE 3 discriminate between nucleic acid from live ‘Ca. L. asi- D. citri.Titersof103 ‘Ca. L. asiaticus’ or less per milli- aticus’ from remnant nucleic acid from dead bacteria. liter could result in false negatives at least ca. 50% of Hence, titers based on qPCR may provide false posi- the time. tives, may overestimate the titer of live pathogen, or D. citri Acquisition of ‘Ca. L. Asiaticus’. There both. Using ethidium monoazide to discriminate are several reports of varying time periods required for between live and dead cells showed that ‘Ca. L. asiat- D. citri to acquire ‘Ca. L. asiaticus.’ Here I differentiate icus’ titers detected with qPCR with plant tissues were two different aspects of what could be considered reduced 20–40% when dead cells were inhibited (Triv- acquisition. Psyllid acquisition of ‘Ca. L. asiaticus’ can edi et al. 2009). A similar study using propidium mona- be defined as initial exposure when the pathogen is first zide to exclude dead ‘Ca. L. asiaticus’ found that qPCR ingested by the psyllid or it can mean when ‘Ca. L. asi- provided ca. 2.2 Â 106 live ‘Ca. L. asiaticus’ per gram of aticus’ subsequently multiplies in the psyllid. It is plant tissue while some infected but asymptomatic important to delineate both types of acquisition plants had no detectable pathogen per gram of plant because some psyllids may not have propagated ‘Ca. L. tissue (Hu 2012). Further, the reliance on providing asiaticus’ although they have been exposed to the ‘Ca. L. asiaticus’ to D. citri using infected plants results pathogen during ingestion. Such psyllids might be con- in wide variations among tested psyllids fed on differ- sidered resistant to ‘Ca. L. asiaticus’ propagation. ent plants and on different plant tissues due to varia- Psyllids that are not exposed to ‘Ca. L. asiaticus’ during tions in ‘Ca. L. asiaticus’ titers between different plants ingestion due to a low titer, brief length of time of and tissues (Li et al. 2009). This is further compounded ingestion, or both, cannot be assessed for acquisition of due to wide variations in the amounts of phloem that propagated pathogen. Therefore, ensuring that psyllids psyllids ingest. Psyllids ingest phloem throughout the have been initially exposed to a measured dose of ‘Ca. nymphal stages through the adult, resulting in varying L. asiaticus’ is essential to characterizing D. citri vector amounts of phloem sap passing through their digestive competence. systems over time. Such variations likely influence vec- D. citri acquire (ingest) ‘Ca. L. asiaticus’ within as lit- tor competence variation among different tests and dif- tle as 15 min after feeding on an infected plant to as ferent D. citri. long as 5 h (Xu et al. 1988). The longer the period that The qPCR methods used to characterize ‘Ca. L. asi- the psyllid is allowed to ingest phloem from an infected aticus’ in plant and psyllid tissues may influence plant the greater the exposure and subsequent ‘Ca. L. reported ‘Ca. L. asiaticus’ titers. For example, qPCR asiaticus’ propagation. D. citri nymphs allowed to feed procedure is to generate a standard curve based on on infected plants resulted in more adult D. citri with known copies of plasmid containing ‘Ca. L. asiaticus’ the bacterium compared with when only the adult was DNA. The curve is used to determine the titer of the allowedtofeed(Inoue et al. 2009, Pelz-Stelinski et al. unknown ‘Ca. L. asiaticus’ DNA. This assumes that the 2010). D. citri containing ‘Ca. L. asiaticus’ that are plasmid DNA and pathogen DNA behave similarly in allowedtofeedonuninfectedplantsfor25dshoweda the qPCR reaction. Though Trivedi et al. (2009) report decrease in the proportion of infected psyllids over this is likely, there may be some conditions where time from nearly 100% infected in one initial group to pathogen DNA amplifies more efficiently than plasmid <10% infected remaining from this same initial group DNA. In addition, multiple copies of the target gene in (Fig. 1;datafromPelz-Stelinski et al. 2010). The reason ‘Ca. L. asiaticus’ will produce higher titers of ‘Ca. L. for the decline in the proportion of infected psyllids asiaticus’ DNA, compared with the standard curve. over time was not determined, but the authors believed There are also limits to the detection of low titers of it suggested that ‘Ca. L. asiaticus’ does not persist in the pathogen using qPCR. Trivedi et al. (2009) D. citri. They suggested that the loss of ‘Ca. L. asiat- observed that low plasmid titers of 2 Â 101 and 2 Â 100/ icus’-infected psyllids over time was due to host aging, ml did not amplify consistently, but the precision in negative effects of ‘Ca. L. asiaticus’ on D. citri, and the plant or psyllid tissue was not provided. absence of ‘Ca. L. asiaticus’ propagation in adult psy- Consider a psyllid that ingests 1,000 ‘Ca. L. asiat- llids. There is great variation in the decline over time icus.’ Would this be detected by standard qPCR techni- between the different groups of psyllids and between ques if 20 DNA templates proved needed for detection two different starting groups on the same day they via qPCR as reported by Trivedi et al. (2009)? Typically, were held on the healthy plants (Fig. 1). A significant the entire DNA extract is suspended in 50 mlfrom negative correlation was observed between percent which 1 ml is used to obtain the template for qPCR. infection and days on a healthy plant (Pelz-Stelinski Hence, even were 100% of the 1,000 ‘Ca. L. asiaticus’ et al. 2010). However, Figure 1, modified from the fig- remaining after the extraction and every single ‘Ca. L. ure provided by Pelz-Stelinski et al. (2010), suggests asiaticus’ copy was amplifiable during qPCR, ca. 44% that there is great variation between days 1–15 and that of the 1-ml aliquots from this extraction will contain less the negative correlation is largely the result of the than 20 ‘Ca. L. asiaticus’ templates based on a Poisson decline at day 20. Why should a precipitous decrease distribution of template DNA among the 1-mlsamples. in infected psyllids occur after 20 d on a healthy plant? Were only 100 templates ingested, the probability of a What does this say about D. citri vector competence? 1-ml sample with 20 or greater templates is 10À14 or 1 Information on the titers of live ‘Ca. L. asiaticus’ in 100 trillion! over time in these insects is essential to explain the There have been no reports on the detection limits decline in infected psyllids. It is possible that some of current qPCR methods for ‘Ca. L. asiaticus’ in infected psyllids at day 1, though positive using qPCR, 4JOURNAL OF ECONOMIC ENTOMOLOGY

Fig. 1. Percent retention of ‘Ca. Liberibacter asiaticus’ in infected D. citri reared as nymphs on infected citrus and placed on healthy citrus plants. The line connecting contiguous data points has been added to emphasize the greater variation between the early incubation periods. (Modified from Pelz-Stelinski et al. 2010). did not have infectious ‘Ca. L. asiaticus.’ Some portions The reasons for the variation in D. citri vector com- of the nucleic acid detected on day 1, or soon there- petence in various experiments unfortunately cannot after, were likely remnants from the ingested inoculum. be determined. Any or some combinations of all of the The length of time that remnant DNA survives or accu- following factors are likely the cause for any observed mulates in D. citri, the length of time needed for live variations: 1) differences in the psyllid populations used ‘Ca. L. asiaticus’ to propagate, and the amount in experiments, 2) differences in the titers of the patho- of ‘Ca. L. asiaticus’ that might be excreted in honeydew gen provided to the psyllids, 3) differences in time must be determined. These factors could all be influ- allowed for feeding, 4) differences in the plant host enced by the environment. If noninfectious DNA sur- physiology, 5) genetic variation in the pathogen, 6) dif- vives for ca. 20 d in the psyllid, some of the initial ferences in conditions of the diagnostic PCR methods, positives in the sample may not have had any ‘Ca. L. 7) variations in the diversity of the bacterial commun- asiaticus’ that multiplied in psyllid tissues, and further, ities in the guts of D. citri, and other possibilities not ‘Ca. L. asiaticus’ in the psyllid gut may decrease by mentioned here. The community of bacteria in D. citri being excreted in honeydew. If the original 100% test- is complex, dynamic, and only beginning to be appreci- ing positive for ‘Ca. L. asiaticus’ on day 1 did not con- ated, although it is already apparent that different tain live pathogen, then the percent of psyllids infected bacterial species are correlated with the presence of with propagating ‘Ca. L. asiaticus’ in this study may be ‘Ca. L. asiaticus’ (Fagen et al. 2012). The effect of vary- only the 10% or so of those remaining with ‘Ca. L. asi- ing the compositions of these communities on D. citri aticus’ at 25 d. It remains unclear whether there could competence is unknown. In view of the host of poten- be a further decline in the percent infected beyond tial unknown variations that influence D. citri vector 25 d, perhaps to zero. competence, there is little useful information to gauge It is possible that ‘Ca. L. asiaticus’ does not multiply which of these contribute to different observations. in the tissues of some D. citri. This possibility deserves Many studies of arthropod vector competence have attention. ‘Candidatus L. asiaticus’ is a phloem-limited found a direct relationship between the titer of the bacterium that has been difficult to culture. Perhaps pathogen provided to the arthropod in the ingested ‘Ca. L. asiaticus’ only multiplies in the lumen of the D. inoculum and its subsequent propagation. The proba- citri alimentary tract that contains phloem. The bacte- bility of acquiring propagating pathogen is directly rium may accumulate in other tissues; it can be passed related to the probability of the vector initially being to some offspring and during mating (Mann et al. exposed to the pathogen which is directly tied to the 2011), but it may not multiply in all psyllid tissues. If titer of infectious pathogen in the original source and ‘Ca. L. asiaticus’ does not multiply in all the tissues of the volume of ingested inoculum (Lord et al. 2006). some D. citri, these psyllids may accumulate detectable Coletta-Filho et al. (2014) observed this with D. citri pathogen in the gut, but over time the bacterium may competence for ‘Ca. L. asiaticus.’ There is no threshold be flushed out in the honeydew and hence the decline of titer below which arthropods cannot be infected if in numbers of positive D. citri over time on healthy one uses a probabilistic approach concerning titer plants observed by Pelz-Stelinski et al. (2010). (Lord et al 2006). A low titer results in more ingested 2015 TABACHNICK:PSYLLID VECTOR COMPETENCE 5 inoculum without sufficient infectious pathogens and uninfected citrus resulted in ca. 40% with ‘Ca. L. asiat- therefore most arthropods are never exposed to enough icus.’ Given the presumed titers of ‘Ca. L. asiaticus’ in pathogens to be detected or propagate. This means symptomatic plants of 6–8 logs/g of plant tissue, one that to observe a positive arthropod with low titer inoc- must explain the almost complete absence of psyllids at ulum might require testing 100s or 1,000s of arthro- 6d with ‘Ca. L. asiaticus’ or evidence of exposure to pods to increase the probability of detecting the rare the pathogen. If D. citri ingests only 1 ml over 6 d they positive. At 103 pathogens/ml, among 1-mlinoculums will have ingested ca. 1,000–100,000 ‘Ca. L. asiaticus’ provided to a psyllid, 38% contain 0 ‘Ca. L. asiaticus’ from plants with titers of 6–8 logs/ml. Possible explana- and only 6% contain 3 or more pathogens assuming tions include that the titer in the plants and the random distribution of the pathogen in phloem. Any amounts fed to the psyllids were too low to ensure inefficiency of qPCR will reduce the number of detec- detectable pathogen in the PCR samples from the psy- tions from samples with a low template copy number llids even after a 7-d incubation period. The incubation as described previously. Most investigators do not test period, even for psyllids ingesting the pathogen on day the large numbers that would be required. This could 1 in this study (a total of 13 d), would have to be too be overcome by performing multiple qPCR tests on short for multiplication to detectable titers, and that every sample to reduce the numbers of false negatives. psyllids that did ingest ‘Ca. L. asiaticus’ excreted most Research is needed to determine the titer of infec- in the honeydew they make before the pathogen tious material in the volume actually ingested by differ- multiplied in the insect. Unfortunately the titers of the ent D. citri and the titer of live infectious ‘Ca. L. pathogen provided to these psyllids were not described; asiaticus’ per volume of psyllid tissue. The use of ‘Ca. there is little information in the literature about the L. asiaticus’ genome equivalents/mg total DNA that is volume of honeydew production, and there is no often provided must assume the psyllid’s contribution information about ‘Ca. L. asiaticus’ in D. citri to the total DNA is constant. Trivedi et al. (2009) honeydew. reported a mean of 3.9 Â 104 genome equivalents/mg Assume that the titer in the phloem ingested by the total DNA in asymptomatic citrus leaf midribs and insects fed by Pelz-Stelinski et al. (2010) was 6 logs/g 5.86 Â 106 genome equivalents/g of tissue, a 2 log dif- or ml of phloem. The amount of honeydew produced ference in titer between the two units of measure. The has been used as a proxy for measuring the amount of latter figure assuming a gram of phloem is equivalent food that is ingested (Boina et al. 2009, Hall et al. to 1 ml leads to the ingestion of 1,000 genome equiva- 2010, Avery et al. 2011). Avery et al. (2011) reported lents in 1 ml of phloem assuming that ‘Ca. L. asiaticus’ that D. citri produced an average of 5.5 honeydew is randomly distributed in phloem. However, reporting drops per day over the first 7 d of feeding and began titers as micrograms of total DNA does not provide the producing honeydew in the first 24 h of feeding. number of ‘Ca. L. asiaticus’ genome equivalents in 1 ml Though the volume of honeydew produced was not of phloem. Hence, psyllids ingesting 5 ml after feeding reported, D. citri produces honeydew once stylets on a symptomatic citurs host, titer of 106 genome reach the phloem (Bonani et al. 2010). In view of equivalents/g tissue, should receive ca. 5,000 genome their feeding activity what can account for the absence equivalents assuming bacteria are randomly distributed of ‘Ca. L. asiaticus’ in psyllids feeding for as long as in phloem! The ability to characterize titers in citrus or 6 d? The production of honeydew using Avery et al. D. citri wouldneedtobereassessedif‘Ca. L. asiaticus’ (2011) is ca. 30–35 drops. Assume that the volumes is nonrandomly distributed, for example, if it aggregates produced are 0.1, 1, or 10 ml of phloem over 6 d. If the in biofilms (Danhorn and Fuqua 2007). Then the prob- titer in the phloem is 106/g and phloem has the same ability of ingesting bacteria in phloem or inoculation by density as water, then 1 ml of phloem will contain1000 psyllids may depend on the distributions of the biofilms ‘Ca. L. asiaticus,’ again assuming ‘Ca. L. asiaticus’ is in the phloem or psyllid. randomly distributed. A gram of phloem is likely less Typically investigators have found ‘Ca. L. asiaticus’ than 1 ml and ‘Ca. L. asiaticus’ may not be randomly titers in citrus on the order of 106–108 genome equiva- distributed. lents/g of plant tissue (Trivedi et al. 2009, Coletta-Filho During the first 6 d of feeding, using the assumptions et al. 2014), though tissues can vary greatly (Li et al. above and ingesting 0.1–10 mlat106/ml, psyllids ingest 2009). Titers in the phloem of different plant tissues 100 to 10,000 ‘Ca. L. asiaticus.’ At 108/ml psyllids ingest have ranged from 14–137,000 bacterial cells/mgoftotal 10,000–1,000,000 ‘Ca. L. asiaticus’! Yet there were no DNA (Tatineni et al. 2008). This information is instruc- psyllids containing ‘Ca. L. asiaticus’ after 6 d of feeding tive for interpreting observations on D. citri vector followed by another 7 d of multiplication of the patho- competence. For example, Figure 2 (modified from gen. Some possible explanations include that 1) the live Pelz-Stelinski et al. 2010)showsthatD. citri adults titer of ‘Ca. L. asiaticus’ in the phloem is less than 106/ allowed to feed on infected citrus for 1–6 d and then ml, 2) ‘Ca. L. asiaticus’ is not randomly distributed in subsequently held for 7 d on uninfected citrus to allow phloem and occurs in biofilms that are not easily for propagation of ingested ‘Ca. L. asiaticus’ resulted in ingested and digested by the psyllid, 3) under the con- no adults with evidence of being exposed to ‘Ca. L. asi- ditions of the study the period of incubation (maximum aticus.’ Adults ingesting phloem between 7–13 d and of 13 d for those ingesting pathogen on day 1 and held held for incubation a further 7 d on uninfected citrus 7 added days) was too short to obtain detectable titers, resulted in nearly 15% with ‘Ca. L. asiaticus,’ and those 4) the method of PCR misses psyllids containing 100 or allowed to feed for >35 d followed by another 7 d on so ‘Ca. L. asiaticus’ as explained above, and 5) many of 6JOURNAL OF ECONOMIC ENTOMOLOGY

Fig. 2. Percent of D. citri positive for ‘Ca. Liberibacter asiaticus’ after feeding on infected citrus for varying acquisition periods. Means of adult psyllids with different letters are significantly different. (Modified from Pelz-Stelinski et al. 2010). the ingested bacteria are not absorbed and quickly pass ‘Ca. L. asiaticus’ in a psyllid population using current through the insect digestion in the honeydew. qPCR may require more than 25 d to assure that there As explained previously, at 103/ml of live ‘Ca. L. asi- is sufficient time to ingest sufficient live pathogen, aticus’ ca. 37% of all 1-ml aliquots of phloem will not allow for multiplication of low titer ‘Ca. L. asiaticus’ in contain a live ‘Ca. L. asiaticus.’ Psyllids ingesting low the psyllid tissues and alimentary system that can be numbers of pathogen, i.e., 1–10, would be missed using detected using current qPCR methods, and may qPCR on the normal small aliquot of the insect homo- require qPCR on several aliquots of each individual to genate. This is consistent with the few positive psyllids detect low titers. on days 1–6 post ingestion, and then only 15% on days Pelz-Stelinski et al. (2010) found no difference 7–13 (Pelz-Stelinski et al. 2010). Among the 15% posi- between the 7–13, 14–20, and 21–27 day groups in tives detected after 7–13 days on infected citrus numbers of psyllids with ‘Ca. L. asiaticus’ and a signifi- followed by 7 d for multiplication were likely some psy- cant increase only between the 21–27, 28–35, and >35 llids that ingested ‘Ca. L. asiaticus’ in the first 6 d with day groups (Fig. 2). Each of these groups contains psy- detectable titers due to a total 14–20 d for multiplica- llids infected during the earlier feeding periods for that tion. Therefore, the absence of psyllids with ‘Ca. L. asi- group with subsequent detectable titer due to the 10 aticus’ observed after feeding on infected plants for days that may be required for detectable propagation. 1–6 d is likely an artifact of the low titer in the ingested In the 7–13 day group, positives would include those inoculum, the likelihood of ingesting small volumes of ingesting ‘Ca. L. asiaticus’ on days 1–10 with detectable phloem, the length of the required incubation period ‘Ca. L. asiaticus’ with the added 7 d on uninfected cit- for detectable ‘Ca. L. asiaticus’ titers, and the methods rus. Those ingesting pathogen on days 11–13 would not of qPCR employed to test the insects. have detectable ‘Ca. L. asiaticus’ until days 21–23 and There are two reasons for the greater detection of would only be detected during the analysis of psyllids infected psyllids with increased lengths of time for ingesting phloem for days 21–27 and so on. As a result ingestion on infected plants. First the insects ingest the percent with detectable ‘Ca. L. asiaticus’ should be more food with increased ingestion duration and there- higher in every subsequent group. The lack of a signifi- fore have encountered more live ‘Ca. L. asiaticus’. cant difference between 7–13, 14–20, 21–27, and Secondly there is greater time for propagation and between 21–27 and 28–34 groups reported by Pelz-Ste- accumulation in the psyllids resulting in an increase in linski et al. (2010) may be due to the low sample sizes the detectable titer and probability of a qPCR positive. that did not distinguish differences and/or there could This is likely the same two reasons why nymphs feeding be differences between psyllid feeding mechanisms on infected plants provide adults with higher infection over time and/or variations in psyllid processing and rates. Unfortunately many reported observations of multiplication of ‘Ca. L. asiaticus’ during this study. uninfected psyllids are likely due in part to these These effects need to be evaluated. and other artifacts from the methods employed as dis- D. citri Latent Period and Ability to Inoculate cussed here. ‘Ca. L. asiaticus’. The latent period is considered the The observations by Pelz-Stelinski et al. (2010) sug- time period from when the vector arthropod ingests gest that to characterize the acquisition of propagating the pathogen to the time when that pathogen can be 2015 TABACHNICK:PSYLLID VECTOR COMPETENCE 7 inoculated into a host. It is not possible to accurately exposed as nymphs were still uninfected (Pelz-Stelinski characterize the latent period or the time it takes for et al. 2010). Though 40–60% of D. citri fed on infected ‘Ca. L. asiaticus’ to colonize and propagate in psyllids plants became PCR positive, only 4–10% of these could without the information about the titer of ingested inoculate ‘Ca. L. asiaticus’ (Pelz-Stelinski et al. 2010). pathogen. The challenges in understanding the causes of The previous discussion of the results in Figure 2 reduced vector competence and being able to interfere suggests that under the conditions of the Pelz-Stelinski with any of the controlling mechanisms enabling inocu- et al. (2010) study, the latent period to detectable ‘Ca. lation by D. citri are great. It is apparent that studies L. asiaticus’ in adult D. citri may be as long as 10–13 d. must be able to accurately characterize the vector com- ‘Candidatus L. asiaticus’ may multiply very slowly in petence phenotype, which will require the ability to the D. citri digestive system or in other psyllid tissues. provide defined accurate titers of live ‘Ca. L. asiaticus’ Assuming, following the discussion above, that 103 bac- known amounts delivered to the psyllid, assess the teria/ml is the minimal titer required to detect ‘Ca. L. effects of ‘Ca. L. asiaticus’ genetic diversity, the influ- asiaticus’ in individual D. citri. It would require ca. 10 ence of temperature and the effects of varying doses of multiplication cycles starting from a single bacterium ‘Ca. L. asiaticus’ at various temperatures on specific or 7 cycles starting from 10 bacteria. Therefore, the aspects of the complex competence phenotype, i.e., gut doubling time for ‘Ca. L. asiaticus’ in D. citri may be infection, salivary gland infection, and inoculation. For ca. once per day. No information is available on the example, many studies maintain the psyllids at a tem- growth characteristics of ‘Ca. L. asiaticus’ in D. citri. perature of 25C. There are numerous examples with ‘Candidatus L. Asiaticus’-Resistant D. other arthropod–pathogen systems demonstrating the citri. Understanding the mechanisms controlling influence of temperature, and also demonstrating that D. citri competence for ‘Ca. L. asiaticus’ is especially the influence of increasing temperature on various important to the efforts to release D. citri incapable of competence traits was not linear (Tabachnick 2013). inoculating ‘Ca. L. asiaticus’ to replace naturally occur- Therefore, observations on susceptibility to infection at ring D. citri. In effect, by reducing or eliminating com- 25C provide little useful information on ‘Ca. L. asiat- petent D. citri one might reduce or eliminate the icus’ at other temperatures and cannot be used to pre- spread of ‘Ca. L. asiaticus.’ dict how different D. citri populations would react at Several approaches to develop incompetent D. citri 25C or any other temperature. The same is likely true are currently being pursued as part of the Nupsyllid for the known different genetic variants of ‘Ca. L. asiat- Project (http://vivo.usda.gov/display/NIFA-0230893- icus’ (Wang and Trivedi 2013). PROJ). Mechanisms or pathways influencing psyllid Whichever method is successful in developing an vector competence could be identified and novel incompetent psyllid, i.e., through artificial selection of agents, i.e., RNAi, proteins, and chemicals found that naturally occurring resistance for being able to inocu- interfere with those mechanism. Comparisons between late or interference of some controlling aspect of psyllid transcriptome analyses of the potato psyllid, Bactericera competence using RNAi or other agents, it will then cockerelli (Sulc), infected with ‘Candidatus L. sol- require understanding of the ability to spread reduced ancaearum’ and D. citri with ‘Ca. L. asiaticus’ were competence to replace natural D. citri populations. It used to identify hundreds of common differentially will then be essential to understand possible effects of expressed genes for insight into conserved controlling interfering with competence on D. citri fitness. Success genes that might be targeted with greater chance of in decreasing the competence of a psyllid population successfully interrupting transmission (Fisher et al. will require knowledge of the adaptive functions of the 2014). Among these were genes influencing biological blocked pathway. adhesion, immune system processes, locomotion, and Arthropod competence for pathogens is likely the reproduction, all believed to have relevance to psyllid consequence of the evolution of vector competence vector competence. mechanisms that have other adaptive functions An alternative approach to reduce vector compe- (Tabachnick 2013). For example, D. citri has five other tence is to identify naturally occurring resistant psyllids congeneric species of Diaphorina that have been and produce them in large enough numbers for release reported on citrus and other closely related plants. to shift the frequencies in natural populations toward Only D. citri and the African psyllid Trioza erytreae reduced competence (Powell and Tabachnick 2014). (del Guercio) are known vectors of both ‘Ca. L. asiat- Naturally occurring psyllids with reduced transmission icus’ and ‘Ca. L. africanus’ (Halbert and Manjunath ability could also be used to identify the causal mecha- 2004). T. erytreae transmits ‘Ca. L. asiaticus’ in the lab- nism controlling naturally occurring resistance in oratory (Massonie et al. 1976) and is the only known D. citri that could then be released into natural popula- vector species of the 10 other species in its group. tions and spread using genetic drive systems, i.e., trans- Therefore, vector competence for ‘Ca. L. asiaticus’ genic and paratransgenic approaches described evolved in two disparate species of psyllids, each with elsewhere (Aksoy 2008, Wang and Jacobs-Lorena near neighbors without this ability. This makes it 2013). Many studies cited above have shown that there unlikely that mechanisms governing competence for a are D. citri without propagation or accumulation of vector-borne pathogen are ancestral to different species ‘Ca. L. asiaticus’ under the conditions of the study. in such disparate genera. It is more likely that the trait For example 60% of the psyllids did not have ‘Ca. L. evolved independently in such distantly related species asiaticus’ after >35 d of feeding, and 40% of the adults (Tabachnick 2013). D. citri competence for ‘Ca. L. 8JOURNAL OF ECONOMIC ENTOMOLOGY asiaticus’ is therefore likely a byproduct of adaptations medium. If ‘Ca. L. asiaticus’ propagation does not for yet unknown functions not necessarily related to occur in psyllid tissues then serial dilutions could be vector competence. This line of reasoning should lead provided directly to the lumen of the alimentary canal to caution in using very different vector–pathogen sys- using enemas as described elsewhere (Klowden 1981). tems to identify useful controlling genes for interrupt- Intrathoracic inoculation could be used to assess ‘Ca. ing inoculation. For example, using B. cockerelli L. asiaticus’ growth characteristics in D. citri tissues. competence for ‘Ca. L. solancaearum’ for transcrip- It is imperative to provide information on the influ- tome comparisons with D. citri competence for ‘Ca. L. ence of the dose of live ‘Ca. L. asiaticus’ and acquisi- asiaticus’ (Fisher et al. 2014) is more likely to identify tion in psyllids under an array of conditions, i.e., both very conserved genes with likely high fitness effects. constant and varying temperatures and different ‘Ca. Interfering with such conserved genes will likely reduce L. asiaticus’ genetic variants. The inability to culture fitness, making it more difficult to spread the interfer- ‘Ca. L. asiaticus’ causes difficulties in providing con- ing agent in nature. stant titers to psyllids and means many inoculation studies must be done in plants that can require months to evaluate. It would be helpful to assess inoculation by Suggestions for Future Research psyllids using the presence of ‘Ca. L. asiaticus’ in dis- The citrus industry urgently requires ways to reduce sected psyllid salivary glands or in psyllid saliva as a the impact of citrus greening. One way is to alter psy- proxy for inoculation that would have to be experimen- llid populations to be incapable of transmitting ‘Ca. L. tally validated. Different psyllid transmission routes asiaticus.’ There are many challenges to overcome due must be considered, i.e., the potential for transmission to the absence of critical information. Issues that have via honeydew, cofeeding, or both. If psyllids directly been reviewed here concerning inoculum titer, qPCR inoculate other psyllids feeding in proximity to one diagnostics, D. citri feeding, characterizing exposure another, then systemic pathogen in phloem is unneces- and propagation, latent period, live versus dead ‘Ca. L. sary for acquisition. Nonsystemic transmission of asiaticus,’ D. citri transmission mechanisms, ‘Ca. L. asi- arthropod-borne pathogens influences pathogen epi- aticus’ multiplication in D. citri tissues, and the influ- demiology (Lord and Tabachnick 2002). ence of honeydew production must be resolved to The challenges to using incompetent D. citri for name just a few. large-scale releases or through genetic drive mecha- One solution to the problem measuring live ‘Ca. L. nisms may be insurmountable with current methods asiaticus’ titers is to employ serial dilutions of plant or (Tabachnick 2003). Hundreds of candidate genes are psyllid tissue to intrathoracically inoculate live unin- being identified between infected and uninfected psy- fected D. citri,asdescribedformosquitoes(Gubler llids (Fisher et al. 2014). Many potential genes or and Rosen 1974). Intrathoracic inoculation or injecting agents that might interfere with an aspect of D. citri pathogen directly into the body of a vector is a very competence may not be useful due to their inability to permissive method to infect the vector because it interfere in nature because of naturally occurring bypasses many barriers to propagation in vectors where genetic and environmental variation. Many of these multiplication of the pathogen in vector tissues has candidate genes will likely have negative fitness conse- been observed. D. citri inoculated with a serial dilution quences on D. citri that will impede their ability to of either plant or psyllid tissue and held for a suitable be dispersed in nature. In addition, genetically modi- incubation period of 10–13 d could then be character- fied organisms (GMOs) are generally unacceptable to ized for propagated ‘Ca. L. asiaticus’ using qPCR. In the public. Selecting a mechanism that is the cause of addition, it will be important to test several aliquots of naturally occurring reduced competence may be tech- every insect with qPCR to reduce the number of false nically easier to accomplish using artificial selection negatives resulting from the Poisson distributions that methods, does not require understanding of the mech- occur with low pathogen titers or the possibility that anism, is not a GMO, and is naturally occurring with ‘Ca. L. asiaticus’ is not distributed randomly in insect likely small negative fitness consequences (Powell and tissues or in the resulting homogenates. The last serial Tabachnick 2014). However, this approach is depend- dilution providing a positive D. citri is the end point to ent on understanding environmental influences on the provide a titer in units of psyllid infectious doses50 per competence phenotype because artificial selection is psyllid following methods described elsewhere (Reed very inefficient when phenotypic variation is largely and Muench 1938). The technique would provide a due to environmental variation. The environmental var- quantifiable measure of live pathogen in any material iation contributing to variation in D. citri vector compe- and could be used to generate standard curves for tence for ‘Ca. L. asiaticus’ even in the laboratory is qPCR. Intrathoracic inoculation can be used as a direct virtually unknown. Many likely contributing factors test for the ability of ‘Ca. L. asiaticus’ to multiply in influencing competence in the laboratory have already psyllid tissues in the absence of phloem. The impor- been presented above. In addition, without a genetic tance of plant phloem as the only medium for ‘Ca. L. drive mechanism large numbers of incompetent psy- asiaticus’ propagation would be supported if the patho- llids would be required for release to have any influ- gen does not multiply after intrathoracic inoculation. ence in shifting frequencies in nature toward reduced Controls would be needed to ensure that live ‘Ca. L. competence. asiaticus’ can be maintained in a serial dilution medium Causes of variation must be resolved to move and perhaps phloem would be needed for the dilution forward toward the goal of an incompetent D. citri. 2015 TABACHNICK:PSYLLID VECTOR COMPETENCE 9

The difficult challenges ahead will be to apply appro- for the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psy- priate methods to identify causes of variation in D. citri llidae). Biocontrol Sci. Technol. 9: 1065–1078. competence, identify the factors that influence these Benyon,L.,L.Zhao,A.Weathersbee,andY.Duan.2008. causes in nature, identify agents that interfere with Nested PCR is essential for the detection of extremely low competence, and develop methods to introduce one or titer of ‘Candidatus liberibacter asiaticus’ from citrus and its vector psyllid Diaphorina citri. Phytopathology 98: S21. a combination of the interfering mechanisms into natu- Boina,D.R.,E.O.Onagbola,M.Salyani,andL.L.Stelin- ral D. citri populations to reduce transmission. The ski. 2009. Antifeedant and sublethal effects of imidacloprid importance of understanding the norms or reaction on Asian citrus psyllid, Diaphorina citri.PestManage.Sci. and the population genetics of vector competence 65: 870–877. genes have been discussed elsewhere (Tabachnick and Bonani, J. P., A. Fereres, E. Garzo, M. P. Mitanda, B. Black 1995; Tabachnick 2003, 2013) and must be Appezzato-Da- Gloria, and R. S. Lopes. 2010. Character- applied to D. citri. The needed information on D. citri ization of electrical penetration graphs of the Asian citrus vector competence will require a substantial effort to psyllid, Diaphorina citri, in sweet orange seedlings. Entomol. have any chance for using vector competence to control Exp. Appl. 134: 35–49. Bove´, J. M. 2006. Huanglongbing: a destructive, newly- greening. Many of the issues presented here could emerging, century-old disease. J. Plant Pathol. 88: 7–37. interfere with progress toward successful control. Some Coletta-Filho,H.D.,M.P.Daugherty,C.Ferreira,and of the issues may be less important to some strategies. J.R.S. Lopes. 2014. Temporal progression of Those researchers involved in the effort must set pru- ‘Candidatus Liberibacter asiaticus’ infection in citrus and dent priorities and reflect on their strategies with the acquisition efficiency by Diaphorina citri. Phytopathology realization that they must carefully evaluate the effect 104: 416–421. of disregarding some of the issues that have been Coy,M.R.,M.Hoffman,H.N.Kingdom,E.H.Kuhns,K.S. raised. Pelz-Stelinski and L. L. Stelinski. 2014. Nested- It is not possible to predict which approach for quantitative PCR approach with improved sensitivity for the detection of low titer levels of Candidatus Liberibacter asiati- developing and using an incompetent D. citri is likely cus in the Asian citrus psyllid, Diaphorina citri Kuwayama. to be successful. A psyllid incapable of inoculating ‘Ca. J. Microbiol. Methods 102: 15–22. L. asiaticus’ will not reduce the damage to an already Da Grac¸a,J.V.,andL.Kirsten.2004.Citrus huanglongbing: infected citrus and the damage done to citrus by the review, present status and future strategies, pp. 229–245. In psyllids themselves. An integrated approach to manag- S. Nagvi (ed.), Diseases of fruits and vegetables, Vol. 1, ing the disease will require improved treatment to Kluwer, Dordrecht, The Netherlands. reduce greening symptoms, replanting with uninfected Danhorn, T., and C. Fuqua. 2007. Biofilm formation and perhaps new greening-resistant citrus varieties, and by plant-associated bacteria. Annu. Rev. Microbiol. 61: continued application of psyllid control measures. 401–422. Fagen,J.R.,A.Giongo,C.T.Brown,A.G.Davis-Richard- son, K. A. Gano, and E. W. Triplett. 2012. Characteriza- tion of the relative abundance of the citrus pathogen Ca. Acknowledgments Liberibacter asiaticus in the microbiome of its insect vector, Diaphorina citri, using high throughput 16s rRNA sequenc- The helpful comments of Barry Alto, Jonathan Day, and Cyn- ing. The Open Microbiol. J. 6: 29–33. thia Lord are greatly appreciated. I thank John Trumble for Fisher,T.W.,M.Vyas,R.He,W.Nelson,J.M.Cicero,M. his assistance in the review process. I greatly appreciated the Willer, R. Kim, G. A. May, J. A. Crow, C. A. Soderland, efforts and thoughtful suggestions provided by three anony- et al. 2014. Comparison of potato and Asian citrus psyllid mous reviewers for educating this inveterate mosquito vector adult and nymph transcriptomes identified vector transcripts biologist about vectors of plant pathogens though with admit- with potential involvement in circulative, propagative Liberi- tedly not 100% success. Their suggestions and ideas greatly bacter transmission. Pathogens 2: 875–907. improved the manuscript. 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