Timothy A. Ebert

Transmission Parameters for Candidatus Liberibacter asiaticus by Asian Citrus Psyllid (Hemiptera: Psyllidae)

ABSTRACT 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 ≈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.

Reexamining the Pooled Sampling Approach for Estimating Prevalence of Infected Insect Vectors

ABSTRACT Our goal was to estimate seasonal changes in the proportion of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), carrying Candidatus Liberibacter asiaticus. Our approach was to test Asian citrus psyllid by using pooled samples. The initial question was about pool size and the consequences of choosing poorly. Assuming no loss in sensitivity when diluting one infected individual with many healthy individuals, then it is recommend that a combination of all the published limits be used: keep the number of pools (n) above 20, the pool size (k) below 100, and the number of infected pools less than half the total number of pools. The most conservative approach to achieving the latter is to optimize pool size given an infection rate (p) such that k = ln(0.5)/ln(1 p). Exceeding these limits increases the probability that all the pools will be infected. If this occurs, then that particular sample will be discarded. Use of multiple pool sizes can be used to manage this risk, but this approach may not always be practical. PooledInfRate is a good program for estimating prevalence, and it is available for free from the Centers for Disease Control and Prevention (CDC). The program provides corrected confidence intervals for prevalence estimates using one or multiple pool sizes. We used a randomization test approach as a contrasting methodology. The bias corrected CDC 95% confidence interval is an upper bound to the “true” 95% confidence interval, and we provide an estimate of the magnitude of the remaining bias in the estimate.

Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real-time PCR

BackgroundHuanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the β-operon, have been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las now allows the design of primers beyond the conserved regions for the detection of Las explicitly.ResultsWe took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR. Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related species Ca. L. americanus (Lam) and Ca. L. africanus (Laf).ConclusionsWe have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB.

Effect of Substrate Voltage on EPG Recordings of Ingestion and Probing Behavior in Diaphorina citri (Hemiptera: Liviidae)

Abstract Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a major pest of citrus production because it is the vector of Candidatus Liberibacter asiaticus (Rhizobiales: Rhizobiaceae), which causes Asiatic huanglongbing, a devastating disease of citrus. Understanding the probing and ingestion behavior of the vector is important in understanding pathogen transmission and possible strategies to reduce disease incidence. We assessed the feeding behavior in D. citri by using electropenetrography, wherein a small electric current is passed through the insect. Changes in circuit voltage are recorded, and patterns of voltage changes are subsequently correlated with specific behaviors. However, different laboratories use different equipment with varying applied voltages. It is axiomatic that there will be some voltage at which there will be a change in the behavior of the insect. Current equipment has a range from 0 to 1300 mV, but studies where voltage was reported were in the range from 20 to 600 mV. The purpose of this study was to determine the behavioral response of D. citri to voltages in this range. Our results demonstrated that 600 mV DC with an input impedance of 109 ohms was below the threshold where D. citris feeding behaviors were affected. Thus, the outcomes of past studies using different voltages should not have been affected. However, in the present study, we did find that choice of host plant altered D. citris feeding behavior; thus, it would be beneficial to standardize the host plant if you were studying the effects of non-host factors that may influence feeding, such as insecticides, interspecific competition, or abiotic conditions.

A new SAS program for behavioral analysis of electrical penetration graph data

We compare three programs for analysis of electrical penetration graph (EPG) data.We provide a dictionary to compare these programs and classify variables.We present an open source program written in SAS for the analysis of EPG data.The new program takes raw data and produces a finished statistical analysis.The program works for any aphid-like insect whose stylet activities are monitored using EPG. Monitoring feeding behaviors of insects whose piercing-sucking mouthparts are inserted into plant tissue is often done by making the insect part of an electronic circuit, using a technique called Electrical Penetration Graph, or electropenetrography (both abbreviated EPG). Fluctuating voltage signals in the circuit are graphed, and resulting waveforms are interpreted by a researcher as specific stylet activities. After measurement of waveforms, data consist of a list of different behaviors and associated durations. These data are further processed to yield hundreds of variables that are compiled and statistically analyzed prior to publication. The goal of this study was to develop a program to make this process more efficient for studies of aphids and related species, given the large quantity of data expected to be generated. Herein, the three major existing programs that perform this function are reviewed. The oldest program (Backus 1.0) both compiles data and calculates a SAS-based statistical analysis; however it only works with the original, recorded variables and is not tailored to aphid studies. The other programs (EPG Calc and the Sarria Excel? workbook) compile a more diverse suite of derived variables suitable for aphids than does Backus 1.0; however, they do not include statistical analyses. A new program (Ebert 1.0) introduced herein uses SAS to calculate the diverse suite of derived variables for aphids, and also provides statistical analysis via powerful mixed-model ANOVA using a single software platform, similar to the Backus program. The code is open source, so that any researcher can adapt this program to deal with behavioral idiosyncrasies of a particular study insect. The new program will be especially valuable for large experiments with many insect subjects.The Backus 1.0 system for classifying variables required some modification in order to deal with all the derived variables for aphids. The new classification system has five levels: Cohort, Insect, Probe, Waveform, and Event. Within each of these levels, variables can be sequential or non-sequential, and these are further subdivided into conditional and non-conditional. These changes will facilitate design of more complex experiments in the future, and the ultimate adaptation of this analysis technique designed around aphids for use with other organisms. There is supplemental material included with the manuscript to assist with understanding the nature of data generated using EPG methods, and the complex task of extracting knowledge from a vast quantity of data generated by these experiments.

Diptilomiopus floridanus (Acari: Eriophyoidea: Diptilomiopidae): Its Distribution and Relative Abundance with Other Eriophyoid Species on Dooryard, Varietal Block, and Commercial Citrus in Florida

Abstract We sampled 526 dooryard, 18 varietal block, and 784 commercial citrus trees in Florida between May 2009 and Apr 2014 for eriophyoid mites including Diptilomiopus floridanus Craemer & Amrine, Aceria sheldoni (Ewing), Aculops pelekassi (Keifer), and Phyllocoptruta oleivora (Ashmead). In total, 1,423 D. floridanus individuals were collected from dooryard citrus trees and 1 each from the lemon cultivar ‘Bearss’ and sweet lime trees from the Florida Citrus Arboretum in Winter Haven. Diptilomiopus floridanus was collected from dooryard citrus in the following counties in Florida: Broward, Collier, Dade, Indian River, Lee, Martin, Palm Beach, Pinellas, Polk, Sarasota, and St. Lucie. The mite was not observed in Charlotte, Hardee, Hendry, Highlands, Manatee, or Okeechobee counties (Florida). Percentages of the eriophyoid mite species collected from dooryard citrus trees and varietal blocks were: A. pelekassi 3.2%, A. sheldoni 9.1%, D. floridanus 17.3%, and P. oleivora 84.2%. The 6 counties with the highest percentages of D. floridanus on dooryard trees were: Collier 36%, Broward 25%, Indian River 25%, Palm Beach 25%, Martin 23%, and St. Lucie 22%. In commercial citrus, A. sheldoni was collected less than 1.0%, A. pelekassi 4.0%, and P. oleivora 75.5%, of the time. Diptilomiopus floridanus was not collected in commercial citrus orchards during this survey. Diptilomiopus floridanus had significantly greater infestation rates on lime and lemon trees compared with grapefruit, tangerine, tangelo, sweet orange, and pummelo trees. However, infestation rates on lime and lemon trees were not significantly different from those on sour orange trees.

Handling Artificially Terminated Events in Electropenetrography Data

Abstract Electropenetrography a.k.a. electrical penetration graph or EPG is a rigorous technique for studying arthropod behavior. Essentially, the arthropod and host are part of an electrical circuit that generates patterns of voltage (waveforms), whose biological meanings are defined by correlation with histology and behavior. EPG is used for studying stylet probing behavior of hemipterans, and blood-feeding arthropods. These results are applied to understanding pesticide action, host plant resistance, and vector-pathogen–host interactions. At the end of all recordings, the arthropod begins a behavior that ends because the scientist stopped recording. An argument for keeping this event in the data has been made based on the assumption that the insect is adapting to laboratory conditions. In this adaptation process, the expected durations of ingestion behaviors will increase as the insect adapts. We show that this assumption can cause problems in data analysis and interpretation of the data. If the assumption is false, then there are more options for analyzing the data. Deleting artificially terminated events can be advantageous, but the best approach needs to consider the biology of the arthropod and align with research objectives.

Erratum for Killiny et al., “A Plant Bacterial Pathogen Manipulates Its Insect Vector's Energy Metabolism”

39. Ghanim M, Fattah-Hosseini S, Levy A, Cilia M. 2016. Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus. Sci Rep 6:33418. https:// doi.org/10.1038/srep33418. Citation Killiny N, Hijaz F, Ebert TA, Rogers ME. 2017. Erratum for Killiny et al., “A plant bacterial pathogen manipulates its insect vectors energy metabolism.” Appl Environ Microbiol 83:e00910-17. https://doi.org/10.1128/AEM .00910-17. Copyright